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 Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 62
Page 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 

Page 2 of 62
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: NORFLURAZON

OFFICE OF PESTICIDE PROGRAMS 

OFFICE OF SCIENCE COORDINATION AND POLICY 

U.S. ENVIRONMENTAL PROTECTION AGENCY 

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NORFLURAZON 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 Relevant 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-Estrogenic 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..........................53 

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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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

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 Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol 

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

HPLC/MS/MS High Pressure Liquid Chromatography/Mass Spectroscopy

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

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 Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

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v 

Abbreviation Terminology 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles 

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

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vi 

Abbreviation Terminology 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Page 1 of 54 

Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 norflurazon interacts with E, A or T hormone pathways, the number and 

type of effects induced, the magnitude and pattern 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 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of evidence (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). The positive ERTA assay result was difficult to 

interpret, given the effect was not inhibited when the cells were treated with a known ER 

antagonist, suggesting a false positive response. In the Tier 1 pubertal assay, decreased pituitary 

weight 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 estrogenrelated effect observed in the mammalian 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-related effects in the Tier 1 and 

Part 158 mammalian studies were only seen at doses that resulted in liver weight changes and/or 

hepatotoxicity, the observed effects are likely secondary resulting from enhanced liver 

metabolism and hormone clearance. 

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Page 2 of 54 

In the FSTRA, effects were only seen in the presence of overt toxicity (decreased feeding/body 

weight). Decreases in eggs laid in the mallard duck study were observed 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 reproduction studies (the effects on avian offspring 

survival/weight are limited in the ability to inform about potential estrogen (and androgen)-

related effects). Therefore, overall, effects on the estrogen-pathway appeared to only occur in 

the presence of effects on body weight and/or decreased feeding. 

For the androgen pathway, norflurazon was negative in the Tier 1 androgen receptor binding and 

steroidogenesis assays. Androgen-related effects in the Hershberger 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 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 clearance 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 androgen-related effects observed in the Part 158 

wildlife studies. 

For the thyroid pathway, in the pubertal female assay, decreased serum T4 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 presence of liver function 

perturbations. 

Although findings of thyroid gland atrophy are identified in the 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 developmental 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 thyroid relate effects were observed in mammals, 

the effects are likely due to perturbation of liver function. The developmental effects observed in 

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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 studies conducted with adult animals but no 

such data exists for potential thyroid 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 observed in dogs at 5.02 mg/kg/day (LOAEL) in a 

chronic toxicity study. This POD is used for deriving 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 based 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 endpoints of concern are based 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 case of norflurazon, although no data exists for 

potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended 

since: 1) the PODs are markedly lower than the 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-related effects were seen in the male pubertal 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 norflurazon are on liver and kidney and that the effects on 

the E, A, and T pathways occur only when the liver is compromised; and 6) the chronic RfD and 

MOEs are protective of the thyroid-related effects 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 expected to impact EPA’s current 

regulatory point of departures and endpoints for human health 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 with both northern bobwhite quail and mallard 

duck are available for norflurazon. In both species, effects on reproductive parameters were 

observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) 

are considered sufficient for evaluating potential reproductive effects to birds from norflurazon 

exposure. Additional testing is not recommended. 

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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 potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary 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 norflurazon interacts with E, A or T hormone pathways, the number and 

type of effects induced, the magnitude and the pattern 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 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of evidence analysis of the potential interaction of 

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).

Norflurazon has a vapor pressure of 2.89 × 10-8 mm Hg at 25°C, and volatilization is not 

expected to contribute significantly to the dissipation of norflurazon in the environment. It has a 

water solubility of 33.7 mg/L at 25°C and an octanol/water partition coefficient of 2.3 (log Pow). 

It is stable to hydrolysis, but does degrade rapidly in water due to photolysis. Norflurazon has a 

whole body bioconcentration factor value in fish of approximately 12. 

The available information considered in determining the potential interaction of norflurazon with 

the E, A, T pathways include submitted EDSP Tier 1 assays and other scientifically relevant 

information (OSRI) such as general toxicity studies and/or other published articles. These data 

are summarized in Sections III.A through III.C. An analysis of the data submitted to the agency, 

using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The 

EDSP Tier 2 studies recommendations are presented in Section V. 

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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 shown below in Table 1. Executive Summaries are 

presented in Appendix 1 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data believed 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 literature and/or data submitted to support pesticide 

registration (e.g., Part 158 guideline studies). The Agency’s review of the initial OSRI is 

provided in the Endocrine Disruptor Review Team on norflurazon (USEPA, 2010). Since then, 

the Agency has also conducted a more recent search of available scientific literature for any 

additional relevant information. Summaries of the Tier 1 assays and the studies cited as other 

scientifically relevant information (OSRI) are presented in Appendix 1 and 2, respectively. 

Additionally, literature/studies considered but not utilized 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 presented at the 2013 FIFRA 

Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how 

the individual lines of evidence are assembled and integrated along two concepts (i.e., 

complementarity and redundancy) within the conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps outlined in the guidance which provide the 

foundation for WoE evaluations. The first step is to evaluate the individual studies for their 

scientific quality and relevance in evaluating potential endocrine interaction(s). The second step 

is to integrate the data along different levels of biological organization while examining the 

extent of complementarity (i.e., the concordance of endpoints within an assay that measures 

multiple endpoints) and redundancy (i.e., the concordance of endpoints/responses across assays) 

in the observed responses across these different levels of biological organization. The third step 

is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to 

evaluate whether additional testing is needed based on the evidence and conclusions described 

above. 

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

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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 include published literature studies as well as 

studies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines 

submitted in support of pesticide registrations. Each 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 specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system, are considered. The collective response of the 

individual endpoints, as well as the conditions under which they were expressed, are considered 

when evaluating an overall indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate 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 support a hypothesis that a chemical 

has the potential to interact E, A or T pathways, are the concepts of complementarity and 

redundancy. These two concepts provide a basis for considering the plausibility, 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 event, such as receptor binding, 

through potential adverse effects in apical endpoints such as sexual development and fecundity at 

the whole organism level. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

After the data have been assembled and integrated, the third step is to characterize the main lines 

of evidence along with the conclusions; this characterization involves three components. The 

first component is whether the data provide relevant, robust and consistent evidence in terms of 

complementarity and redundancy as well as biological plausibility. Second, is at what level of 

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher levels of biological organization?. Finally, under what 

conditions did the responses occur including consideration of whether the responses were 

observed in the presence of overt or systemic toxicity?. The presence of overt and/or systemic 

toxicity introduces uncertainty in the ability to distinguish effects specifically related to an 

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endocrine-related effect from a non-endocrine toxic response. 

