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 

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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: LINURON 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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ii 

TABLE OF CONTENTS 

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

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

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

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

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

B. Other Scientifically Relevant Information (OSRI)......................................5 

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

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

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

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

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

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................25 

IV. Committee’s Assessment of Weight of Evidence..........................................27 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................27 

1. Tier 1 in vivo Assays.............................................................28 

2. OSRI...............................................................................28 

B. Estrogen Pathway...........................................................................30 

C. Androgen Pathway.........................................................................30 

D. Thyroid Pathway...........................................................................30 

E. Conclusions.................................................................................31 

V. EDSP Tier 2 Testing Recommendations....................................................31 

VI. References.......................................................................................33 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Linuron...................................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Linuron........................................11 

 Table 3. Androgenic/Anti-Androgenic Pathway for Linuron......................................18 

 Table 4. Thyroid Pathway for Linuron.............................................................26 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................37 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................43 

APPENDIX 3. References Not Utilized in the Linuron WoE Analysis................................89 

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iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

F2 Second filial generation 

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Abbreviation Terminology 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

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Abbreviation Terminology 

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

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

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Abbreviation Terminology 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Executive Summary 

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

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

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

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

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality were considered 

in this weight of evidence (WoE) assessment. 

In determining whether linuron 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 the dose(s) at which endocrine related responses occurred happened 

concurrently with general systemic toxicity. 

On October 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 analysis of the potential interaction of 

linuron 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) generally 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, although positive results were reported in the literature for ER-related 

responses in vitro, the responses are not supported by the Tier 1 assays (negative ER binding and 

uterotrophic assays) or other literature in vitro assays evaluating ER transactivation assays in 

CHO cells, yeast ER-reporter assays or ER-responsive proliferation assay. Linuron showed an 

equivocal response for aromatase inhibition in vitro. In the female pubertal assay, decreased 

ovarian weight was observed, however there was no corroborating microscopic findings. In 

addition, pituitary weights were also decreased in the study. There were no estrogen-related 

effects noted in the mammalian Part 158 studies. In the Tier 1 FSTRA (with fathead minnow), 

in the absence of overt toxicity, there were alterations in fecundity and fertility at 0.92 mg/L, 

however, these effects are limited in their diagnostic specificity for estrogen-related alterations in 

of themselves; no effects were observed in a Part 158 fathead minnow life-cycle study up to 

0.097 mg/L. In another FSTRA considered as OSRI, plasma vitellogenin concentrations were 

significantly reduced in females; however, no other effects were observed in this study. 

Reductions in egg production (fecundity) were noted in two Part 158 avian reproduction studies, 

but again, these findings are limited in their ability to identify potential estrogen-related 

interactions; decreases in eggshell thickness was observed in the mallard reproduction study, and 

decreased female body weight and food consumption was also observed at this concentration. 

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Overall, while some in vitro assays did indicate potential interaction with the estrogen pathway, 

these findings were not supported by the results of in vivo mammalian and wildlife studies. 

For the androgen pathway, there is consistent complementarity and redundancy of evidence 

across the Tier 1 and OSRI to suggest that linuron acts as an anti-androgen both in vitro and in 

vivo. Linuron in vitro exhibited competitive binding to the AR and activated AR transcriptional 

activity in multiple species in vitro (human, fathead minnow, and rainbow trout). Linuron also 

inhibited ex vivo testosterone production in fetal rat testes. The findings of multiple Hershberger 

assays, male pubertal assays, and reproductive developmental toxicity studies in rats, as well as 

assays measuring the inhibition of androgen-induced spiggin production by female stickleback 

fish kidney cells, support that linuron exhibits anti-androgenic effects in vivo. In mammalian Part 

158 studies, linuron caused changes in testes and epididymal weights and histopathology in the 

testes and epididymides. 

There is convincing evidence of potential interaction of linuron with the thyroid pathway. 

Although no thyroid histopathology effects were observed in any of the Tier 1 or OSRI studies, 

decreases in serum T3 and/or T4 levels were observed across multiple studies including the 

EDSP Tier 1 female pubertal assay, a 12-week female rat thyroid toxicity study, and two 15-day 

adult male rat screening assays considered as OSRI. Additionally, linuron showed weak agonist 

activity to the T3 receptor in two in vitro transcriptional activation assays using the HepG2 cells. 

With regards to the AMA, the assay requirement was satisfied by the mammalian OSRI. 

In summary, for the estrogen pathway, the available data suggests that linuron does not interact 

with the estrogen pathway. For the androgen pathway, linuron appears to act as an anti-androgen 

both in vitro and in vivo. For the thyroid pathway, there was evidence of potential interaction in 

mammals characterized by changes in thyroid hormone levels in the absence of changes in 

thyroid weight or histopathology in multiple studies. 

The current point of departure (POD) of 0.77 mg/kg/day for human health risk assessment is 

based on hematological effects (increased met- and sulf-hemoglobin levels) seen at 3.5 

mg/kg/day (LOAEL) in a chronic toxicity study in dogs. This POD is used for establishing the 

chronic Reference Dose (RfD) used for chronic dietary risk assessment and for calculating 

Margins of Exposure (MOEs) for non-dietary risk assessments. This POD is approximately 65-

fold lower than the lowest dose (50 mg/kg/day) that caused androgen-related effects in studies 

reported as OSRI. Therefore, EDSP Tier 2 studies to investigate androgen-related effects are not 

required. 

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

adult animals, there usually would be a concern that the POD may not be protective of potential 

thyroid toxicity in the young. 

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Therefore, a special thyroid assay in pregnant animals, postnatal, and adult animals is 

recommended. This special study should use a mechanistic approach to generate specific data on 

the thyroid (i.e., the primary target of linuron) to protect the developing nervous system from 

thyroid hormone disrupting chemicals. The specific purpose of this study is to establish a POD 

(i.e., NOAELs and LOAELs or benchmark doses) that may be used for human health risk 

assessment. The POD from this special study would address the concern for the potential ability 

of linuron to disrupt thyroid function in pregnant females and in the fetus or newborn. 

There is evidence for potential interaction with the androgen signaling pathway in fish, based on 

the available fish, mammalian, and in vitro data. While there was limited evidence using fathead 

minnow (effects on fecundity and fertility at 0.92 mg/L, no other androgen-related effects 

observed), anti-androgenic effects were observed in three-spined stickleback fish (≥0.100 mg/L). 

There were no growth, survival or reproductive effects observed in a Part 158 fish life-cycle 

study with fathead minnow up to the highest concentration tested (0.097 mg a.i./L). Given the 

potential for androgen-related effects in fish, the EDSP Tier 2 Medaka Extended One Generation 

Reproduction Test (MEGORT) using Japanese medaka is recommended. 

Non-mammalian data specific to the HPT axis is not available since the test order requirement 

for the Amphibian Metamorphosis Assays was satisfied by the OSRI that demonstrated potential 

interaction with the thyroid pathway. Therefore, the EDSP Tier 2 Larval Amphibian Growth and 

Development Assay (LAGDA) is recommended to add to the understanding of potential adverse 

effects in amphibians. 

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

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

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

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

exposure. Relative to the EDSP, 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 linuron 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 the dose(s) at which endocrine related responses occurred happened 

concurrently with general systemic toxicity. 

On October 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 linuron 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).

Linuron, a phenylurea herbicide has an octanol/water partition (log Kow) of 3.00 and a water 

solubility of 81 mg/L at 20°C. In the environment, linuron is expected to be persistent, 

moderately mobile and semi-volatile. 

The available information considered in determining the potential interaction of linuron 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 considered 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 (EDRT) on linuron (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 to 2013 FIFRA 

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

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

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

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

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

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

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

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

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

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

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

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

above. 

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

EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other 

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 

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

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

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

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

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

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

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

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

endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit 

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

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

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

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

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

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

the 2013 FIFRA SAP meeting that evaluated scientific issues associated with the WoE 

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

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

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

evaluated in terms of primary interactions with the endocrine system vs. secondary effects 

related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 

2013). For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s 

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

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

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

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

weight decreases of ≥10% in mammals. Additionally, other effects including morphological 

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

(e.g., lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

extreme. In some instances, one parameter (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 components described above is 

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

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

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

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

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

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

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

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

which supports a conclusion of whether or not 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. 

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A. EDSP Tier 1 Screening Assays 

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

Table 1. 

Table 1. Tier 1 Screening Assays for Linuron

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine 

cytosol) OCSPP 890.1250 Requirement Satisfied 

(MRID No. 48617402) 

ERα Transcriptional Activation 

Assay (Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied by OSRI 

(Gordon et al., 2004) 

AR Binding Assay (Rat prostate 

cytosol) OCSPP 890.1150 

Requirement Satisfied by OSRI 

(Cook et al. 1993, Lambright et al. 2000, 

and USEPA 2007a) 

Steroidogenesis Assay (Human cell 

line H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement Satisfied by OSRI 

(Wilson et al., 2009) 

Aromatase Assay (human 

recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48617401) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48617403) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied by OSRI 

(Ashby et al., 2004, Freyberger et al., 2007, 

Freyberger et al., 2009, Kang et al., 2004, 

Lambright et al., 2000, Moon et al., 2009, 

Moon et al., 2010, and Tinwell et al., 2007) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied 

(MRID No. 48685501) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied by OSRI 

(USEPA 2007c, USEPA 2007d) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48671701) 

Amphibian Metamorphosis Assay 

(Frog) 

OCSPP 890.1100; 

OECD 231 

Requirement Satisfied by OSRI 

(O’Connor et al. 2002, Seidlova-Wuttke et

al. 2005, and USEPA 2007d) 

Page 16 of 101
LINURON FINAL 

Page 9 of 93 

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

1. Effects on Estrogen Pathway 

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

various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to 

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

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

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

considered further in the WOE assessment. 

In the Tier 1 in vitro ER binding assay, linuron was classified as negative. In the OSRI (Gordon 

et al, 2004) accepted for ERTA, linuron is a very weak inducer of transcription (EC50 of 1.45 × 

10−5

 M compared to 1.55 × 10−11 M for estradiol). In addition, in a yeast assay evaluating 

estrogen receptor activation (Orton et al., 2004), linuron demonstrated anti-estrogenic activity at 

concentrations of 1.9-31.3 μM. In a published study considered as OSRI, linuron was negative 

for ERα and ERβ activation in transfected CHO cells (Kojima, et al., 2004). In a study by 

Vinggaard et al. (1999), linuron was negative for inducing cell proliferation in estrogen-sensitive 

MCF-7 breast cancer cells, and was negative for ER activation in yeast. In the Tier 1 aromatase 

assay, linuron reduced aromatase activity to 68.1% of control activity at the highest soluble 

concentration (10−3.5 M). Since the lowest portion of the curves across the three runs was 

between 50 and 75% activity, linuron was determined to be equivocal for aromatase inhibition. 

The test order requirement for steroidogenesis assay was satisfied by OSRI (Wilson et al., 2009); 

however, estradiol production was not evaluated in the study. 

Linuron did not increase uterine weights in the uterotrophic assay. In the female pubertal assay, 

decreased ovarian weights (↓12%) were noted at the high dose (100 mg/kg/day), but without 

corroborating microscopic findings. A dose-dependent decrease was observed in pituitary gland 

weight (↓15-22%) at both doses tested (50 and 100 mg/kg/day). These effects occurred in the 

absence of overt toxicity. 

In the Tier 1 FSTRA, effects were observed at the highest test concentration (9.1 mg/L), 

including changes in gonadal staging, decreased yolk synthesis, and increased oocyte atresia of 

predominantly pre-vitellogenic follicles and no egg production; however, these effects occurred 

in the presence of overt toxicity (decreased survival and clinical signs of toxicity). Fecundity and 

fertility was also reduced by 50 and 5% at the mid concentration (0.92 mg/L) in the absence of 

overt toxicity. In a FSTRA considered as OSRI with fathead minnow (Marlatt et al., 2013), 

plasma vitellogenin concentrations were significantly reduced in females treated with linuron at 

0.001 and 0.100 mg/L; no other effects were observed in this study. In a Part 158 fish life-cycle 

with fathead minnow, no effects were observed up to 0.097 mg a.i./L. 

