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 61
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 

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EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: BENFLURALIN 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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BENFLURALIN FINAL 

ii 

TABLE OF CONTENTS 

Abbreviations....................................................................................................iii 

Executive Summary............................................................................................1 

I. Introduction.......................................................................................5 

II. Sources of Scientific Data and Technical Information......................................6 

A. EDSP Tier 1 Screening Assays............................................................6 

B. Other Scientifically Relevant Information (OSRI)......................................6 

III. Weight of Evidence (WoE) Evaluation........................................................6 

A. EDSP Tier 1 Screening Assays.............................................................9 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis............................10 

1. Effects on Estrogen Pathway...................................................10 

2. Effects on Androgen Pathway...................................................15 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................20 

IV. Committee’s Assessment of Weight of Evidence..........................................22 

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

1. Tier 1 in vivo Assays.............................................................23 

2. OSRI................................................................................23 

B. Estrogen Pathway...........................................................................24 

C. Androgen Pathway.........................................................................25 

D. Thyroid Pathway...........................................................................25 

E. Conclusions.................................................................................26 

V. EDSP Tier 2 Testing Recommendations.....................................................26 

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

LIST OF TABLES: 

Table 1. Tier 1 Assay for Benfluralin...............................................................9 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Benfluralin ...............................12 

 Table 3. Androgenic/Anti-Androgenic Pathway for Benfluralin..............................17 

 Table 4. Thyroid Pathway for Benfluralin........................................................21 

APPENDIX 1. EDSP Tier 1 Screening Assays........................................................32 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)................................45 

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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 

F2 Second filial generation 

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Abbreviation Terminology 

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 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

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Abbreviation Terminology 

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 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

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Abbreviation Terminology 

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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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 a pesticide chemical 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, whether or not endocrine-related responses occurred at dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On November 13, 2013, the 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 

benfluralin with the E, A or thyroid 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 benfluralin and the estrogen pathway, a positive response for estrogen receptor (ER) agonism 

was observed in the Tier 1 estrogen receptor transactivation assay (ERTA); however, this 

interaction with the estrogen pathway via the ER was not consistent with the negative findings in 

the Tier 1 ER binding and uterotrophic assays. Estrogen-related effects were observed at the 

high dose in the Tier 1 female pubertal assay including decreases in pituitary weight, ovarian 

weight, blotted uterine weight, delayed vaginal opening and increased age at first estrus. All 

estrogen-related findings in the Tier 1 in vivo assay and Part 158 mammalian studies occurred in 

the presence of liver effects including increases in absolute and relative liver weights, and 

significant increases in creatinine, GGT and cholesterol. Additionally, an increase in relative 

ovarian weight in the Part 158 mammalian subchronic rat study occurred at the high dose along 

with significant increases in absolute and relative liver weights. The observed effects are 

consistent with the findings in the mode of action studies which support hepatic enzyme 

induction resulting in enhanced hormone clearance in mammals. 

 

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No effects on the estrogen pathway were observed in the fish short term reproduction assay 

(FSTRA). In a one-generation Part 158 avian reproduction study with bobwhite quail, a decrease 

in eggs laid, as well as effects on offspring survivability and growth, were observed; however, 

these effects coincided with decreases in feed consumption. In the companion mallard duck 

reproduction study, an increase in eggs cracked were reported, but no differences in eggshell 

thinning or other reproductive effects were observed. 

For benfluralin, for the androgen pathway, the only positive findings were seen in the 

Hershberger assay and occurred at the high dose in the presence of overt toxicity. Although the 

presence of overt toxicity confounds the interpretation of the effects on the endocrine parameters 

in the study, a follow-up study to evaluate testosterone clearance showed enhanced 

metabolism/clearance of testosterone at the dose that resulted in a decreased androgen-sensitive 

tissue weights. This suggests that the antiandrogenic effects observed in the Hershberger assay 

are likely due to enhanced testosterone clearance. In the male pubertal assay, decreased serum 

testosterone and decreases in androgen-sensitive tissue weights and epididymal weights were 

seen only at the high dose (400 mg/kg/day). The endocrine-related effects were seen in the 

presence of hepatotoxicity which manifested as increases in absolute (40%) and relative (52%) 

liver weights and an increase (60%) in GGT level. Increased liver weights were also observed at 

lower doses where no androgen-related effects were seen. The only androgen-related effect 

observed in the Part 158 mammalian studies was an increase in testicular weight in the rat 

chronic/carcinogenicity study, which occurred in the presence of overt toxicity. The evidence 

from the mammalian toxicity studies suggest that the androgen-related effects observed are due 

to liver enzyme induction and increased hormone clearance and excretion. In the FSTRA the 

only notable effect in the absence of overt toxicity was an increase in male GSI seen at the 

middle treatment concentration. In the Part 158 avian bobwhite quail study, decrease in 

offspring survivability was observed; however decreased feeding was seen in the parental 

generation. 

For benfluralin, for the thyroid pathway, the effects in the Tier 1 assays were seen in the male 

and female pubertal assays, but no evidence of thyroid activity was seen in the amphibian 

metamorphosis assay (AMA). In the pubertal assays, significant decreases in serum T4 were 

seen at all doses in the male and at the mid and high dose in the female. TSH was significantly 

increased at the mid and high dose in males and high dose only in females. Increases in thyroid 

weight and altered thyroid histopathology were seen only in males at the high dose. All effects 

on the thyroid parameters in the pubertal assays were accompanied by increased relative liver 

weights. Thyroid perturbations were observed in the subchronic rat toxicity study (increased 

thyroid weights) at a high dose that caused significant increases in liver weight. Thyroid 

follicular cell hypertrophy and hyperplasia were observed in both sexes in the chronic/ 

carcinogenicity study in rats. An increased incidence of follicular cell adenomas/ carcinomas 

was observed in this study, but at a dose that was considered to be overtly toxic. 

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In studies that evaluated the mode of action of benfluralin, previously submitted mechanistic 

studies showed that benfluralin induces hepatic microsomal enzyme activity in both sexes in the 

rat. In a 14-day study submitted to support a postulated mode of action for liver and thyroid 

tumors in the rat, benfluralin was shown to induce constitutive androstane receptor (CAR) and 

pregnane X receptor (PXR) related gene transcripts and associated PROD enzyme activities and 

benfluralin also up-regulated T4-specific UGT genes associated with T4 metabolism. These 

effects occurred at a dose that resulted in decreased serum T4 and T3 levels and increased liver 

weight and hepatic hypertrophy. Furthermore, benfluralin is structurally similar to another 

dinitroaniline (trifluralin) which has been shown to induce hepatic UGTs, resulting in decreased 

serum T3 and T4 levels, elevated TSH and altered thyroid histopathology leading to increased 

thyroid tumors in rats (Saghir et al., 2005). 

An evaluation of the Tier 1 and OSRI data shows that the observed effects on the endocrinerelated endpoints for the estrogen, androgen and thyroid pathway occur only when there were 

concurrent increases in liver weight. Based on a WoE, the available data suggests that the antiestrogenic, anti-androgenic and anti-thyroid effects observed in the mammalian in vivo Tier 1 

assays may result from liver enzyme induction and subsequent increased hormone clearance. 

However, there is no convincing evidence for an interaction with estrogen, androgen or thyroid 

pathways in wildlife. 

For benfluralin, the current point of departure (POD) of 0.5 mg/kg/day is based on increased 

histopathological lesions of the kidneys seen in male and in female rats at 5.4 mg/kg/day 

(LOAEL) in a combined chronic toxicity/carcinogenicity study in rats. This POD is used to 

derive the chronic Reference Dose (RfD) used for dietary exposure risk assessments and for 

calculating the Margins of Exposure (MOEs) for non-dietary exposure risk assessments. This 

POD is 50-fold lower than the lowest dose (25 mg/kg/day) that caused thyroid-related effects in 

the Tier 1 male pubertal assay, and 600 to 800-fold lower than the doses that caused estrogenrelated (300 mg/kg/day) or androgenic-related (400 mg/kg/day) effects in the pubertal assays. 

Consequently, the chronic RfD and MOEs derived from current PODs are protective of the 

endocrine-related findings in the Tier 1 in vivo assays and OSRI. In addition the totality of the 

data demonstrated that the endocrine-related effects seen in the mammalian in vivo Tier 1 assays 

may result from liver enzyme induction and subsequent increased hormone clearance. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is NOT 

recommended for benfluralin since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

As there is no convincing evidence for a potential interaction with the estrogen or androgen 

pathway in fish or thyroid pathways in amphibians, no additional testing is recommended. 

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

duck are available for benfluralin. 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 benfluralin 

exposure. Relative to the EDSP, additional testing is not recommended.

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BENFLURALIN FINAL 

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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 a pesticide chemical 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, whether or not endocrine-related responses occurred at dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On November 13, 2013, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) 

of the Office of Pesticide Programs and the Office of Science Coordination and Policy conducted 

a weight-of evidence analysis of the potential interaction of benfluralin 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).

Benfluralin has a low vapor pressure (6.57 x 10-5mm Hg at 25°C), and therefore, volatilization is 

not expected to significantly contribute to the dissipation of benfluralin in the environment. The 

compound has a low water solubility (0.1 ppm at pH 7.00 and 25°C) and has a high 

octanol/water partition coefficient (log Kow = 5.29). It does not hydrolyze but does degrade 

rapidly in water due to photolysis. Benfluralin has a whole body bioconcentration factor value in 

fish of 1,580. Acute toxicity studies in various fish species have shown that benfluralin is highly 

toxic (LC50 values of 0.06 to 1.4 mg a.i./L). 

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

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

relevant information (OSRI) such as general toxicity studies and 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 study 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 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 

assays were submitted as OSRI by the Test Order recipients and/or the public. The agency’s 

review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team 

(USEPA, 2010). Since then, the Agency has also conducted a more recent search of available 

scientific literature for any additional relevant information. Summaries of the available OSRI are 

presented in Appendix 2. 

 

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 hormonal 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 

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 

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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 

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

 

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This uncertainty in distinguishing whether the responses were endocrine-related was discussed at 

the 2013 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 non-endocrine 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 (e.g., 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. 

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 

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which supports a conclusion of whether a substance has the potential to interact with the E, A, 

or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for 

each respective pathway (E, A or T) but are not reported in the table for E, A or T. 

A. EDSP T1 Screening Assays 

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

in Table 1. 

Table 1: Tier 1 Screening Assays for Benfluralin. 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied 

(MRID No. 48614904) 

ERα Transcriptional Activation Assay (Human 

cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48614905) 

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 

Requirement Satisfied 

(MRID No. 48614902) 

Steroidogenesis Assay (Human cell line 295R) OCSPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No. 48614908) 

Aromatase Assay (human recombinant 

microsomes) OCSPP 890.1200 

Requirement Satisfied 

(MRID No. 48614903) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48614909) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No. 48614907) 

Pubertal Female Assay (Rat) OCSPP 890.1450 

Requirement Satisfied 

(MRID No. 48669301) 

Pubertal Male Assay (Rat) OCSPP 890.1500 

Requirement Satisfied 

(MRID No. 48669302) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48614906) 

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 48616704) 

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

1. Effects on Estrogen Pathway 

The potential for benfluralin 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. 

Benfluralin was classified as not interactive (negative) in the in vitro Tier 1 estrogen receptor 

(ER) binding assay, and negative in the steroidogenesis and aromatase assays. A weak positive 

response in the Tier 1 ERTA was observed at benfluralin concentrations of 10−5 and 10−4 M. 

Effects in the Tier 1 female pubertal assay at the high dose (300 mg/kg/day), in the absence of 

overt toxicity, included statistically significant (p<0.05) decreases in ovarian weight [(17% and 

18% for unadjusted and adjusted (for body weight on PND 22)], respectively) and blotted 

uterine weights (21%, adjusted), delayed vaginal opening (3 days, p<0.05), and increased age at 

first estrus. A decrease (p<0.05) in absolute and relative pituitary weights was also observed 

(12 and 19%, respectively) in this assay. Indications of hepatotoxicity at the high dose (300 

mg/kg/day) included: increases (p<0.05) in gamma glutamyl transpeptidase (GGT, 47%); 

cholesterol (↑12%); and creatinine (↑44%); additionally liver weights were significantly 

increased at all doses in this assay (8-37%). 

In the FSTRA, there were no changes in estrogen-related endpoints such as gonadal 

histopathology, gonadal-somatic index (GSI), or plasma vitellogenin (VTG) concentrations. 

While there were no significant differences (p>0.05) in fecundity in the treatment groups 

compared to the negative control, decreases were observed in the low (0.302 μg a.i./L; ↓29%) 

and mid (3.14 μg a.i./L; ↓21%) treatment groups compared to the negative control (high 

treatment group terminated due to overt toxicity); however, a similar decrease was observed in 

the solvent control (↓39%) suggesting the observed decrease was not treatment-related. 

The only estrogen-related effect seen in the Part 158 mammalian toxicity studies was an increase 

in relative ovarian weight (14%, p<0.05) in the subchronic rat toxicity study at 605 mg/kg/day. 