This uncertainty in distinguishing whether the responses were endocrine-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, weight should be placed on signs of endocrine disruption in the 

presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms 

of primary interactions with the endocrine system vs. secondary effects related 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 studies for both human and ecological risk 

assessments. Specifically, in these analyses, the effects that EPA considered to be potential 

evidence of overt toxicity included, but were not limited to: mortality; 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, especially if the effects were 

extreme. In some instances, one parameter (i.e., death or >10% decrease in mammalian body 

weight) was sufficient to consider a dose/concentration 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 decreases 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 effects were generally considered as less severe 

forms of toxicity (e.g., changes in organ weights or blood chemistry). The circumstances for 

which a dose/concentration was considered overtly toxic for a particular study are described in 

Section IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/chemical properties and its 

known modes of action in its overall 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 determining 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 evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available toxicity data into account. 

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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 biologically 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) 

ERα 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) 

Page 16 of 62
NORFLURAZON FINAL 

Page 9 of 54 

B. Effects on Hypothalamic-Pituitary-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 estrogen pathway across the relevant Tier 1 assays are delineated so as 

to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This 

table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in 

the presence of overt toxicity are discussed in the text but are 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 contrast, a response in the ERα 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 supplemental data provided with the ERTA showed the hERα

antagonist ICI 182,780 failed to inhibit the agonist response in the cells treated with norflurazon 

suggesting a false positive response in this assay and the fact that the ER binding assay was 

negative. A weak response also was observed in the steroidogenesis assay with slight increases 

in estradiol production (1.1- to 1.3-fold) at the two highest concentrations tested, 1 and 10 μM. 

In the uterotrophic assay, norflurazon did not induce uterine weight increases. In the female 

pubertal assay, norflurazon also showed no consistent evidence of an estrogenic response. 

Absolute (unadjusted and adjusted) mean pituitary weight was decreased 17 to 18% at the highdose. Additionally, vaginal opening 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 weight (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 norflurazon 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 control (the solvent control 

had a significant 34% decrease in fecundity compared to the negative control). The difference in 

fecundity between the control groups was attributed to an unusually high output of eggs in two 

replicates in the negative control during the last week of the study, although a possible solvent 

effect could not be definitively 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 decreases in male body weight (28%) and length (6%). 

Page 17 of 62
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 reduction egg production (11%) and female and male 

body weight gain were observed at 1000 mg/kg-diet. In the bobwhite quail study, decreases in 

hatchling survival (although not concentration-responsive) at 200 mg/kg-diet was observed. 

There were no effects on mortality, behavior, food consumption, 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.

Page 18 of 62
NORFLURAZON 

Page 11 of 54 

Table 2: Estrogenic/Anti-Estrogenic Pathway for NorflurazonLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Norflurazon1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility/Fecundity 

EDSP Tier 1 Assay 

ER Binding (MRID 48672503) N ERTA (MRID 48672504) 

P4 

Aromatase (MRID48672502) N Steroidogenesis (MRID 48672505) P Uterotrophic (MRID 48618206) N Female Pubertal Rat (MRID 48618204) N N N 

↓17-18% 

(H) 

N ↑2.1 d (H) 

FSTRA (MRID 48686502) NE N N N N 
NORFLURAZON 

Page 12 of 54 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Norflurazon

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Norflurazon1Study Type / Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility/Fecundity 

OSRI 

Developmental toxicity (Rat; 00063621) N N Developmental toxicity (Rabbit; MRIDs 00131151, 00131152) 

 NR N 

Three-generation reproduction (Rat; MRID 00080750) 

NE NE NE NE NE N 

Two-generation reproduction (Rat; MRID 43522301) 

NE NE N NE NE N 

One-generation reproduction (Mouse;MRID 00080751 ) 

NR NR NR NR NR N 

9-mo Chronic toxicity (Rat; MRID 00091056) NE N N NE 2-yr Chronic toxicity (Rat; MRID 00082019) NE N N N Carcinogenicity (Mouse; MRID 00111649) 

NE N 

P8

(H) N 

Chronic toxicity (Dog; MRID 00111618) N N N N Avian Reproduction (Duck; MRID 00052172; 42615302) 

 P9 

Avian Reproduction (Quail; MRID 00039222; 42615301) 

 P11 
NORFLURAZON 

Page 13 of 54 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High tr

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this aabsolute unless otherwise indicated. 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 OSR

column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an X 

Section IV. A 

4. In the ERTA assay, norflurazon had a calculated PC50s of 10–5.3 and 10–5.2 M in the Runs 1 and 2; however, failure of the hER

α antag

the 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 at the high dose included a 100% increase in blood urea nitrogen (BUN) and 65% decrease in 

7. At the high treatment level signs of overt toxicity were observed including reduced feeding and significant decreases in male body w8. 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 la

weight. 

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; fe

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-diet 

P Positive findings (in vitro)/No effect (in vivo) N Negative findings (in vitro)/No effect (in vivo) 

NE Not examined NR Not reported 
NORFLURAZON FINAL 

Page 14 of 54 

2. Effects on Androgen Pathway 

The potential for norflurazon to interact with the androgen pathway are summarized in Table 3. 

The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so 

as to facilitate determination of potential for androgenic, anti-androgenic, or HPG axis effects. 

This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that 

occurred in the presence of overt toxicity are discussed in the text but are not reported in the 

table and not considered further in the WOE assessment

Norflurazon was negative (non-binder) in the Tier 1 in vitro androgen receptor (AR) binding 

assay, and the steroidogenesis assay. Norflurazon was also classified as negative for androgenic 

activity in the Tier 1 in vivo Hershberger. In the anti-androgenic portion of the Hershberger 

assay, high-dose animals had significant decreases in four AST weights (i.e., seminal vesicles 

with coagulating glands, ventral prostate, LABC, and Cowper’s gland) accompanied by a 

decrease in serum testosterone concentration of 31% (NS) in the high-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. Serum testosterone concentration 

was decreased (NS) by 16% in the high- dose animals relative to control. Adjusted weights of 

AST (ventral and dorsolateral prostate and seminal vesicle without fluid) were significantly 

decreased at the high dose. No histopathological 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%, respectively). There was no evidence of effects on male 

reproductive parameters in the existing 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 score) were decreased (11 vs. 29 for negative control) and male 

gonadal development was arrested in three of eight fish. However, these effects occurred in the 

presence of overt toxicity including decreased feeding, decreased body length and decreased 

male body weights (10 and 28%, mid and high). In the Part 158 wildlife studies; decreases in 

hatchling weight/survival were observed in the avian reproduction studies. 