In a Part 158 developmental rat study, increased post-implantation loss and number of 

resorptions (in litters with resorptions) was observed at the highest dose (approximately 50 

Page 17 of 101
LINURON FINAL 

Page 10 of 93 

mg/kg/day). In a developmental rabbit study, spontaneous abortion, decreases in the number of 

live fetuses per litter, and decreased mean fetal body weights were noted at the highest dose 

tested (100 mg/kg/day). The effects in the rat and rabbit developmental studies co-occurred with 

deceased gestational body weight of dams, however it is not clear if the decrease body weight 

was due to decrease weight of uterine contents. In a two-generation reproduction study in Wistar 

rats, the number of implantations was decreased at the highest dose administered (approximately 

56 mg/kg/day) in the presence of overt toxicity (premating body weights decreased in both 

generations and sexes). However, these effects were not observed at comparable doses in another 

2-generation reproductive in CD rats. There were no estrogen-related effects noted in the other 

mammalian Part 158 studies. 

In the Part 158 mallard duck reproduction toxicity study, decreases were noted in egg production 

(30%) and eggshell thickness (7.5%) at the highest dietary concentration (300 mg/kg diet); 

possible effects on embryo viability (viable embryos/eggs set but not statistically significant) 

were also reported at this concentration. Decreased adult body weights were also noted during 

Week 1 at this dose as well as reductions in food consumption. In the Part 158 bobwhite quail 

reproduction study, there was a slight reduction in egg production as well as reductions in 

hatchability and offspring survival at 262 mg/kg-diet, and in the ratio of hatchlings that survived 

to Day 14. In a sheepshead minnow Part 158 early life stage toxicity study, growth and survival 

were decreased at ≥0.766 mg/L, and in a rainbow trout Part 158 early life-cycle study, growth 

was affected at ≥0.042 mg/L with effects on survival at 0.39 mg/L. 

Page 18 of 101
LINURON FINAL 

Page 11 of 93 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Linuron1Study 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 48617402) N ERTA Requirement satisfied by OSRI (Gordon et al., 2004; see below) Aromatase (MRID 48617401) E Steroidogenesis Requirement satisfied by OSRI (Wilson et al., 2009)4Uterotrophic (MRID 48617403) N Female Pubertal Rat (MRID 48685501) NE N ↓12% (H) N 

↓15

%, 

↓22

% 

(L, 

H) 

N N 

FSTRA (MRID 48671701) NE N N N 

Frt: ↓5% 

Fcd: ↓50% 

(M)

OSRI 

ERTA (Gordon et al., 2004) P ERTA (Kojima et al., 2004) N 
LINURON FINAL 

Page 12 of 93 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Linuron1Study 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) 

Yeast ER assay and Frog Ovulatory Response (Orton et al., 2009) 

P P 

Cell Proliferation (Vinggaard et al., 

1999) 

 N 

FSTRA (Marlatt et al., 2013) NE N N N N 

(L

Developmental Toxicity (Rat; MRID 00018167) 

P

 (H) 

Developmental Toxicity (Rabbit; MRIDs 00153867 and 40437201) 

N 

Two-Generation Reproduction Toxicity (Rat; MRID 48046002) 

 N N N N NE N 

Two-Generation Reproduction Toxicity (Rat; MRIDs 41463401 and 41864701) 

 NE NE NE NE NE N 

Chronic Toxicity (Dog; MRID 40952601) 

 NE N N NE NE 
LINURON FINAL 

Page 13 of 93 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Linuron1Study 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) 

Carcinogenicity (Mouse; MRID 00124195) 

 N N N N NE 

Chronic Toxicity/ Carcinogenicity (Rat; MRIDs 00029679, 00029680, and 00164117) 

 NE NE N N NE 

Avian Reproduction Toxicity (Mallard Duck; MRID 42541802) 

P8 

 (H) 

Avian Reproduction Toxicity (Bobwhite Quail; MRID 42541801) 

P10 

 (H) 

Fish Life-cycle Toxicity (Fathead minnow; MRID 49396901) 

 N 

1. Key to responses: Low treatment (L), Medium-low treatment (ML), Medium treatment (M), Medium-high treatment (MH), H

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is 

assay. Changes in weight are absolute unless otherwise indicated. 

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

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 

details see Section IV. A 

4. This study only examined testosterone and progesterone production, not estrogen. 

5. Red material was observed around the nose in 3/6 high-dose (120 mg/kg/day) rats on one day 
LINURON FINAL 

Page 14 of 93 

6. Body weights were decreased (p<0.05) by 10-12% during Days 3-4 in all treated groups. 

7. Survival was decreased and there were clinical signs of toxicity including lethargy and loss of equilibrium in fish at the 9.1 mg

8. Egg production, and egg shell thickness (significant 7.5% decrease) at 300 ppm 

9. There were treatment-related decreases in adult body weight (NS, male and female). 

10. At the high dose (300 ppm), there was a slight reduction in egg production, hatchability, and offspring survival. E Equivocal assay response 

 P Positive findings 

N Negative findings (in vitro)/No effect (in vivo) NE Not examined 
LINURON FINAL 

Page 15 of 93 

2. Effects on Androgen Pathway 

The potential for linuron to interact with the androgen pathway is summarized in Table 3. The 

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

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

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

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

considered further in the WOE assessment. 

The requirements for the Tier 1 AR binding, steroidogenesis, Hershberger and male pubertal 

assays were satisfied by OSRI. In the OSRI AR binding assays using rat prostate cytosol, 

linuron had an IC50 of 64 μM (Cook et al. 1993), 80 μM (USEPA 2007a) and 200 μM 

(Lambright et al. 2000). In a whole cell hAR binding assay, linuron had an IC50 of 

approximately 20 μM (Lambright et al. 2000). Additionally, linuron was found to have similar 

IC50 values in AR binding studies using human, fathead minnow, and rainbow trout AR that 

were considered as OSRI (Wilson et al., 2004 and 2007). Furthermore, in three OSRI AR 

transcription activation studies (ARTA) (Lambright et al., 2000; Freyberger et al., 2010; and 

McIntyre et al., 2000), linuron antagonized AR transcriptional activation induced by DHT and 

methyl-DHT at micromolar concentrations. In OSRI (Wilson et al., 2009) accepted for the Tier 1 

steroidogenesis assay, linuron decreased ex vivo fetal testes testosterone production, and in a study 

examining the effects of linuron on 5α-reductase, linuron inhibited 5α-reductase activity in human 

prostate homogenates with an IC50 of 86 μM. In an in vitro study by Pastoor et al. (1986b), linuron 

reduced the activity of several testicular steroidogenic enzymes and affected Leydig cell response to 

LH.

In an evaluation of androgenic/anti-androgenic activity in vivo, a total of eight Hershberger 

studies were accepted as OSRI (Ashby et al., 2004, Freyberger et al., 2007, Freyberger et al., 

2009, Kang et al., 2004, Lambright et al., 2000, Moon et al., 2009, Moon et al., 2010, and 

Tinwell et al., 2007) and an additional Hershberger validation study (OECD 2009) was 

considered as OSRI. In all of these studies, linuron demonstrated anti-androgenic activity as 

evidenced by consistent reductions in weights of the accessory sex tissues (AST) and 

epididymides. Overt toxicity was noted in only two studies (Freyberger et al., 2009; and Tinwell 

et al., 2007), where terminal body weight were decreased by 12% and 20% at the high dose (100 

mg/kg/day in both studies). Additionally, in a testosterone implant study (Lambright et al., 

2000), linuron administered at 100 mg/kg/day decreased weights of epididymides and AST and 

increased expression of the TRMP2 gene, an androgen-inhibited gene which reportedly peaks 

within five days of castration or anti-androgen treatment. 

In a study that was part of the validation effort for the male pubertal assay (USEPA, 2007c), 

linuron at 50 or 100 mg/kg/day decreased testes, epididymides, pituitary, and accessory sex 

organ weights, and delayed PPS, although overt toxicity (body weights ↓16%) was observed at 

100 mg/kg/day. Similarly in two 15-day adult rat screening assays (O’Connor et al., 2002; and 

USEPA, 2007d), linuron decreased testosterone levels, caused alterations in gonadal pathology, 

Page 23 of 101
LINURON FINAL 

Page 16 of 93 

and decreased epididymides and accessory sex organ weights. However, in these studies, overt 

toxicity (↓≥10% body weight) was observed at the high dose (150 mg/kg/day) in O’Connor et al. 

2002 and at all test doses (≥50 mg/kg/day) in USEPA, 2007d. 

In four studies considered as OSRI (Hotchkiss et al., 2004; McIntyre et al., 2000; Lambright et 

al., 2000; and Wolf et al., 1999), effects on male mammalian parameters were observed. In 

Hotchkiss et al., 2004, effects on male reproductive parameters were observed in a single dosetest and in the presence of overt toxicity (↓maternal body wt). Alterations in testes and 

epididymides histopathology (≥12.5 mg/kg/day) and increase in nipple retention (50 mg/kg/day) 

were observed in McIntyre et al. 2000 in the absence of overt toxicity. In Lambright et al. 2000, 

at the only dose level tested (100 mg/kg/day), there were histopathological changes in the testes 

and epididymis; it is noted in this study that dosing ceased at GD 18 due to decreases in maternal 

body weight gains. In a transgenerational study, Wolf et al. 1999, linuron at 100 mg/kg/day (no 

overt toxicity observed) induced histological abnormalities in the testes and epididymides, 

decreased testes and AST weights, decreased anogenital distance (AGD), increased 

areolae/nipple retention. Additionally, Cook et al. (1993) examined the reproductive 

developmental effects of linuron administered to sexually immature and adult rats. In these 

studies, 200 mg/kg/day linuron caused a decrease in epididymides weight in the immature rats, 

and decreases in AST weights in both immature and adult rats, however, overt toxicity was 

observed in both studies at the only dose tested (↓≥10% body weights) 

In a Part 158 rat (Wistar) study (MRID 48046002), no androgenic or anti-androgenic effects 

were noted at doses up to 53 mg/kg/day; however, decreases in body weights (>10%) and food 

consumption, and increases in spleen weights were noted. In the second Part 158 rat study (Cook 

et al. 1993), a dose of 35.5 mg/kg/day caused decreased testes weights and lesions in the testes 

and epididymides, concurrent with overt toxicity (decreased body weights in both generations). 

In a multi-generation rat reproduction toxicity study by Wolf et al. (1999), linuron at 40 

mg/kg/day decreased testes, epididymides, and AST weights, decreased the cumulative number 

of pups produced, decreased spermatid numbers, and increased age at PPS by 2.8 days. No 

systemic toxicity was noted in this study. 

In a chronic toxicity/carcinogenicity study in rats (MRID 00029679), linuron caused decreases in 

testes weights and pathological changes in the testes and epididymides at doses of 5.1 and 27.1 

mg/kg/day; however, decreased body weight (↓11%) was observed at the high dose 

Pastoor et al. (1986a) examined the effects of linuron on Leydig cell hyperplasia in aged male 

rats. A dose level of 31 mg/kg/day caused discoloration of the testes and an increase in testicular 

adenomas and testicular hyperplasia; however, overt toxicity was noted in these animals 

(decreased body weights >10%). 

In the Tier 1 FSTRA, at the high test concentration (9.1 mg/L), decreased GSI and male tubercle 

scores and alterations in male gonadal histopathology ( increased percentage of spermatogonia) 

were observed. However, these effects occurred in the presence of overt toxicity (decreased 

Page 24 of 101
LINURON FINAL 

Page 17 of 93 

survival and abnormal swimming as well as decreases in male growth parameters). In a nonGuideline FSTRA by Marlatt et al. (2013), no androgen-specific effects of linuron treatment 

were noted; however, the highest concentration tested in this study was 0.1 mg/L. No effects 

were observed in a Part 158 fathead minnow life-cycle study at 0.097 mg/L. 