In a Part 158 avian reproduction study with the northern bobwhite quail reductions in eggs laid, 

embryo viability, hatchling survival, and offspring body weight were observed at 990 ppm 

(increase in % of cracked eggs at this concentration but relatively large % in control as well, no 

effects on eggshell thinning); additionally, eggs laid and embryo viability/hatchlings was reduced 

at the mid concentration (295 ppm) and offspring survival/weight were affected at all 

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concentrations tested (≥96 ppm). While there were no reported treatment-related effects on 

parental survival, body weight, or behavior, food consumption prior to egg laying was 

significantly reduced (p<0.05) at the two lowest treatment concentrations while food 

consumption during egg laying was significantly reduced (p<0.05) at the two highest treatment 

concentrations. In an avian reproduction study with the mallard duck at 975 mg/kg-diet, there 

was a higher incidence (↑11.5%) of cracked eggs in the highest treatment concentration 

compared to control that wasn’t statically significant (p = 0.061), but considered to be 

biologically significant. Eggshell thickness for both cracked and normal eggs was not 

significantly affected by benfluralin exposure; no other treatment-related reproductive effects or 

signs of toxicity were observed. In a Part 158 rainbow trout fish early life-cycle study, larvae 

body length was significantly reduced at ≥5.0 μg a.i./L, and body weight was reduced at ≥14.8 

μg a.i./L.

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BENFLURALIN FINAL 

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Table 2: Estrogenic/Anti-Estrogenic Pathway for Benfluralin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Benflura

Study 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 48614904) N ERTA (MRID 48614905) P Steroidogenesis (MRID 48614908) N Aromatase (MRID 48614903) N Uterotrophic (MRID 48614909) N Female Pubertal Rat (MRID 48669301)

↓21% 

 (H)

↓17-18% 

(H) 

N ↓12-19% (H)

↑2 days 

(first 

estrus) 

(H) 

↑3 days 

(VO) 

(H) 

FSTRA (MRID 48614906) NE N N N N 

OSRI 

Subchronic toxicityRat (MRID 44050001) 

N ↑14% N N 

Subchronic toxicityDog (MRID 43072301) 

N N N N 

Chronic toxicity /Carcinogenicity-Rat N N N N 
BENFLURALIN FINAL 

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Table 2: Estrogenic/Anti-Estrogenic Pathway for Benfluralin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Benflura

Study 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 

(MRID 44050002, 44545001) Chronic toxicity -Dog (MRID 43628702) N N N N Carcinogenicity –Mouse (MRID 41021501) 

N N N N 

Developmental toxicity- Rat (MRID 000147535) 

N 

Developmental toxicity- Rabbit (MRID 42039101) 

 N N 

Two-generation reproduction- Rat (MRID 43628701, 43628702) 

 N N N N N 

Avian ReproductionQuail (MRID 42145502) 

P5 

(L,M,H) 

Avian ReproductionMallard (MRID 42145501) 

P7 

(H) 

1. Key to responses: L= Low treatment, M= Medium treatment, H= High treatment. Arrows (↓ or ↑) indicate the direction of the 

indicates that is parameter is not routinely evaluated or is not applicable in this assay. LW = liver weight, LE = serum levels of lihistopathological changes 

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

in this column. For details see Section IV. A 
BENFLURALIN FINAL 

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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 

details see Section IV. A 

4. Signs of toxicity including mortality, lethargy, loss of equilibrium and bloating were observed with ≥38% of the fish affected. 5. Reduction in eggs laid and embryo viability reported at ≥295 ppm; offspring survival affected at all doses (≥96 ppm). 

6. While there were no reported treatment-related effects on parental survival, body weight, or behavior, food consumption prior to 

significantly reduced (p<0.05) at the two lowest treatment concentrations while food consumption during egg laying was significthe two highest treatment concentrations 

7. Increase in percentage of cracked eggs at 975 mg/kg-diet. P Positive findings (in vitro)/No effect (in vivo) N Negative findings (in vitro)/No effect (in vivo) 
BENFLURALIN FINAL 

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2. Effects on Androgen Pathway 

The potential for benfluralin 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. 

In an evaluation of androgenic/anti-androgenic activity from Tier 1 testing results, benfluralin was 

classified as negative (non-binder) in the in vitro androgen receptor (AR) binding (rat prostate 

cytosol) assay, and the steroidogenesis assay. 

In the Hershberger assay, benfluralin did not induce significant increases in accessory sex tissue 

(AST) weights in the androgenic portion of the assay. However, at the high dose (750 mg/kg/day) 

which was determined to be an overtly toxic dose (↓12% in body weight), animals had significant 

decreases in LABC (27%) and seminal vesicles (33%) weight in the anti-androgenic portion of 

the assay. The changes in androgen responsive tissue weights were accompanied by a nonsignificant decrease (~ 29%) in serum testosterone levels. There were significant treatment-related 

increases (p≤0.5) in absolute liver weight of about 38% and 68% at 250 and 750 mg/kg bw/day 

benfluralin, respectively, in the androgenic portion, and about 39% and 65% in the anti-androgenic 

portion of assay. 

A follow-up study to the Hershberger assay was conducted to examine 14C-testosterone clearance. 

The results confirmed that animals administered 750 mg/kg bw/day benfluralin for ten days had 

similar effects on body weight, body weight gain, and liver and androgen-responsive tissue weights 

as high-dose animals in the definitive Hershberger assay. These animals showed an approximately 

3.7-fold increase in 14C-testosterone clearance compared to the control animals, resulting in lower 

plasma area under the curve (AUC) values (65%) relative to control after ten days of benfluralin 

treatment. These data suggest that testosterone in benfluralin-treated animals was less available to 

interact with androgen-responsive tissues and stimulate/maintain organ weights. The dose of 750 

mg/kg/day was an overtly toxic dose and the effects of benfluralin on testosterone clearance at lower 

doses in the absence of overt toxicity is unclear. 

In the male pubertal assay no changes in androgen-related endpoints were seen at the low or mid 

dose. At the high dose (400 m/kg/day), in the absence of overt toxicity, significant (p<0.05) 

decreases in the weights of the seminal vesicles (32%), ventral prostate (26%), dorsolateral prostate 

(21-22%), LABC (20%) and epididymis (13%) were seen. In addition to the tissue weight 

changes, a 52% decrease (p<0.05) in serum testosterone was also observed at the high dose. 

Absolute and adjusted pituitary weights were decreased at 400 mg/kg bw/day benfluralin, but 

relative pituitary weights were not significantly different. No histopathological lesions were 

Page 23 of 61
BENFLURALIN FINAL 

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observed in the testis, but two animals, in the high dose, were noted to have slightly decreased 

spermatic elements in the epididymis. The age and body weight at preputial separation were not 

affected at any dose level. At 100 mg/kg/day, there were significant (p<0.05) increases in absolute 

(23%) and relative (22%) liver weights. At 400 mg/kg/day, hepatotoxicity was characterized by 

significant (p <0.05) increases in absolute (40%) and relative (52%) liver weights as well as 

increases in serum creatinine (50%) and GGT (60%) levels. 

 

In the FSTRA, there were no changes in androgen-related endpoints such as gonadal histopathology, 

VTG concentrations, or male secondary sex characteristics. There was a statistically significant 

increase (27%) in male GSI at the middle treatment concentration (3.14 μg a.i/L; high concentration 

terminated prior to test termination due to signs of overt toxicity). 

In the mammalian Part 158 studies, the only effect on androgen-related tissue weights was a 48-50% 

increase in testicular weight observed in the rat chronic/carcinogenicity study at a dose (275 

mg/kg/day) that showed overt toxicity. In the Part 158 bobwhite quail reproduction study, 

reductions in embryo viability and/or offspring survival were reported at concentrations of ≥96 ppm; 

however, it is noted that reductions in food consumption were noted in all test concentrations along 

the duration of the study.

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BENFLURALIN FINAL 

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Table 3: Androgenic/Anti-Androgenic Pathway for BenfluralinLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Benfluralin1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histo

pathology

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility/Fecundity 

Age and Weight at 

PPS 

EDSP Tier 1 Assay 

AR Binding (MRID 48614902) N Steroidogenesis (MRID 48614908) N Hershberger (MRID 48614907) N N Male Pubertal Rat (MRID 48699302) 

↓52% 

(H) N N 

↓13 - 

18% 

(H) 

N N 

↓SV, GP, 

DP, VP, 

LABC 

(H) 

N 

FSTRA (MRID 48614906) NE 

GSI: 

↑27% 

(M) 

N N N N 

OSRI 

Subchronic toxicityRat (MRID 44050001) 

N N N N N N 

Chronic toxicity-Rat (MRID 44050002, 44545501) 

↑48 - 

50% 

(H) 

N N N N N 

Chronic toxicity -Dog (MRID 43628702) 

N N N 
BENFLURALIN FINAL 

Page 18 of 53 

Table 3: Androgenic/Anti-Androgenic Pathway for BenfluralinLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Benfluralin1Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes 

Weight/GSI 

Gonad Staging 

and 

Histo

pathology

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility/Fecundity 

Age and Weight at 

PPS 

Carcinogenicity –Mouse (MRID 41021501) 

 N N N N N N 

Developmental toxicity-Rat (MRID 000147535) 

N 

Developmental toxicity- Rabbit (MRID42039101) 

N 

Two-generation reproduction- Rat (MRID 43628701, 43628702) 

N N N N N N N 

Avian Reproduction- Quail (MRID 42145502) 

P5

(L,

ML

H) 

Avian ReproductionMallard (MRID 42145501) 

 N 

1. Key to responses: L= Low treatment, M=Medium treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the respo

indicates that is parameter is not routinely evaluated or is not applicable in this assay. LW = liver weight, LE = serum liver enzymes

size, fertile eggs). 

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

this column. For details see Section IV. A 

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

details see Section IV. A 

4. Signs of toxicity including mortality, lethargy, loss of equilibrium and bloating were observed with ≥38% of the fish affected. 
BENFLURALIN FINAL 

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5. Reduction in embryo viability reported at ≥295 ppm; offspring survival affected at all doses (≥96 ppm) 

6. No reported treatment-related effects on survival or behavior, but food consumption was reduced at two lowest treatments prior t

in two highest treatments during egg production. 

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

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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 that can detect changes in the HPT axis, i.e., 

the pubertal female and male (rat) assays, and the AMA (frog). 

The potential for benfluralin 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. 

In the amphibian metamorphosis assay (AMA), there were no treatment-related effects on 

developmental stage, asynchronous development, nor did it show remarkable thyroid histological 

effects. A significant (p<0.05) 10% increase in 21-day wet weight, relative to the negative 

control, was observed at the lowest 0.0027 mg a.i./L level; however, higher levels were not 

significantly different from the negative control (p>0.05). 

In the Tier 1 male pubertal assay, significant (p<0.05) dose-responsive decreases in T4 (12%, 

29%, and 53%) were seen at 25, 100 and 400 mg/kg/day, respectively. This correlated to a 

corresponding increase in TSH (53% and 105%) at 100 and 400 mg/kg/day, respectively. In 

the Tier 1 female pubertal assay, serum T4 levels were significantly (p<0.05) decreased 13% and 

44% at 75 and 300 mg/kg/day, respectively. A 109% increase in TSH was observed at 300 

mg/kg/day in females. In addition to the changes in thyroid-related hormones, thyroid weights 

(adjusted for body weight on PND 23) were increased 15% at the high dose in males. Increases 

in follicular cell height with a related decrease in colloid area were also observed at the high dose 

in males. The thyroid-related effects observed in the male and pubertal assays occurred at doses 

that also resulted in increased liver weights in both sexes. 

Thyroid perturbations were also seen in Part 158 toxicity studies with benfluralin, including the 

90-day oral toxicity and the chronic/carcinogenicity studies in the rat. Female rats in the 90-day 

oral toxicity study at 605 mg/kg/day significant (p<0.05) increases in absolute and relative 

thyroid gland weight (↑22 and 27%, respectively) were observed, but no histological changes 

were reported. In the chronic/carcinogenicity study, male and female rats at 2500 and 5000 ppm 

(136/275 and 168/331 mg/kg/day in males and females, respectively) had increased thyroid 

follicular cell hypertrophy and hyperplasia, plus increased incidences of combined follicular cell 

adenomas/carcinomas. Both sexes also had increased hepatocellular hypertrophy at these same 

doses, but males also had increased incidences of hepatocellular necrosis and adenomas. 

