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 66
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: TRIFLURALIN

OFFICE OF PESTICIDE PROGRAMS 

OFFICE OF SCIENCE COORDINATION AND POLICY 

U.S. ENVIRONMENTAL PROTECTION AGENCY 

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TRIFLURALIN 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....................................................16 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................21 

IV. Committee’s Assessment of Weight of Evidence..........................................25 

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................25 

1. Tier 1 in vivo Assays..............................................................25 

2. OSRI..................................................................................26 

B. Estrogen Pathway...........................................................................27 

C. Androgen Pathway.........................................................................27 

D. Thyroid Pathway...........................................................................28 

E. Conclusions.................................................................................29 

V. EDSP Tier 2 Testing Recommendations....................................................29 

VI. References.......................................................................................31 

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Trifluralin...................................................................9 

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

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

 Table 4. Thyroid Pathway for Trifluralin ..........................................................23 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................36 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................48 

APPENDIX 3. References Not Utilized in the Trifluralin WoE Analysis............................57 

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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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iv

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 Methods

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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v

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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Page 1 of 58 

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 trifluralin 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 January 8, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

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

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

of trifluralin with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature). 

For the estrogen pathway, trifluralin was negative in the estrogen receptor (ER) binding assay 

which is capable of detecting an interaction of the test compound with the ER (both agonists and 

antagonists). Although the ER transactivation assay (ERTA) was weakly positive (14-18% of 

the response seen with 1 nM 17β-estradiol in three out of four assay runs) at 10−4 M trifluralin, 

there was no evidence of ER agonism (or antagonism) in response to trifluralin treatment in the 

female pubertal assay or uterotrophic assay. Furthermore, the results of the aromatase assay 

indicate that trifluralin does not inhibit aromatase activity. An slight increase (1.2- to 1.4-fold) in 

estradiol production was observed in the steroidogenesis assay at relatively high concentrations 

of trifluralin (10 and 100 μM). In the fish short-term reproduction assay (FSTRA), all effects 

were observed in the presence of overt toxicity, except for a 0.5% decrease in fertility at the low 

concentration for which there were no other effects observed. Trifluralin did not alter estrous 

cyclicity or reproductive organ histopathology in the female pubertal rat assay. 

In the mammalian Part 158 studies, following dietary exposure to rats for 13 weeks, a decrease in 

pituitary weight was observed at doses >40 mg/kg/day. No treatment-related effects on the 

pituitary were observed in the two-generation rat reproduction or in the chronic toxicity studies 

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with rats. The decrease in ovarian weight seen at 337 mg/kg/day in the rat reproduction study 

occurred in the presence overt toxicity. No other estrogen-related effects were observed in the 

mammalian reproduction or developmental studies. Decreases in fecundity were observed in the 

Part 158 sheepshead minnow study in the absence of overt toxicity (≥1.3 μg/L), however, there 

is a lack of diagnostic specificity for this endpoint in of itself regarding potential endocrinerelated effects. A 4% decrease in eggshell thickness and 7% decrease in 14-d offspring body 

weight at 1000 mg/kg-diet in the mallard duck reproduction study was observed; however, no 

other reproductive parameters were affected in this study or the bobwhite quail reproduction 

study. Overall, there is a lack of convincing evidence for a potential interaction with estrogen 

pathway for trifluralin. 

For the androgen pathway, trifluralin was negative for androgen receptor (AR) binding and did 

not alter testosterone production in the steroidogenesis assay. No androgenic effects were seen 

the mammalian Tier 1 in vivo assays at doses that did not cause either systemic toxicity (male 

pubertal assay) or overt toxicity (Hershberger assay). The observed effects in the Hershberger 

assay (decreases in androgen-responsive tissues weights) at the high dose (450 mg/kg/day) 

occurred at a dose that was considered overtly toxic (10% decrease in body weight). In the male 

pubertal assay, decreases in androgen-responsive tissues weights (prostate, Levator anibulbocavernosus and epididymal weights) and decreased serum testosterone were seen at the 

high dose (250 mg/kg/day). This dose caused significant decreases in body weights approaching 

overt toxicity (9%) and increases in absolute (29%) and relative (37%) liver weights. In the 

FSTRA, except for GSI which was increased (p<0.05) dose-dependently at all treatments as well 

as a slight decrease in fertility (0.5%), all other observed effects occurred at a concentration that 

was considered overtly toxic. 

In the mammalian Part 158 studies, following dietary exposure of trifluralin to rats for 13 weeks, 

pituitary weights were decreased at dose levels >40 mg/kg/day. No treatment-related effects on 

the pituitary were observed in the two-generation rat reproduction or in the chronic toxicity 

studies with rats in the absence of overt toxicity. Therefore, as the in vitro assays were negative 

and the findings in the in vivo assays occurred in the presence or approaching overt toxicity, or 

were limited to an isolated effect, there is a lack of complementary evidence within the assays as 

well as a lack of redundancy of evidence across the assays. Hence, trifluralin does not appear to 

potentially interact with the androgen-pathway. 

For the thyroid pathway for trifluralin, in the pubertal assays, dose-dependent decreases in serum 

T4 levels were seen in females at the mid-(75 mg/kg/day) and high (250 mg/kg/day) dose and in 

males at doses of 25, 75 and 250 mg/kg/day. Increased serum TSH levels were observed in both 

males and females at the high dose only. Effects on thyroid histopathology (alterations in 

follicular cell height and colloid area) were also observed in high dose female pubertal rats. All 

thyroid effects in the pubertal assays occurred at doses where increases in liver weights were also 

observed. In the amphibian metamorphosis assay (AMA), while reduced growth and effects on 

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Page 3 of 58 

the development endpoint of normalized hind-limb length was observed, there were no 

corresponding histopathological diagnoses to suggest the developmental is thyroid-related. 

In the mammalian Part 158 studies, following dietary exposure to rats for 13 weeks, pituitary 

weights were decreased at dose levels > 40 mg/kg/day. In the chronic/carcinogenicity study with 

rats, increased thyroid weights and an increase in the incidence of thyroid gland follicular cell 

tumors in males was observed only in the presence of overt toxicity. Additional OSRI (Saghir et 

al., 2008) investigating the mode of action for trifluralin-related thyroid perturbations showed 

that two week treatment of trifluralin in male rats resulted in decreases in serum T3 and T4 and 

elevated thyroid stimulating hormone (TSH). Trifluralin also induced T4-specific hepatic UGT 

activity, and 2-fold increases in bile-flow rate and biliary elimination of T4 also were 

demonstrated. Furthermore, in previously submitted mechanistic studies for benfluralin (a 

dinitroaniline similar to trifluralin), induction of hepatic microsomal enzyme activity was 

observed in both sexes in rats treated with benfluralin. 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 pentaoxyresorufin O-dealkylation (PROD) enzyme activities and 

benfluralin also up-regulated T4-specific uridine diphosphate glucuronosyltransferase (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. 

Based on the profile of liver and thyroid effects seen in the pubertal assays, the available mode 

of action data and the lack of evidence for a potential interaction in the AMA, thyroid effects 

due to trifluralin exposure are likely attributed to enhanced serum T4 clearance due to liver 

enzyme (UGT) induction. 

The conclusion of the WoE evaluation is that trifluralin demonstrates no convincing evidence of 

potential interaction with the estrogen or androgen pathways. The available data suggests the 

thyroid effects observed in the mammalian in vivo studies may result from liver enzyme 

induction and subsequent increased hormone clearance. In the wildlife data, while there is a lack 

of data to support liver enzyme induction which may result in hormone clearance, this mode of 

action may be conserved across other vertebrate taxa. However, there is no convincing evidence 

in the wildlife studies to suggest that there is a potential interaction with E, A or T, as the 

majority of the effects occurred in the presence of overt toxicity and lacked complementarity in 

the absence of overt toxicity. 

In the EDSP Tier 1 in vivo male and female pubertal assays as well as in the OSRI, there was 

evidence for potential interaction with the thyroid pathway in studies conducted with adult 

animals, but no such data exists for potential thyroid toxicity in the young animals. 

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For trifluralin, the point of departure (POD) for risk assessment of 2.4 mg/kg/day is based on 

increased frequency of abnormal stool, decreased body weights and body weight gains and 

toxicity to the hematopoietic system seen at 40 mg/kg/day (LOAEL) in a chronic toxicity study 

in dogs. This POD is used for deriving the chronic Reference Dose (RfD) used for dietary risk 

assessment and for the calculating Margins of Exposure (MOEs) used for non-dietary risk 

assessments. 

In general, since the POD and the toxicity endpoints of concern are based on data obtained from 

adult animals, there is a concern that the POD may not be protective of potential thyroid toxicity 

in the young. However in the case of trifluralin, although no data exists for potential thyroid 

toxicity in the young, a comparative thyroid assay (CTA) is not recommended since: 1) the POD 

is 10-fold lower than the lowest dose (25 mg/kg/day) that caused dose-dependent decrease in 

serum T4 levels in the female pubertal assay; 2) available data suggests the thyroid effects 

observed in the mammalian in vivo studies may result from liver enzyme induction and 

subsequent increased hormone clearance; 3) the CTA would be tested at higher doses and would 

not be likely to yield a POD lower than the level currently used for risk assessment; and 4) the 

chronic RfD and MOEs are protective of the thyroid-related findings seen in the Tier 1 pubertal 

assay. 

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

recommended for trifluralin since additional testing is unlikely to impact the current EPA 

established regulatory endpoints for human risk assessments. 

No fish or amphibian EDSP Tier 2 testing is recommended since there is no convincing evidence 

for an interaction with estrogen, androgen or thyroid pathways in fish or amphibians. 

Additionally, it is noted that life cycle studies which evaluate reproduction with fathead minnow 

and sheepshead minnow are available for trifluralin. 

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

duck are available for trifluralin. In the mallard duck study, effects on reproductive parameters 

were observed; no effects were observed in the bobwhite quail study. 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 trifluralin exposure. Additional testing is 

not recommended. 

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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 trifluralin 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 January 8, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

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

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

of trifluralin 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).

The water solubility of trifluralin is approximately 0.2 mg/L at pH 7 to 9 at 25°C with a 

octanol/water partition coefficient of 5.34 (log Pow). It does not hydrolyze, but does degrade 

rapidly in water due to photolysis. Trifluralin has a low vapor pressure (4.58 x 10-5 mm Hg at 

25°C), and therefore, volatilization is not expected to significantly contribute to the dissipation 

of trifluralin in the environment. Trifluralin has a whole body bioconcentration factor value in 

fish of 1689 to 9586. 

The available information considered in determining the potential interaction of trifluralin 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. This included 

studies published in the open literature and/or data submitted to support pesticide registration 

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

Report of the Endocrine Disruptor Review Team on trifluralin (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. 

Additionally, literature/studies considered but not utilized for the WoE analysis are listed in 

Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria and approach used in the WoE determination on the potential of a 

substance to interact with endocrine-related processes (i.e., E, A or T hormone pathways) were 

as described in the WoE guidance document (USEPA, 2011) and presented at the 2013 FIFRA 

Scientific Advisory Council (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. 

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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 eleven 

assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

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

submitted in support of pesticide registrations. Each study is evaluated for scientific quality and 

relevance for informing interactions with the E, A or T pathway. Additionally, the consistency 

of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple 

endpoints are measured in each assay. 

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the 

relative degree of diagnostic utility of a specific endpoint for discerning whether or not the 

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

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

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

The second step in this WoE process is to formulate hypotheses and integrate the available data 

along different levels of biological organization. Two keys elements in the integration of data, as 

well as characterizing the extent to which the available data support a hypothesis that a chemical 

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

redundancy. These two concepts provide a basis for considering the plausibility, coherence, 

strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are 

meant to evaluate whether or not a chemical can interact with E, A, and T consisting of different 

levels of biological organization from a molecular initiating event, such as receptor binding, 

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher levels of biological organization? Finally, an evaluation of 

conditions where 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 

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

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

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

the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of 

the EDSP Tier 1 screening process. In October, 2013 the SAP stated that, “In summary, the 

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

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

of primary interactions with the endocrine system vs. secondary effects related to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current 

approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological 

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

evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as 

tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight 

decreases of ≥10% in mammals. Additionally, other effects including morphological (e.g., organ 

weights/histopathology), biochemical (e.g., blood chemistry), and other clinical signs (e.g.,

lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

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

weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other 

instances, more than one parameter was needed to determine overt toxicity. For example, in the 

FSTRA, generally, body weight decreases were considered along with other responses when 

assessing potential overt toxicity. Additionally, effects which were considered to be signs of 

systemic toxicity were also captured and these effects were generally considered as less severe 

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

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

Section IV.A. 

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

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

known modes of action in its overall characterization of a chemical’s potential to interact with 

the E, A or T pathway. Adequately addressing the three main components described above is 

fundamental to the WoE process and in determining whether additional data are needed. In 

addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their 

potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or 

conflicting data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, 

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

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The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 

assays and OSRI which are depicted in Tables 2 - 4. These tables contain data that are 

considered scientifically and biologically relevant with regard to a treatment-related effect 

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

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

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

A. EDSP Tier 1 Screening Assays 

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

below in Table 1. 

Table 1: Tier 1 Screening Assays for Trifluralin.

Tier 1 Assays Test Guideline Test Order Status

Estrogen Receptor (ER) Binding Assay 

(Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied (MRID No. 48619704) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OSCPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48619705) 

Androgen Receptor (AR) Binding Assay (Rat 

prostate cytosol) OSCPP 890.1150 

Requirement Satisfied 

(MRID No. 48619702) 

Steroidogenesis Assay 

(Human cell line H295R) 

OSCPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No. 48619708) 

Aromatase Assay (Human 

recombinant microsomes) OSCPP 890.1200 Requirement Satisfied 

(MRID No. 48619703) 

Uterotrophic Assay (Rat) OSCPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48619709) 

Hershberger Assay (Rat) OSCPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No. 48619707) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied 

(MRID No. 48674001) 

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied 

(MRID No. 48674002) 

Fish Short-term Reproduction Assay OSCPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48619706) 

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 48619701) 

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

1. Effects on Estrogen Pathway 

The potential for trifluralin to interact with the estrogen pathway are 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 the 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. 