Additional OSRI studies examined the potential anti-androgenic effects of in vivo and in vitro

linuron exposure on fish by measuring the production of the protein spiggin by female 

stickleback kidney cells. In a study by Jolly et al. (2009), in vitro and in vivo linuron exposure 

significantly inhibited DHT-induced spiggin production. Similar effects (reduction of siggin 

production) were reported in the additional open literature studies [(Katsiadaki et al. (2006); 

Pottinger et al. (2013)], however, purity information for the test substance (i.e., linuron) was not 

provided in those studies, and therefore, these results are not presented in the table below. In a 

Part 158 reproductive toxicity study with the mallard duck and bobwhite quail, effects at the 

highest treatment group (300 ppm) included decreased hatchability, and offspring survival in the 

quail; no androgen-related endpoints were affected in the duck. 

Page 25 of 101
LINURON FINAL 

Page 18 of 93 

Table 3. Androgenic/Anti-Androgenic Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Linuron1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 Assay 

AR Binding Assay satisfied by OSRI (Cook et al. 1993, Lambright et al. 2000, and USEPA 2007a; see Steroidogenesis Assay satisfied by OSRI (Wilson et al., 2009; see below) Hershberger Assay satisfied by OSRI (Ashby et al., 2004, Freyberger et al., 2007, Freyberger et al., 2009, Kang et al., 2004, La

2009, Moon et al., 2010, and Tinwell et al., 2007; see below) 

Male Pubertal Rat Assay satisfied by OSRI (USEPA 2007c and USEPA 2007d; see below) FSTRA (MRID 48671701) NE N N N Frt:↓5% (M) 

OSRI 

AR Binding (Cook et al. 1993) P AR Binding (Lambright et al.2000) 

P 

AR Binding (USEPA 2007a) P AR Binding (Wilson et al., 2004) P 
LINURON FINAL 

Page 19 of 93 

Table 3. Androgenic/Anti-Androgenic Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Linuron1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

AR Binding (Wilson et al., 2007) P ARTA (Lambright et al., 2000) P ARTA (Freyberger et al., 2010) P ARTA (McIntyre et al., 2000) P Fetal Testosterone (Wilson et al., 2009) P Hershberger (Ashby et al., 2004) 

↑31% 

(H) 

↓19% 

(H) 

CG ↓13-29% 

(M, H); 

SV ↓25%, 

PR ↓34% 

 (H) 

Hershberger (Freyberger et al., 2007) 

CG ↓29%, 

LABC ↓43%, 

SV ↓54%, 

VP ↓50% 

(H) 

Hershberger (Freyberger et al., 2009) 

 N N N 
LINURON FINAL 

Page 20 of 93 

Table 3. Androgenic/Anti-Androgenic Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Linuron1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Hershberger (Kang et al., 2004) N 

SV ↓28-47%, VP ↓25-38%, 

CG↓26-39% 

(M, H); 

LABC ↓35%, 

GP ↓20% 

(H) 

Hershberger (Lambright et al., 2000) 

SV ↓44% 

VP ↓37%, 

LABC ↓22% 

Hershberger (Moon et al., 2009) 

VP ↓40-68%, SV ↓48-71%, 

LABC ↓28-50%, 

CG ↓32-55%, 

GP ↓12-19% 

(L, H) 

Hershberger – Stage 3 testing (Moon et al., 2010) 

N ↓17% (H) 

VP ↓22%, SV ↓25%, 

LABC ↓21%, 

CG ↓26% 

(H) 

Hershberger – Stage 4 testing (Moon et al., 2010) 

N ↓21% (H) 

VP ↓23%, SV ↓48%, 

LABC ↓27%, 

CG ↓34% 

(H) 
LINURON FINAL 

Page 21 of 93 

Table 3. Androgenic/Anti-Androgenic Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Linuron1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Hershberger (Tinwell et al., 2007) N N N Hershberger – Phase 2 (OECD, 2009) 

N ↓19% (H) 

VP ↓24-27%, SV ↓26-29%, 

LABC ↓20-23%,

CG ↓28% 

(H) 

Hershberger Phase 3 (OECD, 2009) 

↓24-32% 

(H) 

↓21-27% 

(H) 

VP ↓23%, 

SV ↓32-48%, 

LABC ↓20-27%,

CG ↓34% 

(H) 

Testosterone Implant (Lambright et al., 2000) 

N ↓17% VP ↓33%, LABC ↓24% 

Male Pubertal (USEPA 2007c) N N N ↓(L) N ↓(L) ↓SV, ↓VP, ↓DP, ↓LABC (L) Delaye

(L) 

15-Day Adult Screening Assay (O’Connor et al., 2002) 

↓40% (H) N P (MH, H) ↓9% (H) N PR ↓19%, AST ↓11% (H) 

Reproductive /Development in Male Rats (McIntyre et al., 

2000) 

 N N P (L, M, H) N P (L, M, H) DP ↓11% (H) N 
LINURON FINAL 

Page 22 of 93 

Table 3. Androgenic/Anti-Androgenic Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Linuron1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Reproductive /Development in Male Rats (Lambright et al., 

2000) 

 P P 

Reproductive/ Development in Male Rats (Wolf et al., 1999) 

↓22% P ↓29- 35% P NR 

SV ↓12% (NS), 

VP ↓35%, 

LABC ↓16%, 

GP ↓16%; 

AGD ↓30% 

Two-Generation Reproduction Toxicity (Rat; MRID 48046002) 

N N N N N N N 

Two-Generation Reproduction Toxicity (Rat; MRIDs 41463401 and 41864701) 

N N NE N NE NE N 

Multi-Generation Reproduction Toxicity in Male Rats (Wolf et al., 1999) 

N ↓20% (H) N ↓18% (H) N 

SV ↓14%, 

Cauda 

Epididymides 

↓7% (H) 

P

 (H) 

↑2.8 

(H) 

Chronic Toxicity (Dog; MRID 40952601) 

N N NE N NE 
LINURON FINAL 

Page 23 of 93 

Table 3. Androgenic/Anti-Androgenic Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Linuron1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Carcinogenicity (Mouse; MRID 00124195) 

N N N N N 

Chronic Toxicity/ Carcinogenicity (Rat; MRIDs 00029679, 00029680, and 00164117) 

↑21% 

(M, 

H) 

N NE P (M ) N 

Leydig Cell Hyperplasia in Aged Male Rats (Pastoor, 1986a) 

N P N N 

Biological and Histopathological Effects in Rats (Pastoor, 1986b) 

P N P 

FSTRA (Marlatt et al., 2013) N N N Androgenic/AntiAndrogenic Activity in Fish (Jolly et al., 

2009) 

P 

Avian Reproduction Toxicity (Mallard Duck; MRID 42541802) 

 N 
LINURON FINAL 

Page 24 of 93 

Table 3. Androgenic/Anti-Androgenic Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Linuron1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Testosterone 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Avian Reproduction Toxicity (Bobwhite Quail; MRID 42541801) 

 N 

Fish Life-cycle Toxicity (Fathead minnow; MRID 49396901) 

 N 

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

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in thabsolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), D(PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). 

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 

see Section IV. A 

4. Survival was decreased and there were clinical signs of toxicity including lethargy and loss of equilibrium in fish at the 9.1 mg/L. 

male body weight (26%) and length (8%) were observed at this concentration. 

5. It was stated that at the only dose level tested (100 mg/kg/day), dams were not treated after GD 18 due to a reduction in body wei

6. There were treatment-related decreases in adult body weight (NS, male and female) P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

NE Not examined NR Not reported 
LINURON 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 linuron to interact with thyroid regulation are summarized in Table 4. The 

various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for thyroid or HPT axis effects. This table also includes 

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

overt toxicity are discussed in the text but are not reported in the table and not considered 

further in the WOE assessment. 

Of the Tier 1 assays utilized for assessing thyroid pathway effects, only the female pubertal rat 

assay was submitted. The requirements for the Tier 1 male pubertal and the AMA were satisfied 

by OSRI (USEPA, 2010). In the Tier 1 female pubertal assay, dose-dependent decreases in T4

and pituitary weight were observed, but no corresponding changes were seen in thyroid weight 

or histopathology. The thyroid effects observed in Lech, 2006, (USEPA 2007d) occurred only in 

the presence of overt toxicity. In the validation of the male pubertal assay (USEPA, 2007c), 

serum T4 levels were decreased at the high dose (100 mg/kg/day), TSH was decreased at the low 

dose (50 mg/kg/day), and pituitary weights were decreased at both dose levels; however, the high 

dose animals in this study exhibited signs of overt toxicity. Dose-dependent decreases in both T3

and T4 levels were observed in the 15-day adult rat screening study (O’Connor et al., 2002), with 

no changes in TSH levels or thyroid histopathology or weights; the effects in the high dose 

occurred in the presence of overt toxicity (↓11% body weight). Dose-dependent decreases in T3

and T4 levels were also observed in a thyroid toxicity study (Seidlova-Wuttke et al., 2005) at 

doses of 16.3 and 65.5 mg/kg/day. No overt toxicity was noted in this study. None of the above 

studies showed treatment-related changes in thyroid weight or histopathology, and changes in 

pituitary weight were only noted in the female pubertal assay and the validation trial of the male 

pubertal assay. 

In an in vitro transcriptional activation assays (Hoffman et al., 2009,) using HepG2 cells 

transfected with a luciferase-based reporter gene and a T3-responsive element, linuron was 

reported to have weak agonist activity to the T3 receptor. 

In the Part 158 mammalian long-term toxicity studies conducted with in mice, rats and dogs, no 

treatment-related effects were observed on thyroid weight or histopathology, or on pituitary 

weight or histopathology. 

 

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

treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates 

that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute 

unless otherwise indicated. 

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

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

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

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

P Positive findings 

N Negative findings 

NE Not examined 

Table 4. Thyroid Pathway for Linuron

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Linuron1

Study Type/ 

Literature Citation 

TR Activation Thyroid Weight Thyroid: Gross and Histopathology Serum T3/T4Levels Serum TSH Levels Pituitary Weight Developmental stage (± or asynchronous, HLL)

Growth (BW, BL, SVL) Systemic Toxicity 2

Overt Toxicity 

3 

EDSP Tier 1 Assay 

Female Pubertal Rat 

(MRID 48685501) N N 

T4 ↓27% 

↓48% 

(L, H) 

N 

↓15%, 

↓22% 

(L, H) 

X 

(L, H) N 

Male Pubertal Rat Assay satisfied by OSRI (USEPA 2007c and USEPA 2007d; see below) 

AMA (Frog) Assay satisfied by OSRI (O’Connor et al. 2002, Seidlova-Wuttke et al. 2005, and USEPA 2007d; 

see below) 

OSRI 

TR Transcriptional 

activation (Hoffman et 

al., 2009) 

P

Thyroid Toxicity (Rat; 

Seidlova-Wuttke et al., 

2005) 

 NE NE 

↓T3

↓T4

(L, H)

N NE X

(H) N 

Male Pubertal Assay 

(Rat; USEPA, 2007c) N N N ↓TSH 

(L) 

↓

(L) 

X

(H) 

X 

(H)

15-Day Adult Screening 

Assay (Rat; O’Connor et 

al., 2002) 

 N N 

↓T3

(ML, 

MH,) 

↓T4

(L, ML, 

MH)

N NE 

X

(L, ML, 

MH, H) 

X 

(H)

Two-Generation 

Reproduction Toxicity 

(Rat; MRID 48046002) 

 N N N X

(H) 

X

(H)

Chronic Toxicity (Dog; 

MRID 40952601) N N NE X 

(MH, H) N 

Carcinogenicity (Mouse; 

MRID 00124195) N N N X 

(H) 

X 

(H)

Chronic Toxicity/ 

Carcinogenicity (Rat; 

MRIDs 00029679, 

00029680, and 

00164117) 

 NE N N X 

(M, H) 

X 

(H) 

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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 linuron 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 evaluated scientific issues associated with 

weight of evidence evaluation of the results of the Tire 1 assays stated that “In summary, the 

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

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

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

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

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

OSRI studies are described below. 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).