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Table 4: Thyroid Pathway for Benfluralin

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Benfluralin1 

Study Type/ 

Literature 

Citation 

Serum T4 Level 

Serum TSH Level Pituitary Weight Thyroid Weight Thyroid: Gross and Histopathology Developmental stage or asynchronous & 

HLLGrowth (BW, SVL) Systemic Toxicity Observed2 Overt Toxicity Observed3

EDSP Tier 1 Assays 

Male Pubertal 

Rat (MRID 

48669302) 

↓12%,

29%, 

53% 

(L,M,H) 

↑53%, 

105% 

(M,H) 

↓14% 

H 

↑15% 

H P4

↑LW 

(M,H), 

↑LE 

 (H) 

N 

Female Pubertal 

Rat 

(MRID 

48669301) 

↓13%, 

44% 

(M.H) ↑109% 

(H) 

↓12-

16% 

H 

N N 

↑LW 

(M,H) 

↑LE 

(H) 

N 

AMA (Frog) 

(MRID 

48616704) 

 N N N N 

OSRI 

Two-generation 

reproduction- Rat 

(MRID 

43628701, 

43628702)

N N N X N 

Subchronic 

toxicity-Rat 

(MRID 

44050001) 

N 

↑17 

- 

27% 

N X N 

Chronic toxicity 

- Rat 

(MRID 

44050002, 

44545501) 

N 

↑33 

- 

64% 

P X X 

(H) 

Chronic toxicity -

Dog 

(MRID 

43628702) 

N N N X N 

1. Key to responses: L= Low treatment, M=Medium 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, LE = serum liver enzymes 

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

systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the 

OSRI is indicated by an X in this column. For details see Section IV. A 

4. Increases in follicular cell height with a related decrease in colloid area were observed at the high dose 

P Positive findings 

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N Negative findings 

IV. Committee’s Assessment of Weight of Evidence 

This section of the document will describe the weight of evidence (WoE) determination on the 

potential of benfluralin 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 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 nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and 

OSRI studies are described 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., organ weights or blood chemistry). 

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1. Tier 1 in vivo Assays 

In the Hershberger assays, decreases in bodyweight and bodyweight gain were observed at the 

high dose (750 mg/kg/day) in both the androgenic and antiandrogenic phases. The decreases in 

body weights were 7-11% in the androgenic phase and 10-14% in the anti-androgenic phase. The 

decreases in body weight gains were 49% and 57%, in the androgenic and anti-androgenic 

phases, respectively. In the high dose anti-androgenic group as well as the test group to evaluate 

testosterone clearance, body weights were decreased by 12-20% resulting in a decreased (↓85%) 

cumulative body weight gains. In addition to body weight effects, absolute liver weights were 

significantly increased at the mid (↑38%) and high (↑68%) dose groups in the Hershberger assay. 

Liver weights were similar to controls at the low dose. 

In the male pubertal assays benfluralin, there was no treatment-relate effects on bodyweights or 

body weight gains. In the female pubertal assay, at the high dose (300 mg/kg/day), body weights 

were significantly decreased (8-10%) during PND 22-35, resulting in cumulative decreases in 

body weight gain (15-42%) for each interval up to PND 22-35. Hepatotoxicity was seen in both 

the pubertal assays. In males, absolute liver weight were increased by 22% at the mid dose (100 

mg/kg/day) and by 40% at the high dose (400 mg/kg/day). Relative liver weights were increased 

by 22% and 52% at the mid and high dose groups, respectively. Also, there was an increase 

(60%) in GGT at the high dose only. In females, absolute liver weights were increased by 22% at 

the mid- and by 32% at the high dose. Relative liver weights were increased by 19% and 37% at 

the mid and high dose groups, respectively. Also, there were increases in GGT (47%) and 

cholesterol (12%) levels at the high dose. Creatinine levels were also elevated in males (50%) 

and females (44%) at the high dose, but were not accompanied by histopathological findings or 

significant changes in weight of the kidney. 

In the FSTRA, overt toxicity (mortality, lethargy, loss of equilibrium, bloating) was observed at 

the high test concentration (0.0365 mg a.i./L) with 38% of the fish affected, and as such this 

treatment group was terminated prior to the scheduled test termination. No overt toxicity was 

observed at the lower test concentrations. No treatment-related mortality or clinical signs of 

toxicity were observed in the AMA at the concentrations tested (≥74.4 μg a.i./L). 

2. OSRI 

The liver and the kidneys were the target-organs in the Part 158 studies with benfluralin. In the 

subchronic toxicity study with rats, benfluralin induced dose-related nephritis in males and 

cortical tubular epithelial pigment deposition, increases in absolute and relative liver and kidney 

weights at 20 mg/kg/day, cortical tubular epithelial regeneration, urinary AST increase and liver 

hypertrophy in males at 32 mg/kg/day. In the chronic toxicity study with dogs, in males at the 

high dose (125 mg/kg/day), there were increased absolute and relative liver weights with 

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corroborative increased incidence of minimal to slight centrilobular to diffuse hepatocellular 

hypertrophy. In the two-generation reproduction study, liver effects included dose-dependent 

increases in the absolute and relative weights at 68 and 342 mg/kg/day in the parental animals 

(F0 and F1). Kidney effects observed at these doses included increased incidence of hyaline 

droplets (graded as minimal) in males and chronic progressive nephropathy (graded as slight 

and/or moderate) in males and females. In the chronic toxicity/carcinogenicity study with rats, 

significant increases (p<0.05) in absolute (19-43%) and relative (29-104%) liver weights, were 

observed in both sexes at the mid (136 mg/kg/day) and high (295 mg/kg/day) dose 

groups.Toxicity was corroborated by changes in several serum chemistry parameters (alterations 

in cholesterol, bilirubin, and liver enzymes) and histopathology findings (non-neoplastic and 

neoplastic lesions). Relative thyroid weights were elevated (p<0.05) (+33 to +64% over 

controls) at the mid and high dose groups and correlated with microscopic thyroid lesions (nonneoplastic and neoplastic). Liver and thyroid tumors were seen in both sexes. In the 

carcinogenicity study with mice, there were liver nodules at the mid dose (42 mg/kg/day), 

alterations in a number of liver enzymes (ALSP and alanine aminotransferase) levels, and multi 

focal hyperplasia of the liver and liver tumors in female mice. 

No treatment-related mortality or clinical signs of toxicity were observed in the Part 158 mallard 

duck reproduction study (MRID 42145501). For the bobwhite quail reproduction assay, while 

there were no treatment-related signs of toxicity or mortality, food consumption prior to egg 

laying was significantly reduced (p<0.05) at the two lowest treatment concentrations (96 and 295 

mg/kg-diet (ppm)) while food consumption during egg laying was significantly reduced (p<0.05) 

at the two highest treatment concentrations (295 and 990 ppm). 

In a Part 158 rainbow trout fish early life-cycle study, larvae body length was significantly 

reduced at ≥5.0 μg a.i./L, and body weight was reduced at ≥14.8 μg a.i./L. 

B. Estrogen pathway 

Although the Tier 1 ERTA was positive for ER agonism, interaction with the estrogen pathway 

via the ER was not consistent with the negative findings in the Tier 1 ER binding and 

uterotrophic assays. Estrogen-related effects were observed at the high dose in the Tier 1 female 

pubertal assay including decreases in pituitary weight, ovarian weight, blotted uterine weight, 

delayed vaginal opening and increased age at first estrus. All estrogen-related findings in the 

EDSP Tier 1 in vivo assays and Part 158 mammalian studies occurred in the presence of liver 

effects including increases in absolute and relative liver weights, and significant increases in 

creatinine, GGT and cholesterol. Additionally, an increase in relative ovarian weight in the Part 

158 mammalian subchronic rat study occurred at the high dose along with significant increases 

in absolute and relative liver weights. The observed effects are consistent with the findings in the 

mode of action studies which support hepatic enzyme induction resulting in enhanced hormone 

clearance in mammals. 

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No effects on the estrogen pathway were observed in the FSTRA. In a one-generation Part 158 

avian reproduction study with bobwhite quail, a decrease in eggs laid, as well as effects on 

offspring survivability and growth, were observed; however, these effects coincided with 

decreases in feed consumption. In the companion mallard duck reproduction study, while an 

increase in eggs cracked was reported, no effects on eggshell thinning or other reproductive 

effects were observed. 

C. Androgen pathway 

For the androgen pathway, the only positive findings seen in the Hershberger assay occurred at 

the high dose and in the presence of overt toxicity. Although the presence of overt toxicity 

confounds the interpretation of the effects on the endocrine parameters in the study, a follow up 

study to evaluate testosterone clearance showed enhanced metabolism/clearance of testosterone 

at the dose that resulted in a decreased androgen-sensitive tissue weights. This suggests that the 

antiandrogenic effects observed in the Hershberger assay are likely attributed to enhanced 

testosterone clearance. In the male pubertal assay, decreased serum testosterone and decreases 

in androgen-sensitive tissue weights and epididymal weights were seen only at the high dose 

(400 mg/kg/day). The endocrine-related effects were seen in the presence of hepatotoxicity 

which manifested as increases in absolute (40%) and relative (52%) liver weights and an increase 

(60%) in GGT level. Increased liver weights were also observed at lower doses where no 

androgen-related effects were seen. The only androgen-related effect observed in the Part 158 

mammalian studies was an increase in testicular weight in the rat chronic/carcinogenicity study 

which occurred in the presence of overt toxicity. The evidence from the mammalian toxicity 

studies suggests that the androgen related effects observed are due to liver enzyme induction and 

increased hormone clearance and excretion. In the FSTRA the only notable effect in the absence 

of overt toxicity was an increase in male GSI seen at the middle treatment concentration. In the 

Part 158 avian bobwhite quail study, a decrease in offspring survivability was observed; however 

decreased feeding was seen in the parental generation. 

D. Thyroid pathway 

For the thyroid pathway, the effects in the Tier 1 assays were seen in the male and female 

pubertal assays, but no evidence of thyroid activity was seen in the AMA. In the pubertal 

assays, significant decreases in serum T4 were seen at all doses in the male and at the mid and 

high dose in the female. TSH was significantly increased at the mid and high dose in males and 

high dose only in females. Increases in thyroid weight and altered thyroid histopathology were 

seen only in males at the high dose. All effects on the thyroid parameters in the pubertal assays 

were accompanied by increased relative liver weights. Thyroid perturbations were observed in 

the subchronic rat toxicity study (increased thyroid weights) at a high dose that caused 

significant increases in liver weight. Thyroid follicular cell hypertrophy and hyperplasia were 

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observed in both sexes in the chronic/carcinogenicity study in rats. An increased incidence of 

follicular cell adenomas/carcinomas was observed in this study, but at a dose that was 

considered to be overtly toxic. 

Previously submitted mechanistic studies showed that benfluralin induces hepatic microsomal 

enzyme activity in both sexes in the rat. In a 14-day study submitted to support a postulated 

mode of action for liver and thyroid tumors in the rat, benfluralin was shown to induce 

constitutive androstane receptor (CAR) and pregnane X receptor (PXR) related gene transcripts 

and associated PROD enzyme activities and benfluralin also up-regulated T4-specific UGT 

genes associated with T4 metabolism. These effects occurred at a dose that resulted in 

decreased serum T4 and T3 levels and increased liver weight and hepatic hypertrophy. 

Furthermore, benfluralin is structurally similar to another dinitroaniline (trifluralin) which has 

been shown to induce hepatic UGTs, resulting in decreased serum T3 and T4 levels, elevated 

TSH and altered thyroid histopathology leading to increased thyroid tumors in rats (Saghir et 

al., 2005). 

E. Conclusions 

An evaluation of the Tier 1 and OSRI data show that the observed effects on the endocrinerelated endpoints for the estrogen, androgen and thyroid pathway occur primarily when there 

were concurrent increases in liver weight. A WoE evaluation of the available data suggests that 

the anti-estrogenic, anti-androgenic and anti-thyroid effects observed in the mammalian in vivo

Tier 1 assays may result from liver enzyme induction and subsequent increased hormone 

clearance. However, there is no convincing evidence for an interaction with estrogen, androgen 

or thyroid pathways in wildlife. 

V. EDSP Tier 2 Testing Recommendations 

For benfluralin, the current point of departure (POD) of 0.5 mg/kg/day is based on increased 

histopathological lesions of the kidneys seen in male and in female rats at 5.4 mg/kg/day 

(LOAEL) in a combined chronic toxicity/carcinogenicity study in rats. This POD is used to 

derive the chronic Reference Dose (RfD) used for dietary exposure risk assessments and for 

calculating the Margins of Exposure (MOEs) for non-dietary exposure risk assessments. This 

POD is 50-fold lower than the lowest dose (25 mg/kg/day) that caused thyroid-related effects in 

the Tier 1 male pubertal assay, and 600 to 800-fold lower than the doses that caused estrogenrelated (300 mg/kg/day) or androgenic-related (400 mg/kg/day) effects in the pubertal assays. 

Consequently, the chronic RfD and MOEs derived from current PODs are protective of the 

endocrine-related findings in the Tier 1 in vivo assays or OSRI. In addition the totality of the data 

demonstrated that the endocrine-related effects seen in the mammalian in vivo Tier 1 assays may 

result from liver enzyme induction and subsequent increased hormone clearance. 

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Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is NOT 

recommended for benfluralin since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

As there is no convincing evidence for a potential interaction with the estrogen or androgen 

pathway in fish or thyroid pathways in amphibians, no additional testing is recommended. 

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

duck are available for benfluralin. 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 benfluralin 

exposure. Additional testing is not recommended. 

 

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VI. References 

Adams, E.R., and Jordan, W.H. (1996). Subchronic toxicity studies of benefin (Compound 

054521) administered orally in the diet to Fischer 344 rats for 3 months. Unpublished study 

performed by Lilly Research Laboratories, Greenfield, IN. Laboratory Project ID: R33989, 

R44089, and R29990, February 29, 1996. MRID 44050001. 

Coady K.K., and Sosinski L.K. (2011). Benefin: Evaluation of Benefin in the Human 

Recombinant Aromatase Assay. Unpublished study performed by Toxicology & Environmental 

Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Report No.: 

101143, 04 October 2011. MRID 48614903. 