Trifluralin was negative (or not interactive) in the Tier 1 in vitro estrogen receptor (ER) binding 

and aromatase assays. In contrast, a positive response was observed in the Tier 1 ERα

transcriptional activation assay at trifluralin concentrations of 10−5 and 10−4 M. Additionally, a 

positive response also was observed in the steroidogenesis assay, with slight increases in 

estradiol concentration (1.2- to 1.4-fold) at the two highest concentrations, 10 and 100 μM. 

Trifluralin was negative in both the Tier 1 in vivo uterotrophic and female pubertal rat assays. 

In the FSTRA, fertility was reduced (p<0.05) at the low (↓0.5%; 0.195 μg a.i./L) and high 

(↓4%; 21.5 μg a.i./L) treatments, and there was an 87% decrease in fecundity in the high-dose 

group. Plasma VTG concentrations and GSI in females were not affected, but increased 

incidences of severe oocyte atresia and minimal subacute multifocal ovarian inflammation 

were present at the high dose. However, clinical signs of toxicity also were observed at the 

high dose. Females in the high concentration (5 out of 16 exposed) were observed as either 

bloated, having partial paralysis, a bent tail or all of these signs. Therefore, all responses, with 

the exception of the decrease in fertilization success at the low dose occurred in the presence of 

overt toxicity. 

There were no estrogen-related effects observed in the Part 158 mammalian studies; however, 

decreased pituitary weights were observed at all doses in the subchronic rat toxicity study. 

In a Part 158 fathead minnow fish life cycle study, there were no treatment-related effects on 

reproduction; however, effects on parental survival were observed at ≥ 5.1 μg/L. In a 

second Part 158 fish life cycle study with sheepshead minnow, significant decreases in the 

number of eggs spawned per female was observed at all treatment concentrations (≥1.3 μg/L). 

In this study, parental survival was reduced at 17.7 μg/L (highest treatment) and parental and 

offspring growth and offspring survival were reduced at ≥ 9.6 μg/L. Decreases in growth 

were observed in a Part 158 rainbow trout early life-cycle study at 4.32 μg/L. 

In a Part 158 avian reproduction study with bobwhite quail, no treatment-related effects were 

observed at test concentrations up to 1000 mg/kg-diet. In a second Part 158 avian reproduction 

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study with the mallard duck, there was a statistically significant (p<0.05) 4% reduction in 

eggshell thickness at the highest treatment concentration (1000 mg/kg-diet) in which there was 

also a significant decrease in 14-day survivor body weight. For this study, while there was a 

significant decrease in parental male body weight at the highest treatment, there were no 

apparent overt signs of toxicity, treatment related mortalities or reductions in food consumption 

or female body weight. Additional Part 158 avian reproduction studies with the bobwhite quail 

and mallard duck were available with no treatment-related effects up to test concentrations of 

50 mg/kg-diet.

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Table 2. Estrogenic/Anti-Estrogenic Pathway for Trifluralin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Trifluralin1Study 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 (Frt) 

/ Fecundity (Fcd) 

EDSP Tier 1 Assay

ER Binding (MRID 48619704)

N 

ERTA (MRID 48619705)

 P 

Aromatase (MRID 48619703)

 N 

Steroidogenesis (MRID 48619708)

 P 

Uterotrophic (MRID 48619709)

 N 

Female Pubertal Rat (MRID 48674001) N N N N N N FSTRA (MRID 48619706) NE N N N 

Frt: 

↓0.5%,

(L)

OSRI

Subchronic toxicity (Rat; MRIDs 40138301 and 00151906) 

NR NR NR 

↓

(L, M, 

H) 

Developmental toxicity (Rat; MRID 00152419) 

NR

N

R 
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Table 2. Estrogenic/Anti-Estrogenic Pathway for Trifluralin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Trifluralin1Study 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

2

Age & Weight at VO

2° Sex Characteristics

Fertility (Frt) 

Developmental toxicity (Rabbit; MRID 00152421)

NR NR 

Two-generation reproduction (Rat; MRID 00151901)

N N N N NE N 

Two-generation reproduction (Rat; MRID 00162543)

NE NE N NE NE N 

Two-generation reproduction (Rat; MRID 40405007)

NE N N NE NE N 

Chronic toxicity (Rat; MRIDs 00044337, 00162456, 00162457, 00162458)Chronic toxicity (Mouse; Jaeger, 1986 and MRIDs 00044338, 00158935, 40392313)Chronic toxicity (Dog; MRIDs 42447001, 00151908, 00159618)
TRIFLURALIN 

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Table 2. Estrogenic/Anti-Estrogenic Pathway for Trifluralin

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Trifluralin1Study Type / Literature Citation

ER Binding

ER Activation

Steroidogenesis

Sex Steroid Hormone

Uterine Weight

Ovarian 

Weight / GSI

Ovarian/Gonad 

Staging and 

His

topathology

Pituitary Weight

Estrous Cyclicity

Age & Weight at VO

2° Sex 

Characteristics

Fertility (Frt) 

/ Fecundity (Fcd)

Life-cycle toxicity (Fathead minnow; MRID 05008271)

N 

Life-cycle toxicity (sheepshead minnow; MRID 42449901)

P 

(L, ML

Avian reproduction (Bobwhite Quail; MRID 00131134) 

 N 

Avian reproduction (Mallard Duck MRID 00131132) 

 N 

Avian reproduction (Bobwhite Quail; MRID 40334706)

N 

Avian reproduction (Mallard Duck; MRID 40334704)

P (H)

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

parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. L

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

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 
TRIFLURALIN 

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4. There were five females (out of 16) in the high concentration observed as either bloated, observed having partial paralysis, observ

of these signs. Similar clinical signs were also observed in males at this concentration. 

5. Decreases in number of number of eggs spawned per female at all test concentrations. 6. 4% decrease in eggshell thickness and decrease in 14-d offspring body weight at 1000 mg/kg-diet. 7. While there was a significant decrease in parental male body weight at 1000 mg/kg-diet, there were no apparent overt signs of tox

mortalities, or reductions in food consumption or female body weight 

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

N Negative findings (in vitro) / No effect (in vivo) NE Not examined NR Not reported
TRIFLURALIN FINAL

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2. Effects on Androgen Pathway 

The potential for trifluralin 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 the 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, trifluralin 

was negative (or a non-binder) in the in vitro AR binding assay and there was no effect on 

testosterone production in the steroidogenesis assay. 

In the anti-androgenic portion of Hershberger assay at the high dose (450 mg/kg/day), 

decreases (p<0.05) in three AST weights (i.e., seminal vesicles with coagulating glands, ventral 

prostate, and LABC) and decreased serum testosterone levels (36%; p<0.05) were observed at 

the high dose (450 mg/kg/day). However, bodyweights at this dose were decreased 10% and 

bodyweight gains were decreased 46% indicating overt toxicity. No statistically significant 

effects on AST weights were seen at the low (50 mg/kg/day) and mid dose (150 mg/kg/day) 

groups. In the male pubertal assay, in the high dose, serum testosterone concentration was 

decreased (NS) by 30% and adjusted weights of AST (DP and LABC) and epididymal weights 

were significantly decreased (p<0.5). No histopathological lesions were observed in the testes 

or epididymides. Age and body weight at preputial separation (PPS) were also not affected at 

any dose level. The high dose in this study was approaching an overtly toxic dose as indicated 

by significant decreases (p<0.05) in body weight at post-natal day (PND) 30 and 37 of 11 and 

9%, respectively, with decreases (p<0.5) in body weight gain of 11 to 24%. 

In the FSTRA, with the exception of effects on male GSI and fertility, all effects occurred at the 

highest treatment concentration where overt toxicity was apparent (abnormal 

swimming/partial paralysis in male (5/8) and female fish). Significant increases in GSI was 

observed at all treatments (↑29-39%) and was treatment-dependent. Fertility was decreased 

0.5% at the mid concentration. Additionally, at the high test concentration (21.5 μg a.i./L), 

male nuptial tubercles score were decreased (20 vs. 31 in control) and 17-β estradiol 

concentration in the high-dose males was increased by 78%. Furthermore at this concentration, 

in male gonadal development, there was a shift to the late spermatogenic stage and an 

increased proportion of spermatogonia. 

The only effect on male reproductive parameters in the Part 158 mammalian studies was an 

increase in testes weight (8%) in F1 offspring only in the rat two-generation reproduction 

study. Decreased pituitary weights were observed at all doses in the subchronic rat toxicity 

study. No androgen-related effects were observed in the Part 158 fish or avian studies.

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Page 17 of 58 

Table 3. Androgenic/Anti-Androgenic Pathway for Trifluralin

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

Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight / 

GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility (Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin 

EDSP Tier 1 Assay 

AR Binding (MRID 48619702) N Steroidogenesis (MRID 48619708) N Hershberger (MRID 48619707) N N Male Pubertal Rat (MRID 48674002) N N N 

↓8% 

(M), 

↓11 - 

14% (H) 

N N 

↓12%, 

DP; 

↓13%, 

LABC 

(H) 

N 

FSTRA (MRID 48619706) N 

↑29%, ↑36%, 

(L, M) 

N N 

Frt: 

↓0.5%, 

(L) 
TRIFLURALIN 

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Table 3. Androgenic/Anti-Androgenic Pathway for Trifluralin

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

Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight / 

GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility (Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin 

OSRI 

Subchronic toxicity (Rat; MRIDs 40138301, 00151906) 

 NR NR NR NR ↓ (L, M, H) NR 

Developmental toxicity (Rat; MRID 00152419) NR Developmental toxicity (Rabbit; MRID 00152421) 

NR 

Two-generation reproduction (Rat; MRID 00151901) 

↑8% 

(H, F1) N NE N N NE N 

Two-generation reproduction (Rat; MRID 00162543) 

 NE N NE N NE NE N 

Two-generation reproduction (Rat; MRID 40405007) 

N N NE N NE NE N 

Chronic toxicity (Rat; MRIDs 00044337, 00162456, 00162457, 00162458) Chronic toxicity (Mouse; MRIDs 42447001, 00151908, 00159618) Chronic toxicity (Dog; MRIDs 

42447001, 00151908, 00159618) 

NR NR NR NR NR NR 
TRIFLURALIN 

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Table 3. Androgenic/Anti-Androgenic Pathway for Trifluralin

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

Study Type / Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight / 

GSI 

Gonad Staging 

and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights / 

2° Sex 

Characteristics 

Fertility (Frt) / 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin 

Life-cycle toxicity (Fathead minnow; MRID 05008271) 

 N 

Life-cycle toxicity (sheepshead minnow; MRID 42449901) 

 N 

Avian reproduction (Bobwhite Quail; MRID 00131134) 

 N 

Avian reproduction (Mallard Duck MRID 00131132) 

 N 

Avian reproduction (Bobwhite Quail; MRID 40334706) 

 N 

Avian reproduction (Mallard Duck; MRID 40334704) 

 N 

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

parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. Absensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Levator ani-bulbocavernosus (LABC), Epidi

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

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. Effects at the high dose (450 mg/kg/day) in the anti-androgenic phase of the assay occurred in the presence of overt toxicity (Body 

5. The high dose in this study was approaching an overtly toxic dose as indicated by significant decreases (p<0.05) in body weight at 

and 37 of 11 and 9%, respectively, with decreases (p<0.5) in body weight gain of 11 to 24% 

6. Five males (out of 8) that exhibited either a partial loss of equilibrium and partial paralysis, appearing extremely dark, having a ben

thin). Similar clinical signs were also observed in females at this concentration. 

7. While there was a significant decrease in parental male body weight at 1000 mg/kg-diet, there were no apparent overt signs of toxi

mortalities, or reductions in food consumption or female body weight 
TRIFLURALIN 

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P Positive findings (in vitro) / No effect (in vivo) N Negative findings (in vitro) / No effect (in vivo) NR Not reported 

NE Not examined 
TRIFLURALIN 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 to detect changes in the HPT axis, i.e., 

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

The potential for trifluralin 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. This table also includes HPG-relevant 

findings from the 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 male and female pubertal assays, dose-dependent decreases (25-58%) in T4 

concentrations were seen at the low (25 mg/kg/day), mid (75 mg/kg/day) and high (250 

mg/kg/day) doses. TSH concentrations were elevated (males: ↑74%, NS; females: ↑73%) at 

the high dose only. There was no effect on thyroid weight for either sex, but increases in 

follicular cell height and decreases in colloid area were noted in females at the high-dose. 

In the AMA, there were no histological effects on the thyroid, nor any effects on 

developmental stage. There was an increase of 25 and 18% in normalized hind-limb 

length (HLL) at the low and mid concentration (2.64 and 26.2 μg/L, respectively) and a 20% 

decrease at the high concentration (232 μg/L) compared to the negative control. For this 

endpoint, a marginally significant increase of 16% (p = 0.05) was observed in the solvent 

control compared to the negative control which were of the same magnitude as effects observed 

in the low and mid treatment levels. Additionally, there were significant (p<0.05) increases in 

the solvent control as compared to the negative in the growth measurements including Day 7 

snout-vent length (SVL) and Day 21 body weight. For both of these endpoints, the low and 

middle treatment concentrations were also increased over the negative control (not all effects 

significant at α=0.05), while the high treatment concentration was decreased as compared to the 

negative control. The results of the AMA indicate a reduction of growth of X. laevis tadpoles, 

but there were no corresponding histopathological diagnoses to suggest that this developmental 

delay is thyroid-related. 