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1. Tier 1 in vivo Assays 

Studies were conducted for three of the possible five in vivo Tier 1 assays, including the female 

pubertal assay, the uterotrophic assay and the FSTRA. Signs of systemic toxicity were noted in 

the both the uterotrophic and female pubertal assays and generally consisted of reductions in 

body weight and body weight gains, along with changes in clinical chemistry parameters and 

decreased kidney weight (↓7%) in the pubertal study. No mortality was observed in either study, 

but decreases in body weight of 10-12% in the uterotrophic assay indicated overt toxicity at all 

doses tested (30-120 mg/kg/day). 

In the FSTRA, male survival was reduced by 25% at the highest concentration and male body 

weight was reduced by 22 and 26% at 0.92 and 9.1 mg/L, respectively. Female body weight and 

length were also reduced by 29% and 6%, respectively, at 9.1 mg/L. In addition, lethargy and 

loss of equilibrium in fish at the 9.1 mg/L level indicated that this concentration was overtly 

toxic. 

2. OSRI 

Signs of both systemic and overt toxicity were noted in many of the in vivo OSRI studies, with 

decreases in body weight and body weight gains being the most common finding in many 

studies. In the mammalian toxicity studies, overall signs of toxicity were generally most 

pronounced at doses exceeding 50 mg/kg/day. 

In the nine reports detailing the various Hershberger assays conducted using castrated adult or 

intact juvenile rats, linuron was tested at doses ranging from 3 to 100 mg/kg/day. No mortality 

was noted in any Hershberger study, and when reported, the most common findings were 

reductions in body weight or body weight gain at the highest dose (100 mg/kg/day), with one 

study also reporting increased liver weight at ≥30 mg/kg/day (Ashby et al., 2004). Overt toxicity 

was noted in only two studies (Freyberger et al., 2009; and Tinwell et al., 2007), where terminal 

body weight were decreased by 12% and 20% at the high dose (100 mg/kg/day in both studies). 

In the male pubertal assay (USEPA, 2007c) and the 15-day adult rat screening assays (O’Connor 

et al., 2002 and USEPA, 2007d), systemic and overt toxicity were noted in each study. In the 

pubertal study, body weight was decreased by 7 and 16% at 50 and 100 mg/kg/day, respectively, 

and liver and kidney weights were also decreased at the high dose. In the 15-day adult screening 

assays, which were conducted at linuron doses of 25 to 150 mg/kg/day, body weights were 

decreased at all dose levels with the decreases exceeding 10% at doses of ≥50 mg/kg/day. In 

addition, liver weights were increased in one assay and decreased in the other 15-day assay. 

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In the guideline developmental toxicity studies on rats and rabbits, no mortality was observed 

and the main signs of toxicity were decreases in maternal body weights and pup weights at 

linuron doses ca. ≥50 mg/kg/day. The other five published studies examined the effects of 

linuron on in utero reproductive development of male rats at doses from 12.5 to 100 mg/kg/day 

or on development of immature male rats (Cook et al., 1993) at 200 mg/kg/day. In the in utero

studies, maternal and pup body weights were decreased at dose levels ≥50 mg/kg/day, and pup 

mortality was increased and litter size was decreased, and in the immature rat study, body 

weights were decreased by 27%. 

No systemic or overt toxicity were noted in the thyroid study by Seidlova-Wuttke et al. (2005) in 

female rats at doses up to 65.5 mg/kg/day. 

Four multi-generation reproduction toxicity studies in rat were also evaluated as OSRI, including 

two guideline studies and two published studies (Cook et al., 1993, and Wolf et al., 1999). 

Linuron was administered in the diet at up to 625 ppm (36-54 mg/kg/day) in three of the studies, 

and by oral gavage at up to 40 mg/kg/day in the fourth study. Systemic and overt toxicity were 

noted in the dietary studies and were evidenced by decreases in body weight for both parents and 

offspring at the high dose level (36-54 mg/kg/day) and decreased litter size. 

In the three guideline chronic toxicity/carcinogenicity studies, no overt toxicity was noted at 

doses up to 4.17/3.49 mg/kg/day [M/F] in the chronic dog study, but overt toxicity was noted in 

the high dose groups in both the mouse (261/455 mg/kg/day [M/F]) and rat (27.1/48.3 mg/kg/day 

[M/F]) studies based on decreases in body weight and body weight gain. In addition, all three 

chronic studies showed adverse hematological changes at the highest doses. 

In the two guideline avian reproduction studies, transient decreases were observed in body 

weight of adult ducks during the first week of exposure. No other overt toxicity was noted in 

either study at dietary concentration up to 300 ppm. 

No overt toxicity was noted in a FSTRA (Marlatt et al., 2013) submitted as OSRI, in which 

linuron was tested for androgenic and anti-androgen effects at concentrations of 1-100 μg/L. 

However, both overt toxicity was noted in the two guideline fish early life cycle studies. In the 

sheepshead minnow study, linuron was tested at concentrations from 0.206 to 2.93 mg/L. 

Survival was decreased at the two highest concentrations (1.55 and 2.93 mg/L), and body weight 

and length were decreased at concentrations of ≥0.766 mg/L. In the trout study, linuron was 

tested at seven concentrations from 0.042 to 3.2 mg/L. Survival was decreased at concentrations 

of ≥0.39 mg/L; body weight was decreased at concentrations ≥0.21 mg/L; and body length was 

decreased at all concentrations (≥0.042 mg/L). 

In the study by Pottinger et al. (2013) examining androgen-induced spiggin production, mortality 

was ≤1.5% for female stickleback fish exposed to linuron at 0.25 to 250 μg/L for up to 21 days; 

however, toxicity data from this study was limited. 

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B. Estrogen Pathway 

Although positive results were reported in the literature for ER-related responses in vitro, the 

responses are not supported by the Tier 1 assays (negative ER binding and uterotrophic assays) 

or other literature in vitro assays evaluating ER transactivation assays in CHO cells, yeast ERreporter assays or ER-responsive proliferation assay. Linuron showed an equivocal response for 

aromatase inhibition in vitro. In the female pubertal assay, decreased ovarian weight was 

observed, however there was no corroborating microscopic findings. In addition, pituitary 

weights were also decreased in the study. There were no estrogen-related effects noted in the 

mammalian Part 158 studies. In the Tier 1 FSTRA (with fathead minnow), in the absence of 

overt toxicity, there were alterations in fecundity and fertility at 0.92 mg/L, however, these 

effects are limited in their diagnostic specificity for estrogen-related alterations in of themselves; 

no effects were observed in a Part 158 fathead minnow life-cycle study up to 0.097 mg/L. In 

another FSTRA considered as OSRI, plasma vitellogenin concentrations were significantly 

reduced in females; however, no other effects were observed in this study. Reductions in egg 

production (fecundity) were noted in two Part 158 avian reproduction studies, but again, these 

findings are limited in their ability to identify potential estrogen-related interactions; decreases in 

eggshell thickness was observed in the mallard reproduction study, and decreased female body 

weight and food consumption was also observed at this concentration. Overall, while some in 

vitro assays did indicate potential interaction with the estrogen pathway, these findings were not 

supported by the results of in vivo mammalian and wildlife studies. 

C. Androgen Pathway 

There is consistent complementarity and redundancy of evidence across the Tier 1 and OSRI to 

suggest that linuron acts as an anti-androgen both in vitro and in vivo. Linuron in vitro exhibited 

competitive binding to the AR and activated AR transcriptional activity in multiple species in 

vitro (human, fathead minnow, and rainbow trout). Linuron also inhibited ex vivo testosterone 

production in fetal rat testes. The findings of multiple Hershberger assays, male pubertal assays, 

and reproductive developmental toxicity studies in rats, as well as assays measuring the 

inhibition of androgen-induced spiggin production by female stickleback fish kidney cells, 

support that linuron exhibits anti-androgenic effects in vivo. In mammalian Part 158 studies, 

linuron caused changes in testes and epididymal weights and histopathology in the testes and 

epididymides. 

D. Thyroid Pathway 

There is convincing evidence of potential interaction of linuron with the thyroid pathway. 

Although no thyroid histopathology effects were observed in any of the Tier 1 or OSRI studies, 

decreases in serum T3 and/or T4 levels were observed across multiple studies including the 

EDSP Tier 1 female pubertal assay, a 12-week female rat thyroid toxicity study, and two 15-day 

Page 38 of 101
LINURON FINAL 

Page 31 of 93 

adult male rat screening assays considered as OSRI. Additionally, linuron showed weak agonist 

activity to the T3 receptor in two in vitro transcriptional activation assays using the HepG2 cells. 

With regards to the AMA, the assay requirement was satisfied by the mammalian OSRI. 

E. Conclusions 

For the estrogen pathway, the available data suggests that linuron does not interact with the 

estrogen pathway. For the androgen pathway, linuron appears to act as an anti-androgen both in 

vitro and in vivo. For the thyroid pathway, there was evidence of potential interaction in mammals 

characterized by changes in thyroid hormone levels in the absence of changes in thyroid weight or 

histopathology in multiple studies. 

V. EDSP Tier 2 Testing Recommendations 

In summary, there was no evidence of interaction with the estrogen pathway in mammals. There 

was convincing evidence for potential interaction with the androgen and thyroid pathways in 

mammals in the EDSP Tier 1 assays and OSRI. 

The current point of departure (POD) of 0.77 mg/kg/day for human health risk assessment is 

based on hematological effects (increased met- and sulf-hemoglobin levels) seen at 3.5 

mg/kg/day (LOAEL) in a chronic toxicity study in dogs. This POD is used for establishing the 

chronic Reference Dose (RfD) used for chronic dietary risk assessment and for calculating 

Margins of Exposure (MOEs) for non-dietary risk assessments. This POD is approximately 65-

fold lower than the lowest dose (50 mg/kg/day) that caused androgen-related effects in studies 

reported as OSRI. Therefore, EDSP Tier 2 studies to investigate androgen-related effects are not 

required. 

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

adult animals, there usually would be a concern that the POD may not be protective of potential 

thyroid toxicity in the young. 

Therefore, a special thyroid assay in pregnant animals, postnatal, and adult animals is 

recommended. This special study should use a mechanistic approach to generate specific data on 

the thyroid (i.e., the primary target of linuron) to protect the developing nervous system from 

thyroid hormone disrupting chemicals. The specific purpose of this study is to establish a POD 

(i.e., NOAELs and LOAELs or benchmark doses) that may be used for human health risk 

assessment. The POD from this special study would address the concern for the potential ability 

of linuron to disrupt thyroid function in pregnant females and in the fetus or newborn. 

There is evidence for potential interaction with the androgen signaling pathway in fish, based on 

the available fish, mammalian, and in vitro data. While there was limited evidence using fathead 

minnow (effects on fecundity and fertility at 0.92 mg/L, no other androgen-related effects 

Page 39 of 101
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observed), anti-androgenic effects were observed in three-spined stickleback fish (≥0.100 mg/L). 

There were no growth, survival or reproductive effects observed in a Part 158 fish life-cycle 

study with fathead minnow up to the highest concentration tested (0.097 mg a.i./L). Given the 

potential for androgen-related effects in fish, the EDSP Tier 2 Medaka Extended One Generation 

Reproduction Test (MEGORT) using Japanese medaka is recommended. 

Non-mammalian data specific to the HPT axis is not available since the test order requirement 

for the Amphibian Metamorphosis Assays was satisfied by the OSRI that demonstrated potential 

interaction with the thyroid pathway. Therefore, the EDSP Tier 2 Larval Amphibian Growth and 

Development Assay (LAGDA) is recommended to add to the understanding of potential adverse 

effects in amphibians. 

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

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

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

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

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

Page 40 of 101
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Page 33 of 93 

VI. References 

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northern bobwhite (Colinus virginianus). Wildlife International Ltd., Easton, MD. Sponsored by 

E.I. duPont de Nemours and Company, Newark, DE. Laboratory Project No. 112-271, 

completion date not provided. Unpublished. (MRID 42541801) 

Beavers, J, Foster, J, and Jaber, M (1992) Linuron: a one-generation reproduction study with the 

Mallard duck (Anas platyrhynchos). Wildlife International Ltd., Easton, MD. Sponsored by E.I. 

duPont de Nemours and Company, Newark, DE. Laboratory Project No. 112-272, completion 

date not provided. Unpublished. (MRID 42541802) 

Beck, M (2012) A pubertal development and thyroid function assay of linuron administered 

orally in intact juvenile/peripubertal female rats. WIL Research Laboratories, LLC, Ashland, 

OH. Laboratory Project ID WIL-851004, June 5, 2012. Unpublished. (MRID 48685501). 