Page 22 of 62
NORFLURAZON 

Page 15 of 54 

Table 3: Androgenic/Anti-Androgenic Pathway for NorflurazonLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Norflurazon1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid hormones 

Testes Weight / GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight

Epididymides 

Histopathology 

Pituitary Weight

Accessory Sex Organ 

Weights / 2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at PPS 

EDSP Tier 1 Assay 

AR Binding (MRID 48672501) N Steroidogenesis (MRID 48672505) N Hershberger (MRID 48618203) N N Male Pubertal Rat (MRID 48618205) N N N N N ↓14% (H) 

↓22%, 

DP; 

↓15%, 

VP; 

↓15%, 

SV (H) 

N 

FSTRA (MRID 48686502) NE N 

N N N 

OSRI 

Developmental toxicity (Rat; 00063621) 

N 
NORFLURAZON 

Page 16 of 54 

Table 3: Androgenic/Anti-Androgenic Pathway for NorflurazonLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Norflurazon1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid hormones 

Testes Weight / GSI 

Gonad Staging and 

Histopathology 

Epididymides Weight

Epididymides 

Histopathology 

Pituitary Weight

Accessory Sex Organ 

Weights / 2° Sex 

Characteristics 

Fertility(Frt) / 

Fecundity (Fcd) 

Age and Weight at PPS 

Developmental toxicity (Rabbit; MRIDs 00131151, 00131152) 

N 

Three-generation reproduction (Rat; MRID 00080750) 

 NE NE NE NE NE NE N 

Two-generation reproduction (Rat; MRID 43522301) 

 NE N NE N NE NE N 

One-generation reproduction (Mouse;MRID 00080751 ) 

NR NR NR NR NR NR N 

9-mo Chronic toxicity (Rat; MRID 00091056) 

N N NE NE NE NE 

2-yr Chronic toxicity (Rat; MRID 00082019) N N NE NE N N Carcinogenicity (Mouse; MRID 00111649) 

N N NE NE N NE 

Chronic toxicity (Dog; MRID 00111618) N N NE N N N Avian Reproduction (Duck; MRID 00052172; 42615302) 

 P5 

Avian Reproduction (Quail; MRID 00039222; 42615301) 

 P7 
NORFLURAZON 

Page 17 of 54 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High t

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this aabsolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorbulbocavernosus (LABC), Epididymides (EP); LW=Liver weight 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSR

column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an X 

Section IV. A 

4. 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; fe

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-diet P Positive findings assay response N Negative findings (in vitro)/No effect (in vivo) NE Not examined NR Not reported 
NORFLURAZON FINAL 

Page 18 of 54 

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

The current EDSP Tier 1 battery 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 (rat) 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 effects. This table also 

includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the 

presence of overt toxicity are discussed in the text but are not reported 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 thyroid gland atrophy was observed at the high 

concentration. 

In the Tier 1 female pubertal rat assay with norflurazon, evidence of altered thyroid function 

included decreased total thyroxine (T4) at both doses tested (250 and 500 mg/kg/day) and 

decreased colloidal area at the high dose. Serum TSH levels were unchanged in females. No 

thyroid-related effects were observed in the Tier 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, effects on thyroid weights were observed in a ninemonth chronic oral toxicity study but the effects were not statistically significant in either 

sexes with decreased thyroid 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 microscopic alterations in the thyroid glands were 

reported for this study. Increases in thyroid weights (with no reported histopathological 

effects) were reported for the two year chronic oral toxicity/carcinogenicity study with 

norflurazon in rats in the high-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 and 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). 

Page 26 of 62
NORFLURAZON FINAL 

Page 19 of 54 

Table 4. Thyroid Pathway for Norflurazon.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Norflurazon1

Study Type / 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous) or HLL Growth (BW, SVL)

Systemic Toxicity Observed2 

Overt Toxicity Observed3

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%; 

CC4 

(H) 

N 

AMA (Frog; MRID 

48686501) P5 P6 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 applicable 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 OSRI 

Page 27 of 62
NORFLURAZON 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 reported 

   

Page 28 of 62
NORFLURAZON FINAL 

Page 21 of 54 

IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight 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 recommendations 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 peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (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 biological 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 available 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 perturbations 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 are 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 evaluation of the results of the Tire 1 assays stated that “In summary, 

the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in 

the presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in 

terms of primary interactions with the endocrine system vs. secondary effects related to toxicity 

in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

A. Systemic/Overt toxicity in the in vivo Tier 1 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 below. In addition to the endocrine-related effects described above 

for the in vivo Tier 1 assays and OSRI, other effects 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, more than one parameter was needed to determine 

overt toxicity. Effects which were considered to be signs of systemic toxicity were generally less 

severe forms of toxicity (e.g., changes in organ weights or blood chemistry).

Page 29 of 62
NORFLURAZON FINAL 

Page 22 of 54 

1. Tier 1 in vivo Assays 

Overt toxicity at the high dose (1000 mg/kg/day) in the anti-androgen phase of the Hershberger 

assay manifested as increased incidence of clinical signs (hair loss, dry red material around nose, 

decreased defecation), decreases in body weight (11–24%), body weight gain (90%), and food 

consumption (31%) and increased liver weight (39%). 

In the FSTRA, decreased male body weight and length, 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 other 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/kg/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 at the 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 males and 130 mg/kg/day in females of F0 and F1 

generations. Also increased incidence 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 levels 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 increase 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/carcinogenicity with rats, at the interim sacrifice (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 males and 12% in females) only at the high dose. 

Histopathology showed increased incidence of hemosiderin pigment deposition in the liver of 

both sexes at 52 mg/kg/day. The weight of the left thyroid gland was increased by 30% in males 

at the high dose, while the weight of the right thyroid gland was increased by 12%, 19% and 

32% at the low, mid and high dose groups, respectively. 

Page 30 of 62
NORFLURAZON FINAL 

Page 23 of 54 

In the carcinogenicity study with mice, absolute liver weight was 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). Relative liver weight was increased 43% in males and 19% in 

females at 219 mg/kg/day. Carcinogenic 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 effects (weights and histopathology) 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 effect 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 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 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 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). The positive ERTA assay result was difficult to interpret, given the effect was not 

inhibited when the cells were treated with a known ER antagonist, suggesting a false positive 

response. In the Tier 1 pubertal assay, decreased pituitary weight 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 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-related effects in the Tier 1 and 

Part 158 mammalian studies were only seen at doses 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 presence of overt toxicity (decreased feeding/body 

weight). Decreases in eggs laid in the mallard duck study were observed 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 reproduction studies (the effects on avian offspring 

survival/weight are limited in the ability to inform about potential estrogen (and androgen)-

Page 31 of 62
NORFLURAZON FINAL 

Page 24 of 54 

related effects). Therefore, overall, effects on the estrogen-pathway appeared to only occur in 

the presence of effects on body weight 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 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 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 clearance 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 androgenrelated effects observed in the Part 158 wildlife studies. 

D. Thyroid pathway 

In the pubertal female assay, decreased serum T4 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 presence of liver function perturbations. 

Although findings of thyroid gland atrophy are identified in the 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 developmental findings in the AMA with norflurazon are 

inconsistent with a direct, thyroid-related delay. 

 

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NORFLURAZON FINAL 

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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 thyroid relate effects were observed in mammals, 

the effects are likely due to perturbation of liver 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 studies conducted with adult animals but no 

such data exists for potential thyroid 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 observed in dogs at 5.02 mg/kg/day (LOAEL) in a 

chronic toxicity study. This POD is used for deriving 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 based 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 endpoints of concern are based 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 case of norflurazon, although no data exists for 

potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended 

since: 1) the PODs are markedly lower than the 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-related effects were seen in the male pubertal 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 norflurazon are on liver and kidney and that the effects on 

the E, A, and T pathways occur only when the liver is compromised; and 6) the chronic RfD and 

MOEs are protective of the thyroid-related effects 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 expected to impact EPA’s current 

regulatory point of departures and endpoints for human health 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. 