In a chronic oral carcinogenicity study with trifluralin in rats, an increase in the incidence of 

thyroid gland follicular cell tumors (adenomas plus carcinomas combined) in males was 

observed at the high dose (325 mg/kg/day). In a literature study by Saghir et al., (2008), 

trifluralin was reported to induce thyroid tumors via a UGT-related MoA. After a two-week 

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exposure to trifluralin (441 mg/kg/day), effects on body weight gain (↓20%; p<0.05), serum T3 

(↓17%; p<0.05), serum T4 (↓90%; p<0.05) and TSH (↑37%; NS) were observed. Induction of 

T4-specific hepatic UGT activity and 2-fold increases in bile-flow rate and biliary elimination of 

T4 also were demonstrated. Decreased pituitary weights were observed at all doses in the Part 

158 subchronic rat toxicity study. 

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Table 4. Thyroid Pathway for Trifluralin.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Trifluralin1

Study Type / 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels Serum TSH levels Pituitary Weight 

Developmental stage or asynchronous & HLL 

Growth (BW, SVL,) 

Systemic Toxicity Observed2 

Overt Toxicity Observed3

EDSP Tier 1 Assays

Male Pubertal Rat 

(MRID48674002) N N 

↓25%, 

↓42%, 

↓58% 

(L,M,H)

N N 

LW 

(M,H) 

KE 

(H) 

N 

Female Pubertal Rat 

(MRID 48674001) N 

↑cell 

height, ↓

colloidal 

area (H) 

↓33%, 

↓45% 

(M, H) 

↑73% 

(H) N 

LW, 

KE 

(H)

N 

AMA 

(Frog; MRID 48619701) N 

HLL: ↑18%,

↑25%, ↓17%

(L, M, H)4

Day 7: 

SVL- 

↑9% 

(M), 

↓17% 

(H); 

BW 

↓39% 

(H)5 

N 

OSRI

Subchronic toxicity 

(Rat; MRIDs 40138301, 

00151906) NR NR 

↓

(L, M, 

H) 

X NR 

Developmental toxicity 

(Rat; MRID 00152419) NR NR NR X X 

(M,H, H) 

Developmental toxicity 

(Rabbit; MRID 

00152421) 

NR NR NR X X 

(M, H) 

Two-generation 

reproduction 

(Rat; MRID 00151901) NE N N X N 

Two-generation 

reproduction 

(Rat; MRID 00162543) NE NE NE X N 

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Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Trifluralin1

Study Type / 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels Serum TSH levels Pituitary Weight 

Developmental stage or asynchronous & HLL 

Growth (BW, SVL,) 

Systemic Toxicity Observed2 

Overt Toxicity Observed3

Chronic toxicity 

(Rat; MRIDs 00044337, 

00162456, 00162457, 

00162458) 

NR NR NR X X 

(M, H) 

Chronic toxicity 

(Mouse; MRIDs 

00044338, 00158935, 

40392313) 

NR NR NR X NR 

Chronic toxicity 

(Dog; MRIDs 42447001 

and 00151908/00159618) 

NR NR NR X X 

(H) 

Mechanistic Study 

(Rat; Saghir et al.,2008) T4 ↓90%, 

T3 ↓17% ↑37% NR X N 

1. Key to responses: L=Low treatment, =M 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; KE= serum kidney 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. These results reflect comparison to the negative control. Solvent control HLL was significantly increased 

compared to the negative control and was of similar magnitude as in the low and mid treatment. When 

compared to the solvent control, the low and mid treatments are not significantly different, however the highest 

treatment is significantly decreased. Additionally, while there were no effects on developmental stage in the 

treatments compared to the negative control, there was a significant decrease in stage at the high treatment 

compared to the solvent control. 

5. These results reflect comparison to the negative control. Day 7 SVL and Day 21 body weight solvent control 

was significantly increased compared to the negative control and was of similar magnitude as in the low and 

mid treatment (except for Day 21 body weight compared to mid treatment). When compared to the solvent 

control, the low and mid treatments are not significantly different, however the highest treatment is significantly 

decreased for Day 7 body weight and SVL, Day 21 NHLL, Day 21 SVL and body weight, and Day 21 NF 

stage. 

N Negative findings 

NR Not reported 

NE Not examined 

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IV. Committee’s Assessment of Weight of Evidence 

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

potential of trifluralin to interact with endocrine related processes (i.e., E, A or T hormonal 

pathways) as well as recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

are considered, along with other scientifically relevant information (e.g., 40 CFR Part 158 test 

guidelines and published or publicly available peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and 

OSRI), then performing an integrated analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, coherence, and interrelationships among 

the measured endpoints within and across studies. The available findings from standard 

toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if 

effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively a sequelae of systemic effects. Endocrine modes of action may elicit a number of 

phenotypic consequences other than those evaluated in the Tier 1 assays. 

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to 

whether or not the responses are related through an endocrine pathway, therefore non-endocrine 

toxic responses (including but not limited to mortality or body weight changes) are also 

considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated 

with weight of evidence evaluation of the results of the Tier 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., changes in organ weights or blood chemistry).

1. Tier 1 in vivo Assays 

Systemic toxicity was observed in several of the Tier 1 Assays. In the anti-androgenic phase of 

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the Hershberger assay at the high dose (450 mg/kg/day), overt toxicity manifested as decreased 

(p≤0.05) body weights on Days 7 (↓8%) and 11 (↓10%), and body weight gains (p≤0.05) at each 

measured interval (↓46–65%). Also at the high dose, food consumption was decreased (p≤0.05) 

during the first 4-day interval (Days 1–4; ↓35%) compared to vehicle control. Liver weights 

were increased (p≤0.05, except as noted) by trifluralin treatment in both the androgenic and antiandrogenic portions of the study: by 16% at 150 mg/kg/day; and by 36% at 450 mg/kg/day. 

In the male pubertal assay, absolute and relative liver weights were significantly (p<0.05) 

increased by 29% and 37%, respectively, and serum creatinine level was increased (p<0.05) by 

50% at the high dose (250 mg/kg/day). In the female pubertal assay, absolute and relative liver 

weights were significantly (p<0.05) increased by 17% and 24%, respectively, and serum 

creatinine level was increased (p<0.05) by 100% at the high dose (250 mg/kg/day). 

In the FSTRA, there were five females (out of 16 exposed) in the high concentration observed as 

either bloated, observed having partial paralysis, observed having a bent tail or all of these signs. 

Five males (out of 8 exposed) exhibited either a partial loss of equilibrium and partial paralysis, 

appeared extremely dark, were observed with a bent tail or appeared very thin. 

No treatment-related effects on mortality or clinical behavior signs of toxicity were observed in 

the AMA. 

2. OSRI 

In mammals, the toxicity profile for trifluralin demonstrates that the kidney and the liver are the 

principal target organs in rats and dogs. In subchronic oral studies, liver effects include increased 

liver weights and changes in clinical chemistry parameters. Kidney effects include decreased 

kidney weights, kidney and bladder tumors, increased blood urea nitrogen (BUN), increases in 

total protein, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in the urine. 

Also, protein electrophoresis of urine samples showed the presence of α1-globulin and α2-

globulin. In the kidneys, tubular hyaline casts, minimal cortical tubular epithelial regeneration, 

observed microscopically, and an increased incidence of progressive glomerulonephritis were 

seen. In dogs exposed to trifluralin for 1 year, multifocal cortical tubular cytoplasmic pigment 

deposition was noted in the kidneys of both sexes. In the subchronic studies, blood effects such as 

lower hemoglobin levels and changes in clinical chemistry were reported in rats. In male rats, 

trifluralin was associated with increased incidence of thyroid follicular cell combined adenoma, 

papillary adenoma, cystadenoma and carcinoma tumors. 

In the Part 158 studies with fish, survival in the parental group was decreased at ≥5.1 μg/L in the 

fathead minnow study, and at 17.7 μg/L in the sheepshead minnow study. Parental and offspring 

growth and offspring survival was also affected at 9.6 μg/L in the sheepshead minnow study. 

Reduction in growth were observed at 4.23 μg/L in an early life-cycle study with rainbow trout. 

In the avian reproduction studies, no treatment related effects were observed in bobwhite quail up 

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to 1000 mg/kg-diet. For the mallard duck reproduction study, while a decrease in parental male 

body weight was observed at 1000 mg/kg-diet, there were no apparent overt signs of toxicity, 

treatment related mortalities or reductions in food consumption or female body weight. 

B. Estrogen Pathway 

Trifluralin was negative in the estrogen receptor (ER) binding assay which is capable of detecting 

an interaction of the test compound with the ER (both agonists and antagonists). Although the ER 

transactivation assay (ERTA) was weakly positive (14-18% of the response seen with 1 nM 17βestradiol in three out of four assay runs) at 10−4 M trifluralin, there was no evidence of ER 

agonism (or antagonism) in response to trifluralin treatment in the female pubertal assay or 

uterotrophic assay. Furthermore, the results of the aromatase assay indicate that trifluralin does 

not inhibit aromatase activity. An slight increase (1.2- to 1.4-fold) in estradiol production was 

observed in the steroidogenesis assay at relatively high concentrations of trifluralin (10 and 100 

μM). In the fish short-term reproduction assay (FSTRA), all effects were observed in the 

presence of overt toxicity, except for a 0.5% decrease in fertility at the low concentration for 

which there were no other effects observed. Trifluralin did not alter estrous cyclicity or 

reproductive organ histopathology in the female pubertal rat assay. 

In the mammalian Part 158 studies, following dietary exposure to rats for 13 weeks, a decrease in 

pituitary weight was observed at doses >40 mg/kg/day. No treatment-related effects on the 

pituitary were observed in the two-generation rat reproduction or in the chronic toxicity studies 

with rats. The decrease in ovarian weight seen at 337 mg/kg/day in the rat reproduction study 

occurred in the presence overt toxicity. No other estrogen-related effects were observed in the 

mammalian reproduction or developmental studies. Decreases in fecundity were observed in the 

Part 158 sheepshead minnow study in the absence of overt toxicity (≥1.3 μg/L), however, there is 

a lack of diagnostic specificity for this endpoint in of itself regarding potential endocrine-related 

effects. A 4% decrease in eggshell thickness and 7% decrease in 14-d offspring body weight at 

1000 mg/kg-diet in the mallard duck reproduction study was observed; however, no other 

reproductive parameters were affected in this study or the bobwhite quail reproduction study. 

Overall, there is a lack of convincing evidence for a potential interaction with estrogen pathway 

for trifluralin. 

C. Androgen Pathway 

Trifluralin was negative for AR binding and did not alter testosterone production in the 

steroidogenesis assay. No androgenic effects were seen the mammalian Tier 1 in vivo assays at 

doses that did not cause either systemic toxicity (male pubertal assay) or overt toxicity 

(Hershberger assay). The observed effects in the Hershberger assay (decreases in androgenresponsive tissues weights) at the high dose (450 mg/kg/day) occurred at a dose that was 

considered overtly toxic (10% decrease in body weight). In the male pubertal, decreases in 

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androgen-responsive tissues weights (prostate, LABC and epididymal weights) and decreased 

serum testosterone were seen at the high dose (250 mg/kg/day). This dose caused significant 

decreases in body weights approaching overt toxicity (9%) and increases in absolute (29%) and 

relative (37%) liver weights. In the FSTRA, except for GSI which was increased (p<0.05) dosedependently at all treatments as well as a slight decrease in fertility (0.5%), all other observed 

effects occurred at a concentration that was considered overtly toxic. 

In the mammalian Part 158 studies, following dietary exposure to rats for 13 weeks, pituitary 

weights were decreased at dose levels >40 mg/kg/day. No treatment-related effects on the 

pituitary were observed in the two-generation rat reproduction or in the chronic toxicity studies 

with rats in the absence of overt toxicity. No androgen-related effects were observed in the 

wildlife Part 158 studies. Therefore, as the in vitro assays were negative and the findings in the in 

vivo assays occurred in the presence or approaching overt toxicity, or were limited to an isolated 

effect, there is a lack of complementary evidence within the assays as well as a lack of 

redundancy of evidence across the assays. Hence, trifluralin does not appear to potentially 

interact with the androgen-pathway. 

D. Thyroid Pathway 

For the thyroid pathway for trifluralin, in the pubertal assays, dose-dependent decreases in serum 

T4 levels were seen in females at the mid-(75 mg/kg/day) and high (250 mg/kg/day) dose and in 

males at doses of 25, 75 and 250 mg/kg/day. Increased serum TSH levels were observed in both 

males and females at the high dose only. Effects on thyroid histopathology (alterations in 

follicular cell height and colloid area) were also observed in high dose female pubertal rats. All 

thyroid effects in the pubertal assays occurred at doses where increases in liver weights were also 

observed. In the AMA, while reduced growth and effects on the development endpoint of 

normalized hind-limb length was observed, there were no corresponding histopathological 

diagnoses to suggest the developmental is thyroid-related. 

In the mammalian Part 158 studies, following dietary exposure to rats for 13 weeks, pituitary 

weights were decreased at dose levels > 40 mg/kg/day. In the chronic/carcinogenicity study with 

rats, increased thyroid weights and an increase in the incidence of thyroid gland follicular cell 

tumors in males was observed only in the presence of overt toxicity. Additional OSRI (Saghir et 

al., 2008) investigating the mode of action for trifluralin-related thyroid perturbations showed that 

two week treatment of trifluralin in male rats resulted in decreases in serum T3 and T4 and 

elevated TSH. Trifluralin also induced T4-specific hepatic UGT activity, and 2-fold increases in 

bile-flow rate and biliary elimination of T4 also were demonstrated. Furthermore, in previously 

submitted mechanistic studies for benfluralin (a dinitroaniline similar to trifluralin), induction of 

hepatic microsomal enzyme activity was observed in both sexes in rats treated with benfluralin. 

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 

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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. Data submitted for benfluralin also showed increased testosterone clearance 

accompanied decrease in AST weights in castrated rats treated with benfluralin. 

Based on the profile of liver and thyroid effects seen in the pubertal assays, the available MoA 

data and the lack of evidence for a potential interaction in the AMA, thyroid effects due to 

trifluralin exposure are likely attributed to enhanced serum T4 clearance due to liver enzyme 

(UGT) induction. 