Boeri, R, Magazu, J, and Ward, T (1995). Early life stage toxicity of DPX-Z326-198 (linuron) to 

the sheepshead minnow, Cyprinodon variegates. T.R. Wilbury Laboratories, Inc., Marblehead, 

MA. Sponsored by E.I. du Pont de Nemours and Company, Newark, DE. Laboratory Report ID 

815-DU, April 16, 1996. Unpublished. (MRID 43996502) 

Carakostas, M (1986) Evaluation of clinical laboratory data from two-year rat study with INZ326. Central Research and Development Department, Haskell Laboratory for Toxicology and 

Industrial Medicine, E.I. du Pont de Nemours and Company, Newark, DE. (Re-evaluation of 

data from Haskell Laboratory Report number 100-80 [MRID 00029680], with no new laboratory 

or registrant report or study number provided.) August 13, 1986. Unpublished. (MRID 

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Coder, P (2011) A uterotrophic assay of linuron administered orally in ovariectomized rats. WIL 

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Culik, R (1979) Teratogenicity study of 3-(3,4-dichlorophenyl)-1-methoxy-1-methyl-urea in rats. 

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Everett, R (1980) Two-year feeding study-ChR-CD rats. Haskell Laboratory for Toxicology and 

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Gordon, J, Chu, A, Chu, M, et al. (2004) Detection of estrogen receptor endocrine disruptor 

potency of commonly used organochlorine pesticides using the LUMI-CELLTM bioassay. 

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Hoberman, A, and Staples, R (1985) Developmental toxicity study of INZ-326 administered via 

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Kaplan, A, Smith, L, Koniecki, W, et. al. (1980) Long-term feeding study in rats with 3-(3,4-

dichlorophenyl)-l-methoxy-l-methylurea (Lorox', linuron, INZ-326). Haskell Laboratory for 

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Pont de Nemours and Company, Newark, DE. Laboratory Report No. 100-80, completion date 

not provided. Unpublished. (MRID 00029680) 

Lech, J (2006) Interlaboratory validation of the 15-day adult intact male rat assay with linuron 

and phenobarbital. Charles River Laboratories Preclinical Services, Horsham, PA. Sponsored by 

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This study was also referenced as U.S. EPA, 2007d (MRID#48321201) below. 

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du Pont de Nemours and Company, Haskell Laboratory for Toxicology and Industrial Medicine, 

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reproductive endpoints in the fathead minnow (Pimephales promelas). Comp. Biochem. Physiol. 

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reproduction study in rats. E.I. du Pont de Nemours and Company, Haskell Laboratory for 

Toxicology and Industrial Medicine, Newark, DE. Laboratory Study No. MR 8511-001, March 

29, 1990. Unpublished. (MRID 41463401) 

Mullin, L (1991) Supplemental histopathology data to evaluate eye lesions observed in multigeneration reproduction study No. HLR 20-90. E.I. du Pont de Nemours and Company, Haskell 

Laboratory for Toxicology and Industrial Medicine, Newark, DE. Laboratory Study No. not 

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Orton, F, Rosivatz, E, Scholze, M, et al. (2011) Widely used pesticides with previously unknown 

endocrine activity revealed as in vitro anti-androgens. Environ. Health Perspect. 119(6): 794-

800. 

Pierson, K (1991) Flow-through, 80 day toxicity of DPX-Z326-198 to embryo and larval 

rainbow trout, Onorhynchus mykiss. Haskell Laboratory for Toxicology and Industrial Medicine, 

E.I. du Pont de Nemours and Company, Newark, DE. Laboratory Project ID 538-91, completion 

date not provided. Unpublished. (MRID 42061804) 

Pottinger, T, Katsiadaki, I, Jolly, C, et al. (2013) Anti-androgens act jointly in suppressing 

spiggin concentrations in androgen-primed female three-spined sticklebacks - prediction of 

combined effects by concentration addition. Aquat Toxicol. 140-141: 145-156. 

Rickard, R (1981) Long-term feeding study with 3-(3,4-Dichlorophenyl)-1-methoxy-1-

methylurea (Lorox®; Linuron: INZ-326) in mice. Haskell Laboratory for Toxicology and 

Industrial Medicine, E.I. du Pont de Nemours and Co., Newark, DE. Laboratory Project No. 

3336, April 1981. Unpublished. (MRID 00124195)

Suter, P, Vogel, W, and Wilson, J (1988) Two-generation reproduction study in the rat. RCC 

AG, Itingen, Switzerland. Laboratory Project No. 071504, November 18, 1988. Unpublished. 

(MRID 48046002) 

USEPA (1995) Reregistration Eligibility Decision (RED): Linuron. U.S. Environmental 

Protection Agency, Washington, DC. March, 1995. EPA 738-R-95-003. 

USEPA (2007a) Androgen receptor (AR) binding validation, EDSP integrated summary report. 

U.S. Environmental Protection Agency. November 7, 2007. 

USEPA (2007c) Pubertal development and thyroid function in juvenile male rats, EDSP 

integrated summary report. U.S. Environmental Protection Agency. September 2007. MRID 

48321202.

USEPA (2007d) Interlaboratory validation of the 15-day adult intact male rat assay with linuron 

and phenobarbital. U.S. Environmental Protection Agency. August 27, 2007. MRID 48321201. 

Note: This study was also referenced as Lech, 2006 above. 

USEPA (2010) Linuron - report of the Endocrine Disruptor Review Team – Test Order #: 

EDSP-035506-122. December 16, 2010. Office of Chemical Safety and Pollution Prevention, 

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

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

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

Safety and Pollution Prevention Science Coordination and Policy, U.S. Environmental Protection 

Agency, Washington, DC. 

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

held July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results of EDSP Tier 1 

Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 

Agency, Washington, DC. 

Vinggaard, A, Breinholt, V, and Larsen, J (1999) Screening of selected pesticides for oestrogen 

receptor activation in vitro. MRID 48041733. Food Additives and Contaminants 16 (12): 533-

542. 

Wilga, P (2011) Linuron: human recombinant aromatase assay. CeeTox, Inc. Kalamazoo, MI. 

Laboratory Study No. 9138V-100325AROM. December 7, 2011. Unpublished. (MRID 

48617401) 

Willoughby, J (2011) Linuron: estrogen receptor binding (rat uterine cytosol). CeeTox, Inc., 

Kalamazoo, MI. Laboratory Study No. 9138V-100325ERB, December 8, 2011. Unpublished. 

(MRID 48617402) 

Willoughby, J (2012) Supplemental Information - Laboratory Proficiency Data for ERTA assays 

and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. CeeTox, 

Inc., Kalamazoo, MI. Laboratory Study No. not provided, July 19 2012. Unpublished. (MRID 

48843501) 

Wilson, V, Cardon, M, Thornton, J, et al. (2004) Cloning and in vitro expression and 

characterization of the androgen receptor and isolation of estrogen receptor α from the fathead 

minnow (Pimephales promelas). MRID 48041738. Environ. Sci. Technol. 38: 6314-6321. 

York, D.O. (2012). Linuron – Fish Short-term Reproduction Assay with the Fathead Minnow 

(Pimephales promelas). Performed by Smithers Viscient, Wareham, Massachusetts, Laboratory 

Project No. 13694.6149. Submitted by Tessenderlo Kerley, Inc., Phoenix, Arizona. (MRID 

48671701) 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100) 

Requirement satisfied by OSRI (O’Connor et al. 2002, Seidlova-Wuttke et al. 2005, and USEPA 

2007d); see Appendix 2. 

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

Requirement satisfied by OSRI (Cook et al. 1993, Lambright et al. 2000, and USEPA 2007a); 

see Appendix 2. 

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48617401), linuron (99.4% a.i., Batch #: 

1012A3876) in dimethyl sulfoxide (DMSO; ≤1%) was incubated with human recombinant 

aromatase and tritiated androstenedione ([1β3H(N)]-androst-4-ene-3,17-dione; [3H]-ASDN) for 

15 minutes at 37°C to assess the potential of linuron to inhibit aromatase activity. Linuron was 

tested at logarithmic concentrations from 10–10 M to 10−3

 M in Run 1 and from 10–10 M to 10−3.5

M in Runs 2 and 3, due to precipitation observed at 10−3 M in Run 1. 

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

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

quantified using liquid scintillation counting (LSC). Three independent runs were conducted, 

and each run included a full activity control, a background activity control, a positive control 

series (10−10 M to 10−5

 M) using a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), 

and the test chemical series (10−10 M to 10−3

 or 10−3.5 M) with 3 repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.494 to 0.726 nmol·mgprotein−1

·min−1 for the three test runs, with a mean and standard deviation of 0.598 ± 0.086 

nmol·mg-protein−1

·min−1. Activity in the background controls ranged from 0.20% to 0.31% and 

averaged 0.27% of the full activity controls. The response of the full activity controls and 

background controls was acceptable for each run. 

Results for the positive control were generally within the recommended ranges for the top of the 

curve, bottom curve, Hill slope, log IC50, and coefficient of variation for replicates of each 

concentration within runs. Also, the coefficients of variations (%CVs) for replicates of each 

concentration of linuron within a run were generally within the 15% guideline. For 4-OH 

ASDN, the estimated log IC50 averaged –7.27 M, and the slope was –1.00. 

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For linuron, aromatase activity averaged 0.607 ± 0.071 nmol·mg-protein−1·min−1 at the lowest 

tested concentration of 10–10 M and 0.372 ± 0.023 nmol·mg-protein–1·min−1 at the highest 

successfully tested concentration of 10−3.5 M (highest non-precipitating dose; precipitation was 

observed in Run 1 at 10−3

 M). The data for linuron were not suitable for modeling. The mean 

aromatase activity of 10–3.5 M linuron was 68.1% of control activity, and the best fit mean 

inhibition response curve was a straight line (14% inhibition at 10–4 M and essentially no 

inhibition was observed at other doses). The average lowest portion of the response curve across 

runs was between 50 and 75% activity, indicating an equivocal response at soluble 

concentrations. 

Based on the data from the average response curve, linuron is classified as Equivocal for 

aromatase inhibition in this assay. 

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

In an estrogen receptor (ER) binding assay (MRID 48617402), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiments. 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 radio-labeled reference 

estrogen prior to conducting ER competitive binding experiments. The competitive binding 

experiment measured the binding of a single concentration of [3

H]-17β-estradiol (1 nM) in the 

presence of increasing concentrations of linuron (logarithmic increase from 10−10 to 10−3 M). 

DMSO was used as a vehicle at a final concentration of 2%. The assay included 

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

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

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

following a request by the Agency. The Kd for [3

H]-17β-estradiol was 0.303±0.090 nM, and the 

estimated Bmax was 33.59±0.70 fmol/100 μg protein. The Kd for each run was within the 

expected range of 0.03 to 1.5 nM. 

Precipitation was observed at 10−3

 M for all runs; therefore, the data from the 10−3 M linuron 

concentration were not included in the analysis. In the competitive binding experiment, linuron 

had no effect on the specific binding of radiolabeled estradiol to ER at concentrations of ≤10−4

 M 

in all runs. A mean log IC50 was not calculated for linuron as 50% binding displacement was not 

achieved in any run. The mean log IC50 was −8.9 M for the natural ligand, 17β-estradiol, and 

−5.5 M for the weak positive control, which also had a relative binding affinity (RBA) of 

0.042% compared to the natural ligand. 

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

Receptor Binding Assay. 

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

Requirement satisfied by OSRI (Gordon et al., 2004); see Appendix 2. 