Page 33 of 62
NORFLURAZON FINAL 

Page 26 of 54 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for norflurazon. In both species, effects on reproductive parameters were 

observed. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) 

are considered sufficient for evaluating potential reproductive effects to birds from norflurazon 

exposure. Additional testing is not recommended. 

   

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Page 27 of 54 

VI. References 

Eschbach, B., Aerni, R., Hertl, P., et al. (1994) Two Generation Reproduction 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., Yao, C. (1983a) Dose Range-finding Study of Norflurazon in 

Pregnant Rabbits: Sandoz Project 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—Segment II: Sandoz Project T -1794. (Unpublished study received 

Sep 16, 1983 under 11273-13; submitted by Sandoz, Inc., Crop Protection, San Diego, CA; 

CDL:251279-A). (MRID 00131152) 

Hartman, H.A., Hrab, R. (1972) Compound 9789: Investigation of Teratogenic 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., Carpy, 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., Homestead, FL; CDL: 093995-A). (MRID 00111618) 

Kuhl, A. (2011) Norflurazon — A Dose Range-Finding 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 study in the fathead minnow on norflurazon: Report 

#BW-82-5-1166: Sandoz Project T-1767. Unpublished study received Nov. 15, 1982, under 

11273-13; prepared by EG&G Binomics, submitted by Sandoz, Inc., Crop Protection, San 

Diego, CA; CDL: 248828-A. (MRID 00118047) 

LeBlanc, G. (1982) Early life stage toxicity study in the rainbow trout on norflurazon: Report 

#BW-82-5-1165: Sandoz Project T -1733. Unpublished study received Nov. 15,1982, under 

11273-13; prepared by EG&G Binomics, submitted by Sandoz, Inc., Crop Protection, San 

Diego, CA; CDL: 248829-A. (MRID 00118048) 

Sawhney Coder, P. (2012). Norflurazon — Hershberger Bioassay in 

Orchidoepidiymectomized Rats. Unpublished study by WIL Research Laboratories, LLC, 

Ashland, OH. WIL Research Laboratories, LLC, Ashland, OH. Study No. WIL-639118. 

(MRID 48618203) 

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NORFLURAZON FINAL 

Page 28 of 54 

Sawhney Coder, P. (2012) Norflurazon — Pubertal Development and Thyroid Function in 

Intact Juvenile / Peripubertal Male Rats. Unpublished study by WIL Research Laboratories, 

LLC, Ashland, OH. Laboratory Report No.: WIL-639117. (MRID 48618205) 

Sawhney Coder, P. (2012) Norflurazon — A 14-Day Dose Range-Finding Oral (Gavage) 

Toxicity Study in Juvenile Rats. Unpublished study by WIL Research Laboratories, LLC, 

Ashland, OH. Laboratory Report No.: WIL-639114. (MRID 48618201) 

Sawhney Coder, P. (2012) Norflurazon — 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) Norflurazon — 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., Martin, Kendall, T., and Krueger, H. (2012) Norflurazon — Fish 

Short-term Reproduction Assay with the Fathead Minnow (Pimephales promelas). 

Unpublished study performed by Wildlife International, Ltd., Easton, 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 Detection of Thyroid Active Substances. Unpublished study 

performed by Wildlife International, Ltd., Easton, Maryland. Laboratory report number 

528A-224. Study sponsored by Syngenta Crop Protection, LLC, Greensboro, NC. (MRID 

48686501) 

USEPA. (2010) Norflurazon — Report of the OPP Endocrine Disruptor 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) Endocrine Disruptor Screening Program — Weight of Evidence: 

Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office 

of Chemical Safety and Pollution Prevention. 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 study 

Page 36 of 62
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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. (Unpublished study, including submitter 

summary, received May 8, 1975 under 5F1602; submitted 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. (Unpublished study, including submitter 

summary, received May 8, 1975 under 5F1602; submitted by Sandoz, Inc., Homestead, Fla.; 

CDL:094368-A). (MRID 00080751) 

WARF Institute, Inc. (1975) Compound 9789: Two Year Feeding Study in Rats. Final rept. 

(Unpublished study received May 8, 1975 under 5F1602; submitted by Sandoz, Inc., 

Homestead, Fla.; CDL:094370-A; 094371; 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 under 3G1310; prepared by WARF Institute, 

Inc., submitted by Sandoz, Inc., Homestead, Fla.; CDL: 092233-G). (MRID 00091056) 

WARF Institute, Inc. (1973) Long Term Feeding Study—Rat: 1 Year 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: Two 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) Norflurazon — Human Recombinant Aromatase Assay. Unpublished study 

prepared by CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9047V-100330AROM. 

(MRID 48672502) 

Willoughby, J. (2012) Norflurazon — 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 Information — Laboratory Proficiency Data for 

ERTA Assays and Saturation Binding Data for AR and ER Binding Assays for Assorted 

Chemicals. Unpublished study prepared by CeeTox, Inc., Kalamazoo, MI. (MRID 

48843501). 

Willoughby, J. (2012) Norflurazon — Estrogen Receptor Binding (Rat Uterine Cytosol). 

CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9047V-100330ERB. (MRID 48672503) 

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APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100) 

The 21-day assay (MRID 48686501) of norflurazon (99.5% purity) on amphibian 

metamorphosis of African clawed frogs (Xenopus laevis) was conducted under flow-through 

conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80/control and treatment 

group; 20/replicate) were exposed to negative and solvent (0.04 mL/L dimethylformamide; 

DMF) controls and nominal norflurazon concentrations of 0.10, 1.0, and 10 mg a.i./L. Meanmeasured concentrations were <0.05 (<LOQ; controls), 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 effects are reported based on comparison to the negative control. 

No mortalities were observed in the negative control or treatment groups, and a single mortality 

was observed in the solvent control. Tadpoles with crooked tails were observed for 36 to 54 of 

surviving tadpoles in the controls and treatment groups. In the solvent control and low treatment 

group, one tadpole in each group was observed to be smaller than the other tadpoles; an 

additional tadpole in the low treatment group was observed swimming erratically. 

Day 21 wet weight and snout-vent length (SVL) were significantly increased (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 observed in the low and mid treatment groups; Day 

21 wet weight and SVL in the high treatment group were generally decreased (not significant; 

p>0.05) compared to the negative control. A marginally significant increase (p=0.051; 

Dunnett’s) of 14.5% in Day 7 body weight was observed in the low treatment group compared 

to negative control. 

Norflurazon did not significantly affect (p>0.05) Day 7 or Day 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 asynchronous 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 stage: 54), but the difference was not significant 

(p=0.067; Jonckheere-Terpstra). There were some individuals excluded from the analysis of 

Day 21 growth endpoints because NF stage development 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. 