Coady, K.K., Marino, T.A., Hutchinson, K.L., McClymont, E.L., and Thomas, J. (2011). 

Benefin: The Amphibian Metamorphosis Assay Using the African Clawed Frog, Xenopus laevis. 

Unpublished study performed by Toxicology & Environmental Research and Consulting, The 

Dow Chemical Company, Midland, MI. Laboratory report number 101097. MRID 48614901. 

Cocke, P.J., Gunnoe M.D., and Koenig, G.R. (1990). The Toxicity of Benefin to Rainbow Trout 

(Salmo gairdneri) in a 49-Day Early Life-Stage Study. Performed by Lilly Research 

Laboratories, Greenfield, IN. Laboratory Study No.: F00690. MRID 41613805. 

Dalgard, D. W. (1993). Benefin: 13-Week Oral Toxicity Study in Beagle Dogs. Unpublished 

study prepared by Hazleton Washington, Inc., Vienna, VA. Lab Project No.: HWA 174-135, 

December 28, 1993. MRID 43072301

Koenig, G.R., Jordan, W.H., (1998) A Chronic Toxicity and Oncogenicity Study in B6C3F 1 

Mice given Benefin (EL-11 0, Compound 54521) in the Diet for Two Years. Unpublished study 

performed by Lilly Research Laboratories, Greenfield, IN. Laboratory Project ID: M02785 and 

M02885, December 14, 1988. MRID 41021501. 

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2011). Evaluation of Benefin in an in vitro 

androgen receptor binding assay. Unpublished study performed by Toxicology & Environmental 

Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project ID: 

111097, October 21, 2011. MRID 48614902. 

LeBaron, M.J., Schisler, M.R., Visconti, N.R. (2011). Evaluation of Benefin in an in vitro

Estrogen Receptor Binding Assay. Unpublished study performed by Toxicology & 

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Page 29 of 53 

Environmental Research and Consulting, Dow Chemical Company, Midland, MI. Laboratory 

Project ID: 111123, October 25, 2011. MRID 48614904. 

LeBaron, M.J.; Kan, H.L. (2011) Evaluation of Benefin in an in vitro Estrogen Receptor 

Transcriptional Activation Assay in Human Cell Line HeLa-9903. Unpublished study performed 

by Toxicology & Environmental Research and Consulting, Dow Chemical Company, Midland, 

MI. Laboratory Project ID: 101184, October 20, 2011. MRID 48614905. 

Lebaron, M.J.; Kan, H.L.; Perala, A.W. (2011). Evaluation of Benefin in the In Vitro 

Steroidogenesis Assay. Unpublished study performed by Toxicology & Environmental Research 

and Consulting, The Dow Chemical Company, Midland, Michigan, 48674. Laboratory Study 

No.: 101183, October 14, 2011. MRID 48614908. 

Marino, T.A., K.K. Coady, R.R. Hukkanen, D.W. Louch, N.A. Malowinski, L.K. Sosinski. 

(2011). Benefin: A Fish Short-Term Reproduction Assay with the Fathead Minnow, Pimephales 

promelas. Unpublished study performed by Toxicology and Environmental Research and 

Consulting, The Dow Chemical Company, Midland, Michigan. Laboratory Study No.: 101165, 

September 22, 2011, revised October 17, 2011. MRID 48614906. 

Marty, M.S., Marshall, V.A., Hansen, S.C., et al. (2011). Benefin: Hershberger assay in 

castrated adult male Crl:CD(SD) rats. Unpublished study performed by Toxicology & 

Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. 

Laboratory Project Study ID: 101114, October 17, 2011. MRID 48614907. 

Marty, M.S., Brooks, K.J., and Jeong, Y.C. (2011). Benefin: uterotrophic assay in the immature 

female Crl:CD(SD) rat. Unpublished study performed by Toxicology & Environmental 

Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project ID: 

101116, June 23, 2011. MRID 48614909. 

Marty, M.S., Zablotny, C.L., Hukkanen, R.R. (2012). Benefin: pubertal development and 

thyroid function in intact juvenile/peripubertal female Crl:CD(SD) rats. Unpublished study 

performed by Toxicology & Environmental Research and Consulting, The Dow Chemical 

Company, Midland, MI. Laboratory Study No.: 111073, January 25, 2012. MRID 48669301. 

Marty, M.S., Andrus, A.K., Stebbins, K.E. (2012) Benefin: pubertal development and thyroid 

function in intact juvenile/peripubertal male Crl:CD(SD) rats. Unpublished study performed by 

Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, 

MI. Laboratory Study No.: 111074, January 25, 2012. MRID 48669302. 

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Page 30 of 53 

MacKenzie, K.M. (1985). Rat Teratology Study with Benefin. Unpublished study performed by 

Hazleton Laboratories, Greenfield, Indiana. Laboratory Study No.: 6180-101, June 24, 1985. 

MRID 000147535.

Mercieca, M.D. (1991). Teratology Study in Rabbits with Benefin: Final Report. Unpublished 

study performed by Springborn Laboratories, Inc., Spencerville, OH. Laboratory Study No.: 

3130.9, June 3, 1991. MRID 42039101.

Moore, M.R. (1996). Benefin: Two-Year Dietary Chronic Toxicity/Oncogenicity Study in 

Fischer 344 Rats. Unpublished study performed by Corning Hazleton, Inc. (CHV), Midland, 

Michigan. Laboratory Study ID.: CHV 174-133: DR-0097-3397-005, July 1, 1996. MRID 

44050002. 

Moore, M.R (1998). Benefin: Two-Year dietary Chronic Toxicity/Oncogenicity Study in 

Fischer 344 Rats. Unpublished study performed by Covance Labs. Inc., VA. Laboratory Study 

No.: CHV174-133, DR-0097-3397-005, April 22, 1998. MRID 44545501. 

Murray, A.G., Smith, J.R., and Grothe, D.W. (1991). The Toxicity of Benefin to Mallards in a 

One-Generation Reproduction Study. Performed by Toxicology Research Laboratories, Lilly 

Research Laboratories, Greenfield, Indiana. Laboratory Project No.: A01090. MRID 

42145501.

Murray, A.G., Seacat, J.L., and Grothe, D.W. (1991). The Toxicity of Benefin to Bobwhite in a 

One-Generation Reproduction Study. Prepared by Toxicology Research Laboratories, Lilly 

Research Laboratories, Greenfield, Indiana. Laboratory Project No.: A00690. MRID 

42145502. 

S. Papineni, S., Marty, S.M., Kramer, V.J., Juberg, D.R., Gehen, S., and K. K. Coady. 

Benfluralin: Evaluation of Potential Estrogen, Androgen or Thyroid Effects. Prepared by Dow 

AgroSciences (MRID No.48698801) 

Saghir, S., Charles, G., Bartels, M., Kan, L., Dryzga, M., Brzak, K., Calrk, A. (2008) 

Mechanism of trifluralin-induced thyroid tumors in rats. Toxicology Letters 180, pp. 38-45. 

Trotter, J. (1995). Two-Generation Reproduction Study in Rats with Benefin: Final Report:. 

Unpublished study performed by Hazleton Washington, Inc., Vienna, VA. Laboratory Project 

No.: HWA 174-136, February 9, 1995. MRID 43628701. 

USEPA (2010) Benfluralin: Report of the Endocrine Disruptor Review Team – Test Order #: EDSP 

084301-15 .December 16, 2010. Office of Chemical Safety and Pollution Prevention. U.S. 

Environmental Protection Agency, Washington, D.C. 

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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. 

Walker, M.D. (1995). Benefin: One-Year Oral Chronic Toxicity Study in Beagle Dogs: Final 

Report. Unpublished study performed by Corning Hazleton Inc., Vienna, VA. Laboratory 

Project No.: CHV 174-143: DR-0097-3397-004, April 26, 1995. MRID 43628702. 

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APPENDIX 1. EDSP T1 Screening Assays 

Amphibian Metamorphosis Assay 

The 21-day assay (MRID No. 48616704) of benfluralin (97.5% purity) on amphibian 

metamorphosis of the African clawed frog (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 (0.1 mL/L dimethylformamide; DMF) 

controls and benfluralin at nominal concentrations of 0.004, 0.020, and 0.100 mg a.i./L. Timeweighted average (TWA), measured concentrations were <0.00020 (<LOQ; controls), 0.0027, 

0.0141, and 0.0744 mg a.i./L. The test system was maintained at 21.8 to 22.3°C and a pH of 6.9 

to 7.9. 

There were no significant differences (p>0.05) between the negative and solvent control 

responses for any endpoint. Unless otherwise indicated, all effects are reported based on 

comparison to the negative control. 

No mortalities or clinical signs (i.e., behavioral and other sublethal effects) were observed. Day 

21 wet weight was significantly increased (p<0.05) by 10% in the low treatment group; the mid 

and high treatment groups were not significantly different (p>0.05) from the negative control. 

Normalized (for snout-vent length) hind-limb length (HLL) was comparable for both controls 

and all treatment groups on Day 7 and Day 21. The median NF developmental stage was the 

same for both controls and all treatment groups on both Day 7 (NF stage 54) and Day 21 (NF 

stage 57); no asynchronous development was observed throughout the test. No treatment-related 

effects on thyroid gland histopathology were observed in any treatment group. Mild follicular 

cell hypertrophy was detected in both controls and all treatment groups. 

Androgen Receptor (AR) Binding Assay 

In an androgen receptor (AR) binding assay (MRID 48614902), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR. Prior to conducting the AR competitive 

binding experiments, saturation binding experiments were conducted to demonstrate that the AR 

in the rat prostate cytosol was present in reasonable numbers and was functioning with 

appropriate affinity for the radiolabeled ligand. The saturation binding experiment was 

conducted in duplicate and summarized in a submitted study profile (MRID 48614902). 

The competitive binding assay was conducted to measure the binding of a single concentration of 

[

3

H]-R1881 (10 nM) in the presence of increasing concentrations of benfluralin (logarithmic 

increase from 10-10 to 10-3 M). Ethanol was used as a vehicle at a final assay concentration of 

<3%. The assay included dexamethasone as a weak positive control, and R1881 as the ligand 

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reference standard. Three independent runs were conducted with 3 replicates per concentration 

per run. 

In the saturation binding experiment, the maximum binding capacity (Bmax) was 3.245 fmol/100 

μg protein and the dissociation constant (Kd) was 0.4641 nM. These values were below the 

range of values from the validation studies (7 to 16 fmol/100 μg protein and 0.685-1.57 nM, 

respectively). The Scatchard plot indicated a linear response across the concentrations of ligand 

added. Nonspecific binding as a percent of total binding was less than 20% across the entire 

concentration range in the saturation binding assays (range 6.2-19.8%), with the exception of the 

high concentration (10 nM) in one assay, which was 24.6%. 

The estimated log IC50s for R-1881 and the weak positive control (dexamethasone) were -8.975 

and -4.509 M, respectively. The mean RBA for the positive control was 0.0034%. The solvent 

control responses indicated no drift in the study assay. All performance criteria were met, with 

the exception of the bottom (% binding) of R1881. In Run 2, the bottom was 2.1, just outside the 

recommended range of -2.0 to 2.0%. The range for the other two runs was -1.0 to -1.7, with a 

mean value for the three assays of -1.6%. The reviewers consider this a minor deviation and find 

that the performance criteria were met. 

The lowest percent total [3

H]-R1881 binding at all concentrations of benfluralin was 77%, 78%, 

and 75% resulting in classification for the runs as non-binder, non-binder and equivocal in runs 

1, 2, and 3, respectively. An estimated mean log IC50 for benfluralin could not be determined 

since benfluralin did not displace more that 50% of the radioligand at any concentration. 

Based on the results from the three runs, benfluralin is classified as a Non-Binder in the 

Androgen Receptor Binding Assay. 

Aromatase Assay 

In an in vitro aromatase (CYP 19) assay (MRID 48614903), Benfluralin (97.5% a.i., Lot No. 

ACD13683, TSN100037) was incubated with human recombinant aromatase and tritiated 

androstenedione (1-β [3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in dimethyl sulfoxide 

(DMSO) at concentrations of 0, 10-10, 10-9, 10-8, 10-7, 10-6, 10-5.5, 10-4, or 10-4.5 M for 15 minutes 

to assess the potential of benfluralin to inhibit aromatase activity. Evidence of test material 

insolubility was observed at 10-3 and 10-4 M therefore the highest test concentration analyzed 

was 10-4.5 M. 

Aromatase activity was determined by measuring the amount of tritiated water produced at the 

end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified 

with liquid scintillation counting (LSC). Three runs were conducted, and each run included a 

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full activity control, a background activity control, a positive control series (10-10 to 10-5) with a 

known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the test chemical series (10-10 to 

10-4.5) with three repetitions per concentration. The proficiency evaluation data for the 

investigating laboratory regarding the aromatase assay were not included in the study. 

The response of the full activity controls and background controls were acceptable for each run, 

although the mean background count from assay Run 3 was approximately 100-fold greater 

compared to the previous two assay runs. 