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

The 21-day short-term reproduction assay (MRID 48671701) of linuron with fathead minnows 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish (16 spawning 

groups, 2 males and 4 females in each group; 4.25 months old) were exposed to linuron (99.4% 

purity) at nominal concentrations of 0 (negative control), 0.090, 0.90, and 9.0 mg a.i./L; meanmeasured concentrations were <0.0032 (<LOQ, negative control), 0.099, 0.92, and 9.1 mg a.i./L. 

The test system was maintained at 23 to 25oC and a pH of 7.0 to 7.6. 

Overall mean survival values were 100% for both males and females in the negative control, 

mid, and low treatment groups; 2 males and 1 female died in the high treatment group (88% 

survival). There were changes in coloration and lethargy observed at the high treatment level. In 

addition, the reviewer noted the presence of dark coloration and partial/complete loss of 

equilibrium in males and females of the high treatment group. Male body weight exhibited 

significant reductions (p<0.05) of 22 and 26% at the mid and high treatment levels, respectively, 

compared to the negative control; and female body weight was significantly reduced (p<0.05) by 

29% at the high treatment level compared to the negative control. Female body length was 

significantly reduced (p<0.05) by 6% at the high treatment level, and male length was 

significantly reduced (p<0.05) by 8% at the mid treatment level compared to the negative 

control. 

In the negative control, spawning occurred at least every 4 days in three of the four control 

replicates; mean fecundity was 16 eggs/female/day/replicate (range 13-20 E/F/D); and 

fertilization success averaged 98%. There were no eggs available for assessment at the high 

treatment level; therefore, the high treatment level was excluded from data analysis for the 

endpoints of fecundity and fertility. There were significant reductions in fecundity and fertility 

(p<0.05) of 50% and 5%, respectively, at the mid treatment level. 

Male vitellogenin (VTG) and gonadal-somatic index (GSI) were significantly reduced (p<0.05) 

by 97 and 40%, respectively, at the high treatment level. Male tubercle score was also 

significantly reduced (p<0.05) at the high treatment level compared to the negative control. 

Tubercles were not observed in female fish. 

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Although not analyzed statistically, in male fish, there was an increase in incidence and severity 

of increased proportion of spermatogonia at the high treatment level compared to the negative 

control. In female fish, there were changes in gonadal staging, decreased yolk synthesis, and 

increased oocyte atresia of predominantly pre-vitellogenic follicles at the high treatment level 

compared to the negative control. Plasma sex steroids were not measured. 

Hershberger Assay; (OCSPP 890.1400) 

Requirement satisfied by OSRI (Ashby et al., 2004, Freyberger et al., 2007, Freyberger et al., 

2009, Kang et al., 2004, Lambright et al., 2000, Moon et al., 2009, Moon et al., 2010, and 

Tinwell et al., 2007); see Appendix 2. 

Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48685501), 15 Crl:CD(SD) Sprague-Dawley rats/dose group 

were treated daily via oral gavage with linuron technical (99.4% a.i., Lot # 1012A3876) in corn 

oil at doses of 0, 50 or 100 mg/kg/day from post-natal day (PND) 22 to 42 or 43. Animals were 

examined for vaginal opening (VO) daily beginning on PND 22, and the age and weight at day 

of attainment of VO was recorded. Following sacrifice on PND 42 or 43, blood was collected 

for clinical chemistry analyses, including total thyroxine (T4) and thyroid stimulating hormone 

(TSH), which were analyzed using an electrochemiluminescent immunoassay or 

radioimmunoassay, respectively. Kidney, liver, adrenal gland, thyroid, pituitary, uterus, and 

ovary weights were recorded, and microscopic examinations were performed on the thyroid, 

kidney, ovary, and uterus. 

All females survived to the scheduled euthanasia on Days 42 or 43. No test substance-related 

clinical findings were noted at either dose level at 3 hours post-dose or at the daily examinations 

(~24 hours post-dose). 

There were no significant differences in mean age at VO, body weight at VO, or final body 

weights in the treated groups relative to controls. There were no treatment-related effects on the 

weights of liver, adrenals, uterus (wet or blotted), or thyroid. There were no dose-related effects 

on cycle status at necropsy, mean age at first vaginal estrous, mean cycle length, percent of 

females cycling, or percent of females regularly cycling. No treatment-related microscopic 

lesions were observed on the thyroid, ovary, uterus, or kidney. Serum TSH was not affected by 

treatment. 

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At 100 mg/kg/day, mean body weights were slightly reduced (p≤0.05) by 5-8% for Days 26-29, 

31-39, and 43. Mean cumulative body weight gain was comparable to the controls at the end of 

the study. No effects on bodyweights were observed at 50 mg/kg/day. 

The unadjusted and adjusted kidney weights were decreased (p≤0.05) by 7%, the unadjusted, 

adjusted and relative pituitary weights were decreased (p≤0.05) by 20-22%, and the adjusted 

ovary weights were decreased (p≤0.05) by 12% compared to controls. At 50 mg/kg/day, the 

unadjusted, adjusted, and relative pituitary weights were decreased (p≤0.05) by 13-15%. 

There was a dose-dependent decrease (p≤0.05) in serum T4 levels of 27% and 48% at 50 and 100 

mg/kg/day, respectively. However, there was no associated change in TSH levels or in thyroid 

weight or histology. 

At 100 mg/kg/day, A/G ratio was increased (p≤0.05) by 13%, glucose was decreased (p≤0.05) by 

8%, sodium was decreased (p≤0.05) by 1%, total bilirubin was increased (p≤0.05) by 33%, and 

total protein was decreased (p≤0.05) by 5% compared to the controls. At 50 and 100 mg/kg/day, 

mean AST levels were decreased (p≤0.05) by 29% and 25%, bile acids were increased (p≤0.05) 

by 120% and 92%, mean globulin levels were decreased (p≤0.05) by 7% and 13%, and serum 

urea nitrogen levels were decreased (p≤0.05) by 7% and 13%, respectively. At 50 mg/kg/day, 

calcium was increased (p≤0.05) by 1.7%, chloride was decreased (p≤0.05) by 1%, and potassium 

was decreased (p≤0.05) by 7% compared to the controls. 

The dosing levels tested in this assay are adequate based on the results of a dose range-finding 

study showing body weight loss at 100 mg/kg/day. 

Male Pubertal Assay; (OCSPP 890.1500) 

Requirement satisfied by OSRI (USEPA 2007c and USEPA 2007d); see Appendix 2. 

Steroidogenesis Assay; (OCSPP 890.1550) 

Requirement satisfied by OSRI (Wilson et al., 2009); see Appendix 2. 

Uterotrophic Assay; (OCSPP 890.1600) 

In a uterotrophic assay (MRID 48617403) conducted to screen for potential estrogenic activity, 

linuron (99.4% a.i., Lot # 1012A3876) in corn oil was administered daily via oral gavage to 

groups of 6 ovariectomized Sprague-Dawley rats at dose levels of 0 (vehicle), 30, 60 and 120 

mg/kg/day on post-natal days (PND) 72-74. A positive control group was treated with 17αPage 49 of 101
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ethynyl estradiol (EE) by daily subcutaneous (s.c.) injection at a dose level of 0.003 mg/kg/day. 

All animals were terminated and necropsied approximately 24 hours after the final dose on PND 

75 to determine liver, and wet and blotted uterine weights. 

All animals survived until scheduled termination. Red material was observed around the nose at 

120 mg/kg/day on one day for 3/6 animals compared to 0/6 animals in the other groups. This 

finding remained in a single animal (same day) at 3 hours post-dose. Body weights were 

decreased in all linuron treated groups with significantly decreased body weights at 120 

mg/kg/day (↓10-12%, Days 3-4). Overall body weight gains were decreased (p<0.05) for the 30, 

60 and 120 mg/kg/day groups by 66, 117, and 192%, respectively. Uterine and liver weights in 

the linuron-treated groups were comparable to the controls. Absolute wet and blotted uterus 

weights for the positive EE group were increased (p<0.05) by 797% and 300%, respectively, as 

expected. The high dose tested in this study was adequate based on the observed decreases in 

body weights. 

No statistically significant changes were seen in uterine weight in this assay. Linuron is negative 

in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of linuron with the endocrine system are summarized below. Given the 

breadth of toxicity data available on linuron, the only Part 158 studies discussed in this appendix 

are those that fulfill current guideline requirements and are directly relevant to assessing 

potential endocrine effects (i.e., multiple-generation rodent reproductive studies, development 

studies, chronic toxicity studies, etc.). 

AR Binding 

In a published, multi-component study, Cook et al. (1993) examined the interaction of linuron 

(purity 97%) with the AR. In this study, linuron and four of its metabolites, 1-(3,4-

dichlorphenyl)-3-methoxyurea, 3,4-dichloroanaline, 3,4-dichlorophenylurea; and 1-(3,4-

dichlorophenyl)-3-methylurea), were evaluated for their ability to compete in vitro for binding to 

the androgen receptor. Ventral prostate cytosol was prepared using tissue from rats castrated 24 h 

prior to harvest. The cytosol prep was incubated with [3H]-testosterone in the absence or 

presence of increasing concentrations of the unlabeled ligand. From these data, the concentration 

of ligand which produced 50% displacement (IC50) of [3

H]-testosterone from the androgen 

receptor was calculated. 

The calculated IC50 values were as follows: linuron, 64, 000 nM; l-(3,4-dichlorophenyl)-3-

methoxyurea, 120,000 nM; 3,4-dichloroanaline, 110,000 nM; and l-(3,4-dichlorophenyl)-3-

methylurea, 260,000 nM. The IC50 values for DHT and flutamide were 1.4 nM and 18,000 nM, 

respectively. 

AR Binding 

In a published, multi-component study, Lambright et al. (2000) examined the interaction of 

linuron (purity 99.8%) with the AR binding domain using a rat prostate competitive binding 

assay and a COS whole cell hAR binding assay. 

For the rat prostate cytosol assay, ventral prostate tissue was obtained from 90-day old rats 24 h 

after castration. In four separate replicates, linuron (0, 0.312, 1.0, 3.12, 10.0, 31.2, 100 or 

312 μM) was incubated with pooled prostate homogenate in the presence of [3

H]-R1881 and 

triamicinolone acetate. Nonspecific binding was assessed with 100-fold molar excess of 

unlabelled R1881. Following incubation, ligand-bound receptor was separated from unbound, 

and quantified by liquid scintillation counting. The EC50 of linuron in this assay was ≈200 μM. 

No other data were provided. 

For the whole cell hAR binding assay, COS cells were transiently transfected with the human 

AR expression vector pCMVhAR. Cells were exposed to 5 nM [3

H]-R1881 and linuron (0, 0.5, 

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1.0, 5.0, 10, 15 or 20 μM) 24 h after transfection. Nonspecific binding was assessed with 100-

fold molar excess of unlabeled R1881. Following incubation, the radioactivity of the lysate was 

determined by liquid scintillation counting. The assay was replicated a total of four times. 

Linuron inhibited R1881 AR binding by about 25% at 5 μM (p<0.05) with an EC50 of ≈20 μM. 

No other data were provided. 

AR Binding 

The USEPA (2007a) report summarized data from the validation program for the Tier 1 AR 

binding assay. Linuron (99-99.5% purity) was included in the competitive binding assays 

conducted by the lead laboratory and in the main interlaboratory validation study. In the 

competitive binding assays using the two-parameter model, the mean IC50 for the standard curve 

was 9.42 x 10−10 M. Linuron was analyzed in duplicate at concentrations of 10−8

 to 10−3 M. The 

mean IC50 for linuron was 80,302 ± 14,938.3 nM (−4.10 log M), and the mean IC50 for R1881 

was 0.99 nM (−9.00 log M), for an RBA of 0.001%. The R2 value for the individual curve fit 

was 0.997. 