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There was a slight increase in mild thyroid gland atrophy in the high 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 the treatment groups to the solvent control was 

conducted for all endpoints. At the high treatment level, there was a significant decrease 

(p<0.05) in NF stage at Day 7 and Day 21 as well as significant decreases in Day 7 and Day 21 

wet weight and SVL. There were 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 saturation and competitive 

binding experiments. The competitive binding assay was conducted to measure the binding of a 

single concentration of [3

H]-R1881 (1 nM) in the presence of increasing concentrations (10–10

to 10–3 M or 10–11 to 10–4 M) of norflurazon (99.5±0.5%, Batch #609218). Dimethyl sulfoxide 

(DMSO) was used as the solvent vehicle at a final 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 provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The mean dissociation constant (Kd) for [3

H]-R1881 was 

1.080±0.690 nM, and the mean estimated Bmax was 1.071±0.167 fmol/100 μg protein for the 

single batch of prostate cytosol that was prepared (July 23, 2011). Only one of the three Kd

values was within the range reported in the EPA validation program (0.685 to 1.57 nM), and all 

Bmax 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 Kd estimates among runs.

In the competitive binding experiment, precipitation was observed at norflurazon concentrations 

of 10−3

 M in the first run and at 10−4 M in the second run. Norflurazon did not significantly 

reduce specific binding of the [3H]-ligand to the AR at any soluble concentration tested. A log 

IC50 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. 

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The estimated log IC50s for R1881 and the weak positive control (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 variations between Run 1 compared to Runs 2 and 3 

were likely due to the concentration differences used for R1881 and dexamethasone. The curve 

for dexamethasone was not fully defined in the first run and therefore the calculation for the 

IC50 may be affected. The range of concentrations 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, norflurazon 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 aromatase and tritiated androstenedione ([1β- 3

H(N)]-androst-4-ene-3,17-dione; [3H]-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 concentration of test chemical. Tritiated water was 

quantified using liquid scintillation counting (LSC). Three runs were conducted that included a 

full activity control, a background activity control, a positive control series (10−10 to 10−5

 M) 

with a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN) in duplicate, and the 

norflurazon series (10–11 to 10–3 M) in triplicate. Due to observed 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 IC50, and % CV for 

replicates of each concentration within runs. The estimated log IC50 for 4-OH ASDN averaged 

−7.17 M and the Hill slope was −0.94. 

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For norflurazon, aromatase activity averaged 0.215 ± 0.48 nmol·mg-protein−1

·min−1 at the 

lowest concentration tested 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 concentrations of norflurazon. 

A log IC50 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) Binding Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assay (MRID 48672503), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiments. The saturation 

binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was 

present in reasonable numbers and was functioning with appropriate affinity for the 

radiolabeled reference estrogen prior to routinely conducting ER competitive binding 

experiments. A competitive binding experiment was conducted to measure the binding of a 

single concentration of [3

H]-17β-estradiol (1 nM) in the presence of increasing concentrations 

(10–11 to 10–4 M) of norflurazon (99.5±0.5% purity, Batch #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 included 19-norethindrone as a weak positive control, 

octyltriethoxysilane as a negative control, and 17β-estradiol as the natural ligand reference 

material. 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The single batch of cytosol used for the saturation and 

competitive binding assays was prepared on September 27, 2010. In the saturation binding 

assay, the mean dissociation constant (Kd) for [3

H]-17β-estradiol was 0.081±0.006 nM, and the 

mean estimated Bmax was 52.70±1.05 fmol/100 μg protein. The Kd was within the expected 

range of 0.03 to 1.5 nM. Confidence in these numbers is high due to the goodness of fit and the 

small variation among runs. 

In the competitive binding experiment, precipitation occurred at norflurazon concentrations of 

10–3 M in Runs 1 and 2; therefore, data from this concentration were not used to evaluate 

competitive binding. Norflurazon had no effect on specific binding of [3

H]-17β-estradiol at 

concentrations of ≤10–4 M in all three valid assay runs. Norflurazon displaced less than 25% of 

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the radiolabel estradiol bound to the ER at the highest concentrations successfully tested (10−4

M). 

The performance criteria were met for the assay, and the reference compounds performed as 

expected. Octyltriethoxysilane had no effect on binding; 17β-estradiol and 19-norethindrone 

generally reduced labeled ligand binding at the expected concentrations. The mean log IC50s

were –9.23 M for 17β-estradiol and –5.57 M for 19-norethindrone, and the mean relative 

binding affinity (RBA) was 0.0326% for 19-norethindrone, compared to the natural ligand. 

Although the cytosol preparation (isolated September 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, norflurazon is classified as Not Interactive in the 

Estrogen Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48672504), hERα-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 tests. The experiments were performed using 96-

well plates and each norflurazon concentration was tested in 6 replicates/plate. In addition to 

the required reference compound, ICI 182,780 was added to 2 replicates/plate as a hERα

antagonist. Cells were exposed to the test agents for 24±2 hours to induce reporter (luciferase) 

gene products. Luciferase expression in response to activation of the estrogen receptor was 

measured using a proprietary luciferase assay. A total of nine runs were reportedly conducted 

and results were reported from the two runs judged to be acceptable. 

Cytotoxicity was observed at the highest concentrations tested of 17α-methyltestosterone and 

corticosterone in the first run, and at the highest concentration tested of corticosterone in the 

second run. In both runs the mean responsiveness of cells to 17β-estradiol at concentrations 

≥10–9 M was 132-167% of the response to the positive control (PC; 17β-estradiol at 10–9 M). 

The responsiveness of the cells to 17α-methyltestosterone was below the expected level, 

indicating a decreased sensitivity to very weak agonists. 

Although precipitation of norflurazon was noted at the 10−4

 M concentration, norflurazon 

elicited a clear positive response 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 PC10 threshold. A 

PC50 was calculated for norflurazon of 10–5.3 M in the first run and 10–5.2 M in the second run. 

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However, data were also provided showing that the hERα antagonist ICI 182,780 failed to 

inhibit the response of the cells to norflurazon, while fully inhibiting the response to 17βestradiol. Since the data indicates that the cell’s response to norflurazon may not be due to 

hERα activation but other no-specific response of the assay, norflurazon 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 nominal concentrations of 0 (negative 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 duration 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 test system was maintained at 24.4 to 25.6oC and a 

pH of 7.9 to 8.3. 

Unless otherwise indicated, all effects are reported 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 control, 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 control (median fatpad score: 3). There were no 

other notable observations of secondary sex characteristics or clinical signs for any 

treatment group compared to the controls with the exception that reduced feeding was 

periodically observed from Day 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; Jonckheere 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 male body length of 6% 

at the high treatment level compared to the negative control. Female growth parameters 

were not significantly different (p>0.05) between the norflurazon treatment groups and the 

negative control; however, female body weight in the solvent control was significantly 

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increased (p<0.05) by 11% compared to the negative control. 