The standard curves for 4-OH ASDN met three out of four performance criteria in Run 1, all four 

criteria in Run 2, and only two out of four performance criteria in Run 3. These deviations from 

the guideline recommendations for performance criteria were considered minor by the 

investigators because acceptable Hill slope values and goodness of fit values were obtained for 

all three assay runs. In general, values in Run 3 were more variable compared to the previous 

two assay runs, including a higher background count and higher %CVs for the replicate tubes. 

Aromatase activity was similar across the tested concentration range for benfluralin (10-10 M to 

10-4.5 M). The aromatase activity curves after exposure to benfluralin either did not fit the model 

(Run 1) or were not lower than 75% of full activity (Runs 2 and 3). The log IC50 of the test 

material was -8.0 M. 

Based on the data from the average response curve, and despite the large variability introduced 

by the data from assay Run 3, benfluralin is classified as a Non-inhibitor of aromatase activity in 

this assay. 

Estrogen Receptor (ER) Binding Assay 

In an estrogen receptor (ER) binding assay (MRID 48614904), uterine cytosol from SpragueDawley rats was used as the source of estrogen receptors (ER) to evaluate the potential for 

benfluralin to displace the bound reference estrogen, radiolabeled [3

H]-17β-estradiol from the 

ER. A saturation binding experiment was conducted prior to the competitive binding experiment 

to demonstrate that the ER in the uterine cytosol was present in adequate numbers and 

functioning with the appropriate affinity for the radiolabeled ligand. Saturation binding data 

were not presented in the study report; however, saturation binding data was presented in the 

study profile submitted separately by the test order recipient (study profile, MRID 48675404). 

The 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 of 

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benfluralin (logarithmic increase from 10-10 to 10-3 M). Ethanol was used as a vehicle at a final 

concentration of <3%. The assay included 19-norethindrone as a weak positive control, 

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

material. Three independent runs were conducted with 3 replicates per concentration per run. 

In the saturation binding experiment, the maximum binding capacity (Bmax) was 59.28 fmol/100 

μg protein and the dissociation constant (Kd) was 0.1032 nM. The Bmax fell within the expected 

range of 10 to 150 fmol/100 μg protein and the Kd for the run was within the expected range of 

0.03 to 1.5 nM. Nonspecific binding as a percent of total binding was 1.7%-8.6% across the 

entire concentration range in the saturation binding assay. Only a single run of the saturation 

binding experiment was conducted on the batch of cytosol preparation used for the competitive 

binding experiment. 

For the competitive binding experiment, all performance criteria were met for appropriate 

responses by 17β-estradiol and 19-norethindrone. The log IC50 and RBA could not be 

determined for benfluralin as it did not displace ≥ 50% for the radiolabeled estradiol from the ER 

at any concentration. The bottom of the curve for percent of total binding at the 95% confidence 

interval for benfluralin was 87.7%, 70.2%, and 75.9% in Runs 1, 2, and 3, respectively. 

Based on the results from the three runs, benfluralin is classified as Not Interactive in the 

Estrogen Receptor Binding Assay. 

ERα Transcriptional Activation Assay 

In an estrogen receptor transcriptional activation assay (MRID 48614905) conducted by 

Toxicology & Environmental Research and Consulting, hERα-HeLa-9903 cells cultured in vitro

were exposed to benfluralin (97.5% purity Lot# ACD13683, TSN100037 at concentrations of 

10−4, 10−5, 10−6, 10−7, 10−8, 10−9 and 10−10 M in DMSO (final concentration 0.1%) for 24 hours. 

The experiments were performed using 96-well plates and each benfluralin concentration was 

tested in triplicate (3 wells/plate) in each experiment. Cells were exposed to the test substance 

for approximately 24 hours to induce reporter (luciferase) gene products. Luciferase expression 

in response to activation of the estrogen receptor was measured upon addition of a luciferase 

substrate and detection with a luminometer with acceptable sensitivity.

Benfluralin was tested up to the limits of both cytotoxicity and solubility; 10−3.5 M was both 

cytotoxic to the cells (79.1% viability compared to controls) and precipitated in the cell culture 

media at the end of the 24-hour treatment duration. The positive control and reference chemicals 

induced the appropriate responses, although the assay's responsiveness to 17α-methyltestosterone 

indicated increased sensitivity to weak agonists. The RPCMax (maximum level of response 

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induced by chemical expressed a percentage of the response induced by 1 nM estradiol (E2) on 

the same plate) was 13.3-19.1%. The PCMax was the limit of solubility and cytotoxicity, 10−4

 M. 

Because the RPCMax > PC10 in all three assay runs, benfluralin was considered positive for 

estrogen receptor transcriptional activation in this test system. 

Fish Short Term Reproduction Assay 

The 21-day short-term reproduction assay of benfluralin with fathead minnows (Pimephales 

promelas) was conducted under flow-through conditions. Adult fish (20 spawning groups; 2 males 

and 4 females in each group; 1 group/replicate and 4 replicates/control or treatment; ca. 6 months 

old) were exposed to negative and solvent (0.05 mL/L dimethylformamide; DMF) controls and 

benfluralin (97.5% purity) at nominal concentrations of 0.00033, 0.0033, and 0.033 mg a.i./L; 

time-weighted average (TWA), measured concentrations were <0.0000505 (<LOQ; controls), 

0.000302, 0.00314, and 0.0365 mg a.i./L. The test system was maintained at 24.3 to 25.5o

C and a 

pH of 7.5 to 7.8. 

Unless otherwise indicated in this DER, all effects are reported based on comparison to the 

negative control. 

Overt toxicity (e.g., mortality, lethargy, loss of equilibrium, bloating) was observed among fish 

exposed to the high treatment level with ≥38% of the fish affected; therefore, this treatment group 

was removed from the study and excluded from any subsequent statistical analyses. In the low and 

mid treatment levels, no effect on mortality was observed; overall mortality was 4.2% in the 

negative and solvent controls and low treatment group and 0% in the mid treatment group. No 

treatment-related effects on secondary sex characteristics or clinical signs (i.e., behavioral and 

other sublethal effects) were observed; however, one female in the mid treatment group was 

observed to have slight vertical banding present. Benfluralin exposure was not associated with any 

significant changes (p>0.05) in body weight or length in males or females relative to the negative 

control. 

With the exception of one negative control replicate and two solvent control replicates, spawning 

occurred every 4 days. Fecundity in the negative and solvent controls was 30.7 and 18.8 

eggs/female/day/replicate, respectively (range for negative control: 13.2-49.0; range for solvent 

control: 11.0-26.9). Mean fertilization success in the negative and solvent controls was >99%. No 

significant differences (p>0.05) in fecundity or fertilization success were observed in the 

benfluralin treatment groups relative to the negative control. Plasma vitellogenin (VTG) was not 

significantly different (p>0.05) in males or females compared to the negative control. Gonadosomatic index (GSI) was significantly increased (p<0.05) by 27% in males at the mid treatment 

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level compared to the negative control; there were no significant differences (p>0.05) in GSI in 

females compared to the negative control. Male nuptial tubercle score was not significantly 

different (p>0.05) relative to the negative control; no tubercles were observed in females. 

No treatment-related effects on gonadal histopathology were observed in males. The incidences of 

grade 4 (severe) oocyte atresia (9 or more atretic oocytes in a section) were slightly higher in the 

solvent control group (n=6) and the low treatment group (n=7) relative to the negative control and 

the mid treatment group (n=0 for both). However, the reason for this increase was not clear and 

was deemed not to be treatment related. Five of the six reported incidences of severe atresia in the 

solvent control group occurred in the two replicates in which a decrease in fecundity was observed, 

and all four females in the low treatment replicate that produced no eggs also had severe oocyte 

atresia. 

While not statistically significant (p>0.05), mean fecundity in the solvent control group was 

reduced by 39% compared to the negative control. Therefore, there is uncertainty as to whether 

there were solvent effects in the study. Two of the four replicates in the solvent control had 

fecundity rates of 11 eggs/female/day, and these replicates did not spawn every four days; the 

fecundity rates for these two replicates seven days prior to the exposure period were 27.3 and 20.6 

eggs/female/day, suggesting adequate spawning prior to test initiation. Fecundity in one replicate 

of the negative control was also reduced compared to the pre-exposure period, and the replicate 

did not spawn every four days (13.2 eggs/female/day in the 21-day exposure period versus 31.1 

eggs/female/day in the 7 days prior to the exposure period). Although the observed decline in these 

replicates may be due to biological variation, the possibility of a solvent interference cannot be 

conclusively ruled out. One replicate in the low treatment group did not produce any eggs during 

the exposure period. While not statistically different (p>0.05) from the negative control, decreases 

of 29 and 21% in fecundity in the low and mid concentration groups, respectively, were of a similar 

magnitude as that for the solvent control. Therefore, the results of the study do not suggest a 

significant treatment-related effect on fecundity. 

Hershberger Assay 

A Hershberger Assay (MRID 48614907) was performed to investigate both androgenic and antiandrogenic effects in male rats. To screen for potential androgenic activity, benfluralin (benefin; 

97.5% a.i., lot # ACD13683) in corn oil was administered daily via oral gavage to groups of 

seven 55-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 50, 250, or 

750 mg/kg/day. An androgenic positive control group of 7 castrated rats exposed to 0.4 

mg/kg/day testosterone propionate (TP) by subcutaneous injection was also included. 

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To screen for potential anti-androgenic activity, benfluralin was administered daily via oral 

gavage to groups of seven 55-day old, castrated male Sprague Dawley rats at dose levels of 0 

(vehicle), 50, 250, or 750 mg/kg/day in conjunction with a daily dose of reference androgen TP 

at 0.4 mg/kg/day by subcutaneous injection. An anti-androgenic positive control group of 7 

castrated rats exposed to 0.4 mg/kg/day TP and 3.0 mg/kg/day flutamide (FT) was included. TP 

alone at 0.4 mg/kg/day was used as the anti-androgenic negative control. 

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. In the androgen antagonist portion of the study, blood 

samples were taken prior to sacrifice to evaluate serum testosterone levels in animals treated with 

TP + 0, 50, 250 or 750 mg/kg/day benfluralin for 10 days. 

In addition, a 14C-testosterone clearance experiment was conducted to examine if the decrease in 

androgen-responsive tissue weights were attributed to enhanced clearance of exogenous 

testosterone secondary to hepatic enzyme induction by benfluralin. In this study, two groups of 

seven castrated, jugular vein-cannulated male rats were dosed with benfluralin daily via oral 

gavage to at dose levels of 0 (vehicle) or 750 mg/kg/day in conjunction with a daily dose of 

reference androgen TP at 0.4 mg/kg/day by subcutaneous injection. 14C-Testosterone was 

administered on Days 1 and 10 to measure plasma clearance of 14C-testosterone radioactivity. 

On Day 1, all animals had blood samples collected from the cannulae at 0.08, 0.17, 0.25, 0.5, 1, 

2, 3, and 6 h post-dosing. On Day 10, three controls and four 750 mg/kg/day rats remained 

patent and had blood samples collected as on Day 1, except the 6 h sample was collected via the 

orbital sinus. The non-patent animals only had 6 h samples collected via the orbital sinus. 

Clinical signs, body weight and androgen responsive tissue weights were also evaluated. 

All animals survived until scheduled termination. There were no treatment-related gross 

pathological findings. 

In the androgen agonist assay, 3/7 rats were noted to have decreased feces and 2/7 rats were 

observed with clear perioral soiling at 750 mg/kg/day. Additionally at this dose, body weights 

were decreased (p≤0.05, except not significant [NS] on Day 11) on Days 4, 7, and 11 by 7-11%, 

resulting in a 15.0 g cumulative body weight loss (p≤0.05) for Days 1-4 and decreased (p≤0.05) 

cumulative body weight gains for Days 1-7 (↓76%) and Days 1-11 (↓49%). Cumulative body 

weight gains were also decreased (p≤0.05) by 61% for Days 1-4 in the 250 mg/kg/day group; 

however, body weights in this group were similar to controls, as were cumulative body weight 

gains for Days 1-7 and 1-11. Animals dosed with 0.4 mg/kg/day TP had 6-7% increases 

(p≤0.05) in body weights on Days 7 and 11, resulting in increased (p≤0.05) body weight gains 

for Days 1-4 (↑55%), 1-7 (↑60%) and 1-11 (↑44%). During Days 1-4, food consumption was 

decreased (p≤0.05) by 12-44% at 250 mg/kg/day and above. There were no significant increases 

in any of the five target accessory sex organs of animals dosed with the test compound. Absolute 

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liver weights were increased (p≤0.05) by 38-68% in the 250 mg/kg/day and above groups. 

Animals in the 0.4 mg/kg/day TP group had accessory sex organ weight increases in all five 

accessory sex organs indicating that the assay was responding properly. The %CVs for the 

vehicle controls, 0.4 mg/kg/day TP group, and the 750 mg/kg/day group were compared to the 

performance criteria values as stated in the Guideline. The %CV for the ventral prostate in the 

vehicle controls exceeded the criteria value (70% vs. 45% maximum recommended); however, is 

unlikely that these data would have affected the interpretation of the assay results since no 

increases were observed in the other tissue weights. All other %CVs were less than the 

maximum recommended values.

In the anti-androgen assay, 3/7 rats at 750 mg/kg/day were observed to have decreased feces. 