In the interlaboratory validation studies, three independent runs were conducted by each 

laboratory with a standard, a weak positive control (dexamethasone), and each test chemical; 

each concentration was analyzed in triplicate. Linuron was investigated at concentrations of 

10−10 to 10−3 M. Data were analyzed using the two-parameter model and re-analyzed using the 

four-parameter model. For the four laboratories, the mean log IC50 for the standard curve was 

−8.624 log M, and the mean log IC50 for the dexamethasone was −4.183 log M; RBAs ranged 

0.0004-0.0100%, with a median value of 0.0039%. For linuron, mean log IC50 values from the 

four laboratories ranged −4.05 to −3.85 log M, mean R2 values ranged 0.68-0.97, and log RBAs 

ranged −4.79 to −4.22. Using the four-parameter model, mean log IC50 values for the reference 

standard and dexamethasone were −8.775 and −4.352 log M, respectively; the mean log RBA 

was −4.386. For linuron, the mean log IC50 value was −4.043 log Mand the mean log RBA was 

−4.688. The average curve across runs crossed 50% for all analyses, confirming linuron as an 

AR binder. The authors noted that one run of linuron from each of two laboratories either did not 

converge or produced a poor fit to the standard curve (R2 value of 0.15); however, the other two 

runs in both laboratories had R2

 values >0.5. 

AR Binding 

In a published study by Wilson et al. (2004), the in vitro binding of linuron (99.8% purity) 

fathead minnow androgen receptor was investigated. COS-1 African green monkey kidney cells 

were transiently transfected with a vector containing either fathead minnow AR or hAR for 

comparison. Twenty-four h post-transfection, the cells were incubated with [3

H]-R1881 in the 

presence of increasing concentrations of linuron. Linuron was found to have an IC50 of 

approximately 42 μM (−4.38 log M) in this study. 

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In a similar published study by Wilson et al. (2007), the in vitro binding of linuron (99.8% 

purity) to androgen receptors from fathead minnow, rainbow trout, and humans was investigated. 

COS-1 African green monkey kidney cells were transiently transfected with vectors containing 

either fathead minnow AR, rainbow trout AR, or hAR. Twenty-four h post-transfection, the cells 

were incubated with [3H]-R1881 in the presence of increasing concentrations of linuron. 

Linuron was found to have log EC50 of −4.71 log M for hAR, −4.28 log M for rainbow trout AR, 

and −4.41 log M for fathead minnow AR. The relative binding affinities for linuron for all three 

receptors was <0.009. 

Androgen Receptor Transactivation Assays (ARTA)

In a multi-component study, Lambright et al. (2000) investigated the effects of linuron (purity 

99.8%) on two in vitro transcriptional activation assays, a CV-1 cell hAR transcriptional 

activation assay and an MDA-MB-45J-KB2 cell hAR transcriptional activation assay. In the CV1 cell hAR transcriptional activation assay, CV-1 cells were transiently transfected with the 

pCMVhAR vector and an MMTV-luciferase reporter gene. Twenty-four and 48 hours after 

transfection, cells were exposed to DHT and linuron (0, 0.5, 1.0, 5, 10, 15 or 20 μM). Five to six 

hours after exposure, cells were harvested and luciferase activity was determined using a 

luminometer. The assay was replicated a total of four times. Linuron inhibited DHT-induced 

transcriptional activation at doses as low as 10 μM, which was also the EC50 value. It was not 

clear whether the data came from the exposure which occurred 24 or 48 hours after transfection. 

For the MDA-MB-45J-KB2 cell hAR transcriptional activation assay, MDA-MB-453-KB2 cells, 

which contain endogenous hAR, stably transfected with the MMTV.neo.luciferase gene, were 

exposed to 0.1 nM DHT and linuron (0, 1, 5, 10, 15, or 20 μM in ethanol). The cells were 

incubated for 24 h and the cells harvested. Reactive light units were determined using a 

luminometer. Linuron inhibited DHT-induced luciferase expression at 5 μM and above with an 

EC50 of approximately 10 μM. 

In another published ARTA study by Freyberger et al. (2010), the androgen-sensitive PALM 

cell line (human prostate cancer cell line PC-3 stably transfected with hAR and a luciferase gene 

under the control of mouse mammary tumor virus [MMTV]) was validated for its ability to 

detect androgen agonists and antagonists. Several known androgen agonists and antagonists, 

including linuron (99.7% purity), were tested. Linuron was shown to be a weak antagonist in the 

assay when tested with 0.2 nM methyldihydrotestosterone at concentrations ranging up to 10 

μM. No data were provided. It was stated that most of the antagonists were only weakly active, 

and that the highest concentration of 10 μM was too low to consistently produce a sufficiently 

complete concentration response curve suitable for robust EC50 estimation. 

In a multi-component study by McIntyre et al. (2000), the ability of linuron to activate 

transcription through the human androgen receptor (AR) in vitro was also investigated using a 

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reporter gene assay. Human hepatoma HepG2 cells were transfected with three plasmids: 1) 

human AR; 2) an androgen-responsive MMTV-luciferase (luc) reporter plasmid; and 3) pCMV 

32 β-galactosidase (β-gal) as an internal control for evaluating transfection efficacy and 

cytotoxicity. The transfected cells were rinsed with phosphate-buffered saline (PBS), then treated 

with DMSO (control); DHT at 10−11 to 10−5 M; linuron (99% purity) at 10−7 to 10−5 M; or DHT 

plus linuron at the concentrations listed. The cells were incubated for 24 hours, then rinsed, 

lysed, and assayed for luciferase and β-galactosidase activities. Luciferase activity was 

normalized to β-galactosidase activity. 

The results of the in vitro study indicated that linuron had no detectable AR agonist activity but 

is a competitive AR antagonist. At dosages of 10−6

, 3 × 10−6, and 10−5 M, linuron competitively 

antagonized transcriptional activity induced by DHT in a dose-dependent manner, with a Kd of 

75.8 × 10−8 M. On the basis of these results, linuron was classified as a weak competitive AR 

antagonist. 

ER Transcriptional Activation (ERTA) Assays 

In a published study by Gordon et al. (2004), human ovarian carcinoma (BG-1) cells were 

stably transfected with an estrogen-responsive luciferase reporter gene plasmid (pGudLuc7ere) 

containing the estrogen responsive element (ERE) and luciferase reporter gene to create the 

BG1Luc4E2 cell line (LUMI-CELLTM ER bioassay). This cell line responds to exposure to 

estrogen and estrogenic chemicals in a time-, dose dependent-, and chemical-specific manner 

with the induction of the luciferase gene expression. BG1Luc4E2 cells were grown, plated, and 

incubated for 24-48 h prior to treatment. For dosing, medium containing the test substance 

(dissolved in DMSO) was added to each well (concentrations not specified), and the plate was 

incubated for 24 hours. The cells were then lysed, and the luciferase activity was measured using 

a luminometer. The relative light units (RLUs) for each test substance were compared to those 

induced by the reference standard 17β-estradiol after subtraction of background radioactivity. 

Each compound was tested at least three times on three different sets of plates, and the EC50

value was determined. 

The bioassay yielded an EC50 value of 1.45 x 10−5

 M for linuron, with a relative induction factor 

of 1.07 x 10−6 compared to 17β-estradiol (1.55×10-11 M). 

Receptor Transcriptional Activation 

In a published study by Kojima et al. (2004), 200 pesticides (including linuron; purity 95-100%) 

were evaluated for their potential to interact with human ERα, ERβ and AR using Chinese 

hamster ovary (CHO-K1) cells transfected with the appropriate plasmid. It was reported that the 

chemicals were tested at non-cytotoxic concentrations, but no data were provided. 

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Linuron was negative for ERα, ERβ, and AR transcriptional activation in this test system. No 

data were presented for negative findings. Linuron did exhibit weak anti-androgenic activity, 

with a RIC20 (relative inhibitory concentration to 20% of the activity invoked by 10−10 M 

dihydrotestosterone; DHT) of 2.0 × 10−4

 M and a relative luciferase activity (RLA) of 37% 

compared to DHT (100%). 

Yeast Estrogen Screen (YES), Yeast Androgen Screen (YAS), and Frog Ovulatory 

Response 

In a published study by Orton et al. (2011), 12 environmental chemicals (including linuron 

purity >97%) were screened for estrogen and androgen agonist/antagonist activities using 

recombinant yeast assays (YES and YAS), and a frog (Xenopus laevis) ovulatory response assay. 

The yeast assays used E2 or testosterone as agonists. For the ovulatory response assay, sexually 

mature frogs were anesthetized and euthanized, and the ovaries removed. The ovaries were then 

cut into 10 oocyte fragments, and two fragments were cultured per well in 24-well plates for 20 

h. The medium was removed and the number of ovulated oocytes was counted. A submaximal 

concentration (≈60% of maximal response) of human chorionic gonadotropin (hCG) was 

incubated in conjunction with the pesticides. Medium concentrations of estrogen, testosterone, 

and progesterone were also measured. Linuron was tested at 0.008-15.6 μM in the yeast assays, 

and at 6.25 μM and 62.5 μM in the ovulatory response assay. 

It was stated that none of the chemical tested were agonists in either the YES or YAS assays. 

Linuron demonstrated anti-estrogenic activity at concentrations of 1.9-31.3 μM in the YES 

assay, and anti-androgenic activity at concentrations of 0.98-62.5 μM in the YAS assay. In the 

ovulation assay, 62.5 μM linuron exposure caused an increase (p<0.05) in progesterone (↑70%), 

a slight decrease (NS) in testosterone (↓12%) and E2 (↓8%), and reduced (NS) percent ovulation 

(15.0% treated vs. 93.5% controls). 

ERTA/Cell Proliferation 

In a published study by Vinggaard et al. (1999), 20 pesticides, including linuron (purity 97-

99.9%), were screened for their ability to activate ER in two in vitro assays: induction of cell 

proliferation in the estrogen-responsive human breast carcinoma cell line MCF-7; and response 

of yeast cells (Saccharomyces cerevisiae) stably transfected with the hERα and a reporter 

plasmid encoding for β-galactosidase (Yeast Estrogen Screen; YES). Linuron was tested at a

single concentration of 10 μM in the MCF-7 cell proliferation assay and from 0.2 to 500 μM in 

the YES. Linuron was reported to be negative in both assays (data only reported for MCF-7 

assay results). 

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Fetal Testosterone (Steroidogenesis) Production

In a published study by Wilson et al. (2009), the effects of linuron exposure in utero and in vitro

on fetal testis testosterone production and gene expression were investigated. Time-mated 

pregnant Sprague-Dawley rats were dosed with linuron (99.8% purity) in corn oil by daily oral 

gavage (dose volume 2.5 mL/kg) at dose levels of 0, 12.5, 25, 50, or 75 mg/kg/day from 

gestation day (GD) 13-18. On GD 18, approximately one hour after administration of the final 

dose, dams were anesthetized with CO2 and exsanguinated. Fetal testes were collected and 

incubated for 3 hours in medium; the medium was then collected and analyzed for testosterone 

and progesterone levels. Fetal testes from the male offspring were also examined for gene 

expression of insulin-like peptide 3 (insl3) along with several enzymes in the steroidogenic 

pathway (cyp17a, StAR, and cyp11a) using real-time PCR. 

Additionally, the direct effect of linuron on testosterone and progesterone production by fetal 

testes was evaluated in vitro. Untreated, pregnant Sprague-Dawley dams were euthanized and 

necropsied on GD 18, and the testes were collected from all male fetuses and incubated as 

previously described. In the first experiment, the testes were exposed to increasing 

concentrations of linuron: 0 (ethanol vehicle), 1, 3, 10, 30, 100, or 300 μM. In the second 

experiment, the testes were exposed to 1-(3,4-dichlorophenyl) urea (DCPU), a linuron 

metabolite, at the same concentrations used in the first experiment; a group exposed to 30 μM 

linuron was also included. Ketoconazole (3 μM), a known cyp17 inhibitor, was used as a 

positive control group in both experiments. Medium was collected following 3 hours incubation 

for testosterone and progesterone analysis. 

During the in utero exposure, there were no significant differences between groups in maternal 

survival, maternal body weight, litter loss, or litter size. While all the dams gained weight, body 

weight gains were generally dose-dependently decreased (not significant [NS]) at 12.5 (↓21%), 

25 (↓44%), 50 (↓78%), and 75 (↓71%) mg/kg. When examined on an individual fetal basis, ex 

vivo fetal testes testosterone production was significantly decreased in all treated groups. 