Spawning occurred at least every 4 days (except for one replicate each in the negative and 

solvent control), and mean fecundity in the negative and solvent controls was 28.1 and 18.5 

eggs/female/day/replicate, respectively (range: 22.6 to 31.6 for 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 was a significant decreasing monotonic trend 

(p<0.05) of 34 to 95% compared to the negative control at all treatment levels. Additionally, 

there was a significant reduction in solvent control 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 concentration (0.04 mL/L) was consistent between the solvent 

control and all norflurazon treatment groups. There was a marginally significant reduction 

(p=0.057) of 2.25% in fertility in the mid treatment 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 negative control; there was no significant difference 

(p>0.05) in female VTG between treatment groups and the negative control. Male tubercle 

scores were significantly reduced (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, Jonckheere test) of 13% at the mid treatment 

level compared to the negative control. No tubercles were observed in females. There were 

no significant differences in male or female GSI for any treatment. Sex steroids (testosterone 

and 17β-estradiol) were not measured. 

There was a higher incidence of mild to moderate altered proportions of sperm precursors in 

3 of 8 males in the high treatment group suggesting 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. Therefore, 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 treatment groups (median tubercles scores: 25 and 11, 

respectively) compared to the solvent control (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. 

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Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger assay (MRID 48618203) screening for androgenic activity, norflurazon 

(99.5% a.i., Batch# 609218) in 1% carboxymethylcellulose (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 rats exposed to 0.2 mg/kg/day testosterone propionate (TP) in 

corn oil by subcutaneous (s.c.) injection plus 1% CMC by oral gavage was included. 

To screen for potential anti-androgenic 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/day 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 the negative control group for the anti-androgenic activity. An 

anti-androgenic positive control group consisted of six castrated 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 animals 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, clinical signs included right and left forelimb hair loss at 300 

mg/kg/day and left forelimb, abdominal, and thoracic hair loss, and dried red material around 

nose at 1000 mg/kg/day. Body weights and overall body weight gains for the 300 mg/kg/day 

treatment group were comparable to controls throughout the duration of the assay. Animals 

dosed with 1000 mg/kg/day had decreased (p<0.05) body weights (↓11-15%) on Days 3-9, and 

decreased (p<0.05) overall 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 observed 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 gland; and 48% in glans 

penis. 

In the anti-androgen assay, no clinical 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 the 

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forelimb, neck, abdominal, thoracic, and hindlimb 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 comparable 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 treatment-related effects 

in any of the accessory sex organs of animals dosed with 100 or 300 mg/kg/day norflurazon + 

TP. At 1000 mg/kg/day, accessory sex 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 for 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. However, norflurazon was positive for anti-androgenicity 

in the Hershberger assay as statistically significant 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 control), 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 weight at day of attainment were recorded. After sacrifice 

on PND 42, total thyroxine (T4) and thyroid stimulating hormone (TSH) levels were analyzed 

using an electrochemiluminescent immunoassay for T4 and a magnetic [125I]rTSH 

radioimmunoassay for TSH. Adrenal, liver, kidneys, pituitary, thyroid, and urogenital organ 

weights were recorded and microscopic examination of the ovaries, uterus, thyroid, and kidneys 

were conducted. 

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All animals survived until the scheduled necropsy. No treatment-related findings were 

observed during the detailed physical examinations of the animals. No changes in estrous 

cyclicity were observed, and no treatment-related gross necropsy findings were noted. 

At 250 mg/kg/day and above, body weight gains were decreased (not significant [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 conclusion 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 females; these two animals exhibited absent and 

delayed VO, respectively, and absent and reduced corpora lutea, respectively. It was 

considered that the latter effects were secondary to the body weight 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 increased (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-related as relative weights were not affected. No 

other organ weights were affected by treatment with norflurazon. 

Mean serum T4 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 considered to be treatment-related and was presumed 

to be a response to the reduced T4 levels in this dose group. Thyroid follicular cell height was 

unaffected by treatment. 

The dose levels tested in this assay are adequate 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 Crl: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 control), 185, or 375 mg/kg/day from 

post-natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) daily 

beginning on PND 30 and age and weight at day of attainment was recorded. Following 

sacrifice on PND 53, total serum testosterone, thyroxine (T4), and thyroid stimulating hormone 

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(TSH) levels were analyzed using a chemiluminescent immunoassay for testosterone and T4, 

and radioimmunoassay for TSH. Adrenal, liver, kidney, pituitary, thyroid, and urogenital organ 

weights were recorded and microscopic examinations of the testes, epididymides, thyroid, and 

kidneys were performed. 

All animals survived until the scheduled necropsy. No treatment-related findings were 

observed during the detailed physical examinations of the animals. There were no treatmentrelated changes in serum T4, TSH, or testosterone concentrations. Thyroid follicular cell height 

and colloid area were similar between treated and control groups. No test material-related gross 

or microscopic pathological findings were noted. 

No treatment-related effects on body weights or food consumption were observed. Final body 

weights were decreased (not significant [NS]) by 6-7% in treated 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% during 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 days (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) and relative kidney weight (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 unadjusted pituitary weights (14%, 

p<0.05) and dorsolateral prostate weights (22%, p<0.05); and adjusted 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 weight (14%, p<0.05) and 

relative kidney weight (7%, p<0.05). 

The study report provided normal (historical) ranges 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 performing laboratory. The parameters reported that were 

outside the laboratory normal range are albumin, ALT, AST, bile acids, cholesterol, potassium, 

total protein, triglyceride and 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 adequate based on the decreased final body weights, 

reduced overall body weight gains, and increased liver weights in this study. 

Steroidogenesis Assay; (OCSPP 890.1550) 

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In a steroidogenesis assay (MRID 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 assay concentration of 0.05%. 

Three independent, valid experiments were performed; an initial run was rejected and the 

samples were not analyzed. Testosterone and 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 run of a test chemical 

plate to demonstrate that the assay responded properly to positive control agents at two 

concentration levels; positive controls included the known inhibitor (prochloraz) and inducer 

(forskolin) of estradiol and testosterone production. 

Guideline acceptability recommendations and requirements were met, including lack of 

cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

%CV), and appropriate induction and inhibition with positive controls. 

 

No evidence of cytotoxicity was observed at any tested concentration of norflurazon. However, 

precipitation was observed at the 100 μM concentration in all three runs and at 10 μM in one 

run; therefore, 10 μM was the highest concentration evaluated. There were no statistically 

significant changes in testosterone production following incubation with norflurazon in all three 

runs, although there was a trend toward increased testosterone levels at 1 and 10 μM (1.15- and 

1.26-fold, respectively). Estradiol production was significantly increased in Run 3 at 10 μM 

(1.2-fold) and in Run 4 at 1 μM (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 significant and reproducible increases 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) conducted 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 17α-ethynyl estradiol (EE) in corn oil 

at 0.3 mg/kg/day by oral gavage. Body weights were determined daily. All animals were 

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terminated and necropsied on PND 59 approximately 24 hours after the final dose 

administration to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No significant 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 the 1000 mg/kg dose group compared to controls, 

resulting in a decrease (p<0.05) of 257% in overall (Day 1-4) body weight gains (BWG). Body 

weights in the low-dose (300 mg/kg) group were also decreased (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 the positive control (EE) group were decreased 

(p<0.05) by 9% and 14% on Days 3 and 4, respectively, and overall BWG for the EE animals 

was decreased (p<0.05) by 183% in comparison with the vehicle controls. 

Uterine weights in the norflurazon treated groups were comparable 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. 