E. Conclusions 

The conclusion of the WoE evaluation is that trifluralin demonstrates no convincing evidence of 

potential interaction with the estrogen or androgen pathways. The available data suggests the 

thyroid effects observed in the mammalian in vivo studies may result from liver enzyme induction 

and subsequent increased hormone clearance. In the wildlife data, while there is a lack of data to 

support liver enzyme induction which may result in hormone clearance, this MOA may be 

conserved across other vertebrate taxa. However, there is no convincing evidence in the wildlife 

studies to suggest that there is a potential interaction with E, A or T, as the majority of the effects 

occurred in the presence of overt toxicity and lacked complementarity in the absence of overt 

toxicity. 

II. EDSP Tier 2 Testing Recommendations 

In the EDSP Tier 1 in vivo male and female pubertal assays as well as in the OSRI, there was 

evidence for potential interaction with the thyroid pathway in studies conducted with adult 

animals, but no such data exists for potential thyroid toxicity in the young animals. 

For trifluralin, the point of departure (POD) for risk assessment of 2.4 mg/kg/day is based on 

increased frequency of abnormal stool, decreased body weights and body weight gains and 

toxicity to the hematopoietic system seen at 40 mg/kg/day (LOAEL) in a chronic toxicity study in 

dogs. This POD is used for deriving the chronic Reference Dose (RfD) used for dietary risk 

assessment and for the calculating Margins of Exposure (MOEs) used for non-dietary risk 

assessments. 

In general, since the POD and the toxicity endpoints of concern are based on data obtained from 

adult animals, there is a concern that the POD may not be protective of potential thyroid toxicity 

in the young. However in the case of trifluralin, although no data exists for potential thyroid 

toxicity in the young, a comparative thyroid assay (CTA) is not recommended since: 1) the POD 

is 10-fold lower than the lowest dose (25 mg/kg/day) that caused dose-dependent decrease in 

serum T4 levels in the female pubertal assay; 2) available data suggests the thyroid effects 

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observed in the mammalian in vivo studies may result from liver enzyme induction and 

subsequent increased hormone clearance; 3) the CTA would be tested at higher doses and would 

not be likely to yield a POD lower than the level currently used for risk assessment; and 4) the 

chronic RfD and MOEs are protective of the thyroid-related findings seen in the Tier 1 pubertal 

assay. 

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

recommended for trifluralin since additional testing is unlikely to impact the current EPA 

established regulatory endpoints for human risk assessments. 

No fish or amphibian EDSP Tier 2 testing is recommended since there is no convincing evidence 

for an interaction with estrogen, androgen or thyroid pathways in fish or amphibians. 

Additionally, it is noted that life cycle studies which evaluate reproduction with fathead minnow 

and sheepshead minnow are available for triflurlin. 

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

are available for trifluralin. In the mallard duck study, effects on reproductive parameters were 

observed; no effects were observed in the bobwhite quail study. 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 trifluralin exposure. Additional testing is not 

recommended. 

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VI. References

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Unpublished study prepared by Lilly Research Laboratories. (MRID 41386202) 

Adams, E., Bernhard, N., Jordon, W. (1992) A Chronic Toxicity Study of Trifluralin (Compound 

036352) Administered Orally to Beagle Dogs for One Year (Supp.): Lab Project Number: 

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Bathe, R. (1984) 12-Month Oral Toxicity (Feeding) Study in Beagle Dogs: Trifluralin Substance 

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Beavers, J., Dukes, V., Jaber, M. (1987) Trifluralin Technical: A One-generation 

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Becker, H. (1984) Multiple Generation Study in the Rat: Trifluralin Substance Technical Grade 

(Code: HOE 38474 OH AT210): Project No. 008875. Unpublished study prepared by Research 

& Consulting Co., AG. (MRID 00151901) 

Becker, H. (1985) Trifluralin, Substance Technical Grade: Multiple Generation Study in the 

Rat:Diet Preparation, Stability, and Analysis of the Test Article: 1st Amendment to Report: 

Project No. 008875: Report No. A32777. Unpublished study prepared by Research & 

Consulting Co. AG. (MRID 00159619) 

Byrd, R. (1984) A Teratology Study of Trifluralin (El-152, Compound 36352) Administered 

Orally to Charles River CD Rats: Study R08484. Unpublished study prepared by Lilly Research 

Labs. (MRID 00152419) 

Byrd, R. (1984) A Teratology Study (II) of Trifluralin (El-152, Compound 36352) Administered 

Orally to Dutch Belted Rabbits: Study B01784. Unpublished study prepared by Lilly Research 

Labs. (MRID 00152421) 

Coady, K., Sosinski, L. (2012). Trifluralin: Evaluation of Trifluralin in the Human Recombinant 

Aromatase Assay. Unpublished study prepared by Toxicology & Environmental Research and 

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Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project Study ID: 111053. 

(MRID 48619703) 

Coady, K., et al. (2012). Trifluralin: 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, Michigan 48674. Laboratory 

report number 111016. (MRID 48619701) 

Donaubauer (1986) Chronic Feeding Study (24 Months) in Rats: Trifluralin: Study No. 680: 

Report No. 85.0302. Unpublished study prepared by Hoechst Aktiengellschaft, Pharma 

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Donaubauer (1986) Carcinogenicity Study in Rats (28-month Feeding Study): Trifluralin: Study 

No. 680: Report No. 85.0087. Unpublished study prepared by Hoechst Aktiengesellschaft, 

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Ellgehausen, H. (1984) Determination of Trifluralin Substance TechGrade (Code: HOE 38474 O 

H AT210) in Rodent Feed: Project No. 008875. Unpublished study prepared by Research & 

Consulting Co. (MRID 00151902) 

Emmerson, J., Pierce, E., McGrath, J. (1980) The Chronic Toxicity of Compound 36352 

(Trifluralin) Given as a Component of the Diet to Fischer 344 Rats for Two Years: Studies R-87 

and R-97. Unpublished study received Sep 18, 1980 under 1471-35; submitted by Elanco 

Products Co., Div. of Eli Lilly and Co., Indianapolis, Ind.; CDL:243289-A, 243290. (MRID 

00044337) 

Emmerson, J., Owen, N., McGrath, J. (1980) The Chronic Toxicity of Compound 36352 

(Trifluralin) Given as a Component of the Diet to the B6C3F1 Mouse for 24 Months: Studies 

M9067 and M-9077. Unpublished study received Sep 18, 1980 under 1471-35; submitted by 

Elanco Products Co., Div. of Eli Lilly and Co., Indianapolis, Ind.; CDL:243291-A; 243292; 

243293. (MRID 00044338) 

Fink, R., Beavers, J., Brown, R. (1978) One-generation Reproduction Study—Mallard Duck: 

Compound 36352, Trifluralin: Project No. 151-103. Final Rept. Unpublished study received 

Sep 20, 1983 under 1471-70; prepared by Wildlife International Ltd., submitted by Elanco 

Products Co., Div. of Eli Lilly and Co., Indianapolis, IN; CDL:251256-A. (MRID 00131132) 

Fink, R., Beavers, J., Brown, R. (1979) One-generation Reproduction Study—Bobwhite 

Quail: Compound 36352, Trifluralin: Project No. 151-102. Final report. Unpublished study 

received Sep 20, 1983 under 1471-70; prepared by Wildlife International Ltd., submitted by 

Elanco Products Co., Div. of Eli Lilly and Co., Indianapolis, IN; CDL:251256-C. (MRID 

00131134) 

Hoyt, J. (1986) A One-year Two-generation Reproduction Study in CD Rats Maintained on 

Diets Containing Trifluralin (EL-152, Compound 36352): Studies R06384 and R13984. 

Unpublished study prepared by Lilly Research Laboratories. (MRID 00162543) 

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Jung, J., Ishida, K., Nishihara, T. (2004) Anti-estrogenic activity of fifty chemicals evaluated by 

in vitro assays. Life Sciences, 74:3065–3074. 

Jungbauer, A., Beck, V. (2002) Yeast reporter system for rapid determination of estrogenic 

activity. Journal of Chromatography B, 777:167–178. 

Kojima, H., et al. (2004) Screening for Estrogen and Androgen Receptor Activities in 200 

Pesticides by In Vitro Reporter Gene Assays Using Chinese Hamster Ovary Cells. Environ Health 

Perspect, 112:524–531.

LeBaron, M., Kan, H., Perala, A. (2011) Evaluation of trifluralin in the in vitro steroidogenesis 

assay. Unpublished study prepared by Toxicology & Environmental Research and Consulting, 

The Dow Chemical Company, Midland, MI. Laboratory Study No.: 101187. (MRID 48619708) 

LeBaron, M., Schisler, M., Visconti, N. (2012). Evaluation of trifluralin in an in vitro estrogen 

receptor binding assay. Unpublished study prepared by Toxicology & Environmental Research 

and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project ID: 111124. 

(MRID 48619704) 

LeBaron, M., Schisler, M., Visconti, N.R. (2012). Evaluation of trifluralin in an in vitro 

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

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Macek, K., Lindberg, M., Sauter, S., Buxton, K.S., Costa, P.A. (1976) Toxicity of Four 

Pesticides to Water Fleas and Fathead Minnows. Duluth, Minn.: U.S. Environmental Protection 

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from: NTIS, Springfield, VA; PB-262 912). (MRID 05008271) 

Marino, T., et al. (2011). Trifluralin: A Fish Short-term Reproduction Assay with the Fathead 

Minnow, Pimephales promelas. Unpublished study performed by Toxicology & Environmental 

Research Consulting, The Dow Chemical Company, Midland, Michigan. Lab Study No.: 

101098. (MRID 48619706) 

Martin, M., et al. (2009) Profiling the Reproductive Toxicity of Chemicals from Multigeneration 

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Marty, M., Zablotny, C., Thomas, J. (2012). Trifluralin: Pubertal Development and Thyroid 

Function in Intact Juvenile/Peripubertal Female Crl:CD(SD) Rats. Unpublished study prepared 

by Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 

Midland, MI. Laboratory Report No. 111078. (MRID 48674001) 

Marty, M., Andrus, A., Thomas, J. (2012) Trifluralin: Pubertal Development and Thyroid 

Function in Intact Juvenile/Peripubertal Male Crl:CD(SD) Rats. Unpublished study prepared by 

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

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Midland, MI. Laboratory Report No.: 111079. (MRID 48674002) 

 

Marty, M., Brooks, K. (2012). Trifluralin: Uterotrophic Assay in the Immature Female 

Crl:CD(SD) Rat. Unpublished study prepared by The Dow Chemical Company, Toxicology & 

Environmental Research and Consulting, Midland, MI. Laboratory Project Study ID.: 101144. 

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Marty, M., Marshall, V., Jeong, YC. (2011). Trifluralin: Hershberger Assay in Castrated Adult 

Male Crl:CD(SD) Rats. Unpublished study prepared by Toxicology & Environmental Research 

and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 101133. 

Unpublished. (MRID 48619707) 

Mayer, FL, Jr., Ellersieck, MR, USDOI/FWS Resour Publ No.160: 505 (1986) as cited in the 

ECOTOX database. Available from, as of February 20, 2012: http://cfpub.epa.gov/ecotox/: cited 

in HSDB, Toxnet, NLM. 

Morinaga, H. et al. (2004) A Benzimidazole Fungicide, Benomyl, and Its Metabolite, 

Carbendazim, Induce Aromatase Activity in a Human Ovarian Granulose-Like Tumor Cell Line 

(KGN). Endocrinology, 145:1860-1869. 

Petit, F., et al. (1997) Two complementary bioassays for screening the estrogenic potency of 

xenobiotics: recombinant for trout estrogen receptor and trout hepatocyte cultures. Journal of 

Molecular Endocrinology, 19:321-335. 

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. 

Schutz, Weigand, Kramer (1980) Repeated-dose (3 Months) Oral Toxicity Study of the Active 

Substance HOE 38474 (Code: HOE 38474 O H AT204) Administered in the Feed to Rats: 

Report No. 618/80. Unpublished study prepared by Hoechst Aktiengesellschaft. (MRID 

00151906) 

Schutz, Donaubauer (1986) Combined Chronic Toxicity and Carcinogenicity Study in Rats 

(24/28-month Feeding Study): Summary and Evaluation of the Results: Trifluralin: Study No. 

680: Report No. 86.0092. Unpublished study prepared by Hoechst Aktiengesellschaft, Pharma 

Forschung Toxikologie. (MRID 00162456) 

Soto, A., et al. (1995) The E-SCREEN Assay as a Tool to Identify Estrogens: An Update on 

Estrogenic Environmental Pollutants. Environ Health Perspect, 103 (Suppl 7):113-122. 

Suter, P. (1986) Oncogenicity Study with Trifluralin Active Ingredient Technical (HOE 38474 O 

H AT210) in Mice: Project No. 008853. Unpublished study prepared by Research & Consulting 

Company AG. (MRID 00158935) 

Suter, P., Horst, K., Vogel, W., et al. (1987) Oncogenicity Study with Trifluralin Active 

Ingredient Technical (HOE 38474 OH AT210) in Mice: Supplementary Data in Support of 

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Previously Submitted Study Acc. No. 262516-262520: Laboratory Project No. 008853, Report 

No. A 36197. Unpublished study prepared by Research & Consulting Co. AG. (MRID 

40392313) 

Usher, R. (1986) A Supplementary Report of a Special Urinalysis Study in Fischer 344 Rats 

Maintained on Diets Containing Trifluralin (Compound 36352) for Three Months: Study R04785. 

Unpublished study prepared by Lilly Research Laboratories. 263 p. (MRID 40138301) 

USEPA (2010) Trifluralin- Report of the OPP Endocrine Disruptor Review Team; Test Order#: 

EDSP- 036101-250. December 20, 2010. Office of Chemical Safety and Pollution Prevention. 

U.S. Environmental Protection Agency, Washington, DC. 

USEPA (2011) Endocrine Disruptor Screening Program — Weight of Evidence: Evaluating 

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

Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC. 