Cumulative body weight gains were decreased (p≤0.05, except NS on Day 11) on Days 1-4 

(↓55%), 1-7 (↓37%) and 1-11 (↓23%) at 250 mg/kg/day; however, body weights in this group 

were similar to controls. At 750 mg/kg/day, body weights were decreased (p≤0.05) on Days 4, 

7, and 11 by 10-14%, resulting in a 15.1 g cumulative body weight loss (p≤0.05) for Days 1-4 

and decreased (p≤0.05) cumulative body weight gains for Days 1-7 (↓69%) and Days 1-11 

(↓57%). Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) 

cumulative body weight gains on Days 1-4 (↓22%) and 1-7 (↓19%); however body weights and 

cumulative body weight gains on Days 1-11 were similar to TP controls. During Days 1-4, food 

consumption was decreased (p≤0.05) by 23-50% at 250 mg/kg/day and above, and was 

decreased at 750 mg/kg/day during Days 4-7 and 7-11 (↓13% both periods). 

In the 750 mg/kg/day group, accessory sex organ weight were significantly decreased in two of 

the five target tissues: levator ani-bulbocavernosus (LABC; 33%) and seminal vesicles (27%). 

Non-significant decreases were observed in the remaining three target tissues. Absolute liver 

weights were increased (p≤0.05) by 39-65% in the 250 mg/kg/day and above groups. Animals 

dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) accessory sex organ 

weights in all five accessory tissues to indicate that the test system was responding properly to 

detect androgen antagonists. In rats treated with 750 mg/kg/day + 0.4 mg/kg/day TP, serum 

testosterone levels were decreased (NS) by 29% compared to TP controls. The %CVs for the 

vehicle controls, 0.4 mg/kg/day TP group, and the 750 mg/kg/day group were compared to the 

performance criteria values according to the Guideline. The %CV for the ventral prostate in the 

vehicle controls exceeded the criteria value (70% vs. 45% maximum recommended), but was 

unlikely to have affected the interpretation of the assay results. All other %CV were less than 

the maximum recommended values. 

In the 14C-testosterone clearance experiment, 6/7 rats dosed with 750 mg/kg/day had urine 

perineal staining, and 2/7 rats had red perinasal staining. In the 750 mg/kg/day group, body 

weights were decreased (p≤0.05) by 12-20% on Days 4, 7, and 10; resulting in body weight 

losses of 18.7 g and 1.0 g for Days 1-4 and 1-7, and decreased (p≤0.05) cumulative body weight 

gains for Days 1-10 (↓85%). Animals dosed with 750 mg/kg/day of the test compound had nonPage 47 of 61
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significant decreases in accessory sex organ weights: 17% in Cowper’s gland; 25% in glans 

penis; 37% in LABC; 53% in ventral prostate; and 48% in seminal vesicles. Absolute liver 

weights were increased (NS) by 38% in this group. Rats treated for 10 days with 750 mg/kg/day 

of the test compound cleared 14C-radioactivity 3.7-fold faster (p≤0.05) than the TP-only controls. 

This was a result of the elimination half-life present in the 750 mg/kg/day group. Additionally, 

the 750 mg/kg/day animals had an AUC that was lower (p≤0.05) than the TP-only controls 

(0.014 μg×h×g-1 treated versus 0.039 μg×h×g-1 control). 

Benfluralin 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, benfluralin was positive for anti-androgenicity 

in the Hershberger assay as statistically significant decreases in organ weights were seen in two 

of the five androgen sensitive tissues in the androgen antagonist assay. At 750 mg/kg/day, a 

significant decrease in body weight (12%; p<0.05) and body weight gain (57%; p<0.05) were 

seen after 10 days of treatment indicating overt toxicity at the dose that showed decreases in 

androgen-sensitive tissue weights. Additional data provided on testosterone clearance in 

benfluralin-treated rats in conjunction with the observed increase in liver weights, provide 

evidence that the reduced accessory sex organ weights may be secondary to increased 

testosterone metabolism and clearance. 

Female Pubertal Assay 

In a Female Pubertal Assay (MRID 48669301), groups of 16 Crl:CD(SD) rats were treated daily 

via oral gavage with benfluralin (97.5% a.i.; Lot# ACD13683) in corn oil at doses of 0, 25, 75 or 

300 mg/kg/day from post-natal day (PND) 22 to 42. Vaginal opening (VO) was evaluated daily, 

and age and weight at attainment of VO were recorded. Once VO was completed, vaginal lavage 

samples were collected daily (including the day of necropsy) to evaluate estrus cycle length and 

pattern. After sacrifice on PND 42, blood was collected and a gross necropsy was conducted. 

Clinical chemistry parameters were evaluated, including serum thyroxine (T4) and thyroid 

stimulating hormone (TSH) levels using a radioimmunoassay assay (TSH) or chemiluminescent 

immunoassay (T4). The following organs were weighed: liver, kidneys, adrenals (weighed as 

pairs), ovaries (without oviducts, weighed as pairs), imbibed and blotted uterus (with oviducts 

and cervix), thyroid, and pituitary. Microscopic evaluations were performed on thyroid, kidneys, 

uterus, and typically the right ovary. 

All animals survived until scheduled termination. One 75 mg/kg/day female was removed from 

the study after being dosed twice on one day. All of the 300 mg/kg/day animals had dark yellow 

urine, a typical finding following dinitroaniline herbicide exposure. There were no significant 

treatment-related effects on mean estrous length, percent cycling, percent regularly cycling or 

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body weight at VO. There were no gross findings at necropsy and no histopathological lesions 

in the uterus, ovaries, kidneys or thyroid. 

Animals treated at 300 mg/kg/day had decreased (p<0.05) body weights (↓8-10%) and 

cumulative body weight gains (↓15-42%) through PND 35; terminal body weights largely 

recovered by necropsy at PND 42 (↓4%; NS) with a 6% decrease (p<0.05) in overall body 

weight gain (PND 22-42). Both unadjusted and adjusted mean ages at VO were increased 

(p<0.05) in the 300 mg/kg/day group (PND 37.2-37.3 treated vs. PND 34.4 controls) but not at 

lower dose levels. Age at first estrus was also increased (p<0.05) at 300 mg/kg/day (PND 37.2 

treated vs. PND 35.4) due to the delay in VO. 

Serum T4 levels were decreased (p<0.05) by 13% at 75 mg/kg/day and 44% at 300 mg/kg/day, 

and TSH levels were increased (p<0.05) by 109% at 300 mg/kg/day; however, there were no 

associated changes observed in thyroid weight or histopathology. Other clinical chemistry 

changes in the 300 mg/kg/day animals included: decreased (p<0.05) alkaline phosphatase 

(↓16%) and increased (p<0.05) gamma glutamyl transpeptidase (GGT, 47%), cholesterol 

(↑12%), and creatinine (↑44%). Unadjusted, adjusted, and relative liver weights were increased 

(p<0.05) by 19-23% at 75 mg/kg/day and 32-37% at 300 mg/kg/day, and relative liver weight 

was increased (p<0.05) by 8% at 25 mg/kg/day. At 300 mg/kg/day, decreases (p<0.05) were 

also observed in pituitary weight (unadjusted, adjusted, and relative; ↓12-19%), ovary weights 

(unadjusted and adjusted; ↓17-18%) and blotted uterus weight (adjusted; ↓21%). 

The high dose of 300 mg/kg/day is considered adequate based on decreased body weight gains 

over the course of the treatment period and increased liver weights with concomitant significant 

decreases in alkaline phosphatase, and increases in GGT and cholesterol. 

Male Pubertal Assay 

In a male pubertal assay (MRID 48669302), groups of 16 Crl:CD(SD) rats were treated daily via 

oral gavage with benfluralin (97.5% a.i.; Lot #ACD13683) in corn oil at doses of 0, 25, 100 or 

400 mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined for preputial 

separation (PPS) daily beginning on PND 30, and the age and weight of each animal on the day 

of attainment was recorded. Following sacrifice on PND 53, total serum testosterone, thyroxine 

(T4), and thyroid stimulating hormone (TSH) levels were analyzed using radioimmunoassay 

(RIA) or chemiluminescent immunoassay. The following organs were weighed: liver, kidneys 

and adrenals (weighed as pairs), testes and epididymides (weighed individually), thyroid and 

pituitary (weighed after fixation), ventral prostate, dorsolateral prostate, seminal vesicle with 

coagulating glands, and the levator ani plus bulbocavernosus muscles (LABC). Microscopic 

evaluations were performed on testes, epididymides, thyroid, and kidneys. 

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All rats survived to scheduled termination. There were no treatment related effects on mean age 

and weight at completion of PPS, mean weights of adrenals, kidneys, and testes, and gross and 

microscopic examinations of the testes, epididymides, and kidneys. 

Systemic findings were observed at 400 mg/kg/day. Dark yellow/orange urine, a common 

finding following dinitroaniline herbicide treatment, was observed in all rats. Unadjusted and 

adjusted final body weights were decreased (not significant [NS]) by 8%, resulting in a 9% 

decrease (p<0.05) in overall body weight gains (PND 23-53). Unadjusted, adjusted, and relative 

(to body) liver weights were increased (p<0.05) by 40-52%, unadjusted and adjusted pituitary 

weights were decreased (p<0.05) by 13-14%, and unadjusted and adjusted thyroid weights were 

increased (p<0.05, except unadjusted NS) by 15%. Serum alkaline phosphatase levels were 

decreased (p<0.05) by 40%, and serum total protein, creatinine, and gamma glutamyl transferase 

were increased (p<0.05) by 7%, 50%, and 60%, respectively. Globulin was increased (not 

statistically analyzed) by 22%. Phosphorus, chloride, and potassium levels were decreased 

(p<0.05) by 9%, 2%, and 6%, respectively. 

At 100 mg/kg/day, unadjusted, adjusted and relative (to body) liver weights were increased 

(p<0.05) by 22-23%. Serum alkaline phosphatase levels were decreased (p<0.05) by 17%. 

Phosphorus was decreased (p<0.05) by 9%. 

At 25 mg/kg/day, only relative liver weight was increased (p<0.05), by 7 %. 

There was a dose-dependent decrease (p<0.05) in serum T4 levels of 12%, 29% and 53% at 25, 

100 and 400 mg/kg/day, respectively, and a dose-dependent increase (p<0.05) in serum TSH 

levels of 53% and 105% at 100 and 400 mg/kg/day, respectively. Serum testosterone was also 

decreased (NS) by 52% at 400 mg/kg/day. When one animal with a testosterone level well 

above the group average was excluded from the analysis, the decrease in testosterone at 400 

mg/kg/day was 69% (p<0.05). At 400 mg/kg/day, decreased (p<0.05) unadjusted and adjusted 

accessory sex tissue weights were observed as follows: (i) seminal vesicle + coagulating gland 

with fluid by 32%; (ii) seminal vesicle + coagulating gland without fluid by 25-26%; (iii) ventral 

prostate by 26%; (iv) dorsolateral prostate by 21-22%; (v) LABC by 20%; and (vi) epididymis 

by 13-18%. At 100 mg/kg/day, only adjusted right epididymis weight was decreased (p<0.05), 

by 9%. Animals treated with 400 mg/kg/day had increases in thyroid follicular cell height with 

concomitant decreases in colloid area. A slight decrease in spermatic elements in the right 

epididymis was observed in two animals (#6976 & #6982) at 400 mg/kg/day. 

The dose levels used in this study were considered to be adequate based on markedly increased 

liver weights and significant changes in thyroid hormone levels and histopathology, as well as a 

significant decrease in body weight gain, at 400 mg/kg/day. 

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Steroidogenesis Assay 

In a steroidogenesis assay (MRID 48614908), H295R cells cultured in vitro in 24-well plates 

were incubated with benfluralin (97.5% purity, Lot # ACD13683, TSN100037) at logarithmic 

concentrations from 10-10 to 10-4 M in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was 

used as the vehicle, at a final concentration of 0.1%. 

Testosterone and estradiol levels were measured with LC/APPI-MS/MS. Three independent 

experiments were performed. A Quality Control (QC) plate was run concurrently with each 

independent run of the test chemical plate to demonstrate that the assay responded properly to 

positive control agents at two concentration levels; positive controls included the known inducer 

(forskolin) and inhibitor (prochloraz) of estradiol and testosterone production. 

Benfluralin was tested at concentrations up to 10-4 M with no evidence of precipitation or 

cytotoxicity. Although the final assay run was conducted with H295R cells from passage 11.5 

(one passage higher than the recommended maximum of 10), the guideline requirements for CV 

≤30% between replicate wells within a plate and between plates were met for all three assay 

runs. Estradiol production was statistically increased 1.2-fold in two of three independent assays 

at 10-4 M and by 1.3-fold in one assay at 10-5 M, but no dose-response was observed. Slight 

decreases in testosterone and estradiol production in a single run were also not considered of 

biological relevance as statistically significant differences at concentrations that do not follow a 

dose-response curve may be due to random effects. 

Based on the hormone responses in three independent runs, benfluralin treatment did not result in 

statistically significant and reproducible alterations in testosterone or estradiol production. 