 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Prenatal Development 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 through 15 inclusive. Insemination 

was by natural means with stock males. Maternal 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 intermediate dose and ↓17% at 

the high dose), and for the entire study period (↓12% at the low and intermediate 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 maternal 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 changes were seen in gravid uterine weight, total implantation, fetal 

viability, or sex ratio. Cesarean data were unremarkable. Developmental toxicity was 

suggested at the high dose in the form of increases (NS) in bipartite thoracic vertebrae (10th–

13th) and rudimentary 14th ribs. However, these increases were considered minor and 

secondary to maternal effects in the high-dose group. Therefore, the NOAEL for 

developmental toxicity is 400 mg/kg/day 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 Corporation, Perkasie, PA) were administered 0, 10, 30, or 60 

mg/kg/day norflurazon technical (98.4%, Lot/Batch #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. Treatment-related decreases in body weight gain were observed at 

all doses in maternal rabbits. Body weight gain was decreased 32%, 50%, and 156% during the 

dosing period, and 17%, 39%, and 57% overall in the low-, mid-, and high-dose animals. 

Gravid uterine weight data were not reported. The NOAEL for maternal toxicity is 30 

mg/kg/day, and the LOAEL is 60 mg/kg/day, based 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. Developmental toxicity was suggested by increases (p<0.05) in the fetal 

incidence of incompletely ossified frontal bones, 16th caudal vertebrae, unossified first 

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dose groups, and unossified proximal epiphysis of the tibia in the low- and high-dose groups. 

Litter incidence was increased for incompletely ossified frontal bones and unossified middle 

phalanx of the fifth digit of the forelimb in the mid- and high-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 skeletal 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/sex/control; Sprague-Dawley, Madison, WI) were administered 

norflurazon technical (98.8% purity, Lot/Batch #004) in the diet. The F0 generation of rats was 

administered norflurazon at nominal doses of 0, 125, 250, and 500 ppm (0, 6.25, 12.5, and 25 

mg/kg/day} for 18 weeks prior to mating to produce the F1a and F1b generation. Subsequent 

to this, the F1a generation was 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 the F2a and F2b generations. The F2a animals 

continued to receive norflurazon at these doses, and 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 survival and clinical signs in any generation of parental rats in 

this study. Body weights on LDs 4 and 21 in dams of F1a, F1b, and F2a litters were reported, 

but no consistent treatment-related effects were observed. No other body weight data were 

reported. However, subchronic and chronic toxicity studies conducted 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 norflurazon 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. The 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 toxicity is 51.25 mg/kg, and the LOAEL was not 

determined. 

This study is classified as unacceptable/guideline due to the lack of parental systemic 

toxicity at the doses tested, including the high dose (51.25mg/kg/day). 

Two-Generation Reproduction in Rodents (Rat)

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In a two-generation reproduction study (MRID 43522301), male and female Wistar Crl:(WI) 

br rats (25/sex/dose; Charles River WIGA GmbH, Sulzfeld, Switzerland) received norflurazon 

technical (98.1% purity, Lot/Batch #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, and 138.6 mg/kg/day for F1 males; and 17.1, 81.7, 

and 173.0 mg/kg/day for F1 females). The F0 animals were pretreated with norflurazon for 70 

days prior to mating followed by treatment during 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 liver and kidneys were weighed. 

Organs/tissues collected for microscopic evaluation at study termination were the liver, 

kidneys, pituitary, testes with epididymides, prostate, seminal vesicles with 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 study, and body weight gain for F1 male parental rats was 

decreased by 31% for Weeks 0–31 of the study. Significant decreases in F1 male body weights 

were <10% (6 to 9%). No effects on food consumption were noted. During gestation and 

lactation, body weight gain in F0 female rats at the second mating was decreased 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 lactation were observed for the F1 females. The 

absolute and relative weights of the liver and kidneys were generally increased (p<0.01 or 

p<0.05) in both F0 and F1 high-dose male and female parental rats, and were accompanied by 

an increased incidence of hepatocellular hypertrophy in both sexes, and an increased incidence 

of single cell necrosis in female rats. At the intermediate dose, liver and kidney weights were 

also increased (p<0.01 or p<0.05) in parental males (and F1 female kidney weights), with an 

increase in the incidence of hepatocellular hypertrophy similar to that observed in the high-dose 

animals. 

Based upon the significant increases in liver and kidney weights observed in both 

generations of parental rats and the increased incidence of hepatocellular hypertrophy in 

these animals, the LOAEL for systemic toxicity is 750 ppm (50.8 mg/kg/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 F1 

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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 performance showed decreased (p<0.05) mean pup weight in the 

high dose, F2b litter pups on LD 21 for males and for females on LDs 4 and 21. Lactation 

index was observed to be decreased (NS) at the high dose (59% 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 generations. Mean pre-coital interval was 

decreased (NS) at the high dose for the first litter of the F0 parental generation, and for both 

litters of the F1 parental generation. Weight gain was affected in the F2b pups, with an 18% 

decrease (significance not reported) observed in the high-dose pups on LDs 4–21; the number 

of live pups also was decreased (p<0.01) in this group. Liver and 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 liver weights only). Occasional increases in relative liver and 

kidney weights were noted in the mid-dose pups (all female groups and F2b males). 

In the F1 and F2 litters, reproductive toxicity was demonstrated at the high dose by decreased 

weight gain in F2b litter pups (18%) for LD 4–21, and by significant increases in absolute and 

relative weights of the liver and kidneys for male and female pups. There were no apparent 

detrimental effects of norflurazon on reproductive performance in this study. Therefore, the 

reproductive LOAEL is 1500 ppm (102.5 mg/kg/day for F0 males; 129.7 mg/kg/day for F0 

females; 138.6 mg/kg/day for F1 males; and 173.0 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 for 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 reproductive 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 norflurazon 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 generation of mice received norflurazon for 18 weeks 

prior to mating to produce the F1a litter. The parent animals were sacrificed after 

weaning the F1a litter. Animals from the F1a litter were subsequently used in a two-year 

chronic feeding study (MRID 00111649). 

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There were no significant treatment-related systemic or reproductive effects reported in 

parental mice or in the F1a offspring at any dose tested in this study. Therefore, the NOAEL 

for systemic toxicity is 48.5 mg/kg, and the systemic LOAEL was not determined. 

Additionally, the NOAEL for reproductive toxicity is 48.5 mg/kg and the LOAEL was not 

determined. It was concluded that the doses used in the present study were inadequate for 

assessment of the reproductive toxicity of norflurazon in mice. 

This study is classified as unacceptable/guideline 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 lactation, and the lack of any effect 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 technical (98.8% purity, Lot/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 macroscopic and/or microscopic 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 norflurazon on survival, body weight, body weight gain, 

food consumption, or food efficiency, in male or female rats at any dose 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 female rats (many tubules in males and few tubules in females) 

was increased, as was the incidence of medullary 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 was 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, there were no reported microscopic 

alterations in the thyroid at any dose. Gonadal weight in female 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 females, and the microscopic changes 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 increased at the intermediate and high doses in 

both sexes, there were no correlated histopathological effects. Therefore, effects on thyroid 

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weight, although related to treatment, do not indicate a toxic effect. 