Westen, H. (1984) Multiple Generation Study in Rat: Trifluralin Substance Technical Grade 

(Code: HOE 38474 O H AT210): Pathology Report Part II: Project No. 008875. Unpublished 

study prepared by Research & Consulting Co., AG. (MRID 00151903) 

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APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100) 

The 21-day assay (MRID 48619701) of trifluralin (97.1% 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 a negative control and solvent control (0.1 mL/L dimethyl 

formamide, DMF) and nominal trifluralin concentrations of 0.00300, 0.0300, and 0.300 mg a.i./L. 

The reviewer-calculated time-weighted average (TWA), measured concentrations were 

<0.000181 (<LOQ; controls), 0.00264, 0.0262, and 0.232 mg a.i./L. The test system was 

maintained at 21.3 to 22.9°C and a pH of 7.1 to 7.4. 

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

negative control. 

No treatment-related mortalities or clinical signs were observed during this study. On Day 6, one 

tadpole in the high treatment group was observed resting at the surface. In the same vessel, one 

tadpole was observed with abnormal suspension and appeared to have internal bleeding on Days 

12 and 13 and was observed resting on the surface on Day 14. These observations were not 

considered to be treatment-related and may have been due to physical harm to a single tadpole. At 

test termination, spinal curvature (bent tail) was noted in both controls and in all treatment groups; 

the percentage of affected tadpoles ranged from 2 to 13%. 

Day 7 body weight and snout-vent length (SVL) were significantly reduced (p<0.05) by 17 and 

39%, respectively at the high treatment level, while Day 7 SVL was significantly increased (p 

<0.05) by 9% at the mid treatment level relative to the negative control. Day 21 body weight and 

SVL were comparable (p>0.05) between trifluralin treatment groups and the negative control. 

Trifluralin had no effect (p>0.05) on Day 7 normalized (for snout-vent length) hind-limb length 

(HLL). Day 21 normalized HLL was significantly increased (p<0.05) in the solvent control and 

low and mid treatment levels by 16, 25, and 18%, respectively, compared with the negative 

control. Day 21 normalized HLL was significantly reduced (p<0.05) by 20% at the high treatment 

level compared to the negative control. There were no effects on Day 7 or Day 21 NF 

developmental stage. No asynchronous development was observed. 

There were no apparent treatment-related effects on thyroid histopathology. Follicular cell height 

and area were not determined. 

A total of 21 individuals were excluded from the analysis of Day 21 growth endpoints because NF 

stage exceeded 60 (“late stage”): these included one tadpole from the negative control, four from 

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the solvent control, seven and nine tadpoles in the low and mid treatment levels, respectively, and 

none in the high treatment level. While the occurrence of late stage tadpoles was not analyzed 

statistically, the pattern of occurrence is at least superficially consistent with the observations of 

greater Day 21 normalized HLL in the lower two treatment levels (suggesting advanced 

development) but smaller Day 21 normalized HLL at the highest treatment level when compared 

to the negative control. 

Statistically significant solvent effects were observed compared to the negative control. For Day 

21 normalized HLL, a significant increase (p=0.05) was observed in the solvent control which 

was similar in magnitude to the increase observed in the low and mid treatment groups. When 

compared to the solvent control, there were no differences at the low and mid treatment groups for 

Day 21 normalized HLL; however, Day 21 normalized HLL at the high treatment level was 

significantly reduced (p<0.05). Additionally, there were significant increases (p<0.05) in the 

solvent control relative to the negative control in Day 7 SVL and Day 21 body weight. For both of 

these endpoints, the low and mid treatment levels were also increased relative to the negative 

control (not all effects significant at p=0.05), while the high treatment level was decreased relative 

to the negative control. Comparisons to the solvent control showed significant reductions (p<0.05) 

for Day 7 SVL and Day 7 body weight in the high treatment group. Given the significant effect 

(p<0.05) at all treatment levels for Day 21 normalized HLL which cannot be explained 

completely by solvent interference, there is evidence to suggest that trifluralin may affect 

developmental endpoints associated with reduced growth in tadpoles exposed to the treatment 

levels and conditions of this study. 

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

In an androgen receptor (AR) binding assay (MRID 48619702), ventral prostate cytosol 

homogenate from Sprague Dawley rats was used as the source of AR to perform a competitive 

binding experiment, which was conducted to measure the binding of a single concentration of 

[

3

H]-R1881 (1 nM) in the presence of increasing concentrations (logarithmic increase from 10−10

to 10−3

 M) of trifluralin (% purity: 97.1; Lot#: DKO-B916-33, TSN100656). Ethanol was used as 

solvent at a final assay concentration of <3%. A total of 3 runs were performed, and each run 

included dexamethasone as a weak positive control, and R1881 as the ligand reference standard. 

Although no data from a saturation binding experiment were provided, a saturation binding 

experiment was carried out by the performing laboratory with this batch of cytosol (MRID 

48614301). A summary of these results indicate that acceptable responsiveness was determined 

for all parameters. 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 (Bmax expected range = 7 to 16 

fmol/100 μg protein and the Kd expected range = 0.685 to 1.57 nM. Nonspecific binding as a 

percent of total binding was >20 % across the entire concentration range for the saturation binding 

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assay. Only a single run of the saturation binding experiment was conducted on the batch of 

cytosol preparation used in the competitive binding experiment. 

In the competitive binding experiment, the estimated mean log IC50s for R1881 (−8.996 M) and 

the weak positive control, dexamethasone (−4.522 M), were within the expected ranges. The 

mean RBA for the weak positive control was 0.0034%. Confidence in these numbers is high due 

to the small variation. The solvent control responses indicated no drift in the study assay. All 

performance criteria were generally met. 

Trifluralin had no effect on specific binding of the [3

H]-ligand to the AR; therefore, a log IC50 and 

RBA could not be calculated. 

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

Receptor Binding Assay. 

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP19) assay (MRID 48619703), trifluralin (97.1% a.i. Lot # DKOB916-33, TSN100656) was incubated with human recombinant aromatase and tritiated 

androstenedione ([1β3

H(N)]-androst-4-ene-3,17-dione; [3H]-ASDN) in 1% dimethyl sulfoxide 

(DMSO) at log concentrations from 10−10 to 10−4.5 M for 15 minutes to assess the potential of 

trifluralin to inhibit aromatase activity. 

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

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

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

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

 M) using a 

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

10−4.5 M) with three repetitions per concentration. At higher tested concentrations (10−4, 10–3.5, 

and 10−3

 M), trifluralin was visibly insoluble. 

Aromatase activity in the full activity controls ranged from 0.133 to 0.225 nmol·mgprotein−1

·min−1 for the three test runs, with a mean and standard deviation of 0.192 ± 0.043 

nmol·mg-protein−1

·min−1. The response of each full activity control within a run was between 90 

to 110% of the average full activity. Activity in the background controls was 0.23 to 0.31% of the 

full activity controls. 

For the positive control substance (4-OH ASDN), aromatase activity results were within the 

recommended ranges for the performance criteria. The estimated log IC50 for 4-OH ASDN 

averaged −7.27 M and the Hill slope was −0.93. 

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For trifluralin, aromatase activity averaged 0.198 ± 0.030 nmol·mg-protein−1

·min−1 at the lowest 

tested concentration (10−10 M) and 0.191 ± 0.029 nmol·mg-protein−1·min−1 at the highest soluble 

concentration tested (10−4.5 M). As a percent of the control, aromatase activity for trifluralin was 

≥96.50% at all concentrations from 10-10 to 10-4.5 M. 

Based on the data from the average response curve, trifluralin is classified as a Non-inhibitor of 

aromatase in this assay. 

Estrogen Receptor (ER) Binding Assay; (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48619704), uterine cytosol from Sprague 

Dawley rats was used as the source of ER for a competitive binding experiment, which was 

conducted to measure the binding of a single concentration of [3

H]-17β-estradiol (1 nM) in the 

presence of increasing concentrations (logarithmic increase from 10−10 to 10−3

 M) of trifluralin 

(97.1% purity; Lot#DKO-B916-33, TSN100656). Ethanol was used as the solvent vehicle at a 

final concentration of <3%. A total of three runs were performed, and each run included 19-

norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17-βestradiol as the natural ligand reference material. 

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

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

radiolabeled reference estrogen prior to routinely conducting ER competitive binding 

experiments. Data from the saturation binding experiment were not included in the original 

report; however, the binding data were reported in a separate report for the batch of cytosol that 

was utilized in the assay. 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 was within the expected range of 10 to 150 fmol/100 μg protein, and the Kd was within the 

expected range of 0.03 to 1.5 nM. Nonspecific binding as a percent of total binding was 1.7% to 

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. 

 

In the competitive binding experiment, the estimated mean log IC50 was −8.934 M for 17βestradiol and −5.442 M for the weak positive control. The mean Relative Binding Affinity (RBA) 

was 0.032% for the weak positive control. 

Trifluralin had no effect on specific binding of the [3

H]-ligand to the ER; therefore, a log IC50 and 

RBA could not be calculated for trifluralin. Specific binding was >75% at all concentrations of 

trifluralin tested. 

Based on the results of the three runs, trifluralin is classified as Not Interactive in the Estrogen 

Receptor Binding Assay. 

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

In an estrogen receptor transcriptional activation assay (MRID (48619705), hERα-HeLa-9903 

cells cultured in vitro were exposed to trifluralin (97.1%, Lot #s DKO-B916-33, TSN100656) at 

logarithmically increasing concentrations from 10-10 to 10-4 M in DMSO. Four independent runs 

were performed using 96-well plates; each concentration was tested in 3 wells/plate. Cells were 

exposed to the test agent for about 24 hours to induce reporter (luciferase) gene products. 

Luciferase expression in response to activation of the estrogen receptor was measured after 

addition of luciferase substrate and detection with a luminometer. 

Trifluralin was tested up to the limit of solubility, 10-4 M. Performance criteria were generally 

met for all reference chemicals, except values for 17α-methyltestosterone were consistently lower 

than the expected range, indicating increased sensitivity of the assay to weak agonists. Trifluralin 

RPCMax was <0%, 18%, 16.6% and 13.7% in runs 1, 2, 3, and 4, respectively; associated PCMax

was 10-4 M. The response was dose-dependent. Because the RPCMax > PC10 in three out of four 

assay runs, trifluralin was considered positive for estrogen receptor transcriptional activation in 

this test system. 

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

The 21-day short-term reproduction assay (MRID 48619706) of trifluralin with fathead minnows 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish (20 spawning 

groups; 2 males and 4 females in each group; 6 months old), were exposed to trifluralin (97.1% 

purity) at nominal concentrations of 0 (negative control), 0 (solvent control; 0.05 mL/L 

dimethylformamide; DMF), 0.000350, 0.00350, and 0.0350 mg a.i./L; time-weighted average 

(TWA), measured concentrations were <0.00003 (<LOQ, controls), 0.000195, 0.00198, and 

0.0215 mg a.i./L. The test system was maintained at 24.5 to 25.6o

C and a pH of 7.4 to 7.6. 

Comparisons between the negative and solvent control groups revealed no significant differences 

(p>0.05) for any endpoint. Unless otherwise indicated in this DER, all effects are reported based 

on comparison to the negative control. 

There was no significant effect (p>0.05) on fish mortality. Clinical signs of toxicity were present 

at the high treatment level, with five males showing partial loss of equilibrium, very thin bodies, 

partial paralysis, and/or a bent spine. The same number of females appeared to be bloated and/or 

partially paralyzed with bent tails. These clinical effects were in contrast to no more than two fish 

from the control groups appearing extremely dark in color or aggressive. Female body weight was 

significantly increased (p<0.05; Dunnett’s) by 10% in the high treatment group compared to the 

negative control. Male body weight was not significantly different (p>0.05) from the negative 

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control. Total length for males and females was not significantly different (p>0.05) from the 

negative control. 

Average spawning frequency and fecundity in controls were at least every 4 days (except for one 

replicate in the solvent control) and 36 eggs/female/reproductive day (range: negative control, 

24.3-50.6; solvent control, 17.2-41.5 egg/female/reproductive day), respectively; fertilization 

success in both controls was 99%. Fertility was significantly reduced (p<0.05; JonckheereTerpstra) by 0.5 and 4% at the low and high treatment levels, respectively, relative to the negative 

control. Fecundity was significantly decreased (p<0.05; Mann Whitney U) by 87% at the high 

treatment level compared to the negative control. 

Male nuptial tubercle scores were significantly reduced (p<0.05; Jonckheere-Terpstra) (p<0.05) at 

the high treatment level (median tubercle score: 20) relative to negative control (median tubercle 

score: 31); no tubercles were noted for females. There was a significant monotonic decrease 

(p<0.05; Jonckheere-Terpstra) of the male gonado-somatic index (GSI) at all treatment levels, 

ranging from 29% (low treatment) to 39% (high treatment) relative to the negative control. 

Female GSI was not significantly different (p>0.05) between the negative control and treatment 

groups. Observations of additional secondary sexual characteristics were reported but results were 

not presented. 

Plasma vitellogenin (VTG) was not significantly different (p>0.05) for males or females. There 

was a significant increase (p<0.05; Jonckheere-Terpstra) of 78% in male 17β-estradiol at the high 

treatment level relative to the negative control. Male plasma testosterone (T) was not significantly 

different (p>0.05). Plasma 17β-estradiol and testosterone were not measured in females. 

Male gonadal development was altered at the high treatment level with a greater proportion of 

testes categorized at the late spermatogenic stage compared to the mid-spermatogenic stage of 

testes in control fish and those exposed to lower levels of trifluralin. For females, there was 

increased incidence of severe increased oocyte atresia in the high treatment group. Finally, there 

was an increase in minimal subacute multifocal ovarian inflammation in females exposed to the 

high treatment level. 

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger assay (MRID 48619707) screening for androgenic activity, trifluralin (97.1%, lot 

no. DKO-B916-33, TSN100656) in corn oil was administered daily via oral gavage to groups of 

55-day old castrated male Sprague Dawley rats (7/group) at dose levels of 0 (vehicle), 50, 150, or 

450 mg/kg/day for 10 consecutive days. An androgenic positive control group consisted of seven 

castrated male rats exposed to testosterone propionate (TP) at 0.4 mg/kg/day via subcutaneous 

(s.c.) injection in conjunction with corn oil via oral gavage. 