Uterotrophic Assay 

In an uterotrophic assay (MRID 48614909) conducted to screen for potential estrogenic activity, 

Benefin (97.5% a.i., lot # ACD13683) in corn oil was administered daily via oral gavage to 

groups of six immature female Sprague-Dawley rats at dose levels of 0 (vehicle), 0 (untreated), 

25, 75, or 200 mg/kg/day on post-natal day (PND) 19-21. A positive control group was treated 

with 17α-ethynyl estradiol (EE) by daily oral gavage at a dose level of 10 μg/kg/day. All 

animals were terminated and necropsied approximately 24 hours after the final dose on PND 22 

to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed in 

animals for any dose groups. No precocious vaginal opening was observed in the test animals. 

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Body weights in the dose groups were comparable to the controls throughout the duration of the 

assay. At a dose of 200 mg/kg/day, body weight gains were decreased by 30% compared to the 

controls. .Uterine weights in the dose groups were comparable to the controls. Absolute wet and 

blotted uterus weights for the 10 μg EE group were increased (p<0.05) by 811% and 466%, 

respectively. 

No statistically significant changes were seen in uterine weight in this study. Benfluralin was 

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

90-Day (Subchronic) Oral Toxicity in Rodents (Rat) 

In three separate subchronic toxicity studies (MRID 44050001), benfluralin (~95.6% purity/a.i., 

Lot/Batch #231EF4) was administered to rats in the diet at concentrations ranging from 0.005 to 

0.75% for 3 months. In Study R33989, 15 rats/sex/dose were fed benfluralin at nominal dietary 

concentrations of 0, 0.025, 0.11 or 0.5% (0, 17, 74 or 341 mg/kg/day for males and 0, 20, 94 or 

395 mg/kg/day for females) for 3 months. In Study R44089, 15 rats/sex/dose were fed diets 

containing nominal concentrations of 0 or 0.75% benfluralin (0 or 522 mg/kg/day for males, and 

0 or 605 mg/kg/day for females) for 3 months. In Study R29990, 15 males/dose were fed diets 

containing benfluralin at nominal concentrations of 0, 0.005, 0.05 or 0.5% (0, 3, 32 or 322 

mg/kg/day) for 3 months. 

In Study R33989, males at 341 mg/kg/day had increased absolute adrenal (10%) and thyroid 

(18%) gland weights and both sexes had increased relative adrenal (10-12%) and thyroid (17-

22%) gland weights. In study R44089, males (522 mg/kg/day) and females (605 mg/kg/day) 

exhibited increased relative adrenal gland weights (males, 11%, and females 9%). Treated 

females also had significant (p<0.05) increase in absolute and relative thyroid weights (22% and 

27%, respectively) and relative ovary (14%) weights when compared to controls. No treatmentrelated histopathological findings were seen in the testes, epididymides, seminal vesicle, 

prostate, ovaries, uterus, cervix, vagina, or mammary, adrenals, pituitary, and thyroid glands in 

either sex in any study. 

Concentration-dependent hepatic changes observed at approximately 0.025% included increased 

absolute and relative liver weights and minimal to slight hepatocellular hypertrophy. The 

absolute and relative liver weights in both sexes were increased at ≥0.025% (0.025 to 0.75%) 

treatment doses. Livers of rats in the ≥0.025% treatment groups exhibited mild diffuse 

centrilobular hypertrophy. Enlarged livers were noted in the 0.11% group males, and in both 

sexes treated at 0.5 and 0.75%. Hepatic microsomal enzyme activities were increased in both 

sexes treated at ≥0.11%. Concentration-dependent nephrotoxic effects were observed in all 

treatment groups. Body weights were significantly (p<0.05) decreased at 0.75% (<10%), and 

food consumption was significantly (p<0.05) decreased at ≥0.5%. Under the conditions of these 

studies, the subchronic LOAEL for these studies is 3.23 mg/kg/day (0.005% benfluralin), based 

on hyaline droplet formation in the kidneys of males at this dose level. A NOAEL was not 

established. 

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90-Day (Subchronic) Oral Toxicity in Non-Rodents (Dog) 

Male and female beagle dogs were orally dosed (capsules) with benfluralin (95.8% purity/a.i., 

Lot/Batch #ACD13683) at 0, 5, 25 or 125 mg/kg/day for 13 weeks (MRID 43072301). Weights 

were obtained for testes, ovaries, pituitary and thyroid; histopathological examinations were 

conducted on the testes, epididymides, prostate, ovaries, uterus, vagina, mammary gland, and 

pituitary and thyroid glands. No significant, treatment-related effects were observed on body 

weight throughout the study. Significant findings consisted of increased absolute and relative 

liver weights in high-dose males. This observation correlated with the increased incidence of 

minimal to slight centrilobular to diffuse hepatocellular hypertrophy. Since clinical pathology 

findings did not indicate any hepatotoxicity, the observed changes in liver weight and 

histopathological findings are suggestive of an adaptive, rather than toxic, effect. Other 

histopathological findings included increased splenic pigment of mid- and high-dose animals. 

Based on the results of this study, the LOAEL was 25 mg/kg/day and the NOAEL was 5 

mg/kg/day. 

Prenatal Development in Rodents (Rat) 

In a developmental toxicity study (MRID 000147535), benfluralin (95.3% purity/a.i., Lot/Batch 

#219EF4) was administered to 25 Crl:CD (SD) rats/group by gavage at 0, 50, 225, 475 or 

1000 mg/kg/day in 10% acacia from gestation day (GD) 6 through 15. Body weights were 

determined on GD 0, 6, 11, 16, and 20 (prior to sacrifice); food consumption was also measured. 

Uterine contents were evaluated at sacrifice (GD 20). Maternal body weights were significantly 

(p>0.05) decreased on GD 11 (96% of control) and 16 (96% of control) at ≥475 mg/kg/day and 

body weight gain was significantly decreased on GD 6 to 11 (48% of control) at the same dose 

levels. Food consumption was also significantly depressed on GD 6 to 11 (81-85% of control) 

and on GD 11 to 16 (93% of control) at the same doses. At necropsy, dilated renal pelvises and 

enlarged hepatic lobes were noted at all doses. No consistent, treatment-related differences in 

the intrauterine parameters between the treated and control groups were observed. The number 

of litters examined in the dose and control groups were 24 or 25. At 225 mg/kg/day, a 

statistically significant increased number of male fetuses with unossified sternebrae were found 

affecting 14, 16, 15, 15 and 21 litters at 0, 50, 225, 475, and 1000 mg/kg/day, respectively. In 

addition, at 225 mg/kg/day, a statistically significant increased number of fetuses were seen with 

dark brown-red areas on the liver (0, 0, 5, 1, 4 1itters at the above respective doses). The 

maternal NOAEL is 225 mg/kg/day and the maternal LOAEL is 475 mg/kg/day based on body 

weight decreases on GDs 11 and 16, and the body weight gain decrement between GD 6 to 11. 

The developmental NOAEL is 1000 mg/kg/day and a developmental LOAEL was not 

determined. 

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Prenatal Development in Non-Rodents (Rabbit) 

In a developmental toxicity study (MRID 42039101), benfluralin (95.6% purity/a.i., Lot/Batch 

#231EF4) was administered to 20 New Zealand White rabbits/group at 0, 25, 50, 100 or 

225 mg/kg/day by gavage from gestation day (GD) 6 through 18. Body weights were 

determined on GD 0, 6, 9, 12, 15, 19, 24, and 29 (prior to sacrifice); food consumption was also 

measured. On Day 29, uterine contents were evaluated. Maternal body weight gain was 

significantly decreased (p<0.01; 6% of control) between GD 6-19 in the 225 mg/kg/day does, 

and was nominally reduced (68% of control) during the same period in the 100 mg/kg/day 

animals. One maternal death and 3 abortions occurred at 225 mg/kg/day. In addition, incidences 

of few (decreased) feces were seen in the two top dose groups (6 and 6, respectively, versus 0 

control). Food consumption was reduced between GD 6-19 in the two highest dose groups 

(p<0.01; 70% of control at 225 mg/kg/day), and may have been related to the body weight gain 

depression, as well as the few feces. No consistent, treatment-related differences in the 

intrauterine parameters between the treated and control groups were observed. The maternal 

NOAEL was determined to be 100 mg/kg/day and the maternal LOAEL was 225 mg/kg/day 

based on dose-related nominal body weight gain decrement, few feces, and reduced food 

consumption. The developmental NOAEL is 225 mg/kg/day and a developmental LOAEL was 

not determined. 

Two-Generation Reproduction in Rodents (Rat) 

In a two-generation reproduction study (MRID 43628701 and 43628702), Sprague·Dawley 

CAL: CD BR rats (30/dose/sex) were fed test diets containing benfluralin (95.8% purity/a.i., 

Lot/Batch #ACD13683) at nominal concentrations of 0 (basal diet), 100, 1000 or 5000 ppm 

[males (premating), F0: 0, 7.2, 68.1, or 342 mg/kg/day, F1: 0, 7.3, 74.4, or 401 mg/kg/day; 

females (premating), F0: 0, 8.8, 80.0, 392 mg/kg/day, F1: 0, 8.6, 87.3, or 437 mg/kg/day] during 

premating, gestation and lactation periods. No treatment-related deaths or clinical signs were 

observed during the study. At 5000 ppm, decreases in mean body weights were observed during 

the premating periods for F0 (↓10%, males; ↓10.4%, females) and F1 (↓20.5%, males; ↓15.9%, 

females) animals, gestation periods for F0 (↓16.7%) and F1 (↓18.5%) females, and lactation 

periods for F0 (↓13.1 %) and F1 (↓12.1 %) females. Mean body weight gains were decreased at 

5000 ppm during the premating periods for F0 (↓17.2%, males; ↓27.7%, females) and F1 

(↓14.6%, males; ↓2.0%, females) animals and gestation periods for F0 (↓28.4%) and F1 

(↓21.3%) females; during lactation increases were noted in mean body weight gains for F0 

(↑18.5%) and F1 (↑4.94-fold) females. The decreases in mean body weights were accompanied 

by significant decreases in food consumption during the premating, lactation, and gestation 

periods. Mean litter sizes of F0 and F1 animals in the 5000 ppm group were significantly 

decreased and lower than the mean (range) historical control value of 14.37 (13.46 to 15.67). All 

reproductive parameters of treated animals were generally comparable to control. 

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At necropsy, treatment-related liver and kidney effects were noted. Liver effects included dosedependent increases in the absolute and relative weights of livers of 1000 and 5000 ppm parental 

animals (F0 and F1), and hepatocellular enlargement in 1000 ppm (F1) and 5000 ppm (F0 and 

F1) animals. Treatment-related litter effects included deceases in mean litter size of high-dose, 

F0 and F1 maternal animals, and the weaning index of F1 pups. An increased incidence of 

dead/missing pups was also observed in mid- and high-dose F0 litters and high-dose F1 litters. 

All other litter parameters of treated animals were comparable to control values. For both the F1 

and F2 generations, 1000 and 5000 ppm male and female pups had significant (p<0.05 or 0.01 at 

1000 ppm; p<0.01 at 5000 ppm) decreases in mean body weights throughout the lactation period. 

The gross and necropsy examinations of F1 pups did not reveal any treatment-related effects. 

Based on the findings (increased absolute and relative liver weights, liver and kidney 

histopathology, and decreased mean pup body weight), a LOAEL for parental/systemic and 

offspring toxicity was established at 1000 ppm [Premating: 68.1 mg/kg/day (males) and 80.0 

mg/kg/day (females)] and a NOAEL at 100 ppm [Premating: 7.2 mg/kg/day (males) and 8.8 

mg/kg/day (females)]. There were no treatment-related effects on fertility or organs of 

reproduction. 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat) 

In a chronic toxicity/oncogenicity study (MRID 44050002 and 44545501), benfluralin (95.8% 

purity/a.i., Lot/Batch #ACD13683) was administered to Fischer 344 rats (60/sex/dose) in the diet 

at nominal dose concentrations of 0, 10, 100, 2500, and 5000 ppm (0, 0.5, 5.4, 136.3, and 274.8 

mg/kg body weight/day for males; 0, 0.7, 6.8, 167.9, and 331.3 mg/kg body weight/day for 

females) for up to 2 years. Of these rats, 10/sex/dose were sacrificed at 12 months. Organ 

weights were obtained for testes, ovaries, and thyroid (at a minimum); histopathology was 

conducted on the testes, ovaries, uterus, and thyroids. Survival was significantly reduced 

(p<0.05) in males at 100, 2500, and 5000 ppm to 62, 66, and 64%, respectively, at study 

termination. However, the mortality rate did not reach statistical significance in males at any 

dose until the final week of the study. The only treatment-related clinical sign of toxicity was a 

yellowing of the skin in 97 and 100% of 2500 and 5000 ppm females, respectively, and yelloworange coloration of the eyes of both sexes at these doses. At study termination, body weights 

were significantly (p<0.05) lower than control in males (↓8%) and females (↓18%) at 2500 ppm 

and in males (↓17%) and females (↓28%) at 5000 ppm. Body weight gains were reduced 

(p<0.05) at 2500 and 5000 ppm throughout the second year of the study. 