This study is classified as core supplementary, based on the fact that this study satisfies no 

specific Guideline requirement under Subdivision F guidelines. However, this study provides 

useful toxicity data on norflurazon in rats after 39 weeks of exposure. 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

In a two-year carcinogenicity study (MRID 00082019, interim report MRID 00111617) 

male and female SD rats (85 rats/sex/dose and 170 rats/sex/control; Sprague-Dawley, 

Madison, WI) were administered norflurazon technical (98.8% purity, 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 macroscopic and/or microscopic evaluation at study 

termination included the liver, pituitary, adrenals, mammary gland, thyroids, kidneys, testes, 

prostate, uterus, and ovaries. The liver, kidneys, gonads, pituitary, adrenals, and thyroids 

were weighed at Week 52 (n=10/sex/dose) and at study termination (Week 104). 

No effects of norflurazon were evident for survival (except for an increase in mortality in the 

mid- and high- dose males at Week 104), body weight, 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 female 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. Thyroid 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 also was increased in male rats 

(terminal sacrifice + dying on test) at this dose. The incidence of tubular casts was increased in 

high-dose females that survived to study termination. Other microscopic alterations observed 

in high-dose animals included an increased incidence of parathyroid hyperplasia (both sexes), 

hemosiderin pigment deposition in the spleen (males only) and liver (both sexes), and 

endrometritis and squamous metaplasia of the uterus (females). 

Based on the increased kidney weight and accompanying histopathologic changes, as well 

as increases in liver weight (males and females) and thyroid weight (males), the LOAEL is 

considered to be 1025 ppm (51.25 mg/kg/day) and the NOAEL is 375 ppm (18.75 

mg/kg/day). There was no evidence of carcinogenicity in response to dietary administration 

of norflurazon in this study. 

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This chronic toxicity study is classified acceptable (core minimum) 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, and 218.8 mg/kg/day; intake data were not 

provided) for 100–104 weeks. Organs/tissues collected for macroscopic and/or microscopic 

evaluation at study termination included the liver, pituitary, adrenals, mammary gland, 

thyroids, kidneys, testes, prostate, uterus, and ovaries. The liver, kidneys, gonads, pituitary, 

adrenals, and thyroids were weighed at study termination (Week 104). 

No significant effects were observed on survival, body weight, body weight gain, clinical 

toxicity, or food consumption at any dose 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 female mice, respectively. No statistical significance of liver 

effects was noted. Increased incidences of enlarged spleen, nephritis, swollen/enlarged liver, 

and nodular enlargement of the liver were observed in high-dose males, 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, cystic 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/kg/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 incidence of non-neoplastic pathology in the liver, 

spleen, and kidney in male mice. Carcinogenic potential was evidenced by an increased 

incidence of hepatic adenoma and combined adenoma/carcinoma 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 administered 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, 

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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 macroscopic and/or microscopic 

evaluation, included the liver, pituitary, adrenals, thyroids, kidneys, 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 with 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 weight in both sexes, and the 

NOAEL is 50 ppm (1.53 mg/kg/day in males; 1.58 mg/kg/day in females). 

This chronic toxicity study is classified acceptable (core minimum) 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 mallard studies determined an NOAEC of 40 

mg ai/kg-diet. In the first mallard study (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 concentrations of 40, 200 and 1000 mg 

ai/kg-diet. The NOAEC was the lowest concentration tested, based on 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. 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 mortality, behavior or food 

consumption. 

In the first quail study (MRID 00039222), there 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 control; however, this 

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effect was not concentration-responsive. Number 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 mortality, behavior or food 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 concentrations 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 determined by GLC-ECD. The embryo and larvae exposure was 

initiated within 48 hours after egg fertilization, and continued through 30 days post-hatch. 

Sixty embryos were used in each test chamber and two test chambers per control, solvent 

control, and nominal test concentration were utilized. Forty larvae were utilized in replicate 

tanks (2) for each test concentration. 

Percent hatch was not significantly different between treatment and control groups. No 

toxicological signs were observed at concentrations ≤1.1 mg/L. Survival of larvae exposed to 

mean concentrations ≥4.2 mg/L norflurazon was significantly (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 weight of larvae exposed to 4.2 mg/L norflurazon was significantly 

reduced, compared to control and solvent control larvae, and average wet weight of larvae 

exposed to 2.1 mg/L was significantly reduced compared to control larvae only. The MATC of 

fathead minnows exposed to norflurazon during embryo 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 norflurazon (96.6% purity; Batch/Lot 

not reported) at concentrations 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 levels were present at or above the stated 

concentration as determined by GLC-ECD. The 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 control, solvent control, and nominal 

test concentration were utilized. An unknown number of larvae were utilized in replicate tanks 

(2) for each test concentration. 

The percentage viability of rainbow trout embryos was unaffected by exposure to mean 

norflurazon concentrations up to 11.2 mg/L. The percentage hatch of viable embryos 

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exposed to mean concentrations ≥5.5 mg/L norflurazon was significantly reduced compared 

to control and solvent control embryos. 

Embryos began hatching on Day 23 of exposure. None of the embryos exposed to 11.2 mg/L 

norflurazon hatched successfully, and larvae exposed to 5.5 mg/L norflurazon began dying 

after hatching with no larvae surviving after Day 9 post-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. Lethargic behavior and mortality also were observed 

among larvae exposed to 1.5 mg/L norflurazon through Day 15 post-hatch, after which 

surviving larvae no longer appeared lethargic and none died. Larvae 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 norflurazon were significantly reduced, and also 

appeared to have light pigmentation as compared to larvae in both control 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 control and solvent control larvae. Based on the 

reduced percentage survival and reduced growth 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. 

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APPENDIX 3: References Not Utilized in the Norflurazon WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant information (OSRI) was 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 registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE 

analysis for Norflurazon but were not utilized due to one or more of the following reasons: 1) the 

article was not available in English; 2) the compound of interest was not used 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 adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) 

the study dealt only with non-EDSP assay development; 12) no specific endocrine-related 

endpoints were assessed in the study; and 13) the study contained only data on invertebrates. 

Fogleman, R.W. (1971) Evaluation of the Subchronic (90-day) Toxicity Effects of SAN-9789 in 

Rats: Contract No. 120-546-9-70. Final report. (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: Reproduction Studies in Rats and Mice: 

Volume I: Reproduction Studies on Rats. Final report. (Unpublished study, including submitter 

summary, received May 8, 1975 under 5F1602; submitted by Sandoz, Inc., Homestead, Fla.; 

CDL: 094369-A). (MRID 00080750) 

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WARP Institute, Incorporated (1975) Compound 9789: Reproduction Studies in Rats and Mice: 

Volume 2: Reproduction Studies in Mice. Final report. (Unpublished study, including submitter 

summary, received May 8, 1975 under 5F1602; submitted by Sandoz, Inc., Homestead, Fla.; 

CDL: 094368-A). (MRID 00080751) 

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