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To screen for possible anti-androgenic activity, additional groups of 55-day old castrated male 

Sprague Dawley rats (7/group) were dosed daily via oral gavage with trifluralin in corn oil at 50, 

150, or 450 mg/kg/day for 10 consecutive days in conjunction with a daily s.c. injection of TP at 

0.4 mg/kg/day. The same seven animals used as positive controls in the androgenic portion of the 

study served as negative controls in the anti-androgenic portion of the study, and seven castrated 

male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day for 10 days in conjugation 

with a daily s.c. dose of TP (0.4 mg/kg/day) served as positive controls. 

For both components of the assay, body weights and food consumption were determined daily. 

The animals were terminated approximately 24 hours after the final dose administration. At 

necropsy, the five androgen-dependent tissues (seminal vesicles with coagulating glands, ventral 

prostate, levator ani-bulbocavernosus (LABC), Cowper’s glands, and glans penis), liver, adrenal 

glands, and kidneys were excised, examined macroscopically, and weighed. Serum testosterone 

levels were measured in the anti-androgen assay. 

No treatment-related effects were observed on mortality, clinical signs of toxicity, or gross 

pathology. 

In the androgen agonist assay at 450 mg/kg/day, body weights were decreased (not statistically 

significant [NS]) throughout the study (↓6–8%), compared to vehicle controls, and body weight 

gains were decreased (p≤0.05) at each measured interval (Days 1–4, 1–7, and 1–11; ↓31–88%). 

In the anti-androgen assay at 450 mg/kg/day with TP, compared to negative controls, decreased 

(p≤0.05) body weights were observed on Days 7 (↓8%) and 11 (↓10%), and body weight gains 

were decreased (p≤0.05) at each measured interval (↓46–65%). Overall body weight gains (Days 

1-11) were decreased (p≤0.05) at 150 mg/kg/day with TP (↓22%). All other body weights and 

body weight gains in the trifluralin-treated groups were similar to controls in both the androgen 

agonist and anti-androgen assays. 

In the androgen agonist assay at 450 mg/kg/day, food consumption was decreased (p≤0.05) during 

the first 4-day interval (Days 1–4; ↓35%), compared to vehicle controls. In the anti-androgen 

assay at 450 mg/kg/day with TP, compared to negative controls, decreased food consumption was 

observed during each interval, Days 1–4 (↓31%; NS), 4–7 (↓12%; p≤0.05), and 7–11 (↓14%; 

p≤0.05). At 150 mg/kg/day with TP, food consumption was decreased (p≤0.05) by 8% during 

Days 7–11, compared to negative controls. All other food consumption values in the trifluralintreated groups were similar to controls in both the androgen agonist and anti-androgen assays. 

Liver weights were increased (p≤0.05, except as noted) by trifluralin treatment in both the 

androgenic and anti-androgenic portions of the study, by 16% at 150 mg/kg/day (NS without TP) 

and by 28 and 36% at 450 mg/kg/day (with and without TP, respectively). 

For the androgenic portion of the study, there were no treatment-related effects on the weights of 

any of the accessory sex tissues (seminal vesicles, ventral prostate, LABC, Cowper's glands, or 

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glans penis), adrenals, or kidney at any dose of trifluralin. 

For the anti-androgenic portion of the study at 450 mg/kg/day with TP, decreases (p≤0.05) were 

noted in the weights of the seminal vesicles with coagulating glands (↓21%), ventral prostate 

(↓39%), and LABC (↑21%). Compared to negative controls, serum testosterone was decreased by 

27% (NS) at 150 mg/kg/day with TP and by 36% (p≤0.05) at 450 mg/kg/day with TP. Body 

weights were decreased ≥ 10% at the dose (450 mg/kg/day) where the effects on the androgensensitive tissue weights were observed, indicating overt toxicity at this dose. Furthermore, 

The increase in liver weight, significant decrease in serum testosterone level, and results from 

previously performed studies, suggest that the anti-androgenic effects of trifluralin may be 

secondary to enhanced testosterone metabolism and clearance rather than direct interaction with 

the androgen receptor. 

TP administration produced increases (p≤0.05) of 20–48% in body weight gains at all intervals 

(Days 1–4, 1–7, and 1–11) and an increase in body weight compared to vehicle controls at Day 11 

(↑8%). Food consumption was increased (p≤0.05) by 16 and 18% in TP-treated animals relative 

to vehicle controls during Days 4–7 and 7–11, respectively. Only a minor increase (NS) in food 

consumption was noted in the TP-treated animals during the first interval, Days 1–4. For the TP 

positive control group, there were treatment-related increases (p≤0.05) in the weights of the 

seminal vesicles with coagulating glands (↑1452%), ventral prostate (↑1328%), LABC (↑199%), 

Cowper’s glands (↑580%), and glans penis (↑66%), indicating that the test system was sensitive to 

an androgenic response. All these findings were expected androgenic responses of the positive 

control. 

There was no effect of flutamide on body weights, body weight gains, or food consumption. For 

the flutamide positive control group, there were treatment-related decreases (p≤0.05) in weights 

of the seminal vesicles with coagulating glands (↓70%), ventral prostate (↓66%), LABC (↓44%), 

Cowper’s glands (↓57%), and glans penis (↓14%) compared to weights in the negative control 

group. These data indicate that the test system was sensitive to an anti-androgenic response. 

In the anti-androgen assay, all %CV levels were less than the guideline specified maximums. In 

the androgen agonist assay, only the ventral prostate in the vehicle control group did not meet the 

guideline criteria for maximum %CV (49.0% vs 45% maximum). 

Trifluralin 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. Trifluralin was positive for anti-androgenicity in the 

Hershberger assay based on statistically significant decreases in organ weights seen in three of the 

five androgen sensitive tissues in the androgen antagonist assay. However, the target organ 

weight reductions were observed at a dose (450 mg/kg/day) that exhibited overt toxicity 

[decreased body weights (10%) and body weight gains (46%)], which confounds the 

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interpretation of the study. 

Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48674001), 16 Crl:CD(SD) rats/dose group were treated 

daily via oral gavage with trifluralin (97.1% a.i., Lot # DKO-B916-33, TSN 100656) in corn oil at 

doses of 0, 25, 75 and 250 mg/kg/day from post-natal day (PND) 22 to 42. Animals were 

examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of 

attainment were recorded. After sacrifice on PND 42, blood was collected for clinical chemistry 

analyses, including analysis of total thyroxine (T4) and thyroid stimulating hormone (TSH) using 

immunoassays. Liver, pituitary, adrenals and urogenital organs were collected and weighed, and 

microscopic examinations were performed on the ovaries, uterus, thyroid, and kidneys. 

No treatment-related effects were observed on survival, clinical signs, age of attainment of VO, 

body weight at VO, final body weight, or gross or histological pathology. No treatment-related 

effects were noted on cycle status at necropsy, mean age at first estrus, mean cycle length, percent 

cycling, or percent regularly cycling. 

At 250 mg/kg/day, body weights were decreased (p<0.05) by 7% on PND 28 and 8% on PND 32. 

The mean terminal body weight on PND 42 was decreased (not statistically significant; NS) by 

5%. Body weight gains were decreased (p<0.05) over the intervals from PND 22-25 (30%), PND 

22-28 (21%), and PND 22-32 (17%). After this initial decrease, the body weights of recovered 

slightly such that the decrease in body weight gains over the entire dosing period (PND 22-42) 

was 8% (NS). This effect on body weight was transient and not considered adverse; there was no 

evidence that the delayed growth impacted sexual development. There were no effects on body 

weights or body weight gains at the low or intermediate dose. 

Absolute, adjusted, and relative liver weights were increased (p<0.05) at the intermediate dose by 

11-13% and at the high dose by 17-24%. There was no gross or clinical chemistry evidence to 

support hepatotoxicity. There were no other effects of treatment on organ weights. 

Serum T4 was decreased (p<0.05) in the mid- and high-dose groups by 33% and 45%, 

respectively. TSH was increased (p<0.05) by 73% at the high dose. No effect was noted on 

thyroid weight. In the thyroid at the high dose, increased (p<0.05) incidences of higher follicular 

cells (Grade 3; 8/16 treated vs 1/16 controls) with correspondingly smaller colloid areas (Grade 3; 

8/16 treated vs 1/16 controls) were observed. No differences in these parameters were observed 

in the low- and mid-dose groups. 

The high dose was considered to be an adequate dose based on increased serum creatinine, 

increased liver weights, and changes in serum T4 and TSH levels. 

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Male Pubertal Assay; (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48674002), 16 Sprague Dawley Crl:CD(SD) rats/dose group 

were treated daily via oral gavage with trifluralin (97.1% a.i., Lot # DKO-B916-33, TSN100656) 

in corn oil at doses of 0, 25, 75 or 250 mg/kg/day from post-natal day (PND) 23 to 53. Animals 

were examined for preputial separation (PPS) daily beginning on PND 30, and age and weight at 

day of attainment was recorded. Following sacrifice on PND 53, blood was collected for analysis 

of clinical chemistry parameters, including total serum testosterone, thyroxine (T4), and thyroid 

stimulating hormone (TSH), which were analyzed using immunoassays. Liver, pituitary, 

adrenals, thyroid, and urogenital organ weights were recorded and microscopic examinations were 

performed on the testes, epididymides, thyroid, and kidneys. 

No treatment-related effects were observed on mortality, clinical signs, or gross pathology. Age 

and body weight at PPS in the treated groups were similar to controls. In the thyroid, colloid area 

and follicular cell height scores were similar in the treated groups to the control group. No 

treatment-related findings were noted in the epididymis and testis. 

Body weights and body weight gains at 250 mg/kg/day were not significantly (NS) different from 

controls on PND 53; however, body weights at this dose were decreased on PND 30 and 37 by 11 

and 9%, respectively (p<0.05), and body weight gains were decreased (p<0.05) for the periods 

PND 23-30, 23-37, and 23-42 by 24%, 14%, and 11%, respectively. Although this effect on body 

weight was transient and not considered adverse, the delayed growth could affect sexual 

development. There were no effects on body weights or body weight gains at 25 or 75 

mg/kg/day. 

Absolute, adjusted, and relative liver weights were increased (p<0.05) by 13-16% at 75 

mg/kg/day and by 29-37% at 250 mg/kg/day. At 25 mg/kg/day, relative liver weights were 

increased (p<0.05) by 6%. There was no gross or clinical chemistry evidence to support 

hepatotoxicity, and histology was not performed. The slight effect at 75 mg/kg/day and the 

substantial effect at 250 mg/kg/day are considered equivocal findings, because this effect could be 

an adaptive response. 

At 250 mg/kg/day, decreased (p<0.05) adjusted organ weights were noted in the following 

androgen-sensitive tissues: dorsolateral prostate (12%), levator ani-bulbocavernosus muscle 

complex (LABC, 13%), left epididymis (14%), and right epididymis (11%). Similar effects were 

noted on the unadjusted weights of these organs. Additionally, a minor decrease (8%; p<0.05) 

was noted in the right epididymis at 75 mg/kg/day. These effects were slight and may have been 

related to the delayed growth observed at 250 mg/kg/day. 

Serum T4 levels were decreased (p<0.05) dose-dependently by 25, 42, and 58% at 25, 75 and 250 

mg/kg/day, respectively. At 250 mg/kg/day there also appeared to be an effect on serum TSH 

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(increased by 74%) and testosterone (decreased by 30%); however, neither difference was 

statistically significant, nor were there any effects observed on thyroid weight or histopathology. 

At 250 mg/kg/day, creatinine was increased (p<0.05) by 50%, which was consistent with the 

probe study. Alkaline phosphatase (ALP) was slightly decreased (p<0.05) by 17%; however, the 

toxicological significance of a slight decrease is unclear. Calcium and sodium were also 

increased (p<0.05) by 4% and 1%, respectively, at 250 mg/kg/day, but these minor effects were 

not considered adverse. 

Increased hyaline droplet formation within the kidney proximal convoluted tubule (multifocal) 

was observed in the 25 (13/16, very slight severity), 75 (13/15, very slight), and 250 (15/15, very 

slight to slight) mg/kg/day groups compared to the control (1/16, very slight). However, this 

finding was not associated with degenerative or cytotoxic changes in the kidneys. This was the 

only treatment-related histological lesion, and there was no other evidence of kidney toxicity. 

There were no treatment-related histopathological changes in the thyroid, testes, or epididymides. 

The high dose was considered to be an adequate dose based on increased serum creatinine, 

increased liver weights, and decreased serum T4 levels. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48619708), H295R cells cultured in vitro in 24-well plates were 

incubated with trifluralin (97.1% purity; LOT # DKO-B916-33, TSN100656) at log 

concentrations from 0.0001 to 100 μM for 48 hours in triplicate in three independent experiments. 

Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.1%. 

Testosterone and estradiol levels were measured using LC-APPI-MS/MS. A Quality Control 

(QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate 

that the assay responded properly to positive control agents at two concentration levels. The 

positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of 

estradiol and testosterone production. 

Guideline acceptability recommendations and requirements were generally met, including lack of 

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

%CV), and appropriate induction and inhibition with positive controls. The exceptions were the 

minimum basal hormone production level of 500 pg/mL testosterone was not met in the solvent 

control (SC) in the testosterone assay for 2 runs (491 and 454 pg/mL), and cells with a passage 

number of 11.5 instead of 10.5 were used in a single run. 

No effects of trifluralin were observed on testosterone levels. Decreased (p≤0.05) estradiol levels 

were observed at 0.0001 μM trifluralin in Runs 1 and 2 (0.8-fold the SC) and at 0.001 μM in Run 

2 (0.9-fold). These changes were minimal, unrelated to dose, and not reproducible across all three 

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runs; therefore, the apparent effect on decreased estradiol level was not considered treatment 

related. However, statistically significant increases (p≤0.05) of estradiol levels were noted at 10 

and 100 μM in all runs (1.2–1.4-fold). Although the magnitude of increase was slight, these 

effects of increased estradiol levels were reproducible and were considered treatment related. 