Alterations in cholesterol, bilirubin, and liver enzymes at the two highest doses correlated with 

liver enlargement and an increased incidence of liver lesions. Significant increases (p<0.05) in 

absolute (↑19-43%) and relative (↑ up to 2-fold) liver weights versus controls were observed in 

both sexes at 2500 and 5000 ppm, and hepatic toxicity was corroborated by serum chemistry and 

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histopathology findings. Relative thyroid weights were elevated (p<0.05; ↑33 to 64% over 

control) at 2500 and 5000 ppm, and correlated with microscopic thyroid abnormalities. At 

terminal sacrifice, gross pathology findings included enlarged testes and uterine cysts in males 

and females dosed at 5000 ppm. At 12 months (interim sacrifice), increases in non-neoplastic 

lesions included thyroid follicular cell hypertrophy in the 5000 ppm males and females and 

hepatocellular hypertrophy in 5000 ppm males and females, and in 2500 ppm males. At the 

terminal sacrifice, treatment-related non-neoplastic lesions included the same thyroid and liver 

lesions observed in the interim sacrifice with increasing frequency and severity. In addition, 

hepatic necrosis and thyroid follicular cell hyperplasia were increased in males and females at 

5000 ppm. Thyroid and liver abnormalities correlated with increased tumor incidences at the 

high dose. 

The NOAEL for chronic toxicity was 10 ppm for males and females (0.5 mg/kg/day and 0.7 

mg/kg/day, respectively), based upon an increased incidence of histological lesions of the kidney 

at 100 ppm. The LOAEL was 100 ppm (5.4 mg/kg/day for males and 6.8 mg/kg/day for 

females). The two highest doses, 2500 and 5000 ppm exceeded the MTD as demonstrated by 

decreased weight gain throughout the second year of the study. Under the conditions of this 

study, benfluralin was carcinogenic in both sexes. The test substance induced hepatocellular 

carcinoma/adenoma in male rats, and thyroid follicular adenoma/carcinoma in males and 

females, at 2500 and 5000 ppm. 

Carcinogenicity in Rodents (Mouse) 

In a mouse oncogenicity study (MRID 41021501), benfluralin (95.25% purity/a.i., Lot/Batch 

#231EF4) was administered in the diet to B6C3F/Crl mice (60/sex/group) for up to two years at 

0, 0.005, 0.03, or 0.15% (equivalent to 0/0, 6.0/6.9, 36.4/41.8, and 184.7/223.5 mg/kg/day [M/F], 

respectively). This mouse study consists of two replicate studies run concurrently in which 30 

mice/sex/group in each study were dosed as stated above. Histopathological examinations were 

conducted on the testes, epididymides, prostate, seminal vesicle, ovaries, uterus, pituitary, and 

thyroids; it is not clear from the DER if any of these tissues were weighed prior to pathology 

examinations. 

Mortality, clinical signs, and food consumption for both sexes at all doses were unaffected by 

treatment with benfluralin. No treatment-related findings were observed in the 0.005% dose 

group. Body weights and weight gain were equivocally decreased in males and females of all 

dose groups, except for female body weight (↓8%) and body weight gain (↓11%; p≤0.05) that 

was considered treatment-related, and potentially toxicologically significant, in the highest dose 

group. Male body weight and body weight gain values in all dose groups were generally less 

than control at all time periods (↓ ≤8%). 

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No significant, treatment-related effects on organ weights were observed, except for increased 

absolute and relative liver weights in all high-dose animals. An indication of hepatotoxicity in 

males was demonstrated by a nominal increase in multifocal hepatocellular hyperplasia (7/60 

versus 1/60 in control) at the high dose. Apart from nominally depressed body weight, 

nominally increased incidence of obstructive urological syndrome, and multifocal hepatocellular 

hyperplasia in males at the high dose, no other indications of toxicity in male mice were seen. In 

females in the 0.03% dose group, the incidence of liver nodules was slightly elevated (12/60 

treated versus 7/60 in control). At 0.15%, toxicity was observed in the liver of females as 

follows: at termination, an increased incidence of liver nodules (25/59 treated versus 7/60 in 

control); an increased incidence of minimal to moderate focal hyperplasia (20/59 treated versus 

6/60 in control); and an increase in slight to moderate multifocal hyperplasia (6/59 treated versus 

1/60 in control). 

The incidences of hepatocellular adenoma and carcinoma were not significantly different from 

control at any treatment level, but showed a trend to increase in the high-dose animals. The 

gross and microscopic findings, such as increased liver nodules and hypertrophy that were 

observed, demonstrated that benfluralin exposure appears to affect the morphology and growth 

of hepatocytes. 

The LOAEL is 41.8 mg/kg/day based on microscopic and macroscopic liver changes. The 

NOAEL for females is 6.9 mg/kg/day. The LOAEL for males is184.7 mg/kg/day based on the 

slight toxicity signals observed in male mice. The NOAEL for males is 36.4 mg/kg/day. 

Chronic Oral Toxicity in Non-Rodents (Dog) 

In a chronic toxicity study (MRID 43628702), male and female beagle dogs (4/sex/group) were 

dosed by capsule with benfluralin (95.8% purity/a.i., Lot/Batch #ACD13683) at 0, 5, 25, or 125 

mg/kg body weight/day for one year. Weights were obtained for testes, ovaries, pituitary, and 

thyroid; histopathological examinations were conducted on the testes, epididymides, prostate, 

ovaries, uterus, vagina, mammary gland, pituitary, and thyroids. 

With the exception of one high-dose female, which was sacrificed in moribund condition, all 

animals survived to terminal sacrifice without any adverse clinical signs. The mean body 

weight, food consumption, gross pathology, and organ weights of treated animals were 

comparable to control values. Histopathology observations at terminal sacrifice revealed 

increased incidences of sinusoidal cell pigment in the livers of mid- and high-dose females, and 

high-dose males. Based on the findings of liver histopathology, a LOAEL of 125 mg/kg/day was 

established in males and 25 mg/kg/day in females. A NOAEL of 25 mg/kg/day was established 

in males and 5 mg/kg/day in females. 

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Mechanism of Action Study (Rat)

The current study (Saghir et al., 2008) was designed to determine the mechanism of thyroid 

hyperactivity after trifluralin exposure. Groups of 15 male Fischer 344 rats were exposed to 

trifluralin (0 or 6500 ppm) in the diet for two weeks; a positive control 

group was administered phenobarbital (PB; 75 mg/kg/day) by oral gavage. The time-weighted 

average daily intake of trifluralin was 441 ± 77 mg/kg/day in the treatment group. After 

dosing for two weeks, ten rats/group were sacrificed, sera was collected for T3, T4, and TSH 

concentration analyses by radioimmunoassay, and livers and thyroids were harvested and 

weighed. The livers were also analyzed for selected T4-specific uridinediphosphate 

glucuronosyltransferase (UGT) gene expression and total UGT enzyme activity. The bile 

ducts from the remaining rats from each group were cannulated at the end of the dosing period, 

and bile was collected on ice for two hours. Bile samples were analyzed for T3, T4, and 

reverse-T3 by an HPLC/MS/MS method. 

Body weight gain was decreased by 20% (p≤0.05) in the trifluralin-treated rats compared to 

negative control and PB-treated rats. Liver and thyroid weights also were increased in the 

trifluralin- and PB-treated animals; all increases were significant (p≤0.05) except for the thyroid 

after trifluralin exposure. In the trifluralin-treated rats, serum T3 and T4 concentrations were 

decreased by 17% and 90%, respectively (compared to negative control), and TSH 

concentration was increased by 37%. PB treatment decreased T4 (23%) and increased TSH 

(53%) concentrations, but had no effect on T3 concentration. Trifluralin treatment induced total 

hepatic UGT enzymes by 2.4-fold, as well as mRNA expression of selected hepatic UGT 

isozymes (UGT1A1, 1.4-fold; UGT1A6, 6.4-fold; UGT2B1, 3.7-fold). The total amount of T4 

in bile was increased >2-fold in trifluralin-treated rats. Trifluralin treatment also increased bile 

flow, with a 3.2-fold increase in the biliary elimination of conjugated T4 and a 63% increase in 

conjugated T3. Based on these data, decreases in total serum T3 and T4 concentrations in 

trifluralin-treated rats were due to enhanced peripheral metabolism (increased conjugation by 

(UGTs) and increased bile flow resulting in compensatory increases in TSH synthesis and 

secretion. Continued, and increased, concentrations of TSH after chronic exposure would cause 

stimulation of the thyroid gland, resulting in cellular hypertrophy, proliferation, and 

predisposition to the development of follicular cell tumors. 

Chronic Fish Early Life Stage Toxicity Test 

In a 49-day chronic fish early life stage (ELS) toxicity study with rainbow trout (MRID 

41613805), fish were exposed to mean measured benfluralin concentrations of negative control, 

solvent control, 0.8, 1.9, 5.0, 14.8, and 45.5 μg a.i/L. Time to hatch was similar (5 to 8 days) in 

the controls and the treatment concentrations. Percent hatch ranged from 95-100% on the 

controls and all treatment concentrations. All larvae in the highest treatment concentration (45.5 

μg a.i/L) died by day 33 post-hatch and so were subsequently excluded from further statistical 

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analysis. Clinical signs of toxicity included hyperactivity and prostrate position was observed in 

the two highest treatment concentrations (14.8 and 45.5 μg a.i/L) and no treatment related 

deformities were observed. At test termination, mean length was significantly (p<0.05) reduced 

at the 5.0 and 14.8 μg a.i/L treatment levels, and blotted wet weight was significantly (p<0.05) 

reduced at the 14.8 μg a.i/L treatment concentration when compared to the pooled controls. 

Based on effects on mean length and mean wet weight, the NOAEC and LOAEC for this study 

were determined to be 1.9 and 5.0 μg a.i/L, respectively. 

Chronic Avian Reproduction Studies (Mallard duck and Northern Bobwhite quail) 

In a chronic avian reproduction study with the mallard duck (MRID 42145501), benefin 

(benfluralin) was exposed to birds in the diet at mean measured concentrations of control, 97, 

288, and 975 ppm. One incidental mortality occurred in each of the control and two lowest 

treatment concentrations. There were no clinical signs of toxicity observed in the control or any 

of the treatment concentrations. Necropsy of all mortalities and sacrificed birds revealed no 

compound-related gross or microscopic lesions. There were no significant (p>0.05) treatment 

related effects on body weight yet there were significant (p<0.05) decreases in mean food 

consumption at the two lowest treatment concentrations when compared to the negative control. 

These decreases in mean food consumption were not accompanied by a significant decrease at 

the highest treatment concentration and therefore the study author did not attribute this effect to 

benfluralin exposure. 

When compared to the negative control, there were no statistically significant differences in all 

measured reproductive parameters. There was a high incidence of cracked eggs in the highest 

treatment concentration (11.5%) but this was not shown to be significantly different from the 

control (p = 0.061). The study reviewer determined this effect to be biologically significant. 

Eggshell thickness for both cracked and normal eggs was not significantly affected by 

benfluralin exposure. Similarly, offspring body weight was not significantly different in the 

treatment groups when compared to the negative control. 

The NOAEC and LOAEC for this study were determined to be 288 and 975 ppm, respectively, 

due to an increase in incidence of cracked eggs at the highest treatment concentration (study 

reviewer-determined). 

In another chronic avian reproduction study with the northern bobwhite quail (MRID 

42145502), birds were exposed to benfluralin in the diet at mean measured concentrations of 

control, 96, 295, and 990 mg/kg-diet (ppm). There were no clinical signs of toxicity observed in 

the study in the control or any of the treatment concentrations. Nine bobwhite quail died during 

the course of the study yet these deaths were not attributed to treatment. One female in the 

middle treatment group was sacrificed due to pen-related injuries. Necropsy of all deaths and 

sacrificed birds revealed no findings of compound related gross or histopathological lesions. 

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Based on the study-reviewer’s results (based in part on Table A in the Data Evaluation Record), 

when compared to the control group, there was a significant reduction in the highest treatment 

concentration (990 ppm) for eggs laid, viable embryos of eggs set, normal hatchlings of live 3-

week embryos and eggs set, and 14-day old survivors. Additionally, the percentage of cracked 

eggs was significantly higher (p<0.05) at the highest treatment concentration (16.2%) when 

compared to the control group (8.5%). The study authors provided no explanation for the high 

percentage of cracked eggs in the control group. 

Additionally, there appears to be a significant reduction in eggs laid, viable embryos and 

hatchlings of egg set, and reductions in 14-d offspring survival at the mid concentration (295 

ppm). There were no significant (p>0.05) differences in eggshell thickness in the treatment 

concentrations when compared to the control. The study author reported that mean body weight 

and mean body weight gain were significantly reduced (p<0.05) at the highest treatment 

concentration when compared to the control prior to egg laying; however, the reviewer analysis 

did not detect any differences in body weights in the overall study duration. During the 

preproduction period, there were significant but slight reductions in food consumption between 

the control and all treatment levels. A significant decrease in food consumption during the 

production period was noted at the two higher test concentrations. Analysis of two-week old 

survivors per eggs set and 14-day survivor weight showed a significant (p<0.05) reduction at all 

treatment concentrations when compared to the negative control (≥96 ppm). Consequently, a 

NOAEC could not be determined based on these effects occurring at all treatment 

concentrations. 

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