Based on hormone responses in each of the independent runs, trifluralin treatment resulted in 

statistically significant and reproducible increases in estradiol production, but trifluralin treatment 

did not result in statistically significant and reproducible alterations in testosterone production. 

Uterotrophic Assay; (OCSPP 890.1600) 

In an uterotrophic assay (MRID 48619709) conducted to screen for potential estrogenic activity, 

trifluralin (97.1% a.i., Lot # DKO-B916-33, TSN100656) in corn oil was administered daily for 3 

consecutive days via oral gavage to groups of six immature female Sprague Dawley rats at dose 

levels of 0 (vehicle), 25, 75, or 225 mg/kg/day on post-natal days (PND) 19-21. A positive 

control group was treated with a daily dose of 17α-ethynyl estradiol (EE) at 10 μg/kg/day by oral 

gavage. Body weights were determined daily. All animals were examined for precocious vaginal 

opening, and then terminated and necropsied on PND 22 approximately 24 hours after the final 

dose to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. No clinical signs of toxicity were observed, and 

no precocious vaginal opening was noted in the trifluralin-treated females. At 225 mg/kg/day, 

overall body weight gains were decreased (p<0.05) by 34%. Body weights in the low- and middose groups, and the positive control group were comparable to the vehicle controls throughout 

the duration of the assay. Uterine weights in the trifluralin treated groups were comparable to the 

vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) group 

were increased (p<0.05) by 707% and 396%, respectively, as expected.!!

The doses tested were determined to be adequate based on a 34% decrease in overall body weight 

gain in the high-dose group. 

No statistically significant changes were seen in the uterine weight in this assay. Trifluralin 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)

A subchronic oral toxicity study (MRID 40138301) was conducted in Fischer 344 rats with 

trifluralin (purity and Lot/Batch not reported) concentrations of 0.005, 0.02, 0.08, 0.32 or 

0.64% in the diet. The NOAEL was 0.005% (50 ppm or 2.5 mg/kg/day). The LOAEL was 

0.02% (10 mg/kg/day) based on increased hyaline droplet formation in cortical cells, increased 

total urinary protein excretion, and changes in urine color and clarity. Many of the effects 

were reversible within a six-week recovery period. 

A second subchronic oral toxicity study (MRID 00151906) in Wistar rats and trifluralin (purity 

and Lot/Batch not reported) doses of 0, 800, 2000, or 5000 ppm indicated that the NOAEL was 

less than 800 ppm (40 mg/kg/day) due to reductions in relative liver and pituitary gland weights 

at all doses tested. 

Prenatal Development in Rodents (Rat)

In a prenatal development study in the rat (MRID 00152419), Charles River rats were 

administered gavage doses of 0, 100, 225, 475 or 1000 mg/kg/day of trifluralin (purity and 

Lot/Batch not reported) on gestation days 6-15. The maternal toxicity LOAEL was 475 

mg/kg/day due to reduced weight gain and food consumption; the maternal toxicity NOAEL was 

225 mg/kg/day. The developmental toxicity LOAEL was 1000 mg/kg/day due to reduced mean 

fetal body weight; the developmental toxicity NOAEL was 475 mg/kg. No teratogenic effects 

occurred in the rat. 

Prenatal Development in Non-Rodents (Rabbit)

In a prenatal development study in the rabbit (MRID 00152421), Dutch Belted rabbits were 

given oral doses of 0, 100, 225 or 500 mg/kg/day of trifluralin (purity and Lot/Batch not 

reported) on gestation days 6-28. The maternal toxicity LOAEL was 225 mg/kg/day due to 

anorexia, cachexia and resulting abortion; the maternal toxicity NOAEL was 100 mg/kg/day. 

The developmental toxicity LOAEL was 500 mg/kg/day based on depressed fetal weight and an 

increased number of fetal runts; the developmental toxicity NOAEL was 225 mg/kg/day. No 

teratogenic effects occurred in the rabbit. 

Two-Generation Reproduction in Rodents (Rat)

In a multi-generation reproduction toxicity study (MRIDs 00151901, 00151902, 00151903, and

00159619), trifluralin (>99% purity; Lot/Batch #AT 210) was administered continuously in the 

diet to outbred Wistar KFM-Han SPF quality rats at nominal doses of 0, 200, 650, or 2000 ppm 

(equivalent to approximately 0, 10, 32.5 and 100 mg/kg/day; calculated by reviewers using 1 ppm 

= 0.05 mg/kg). The P animals (30/sex/dose) were given test article diet formulations for 80 days 

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prior to mating to produce the F1a litters. Ten days after weaning of the F1a litters, a second 

mating of the P generation (with different pairs) was conducted to produce the F1b litters. After 

weaning, 26 rats/sex/dose from the F1b litters were selected to be F1 parents, and were given the 

same diet concentration as their dams for at least 100 days prior to mating. F1 parents were bred 

(siblings not paired) to produce F2a and F2b litters by using the same procedures as described for 

their parents. 

Parental mortalities included one mid-dose P generation male and one F1 generation female each 

from the control, mid-, and high-dose groups. The death for the mid-dose female was attributed 

to acute renal failure; no cause of death was stated for the other mortalities. Yellow discoloration 

of the urine was reportedly dose-dependently increased; however, no data were provided. Yellow 

discoloration of the adipose tissue, characteristic of dinitroaniline treatment, also was noted in the 

mid-dose females and high-dose males and females of both generations. Body weight was 

decreased (p≤0.05) in the F1 females during pre-mating (6-15%), gestation (7-9%), and lactation 

(4-9%) for both litters. Terminal body weight in F1 males and females was decreased (p≤0.05) 

by 6%. Decreases in food consumption were seen occasionally in parental animals during 

acclimation and in females during gestation, but were more prevalent in dams during lactation. 

Relative (to body) liver weights were increased (p≤0.01) in the mid-dose P males, and F1 males 

and females (6 to 10%), and high-dose P and F1 males and females (16-27%). In the parental 

males, relative kidney weights were increased (p≤0.01) 6 to 14% at all doses in the P generation, 

and increased 10% at the mid- and high-dose in the F1 generation. Relative testes weights were 

increased 8-13% (p≤0.05) at the mid-dose (F1 generation) and at the high-dose in both 

generations. 

The only finding at 200 ppm was an increase (p≤0.01) of 6% in relative kidney weight in the P 

generation males. The LOAEL for parental toxicity is 650 ppm (nominally equivalent to 32.5 

mg/kg/day) based on increased lesions of the renal proximal tubules in the F1 females and 

increased relative (to body) weight of the liver and kidney (males). The NOAEL is 200 ppm 

(nominally equivalent to 10 mg/kg/day). 

There was no adverse effect of treatment on pup mortality. Pup weights were decreased (p≤0.05) 

5 to 12% in the mid- and high-dose F1 litters; (ii) decreased (p≤0.05) 3 to 8% in the high-dose F2a 

litter; and decreased (p≤0.05) 2 to 7% in the mid- and high-dose F2b litters. Litter size was 

decreased (p≤0.05) by 13-16% in the high-dose F2a litter on PND 0, and in the high- dose F2b 

litter on PND 0 and 21. Relative liver weights were increased (p≤0.05) in the mid-dose F2b 

females, and in the high-dose F2a animals and F2b females (6-13%). Relative kidney weights 

also were increased (p≤0.01) 5% in the high-dose F2b females, and relative testes weights were 

increased 8% in males in both F1 litters. There were no effects of treatment in the low-dose pups. 

The LOAEL for offspring toxicity is 650 ppm (nominally equivalent to 32.5 mg/kg/day) based on 

decreased pup weights in both generations and increased relative to body liver weight in the F2b 

females. The NOAEL is 200 ppm (equivalent to 10 mg/kg/day). 

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There were no effects of treatment on precoital interval, gestation length, behavior of dams 

during parturition and lactation, pup mortality, or the proportion of parents that mated, became 

pregnant, delivered, or reared litters to weaning. A LOAEL for reproductive toxicity was not 

observed; the NOAEL for reproductive toxicity is 2000 ppm (nominally equivalent to 100 

mg/kg/day). 

Two-Generation Reproduction in Rodents (Rat)

In a multi-generation reproduction toxicity study (MRIDs 00162543 and 44135107), trifluralin 

(96.4% purity; Lot/Batch #554AP2) was administered continuously in the diet to outbred 

Crl:CD (SD) rats at nominal doses of 0, 200, 630, or 2000 ppm (0, 15, 47 and 148 mg/kg/day). 

The P animals (25/sex/dose) were given test article diet formulations for 70 days prior to mating 

to produce the F1a litters. Females kept progeny (Fla) for 21 days postpartum; after weaning, 

25/sex/group of F1a animals were selected to become F1 parents. One weanling/sex/litter or the 

remaining pups was given a gross necropsy. The P parental animals were again mated at 25 

weeks of age and allowed to deliver and rear the F1b generation for 21 days postpartum. All 

Flb pups were sacrificed and given a gross necropsy at weaning. At 36 weeks of age, all P 

parents were killed and given a gross necropsy, and reproductive tissues and kidneys were 

collected for histopathological evaluation. 

All animals survived the study, except for the death of a single P male in the low-dose group due 

to urolithiasis on Study Day 126. No treatment-related clinical effects were present, except for a 

reported dose-dependent, yellow discoloration of the urine. Decreased body weights and body 

weight gains were seen in the high-dose P males; F1 males also had decreased body weights and 

body weight gains during the growth period in the mid- and high-dose groups, but only highdose male were affected during the reproductive period. Decreased body weights and body 

weight gains were also seen in the high-dose P and F1 females. Statistical significance was not 

reported, and body weight/body weight gain data were not available for review. A significant 

(p<0.05) decrease in food consumption was noted for high-dose P males and females. 

No treatment-related effects on fertility, mating performance, number of pregnant females, 

gestation length/survival, litter size, pup survival, or sex ratio were observed. An apparent, dosedependent decrease in pup body size and weight was noted. A LOAEL for reproductive toxicity 

was not observed; the NOAEL for reproductive toxicity is 2000 ppm (equivalent to 148 

mg/kg/day). The LOAEL for offspring toxicity is 630 ppm (equivalent to 47 mg/kg/day) based 

on small pup size in 3 litters. The NOAEL is 200 ppm (equivalent to 15 mg/kg/day). 

Histopathological examinations were conducted on selected tissues (i.e., vagina, uterus, ovaries, 

mammary glands, testes, epididymis, seminal vesicles, prostate, kidneys, and all gross lesions) 

from control and high-dose animals. No treatment-related histopathological effects were 

observed. The LOAEL for parental toxicity is 630 ppm (equivalent to 47 mg/kg/day) based on 

decreased body weight and food consumption. The NOAEL is 200 ppm (equivalent to 15 

mg/kg/day). 

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Two-Generation Reproduction in Rodents (Rat)

In a multi-generation reproduction toxicity study (MRID 40405007), trifluralin (97.3% purity; 

Lot/Batch #5320) was administered continuously in the diet to CD(CRL) rats (25/sex/dose) at 

nominal doses of 0, 50, 450, or 4000 ppm (equivalent to 3.9/4.7, 35/42, 295/337 mg/kg/day, 

M/F). The P animals were given test article diet formulations for 10 weeks prior to mating to 

produce the F1a litters. In a sub-study to evaluate dominant lethal effects, a second mating was 

conducted with treated males and untreated females of the P generation to produce the F1b 

litters. After weaning, 25 rats/sex/dose from the F1a litters were randomly-selected 

(one/sex/litter, when possible) to become the parents of the F2 generation, and were given the 

same concentration test formulation as their dams for at least 10 weeks prior to mating. There 

were no effects of treatment on parental survival, clinical signs, or pup sex ratio. 

Body weights were decreased (p≤0.05) during pre-mating at the high-dose in the P generation 

males and females (↓6 to 15%) and F1 males and females (↓15 to 25%), and during gestation and 

lactation in the P (↓7 to 19%) and F1 (↓18 to 24%) dams. Body weight gain was consistently 

decreased (p≤0.05) during pre-mating in the P males, during Week 1 in the P females and F1 

males, and during Weeks 1, 5, and 10 in the F1 females. Food consumption was decreased 

(p≤0.05) in the P generation males and females (↓8 to 20%) and F1 males and females during 

pre-mating (↓13 to 30%) and during gestation and lactation in the P (↓13 to 28%) and F1 (↓21 to 

32%) dams. Food efficiency during pre-mating was decreased (p≤0.05) consistently in the P 

males, and occasionally in the P and F1 females. Absolute (↓20%) and relative to body (↓10 to 

11%) ovary weights were decreased (p≤0.05) in both generations. Yellow discolored adipose 

tissue, characteristic of dinitroaniline treatment, was observed in the P and F1 males and females 

(23-24/25 treated versus 0/25 control). Findings of denser stroma of the endometrium were 

observed in the P and F1 females (14-15/25 treated versus 0/25 control), indicating uterine 

atrophy. 

Only minor and/or transient decreases in body weight and food consumption in mid-dose F1 

males were observed. Yellow-colored adipose tissue was observed in mid-dose P females (2/25 

treated versus 0/25 control), and is characteristic of dinitroaniline treatment. There were no 

treatment-related effects at the lowest dose. 

The LOAEL for parental toxicity is 4000 ppm (equivalent to 295/337 mg/kg/day, M/F) based on 

decreased body weight, body weight gain, food consumption, and food efficiency in males and 

females of both generations; and uterine atrophy and decreased ovary weight in both generations. 

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Overt toxicity was apparent at this dose as demonstrated by the body weight decreases of ≥10%. 

The NOAEL is 450 ppm (equivalent to 35/42 mg/kg/day, M/F). 

In the high-dose group, fetal viability (86% treated versus 94% control), neonatal viability index 

(85% treated versus 97% control) and litter viability (80% treated versus 100% control) were 

decreased (p≤0.05) in the P generation (F1a litters). Lactation index also was decreased (NS) in 

these animals. The number of implantation sites were decreased (p≤0.05) in the F1a (↓9%) and 

F2 (↓18%) litters, resulting in reductions (p≤0.01) in the number of newborn pups (↓17 to 22%) 

and litter size on post-natal days (PND) 1 and 4 (↓17 to 25%). Litter size remained decreased 

(NS) throughout the remainder of lactation in both generations. Pre-implantation loss was 

increased (15.5% treated versus 9.8% control; p≤0.05) in the F1b litters (mating of treated males 

with untreated females), yielding a decreased (↓11%; p≤0.05) number of live fetuses. The 

LOAEL for reproductive toxicity is 4000 ppm (equivalent to 295/337 mg/kg/day, M/F) based on 

decreased ovary weights in both generations; decreased lactation index in the F1a pups; and 

decreased number of implantation sites, newborn pups, litter size, and pup weights in both 

generations. The NOAEL is 450 ppm (equivalent to 35/42 mg/kg/day, M/F). 

Pup weights in the high-dose group were decreased (p≤0.05) throughout lactation in the F1a 

litters (↓15%) and on PND 14 and 21 in the F1a litters (↓74 to 81%). There were no offspring 

effects observed in the low- and mid-dose animals. The LOAEL for offspring toxicity is 4000 

ppm (295/337 mg/kg/day M/F), based on decreased fetal, neonatal, and litter viability; decreased 

pup weights in F1a litters; and decreased number of newborn pups, litter size, and pup weights in 

both generations; and decreased number of implantation sites. The NOAEL for offspring toxicity 

is 450 ppm (35/42 mg/kg/day, M/F). 

Chronic Oral Toxicity/Carcinogenicity in Rodents (Rat)

A chronic oral carcinogenicity study (MRID 00044337) was conducted with trifluralin (purity 

and Lot/Batch not reported) in Fischer 344 rats with dietary doses of 0, 813, 3250 or 6500 ppm 

for two years. The high dose (325 mg/kg/day) resulted in significant increases of combined 

malignant and benign urinary bladder tumors in females. An increase in the incidence of 

carcinomas of the renal pelvis was seen in all dose groups of males. In addition, an increase in 

the incidence of thyroid gland follicular cell tumors (adenomas plus carcinomas combined) in 

males was observed at 6500 ppm. 

Additional rat bioassays included: a two-year study in SD rats at trifluralin doses of 0, 200, 1000 

or 2000 ppm; a 78-week study in Osborne-Mendel rats at trifluralin doses of 0, 3250 or 6500 ppm 

(conducted by the National Cancer Institute); and a two-year study in Wistar rats at trifluralin 

doses of 0, 200, 800 or 3200 ppm (MRID 00162456, 00162457, 00162458). In the last study, 

the systemic LOAEL is 800 ppm (40 mg/kg/day) based on body weight changes; the NOAEL is 

200 ppm. 

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Mechanism of Action Study (Rat)

A significant increase in thyroid follicular cell tumors was observed in male rats after oral 

administration of trifluralin in a 2-year chronic toxicity/carcinogenicity study with Fischer 344 

rats (MRID 00044337). 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. 

Carcinogenicity in Rodents (Mouse)

A 78-week carcinogenicity study (Jaeger, 1986) was conducted by the National Cancer 

Institute with B6C3F1 mice at trifluralin (purity and Lot/Batch not reported) doses of 0, 2375 or 

5000 ppm. The incidence of liver carcinomas, alveolar-bronchiolar adenomas, and squamouscell carcinomas of the forestomach in the female mice was suspected to be due to contamination 

of the test compound with N-nitroso-di-n-propylamine. 

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A two-year study conducted in B6C3F1 mice (MRID 00044338) at trifluralin (purity and 

Lot/Batch not reported) doses of 0, 563, 2250 or 4500 ppm, found no tumors due to the test 

compound. 

A two-year study in NMRI mice (MRIDs 00158935 and 40392313) used purified trifluralin 

(purity and Lot/Batch not reported) at doses of 0, 50, 200 or 800 ppm. Increased liver weights 

were noted in males at the mid- and high dose (30 and 120 mg/kg/day) and in females at the high 

dose. The systemic LOAEL was 200 ppm (30 mg/kg/day) in male mice and 800 ppm (120 

mg/kg/day) in females. The systemic NOAEL was 50 ppm (7.5 mg/kg/day) in male mice and 

200 ppm (30 mg/kg/day) in females. No treatment-related tumors were observed. 

Chronic Oral Toxicity in Non-Rodents (Dog). 

In a chronic oral carcinogenicity study in the beagle dog (MRID 42447001), animals were fed 

trifluralin (purity and Lot/Batch not reported) by capsule at doses of 0, 0.75, 2.4 or 40 mg/kg/day 

for one year. The NOAEL was 2.4 mg/kg/day and the LOAEL was 40 mg/kg/day based upon 

reduced body weight; decreased red cells and hemoglobin concentrations; increased 

thrombocyte, methemoglobin, cholesterol and triglyceride concentrations; and increased liver 

weight. 

In a second chronic oral toxicity study in the beagle dog (MRIDs 00151908 and 00159618), 

trifluralin (purity and Lot/Batch not reported) doses of 0, 30, 150 or 750 ppm were fed in the diet 

for one year. The NOAEL was 30 ppm (0.75 mg/kg/day). The LOAEL was 150 ppm (3.75 

mg/kg/day) based upon increased liver weight and methemoglobin concentration. Observations at 

the high dose included: decreased weight gain, decreased RBC concentration, and increased 

concentrations of methemoglobin, serum lipids, triglycerides, and cholesterol. 

Fish Early-Life Stage Toxicity (OPPTS.850.1400)

Rainbow trout [Salmo gairdneri (sic); Oncorhynchus mykiss] were exposed to trifluralin 

(99.86% purity; Batch/Lot not reported) at mean measured concentrations of negative control, 

solvent control, 0.59, 1.14, 2.18, 4.32, and 8.81 μg a.i/Lin a 48-day early life stage test (MRID 

41386202). The time to hatch, hatchability of the eggs, and larval survival were not 

significantly different (p>0.05) from the negative control and there were no treatment-related 

behavioral effects observed in larvae in all concentrations other than the highest tested. The 

NOAEC (μg/L), and the LOAEC, based on significant (p<0.05) reductions in total length and 

body weight, were 2.18 and 4.32 μg a.i/L, respectively. 

Fish Life-Cycle Toxicity (OPPTS 850.1500)

In a fish full life cycle study conducted with fathead minnows (Pimephales promelas), fish were 

exposed to mean measured concentrations of trifluralin (97% purity; Batch/Lot not reported) of 

negative control, 1.5, 1.9, 5.1, 8.2, and 16.5 μg a.i/L (MRID 05008271). After 30 days, survival 

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in the highest treatment concentration was 67% that dropped to 0% after 60 days. While mean 

survival ranged from 89-91% in 30-day and 60-day time points, after 61 weeks of exposure, 

mean survival was 0% in the 8.2 μg a.i/L treatment concentration. Similarly, the middle 

treatment concentration of 5.1 μg a.i/L had mean survival of 30% by test termination at 61 

weeks. Total length and weight measurements were therefore discarded for the 60-day and 61 

week time points for the highest treatment concentration and for the 61-week time point for the 

second highest treatment concentration. The mean number of spawning individuals increased 

from control up to the 1.9 μg a.i/L but then dipped at the 5.1 μg a.i/L. There were no spawning 

females at the two highest treatment concentrations. The mean number of eggs spawned per 

female in the lowest treatment group was 20 as compared to 142 for the control and 84 and 329 

for the 1.9 and 5.1 μg a.i/L treatment concentrations, respectively. Survival in fish in the 1.9 and 

5,1 μg a.i/L treatment concentrations averaged 56% and 72%, respectively for 30 days and 56 

and 61%, respectively for 60 days. The NOAEC and LOAEC for this study based on effects to 

parental survival was 1.9 and 5.1 μg a.i/L, respectively. 

Sheepshead minnows (Cyprinodon variegatus) were exposed to trifluralin (99% purity; 

Batch/Lot not reported) at mean measured concentrations of control, solvent control, 1.3, 4.8, 

9.6, 17.7, and 34.1 μg a.i/L in a 166-day fish full life stage test (MRID 42449901). There 

was 100% mortality observed in the highest treatment concentration (34.1 μg a.i/L), and 

therefore this treatment concentration was removed from further statistical analysis. Parental 

fish at the two highest treatment concentration exhibited loss of equilibrium after 14 days 

post hatch and many were found laying on the bottom of the aquaria. Parental fish at the 4.8 

and 9.6 μg a.i/L mean measured concentrations has darkened coloration in the caudal 

peduncle area. Juveniles in the F1 generation at 12 days post hatch in the second highest 

treatment concentration exhibited a loss of equilibrium and appeared sluggish. This study 

was determined to be scientifically sound but there were no raw data provided for the 

reviewer to conduct an independent statistical analysis and therefore is classified as 

supplemental. The only parameter that could be statistically verified by the reviewer was the 

number of eggs spawned per day per female which showed significant reductions (p<0.05) at 

all treatment concentrations and therefore a NOAEC could not be determined and the 

LOAEC was 1.3 μg a.i/L. 

Avian Reproduction Test (Quail) (OCSPP 850.2300)

In a one-generation avian reproduction test with bobwhite quail (MRID 00131134), 

trifluralin (99.6% purity; Batch/Lot not reported) was administered at nominal dietary 

concentrations of 5 and 50 ppm (mean measured concentrations). NOAEC and LOAEC 

values were not determined; it was reported that trifluralin exposures <50 ppm would not 

significantly affect reproductive success. While the study is classified as acceptable, the 

percentage of cracked eggs in the negative control was 9.7% as a percentage of eggs laid. 

EPA guidance for the bobwhite quail states that typically only 0.6 – 2.0% of eggs laid are 

cracked for this species. 

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In another one-generation avian reproduction test with bobwhite quail (MRID 40334706), 

trifluralin (96% purity; Batch/Lot not reported) was administered at nominal concentrations in 

the diet of control, 250, 500 and 1000 ppm (mean measured concentrations, (mg/kg/day), were 

not reported). No treatment-related effects were observed (DP barcode 417055). 

Avian Reproduction Test (Duck) (OCSPP 850.2300)

In a one-generation avian reproduction test with mallard ducks (MRID 00131132), trifluralin 

(99.6% purity; Batch/Lot not reported) was administered at nominal dietary concentrations and 

5 and 50 ppm (mean measured concentrations). NOAEC and LOAEC values were not 

determined; however, an increase in cracked eggs (2.4%) was observed at the highest trifluralin 

treatment concentration (50 ppm) and was not determined to be significant (p>0.05). A 

reevaluation of this study in 2010 stated that subsequent avian repro studies (MRIDs 40334704 

and 40334706) did not show the cracked eggs effect in concentrations up to 1000 mg a.i./kgdiet and that the increase in cracked eggs for MRID 00131132 for this study was not 

biologically significant and the NOAEC should be established at 50 mg a.i./kg-diet. 

In another one-generation avian reproduction test with mallard ducks (MRID 40334704), 

trifluralin (96% purity; Batch/Lot not reported) was administered at nominal concentrations in the 

diet of control, 250, 500 and 1000 ppm (mean measured concentrations). The NOAEC was 

determined to be 500 ppm, and a LOAEC was 1000 ppm based on reductions in eggshell 

thickness, 14-day survivor body weight, at the highest treatment concentration. The percent 

reductions from the negative control in eggshell thickness and 14-day survivor body weight at the 

highest treatment concentration were 4 and 7%, respectively. There were no apparent overt signs 

of toxicity, treatment related mortalities, or reductions in food consumption or female body 

weight. 

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APPENDIX 3: References Not Utilized in the Trifluralin WoE Analysis 

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

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

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

“other scientifically relevant information (OSRI) was provided in the Report of the Endocrine 

Disruptor Review Team (USEPA, 2010). 

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies 

previously submitted to the agency for registration/reregistration, published literature articles 

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

scientific literature for any additional relevant information for their weight of evidence (WoE) 

evaluations. These articles were evaluated in accordance with the agencies Evaluation Guidelines 

for Ecological Toxicity Data in Open Literature, May 2011 

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

eregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance for 

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

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

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

for Trifluralin but were not utilized due to one or more of the following reasons: 1) the article was 

not available in English; 2) the compound of interest was not used in the study; 3) the test material 

was not adequately described; 4) a formulated end-use product or mixture of chemicals was 

utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the 

experimental conditions were not adequately described; 7) only an abstract of the study was 

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

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

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

dealt only with non-EDSP assay development; 12) no specific endocrine-related endpoints were 

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

Cecconi, S., Rossi, G., Carta, G., Di, L.G., Cellini, V., Canipari, R., and Buccione, R. (2013) 

Effects of trifluralin on the mouse ovary. Environ Toxicol. 28(4):201-206. 

Orton, F., Lutz, I., Kloas, W., Routledge, E. (2009) Endocrine Disrupting Effects of Herbicides 

and Pentachlorophenol: In Vitro and in Vivo Evidence. Environ. Sci. Technol., 43:2144–2150. 

Nishihara, T., et al. (2000) Estrogenic Activities of 517 Chemicals by Yeast Two-Hybrid Assay. 

Journal of Health Science, 46(4): 282–298. 

Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N., and Serrano, P.O. (1995) 

The ESCREEN assay as a tool to identify estrogens: An update on estrogenic environmental 

pollutants. Environmental Health Perspectives 103 (Suppl. 7):113-122. 

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Weir, S.M., Yu, S., and Salice, C.J. (2012) Acute toxicity of herbicide formulations and chronic 

toxicity of technical-grade trifluralin to larval green frogs (Lithobates clamitans). Environ Toxicol 

Chem. 31(9):2029-2034. 

Worth, H.M., et al. (1966) Effect of Trifluralin Treatment on Reproduction in Rats and Dogs. 

Unpublished study prepared by Lilly Research Laboratories. (MRID 00083646) 

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