However, when evaluated on a litter basis, ex vivo testosterone production was significantly 

reduced (p<0.01) only at 50 and 75 mg/kg (↓≈25%). Progesterone production in the treated 

groups was not different from controls, indicating that linuron directly inhibited testosterone 

production in the absence of overt cytotoxicity. The mRNA levels of insl3, cyp17a, StAR, and 

cyp11a in the linuron-treated groups were comparable to controls. 

Testosterone production from the fetal testes was decreased (p<0.05) after in vitro exposure to 

linuron at 30 μM and above and to DCPU at 300 μM. In contrast, there were no effects of 

treatment on progesterone production in either experiment, indicating that linuron can directly 

inhibit testosterone production in the absence of cytotoxicity. Ketoconazole resulted in 

significantly decreased (p<0.0001) testosterone production, demonstrating the expected response 

to the positive control in both experiments. 

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Thyroid Receptor Transcriptional Activation Assays

In a published study by Hoffman et al. (2009) linuron (purity 97%) was tested for hormone 

agonism and antagonism in a thyroid hormone responsive luciferase-based reporter gene assay. 

Human hepatoma HepG2 cells were transfected with T3-responsive element (TRE) reporter 

plasmid [p(DR4)2-SV40-Iuc+] and either a TRα1 or a TRβ1 expression plasmid (pRS-TRα1 or 

pRS-TRβ1). Test chemicals, including the natural ligand T3 and linuron, were tested for TR 

agonism or antagonism in vitro. Linuron was tested at concentrations from 10−11 to 10−5

 M. 

Linuron was reported to be positive with an LOEC of 1 μM. Linuron gave a 1.7 fold induction in 

the agonist assay and only a 1.2 fold induction in the antagonist assay. 

Thyroid Toxicity 

In an published study, Seidlova-Wuttke et al. (2005), tested five chemicals, including linuron 

(purity not reported), for effects on serum lipids and pituitary hormones. Female Sprague 

Dawley rats were ovariectomized at 3 months of age, weighed, and randomly assigned to 

experimental groups (n=12). The test compounds were administered daily with the food for 12 

weeks. The following test groups were established: an ovx control; E2 at 1.2 mg/kg/day (327 

μg/animal/day) as the positive control; and the five endocrine active test substances (including 

linuron) at low and high doses. Linuron was administered at nominal doses of 16.3 and 65.5 

mg/kg/day (4.4 and 17.7 mg/animal/day). 

Prior to the beginning of dosing and at 12 weeks, the animals were anaesthetized, and the size of 

the paratibial fat deposit (lower hind leg) was recorded using quantitative computer tomography. 

Following the last scan, the animals were sacrificed by decapitation, and blood was collected 

from the trunk. The following serum parameters were measured: serum lipids including leptin, 

triglycerides, and low and high density lipoproteins (LDL and HDL); and pituitary hormones 

including LH, TSH, T4, and T3. Serum LH, TSH, leptin, and thyroid hormones were measured 

using radioimmunoassay kits, and serum cholesterol, HDL, LDL and triglycerides were 

measured using commercially available kits and a Hitachi autoanalyzer. 

No mortality was reported, and all animals were reported to be behaviorally and physically 

normal. When compared to the ovx control, E2 decreased water and food intake resulting in 

lower (p<0.05) body weight gain by the end of the study. For linuron, water consumption was 

increased (p<0.05) in the 16.3 (↑8%) and 65.5 (↑14%) mg/kg/day groups; food consumption was 

reduced in the 16.3 (↓7%; NS) mg/kg group and the 65.5 (↓26%; p<0.05) mg/kg/day group; and 

bodyweight gains were reduced (p<0.05) in the 16.3 (↓21%) and 65.5 (↓61%) mg/kg/day groups. 

E2 completely prevented the increase in the paratibial fat depot observed in ovx controls, and 

linuron prevented increased fat accumulation in the 65.5 mg/kg group. For the serum lipids, 

treatment with E2 produced the lowest serum leptin and cholesterol concentrations, and reduced 

(p<0.05) LDL and HDL; serum triglyceride concentrations increased. For linuron, serum leptin 

concentrations were dose-dependently reduced (p<0.05) , and serum cholesterol, LDL, and 

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triglyceride concentrations were reduced (p<0.05) at 65.5 mg/kg; serum HDL concentrations 

were increased (p<0.05) at 16.3 mg/kg. For the pituitary hormones, serum TSH levels were not 

significantly affected by any of the test substances, although treatment with E2 showed a 

tendency for increased TSH. Serum T4 levels were unaffected by E2, but T3 and LH levels were 

reduced (p<0.05). Dose-dependent reduction of serum T4 levels was observed for linuron 

(p<0.05 for the high dose), T3 levels were reduced (p<0.05) in both dose groups, and LH levels 

were reduced (p<0.05) in the 65.5 mg/kg group. 

Results for the pituitary hormones from in vivo testing suggest that linuron may have a direct 

effect within the thyroid gland as serum TSH levels were not affected, but a dose-dependent 

reduction was seen for T3 and T4. The decrease in thyroid hormone production also suggested 

that linuron may have additional effects in the HTP axis. 

Hershberger Assay

In a published study by Ashby et al. (2004), the anti-androgenic effects of linuron were 

compared in a standard castrated adult male Hershberger assay and an intact immature male 

assay. The castrated rats (Alpk:APfSD; 4-6 rats/group) used in the Hershberger bioassay studies 

were 6 weeks old. Rats used in the stimulated weanling studies were PND 21-22. Testosterone 

proprionate (TP), the stimulating androgen, was administered by s.c. injection at 0.5 mL/kg to 

the castrates or by gavage at 2 mL/kg to the weanlings. Rats were given 10 consecutive doses of 

TP, or 10 consecutive doses of the synthetic androgen 17-methyltestosterone (17MT), and a coadministered second oral dose of the chemical under investigation (linuron), or the positive 

control anti-androgen flutamide. Preliminary weanling studies investigated three doses of TP 

(0.4, 1, or 2 mg/kg/day) and two doses of finasteride (1 or 25 mg/kg/day) or flutamide 

(3 mg/kg/day); subsequent weanling studies used TP at 1 mg/kg/day. Studies were terminated 24 

hours after the last day of treatment. Rats were euthanized by cervical dislocation under 

anesthesia. At necropsy, the following tissues were removed and weighed: liver, kidneys, 

Cowper’s glands (CG), levator ani and bulbocavernosus muscles (LABC), seminal vesicles (SV) 

with coagulating glands, and prostate. In addition, the adrenal glands were removed from the 

Hershberger and 17MT weanling studies, the glans penis (GP) from the Hershberger studies, and 

the testes and epididymides from the weanling assays. 

Linuron (purity not reported) suspended in tocopherol-stripped corn oil was administered by oral 

gavage (dose volume 5 mL/kg) at doses of 3, 10, 30, or 100 mg/kg/day for 10 days with 

1 mg/kg/day of TP. Linuron caused a 13-29% decrease (p<0.05) in CG weights at 

≥30 mg/kg/day, and caused decreases (p<0.05) in the following accessory sex tissue (AST) 

weights at 100 mg/kg/day: epididymides (↓19%); SV (↓25%); and prostate (↓34%). At 100 

mg/kg/day, linuron also caused a 31% increase (p<0.05) in testes weight. Linuron increased 

(p<0.05) liver weights at 30 mg/kg/day and above (↑2-10%) but these minor increases were not 

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dose-dependent. Administration of the anti-androgen flutamide at 3 mg/kg/day caused similar 

changes in these tissues that were greater in magnitude. 

Hershberger Assays 

In a published study (Freyberger et al.2007) to validate the OECD Phase 3 Hershberger 

bioassay for anti-androgenic potential, male Wistar rats (Hsd/Cpb:WU) were castrated at PND 

40, acclimated for one week, and randomly assigned to the experimental groups (6 rats/group). 

Test compounds, including linuron (purity 99.7%), were formulated in corn oil and administered 

for 10 consecutive days using dosing volumes of 0.5 mL/kg for s.c. dosing of TP and 5 mL/kg 

for oral gavage dosing of the remaining compounds. Linuron was not tested for androgenic 

potential. To test for anti-androgenicity, castrated rats were supplemented with TP at 0.4 

mg/kg/day and administered with the test compounds. The study included the following dose 

groups: 1) corn oil as the vehicle control; 2) a positive control of flutamide (FLUT; 3 mg/kg); 3) 

negative controls of 4-nonylphenol (NP; 160 mg/kg) and 2,4-dinitrophenol (DNP; 10 mg/kg); 

and 4) linuron (10 and 100 mg/kg). The dose levels for linuron were selected to represent a no or 

low effect level and a clear effect level, respectively. One day after the last treatment, the rats 

were anaesthetized and sacrificed by exsanguination. The liver and the following AST were 

collected, trimmed of fat and adjacent tissue, and weighed: ventral prostate (VP), SV, LABC, 

CG, and GP. The liver and VP were immediately frozen until further analysis. Gene expression 

analysis in the prostate was conducted using Taqman® Real-time polymerase chain reaction 

analysis according to a published method, using primers designed for ODC1 and PBPC3, and 

using 18S rRNA as an internal control. 

No clinical signs, mortalities or effects on food intake were observed for linuron during the 

study. Decreased body weight gain (↓31%) and final body weight (↓5%) relative to the TPcontrol were observed for linuron at 100 mg/kg; however, these changes were not statistically 

significant. When compared to TP-control, the high dose of linuron significantly (p<0.01) 

decreased the weight (relative to body weight) of all androgen-sensitive tissues (↓29-54%) 

except GP; relative liver weights were not affected by treatment. The positive control, FLUT, 

also decreased all relative AST weights (↓21-56%). No effects were observed for the negative 

controls NP and DNP on body weights or organ or tissue weights. In the androgen-responsive 

gene expression analysis, the high dose of linuron and FLUT slightly but significantly (p≤0.5) 

increased ODC1 expression, and the low dose of linuron increased PBPC3 expression (p≤0.01). 

In a published study (Freyberger et al. 2009) to validate the Hershberger bioassay using 

juvenile intact male (stimulated weaning) rats, male Wistar (Hsd/Cpb:WU) pups at PND 19 were 

acclimated for three days and randomly assigned to the control and treatment groups (6 

rats/group). For dosing, the test compounds, including linuron (purity 99.7%), were formulated 

in corn oil, and administered for 10 consecutive days using dosing volumes of 1 mL/kg for s.c. 

dosing of TP and 5 mL/kg for oral gavage for the remaining compounds. To assess antiPage 59 of 101
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androgenic potential, intact rats were supplemented with TP at 1 mg/kg/day and administered the 

other test compounds. The study included the following dose groups: 1) corn oil (vehicle 

control); 2) TP only at 1 mg/kg at the control, 3) FLUT at 3 mg/kg plus TP (positive control); 4) 

NP at 160 mg/kg plus TP (negative control); 5) DNP at 10 mg/kg plus TP (negative control); and 

5) linuron (99% purity) at 10 or 100 mg/kg plus TP. The linuron dose levels were expected to 

result in a no/low effect level and a clear effect level, respectively. One day after the last 

treatment, the rats were anaesthetized and sacrificed by exsanguination. The liver and the 

following AST were collected, trimmed of fat and adjacent tissue, and weighed: testes, 

epididymides, VP, SV, LABC, and CG. Liver samples were immediately frozen until further 

analysis. 

No mortality was observed through the treatment period. When compared with the vehicle-only 

control, administration of TP alone resulted in slightly increased body weight gain and final body 

weight (NS). TP treatment increased absolute weights for the VP, SV, LABC, CG and 

epididymides, and decreased relative testes weights (p<0.01). Liver weights were not affected by 

treatment. Relative organ and tissue weights were not reported. The positive control, FLUT, 

significantly (p<0.01) decreased all absolute and relative AST weights (↓32-64%) and increased 

(p<0.05) testicular weight (↑45%) compared to the TP-control. The negative control NP 

decreased absolute but not relative SV weights compared to the TP-control. No effects of 

treatment were observed for the DNP negative control. 

For the 100 mg/kg linuron group, clinical signs (digging and cleaning movements and salivation) 

were observed. Body weight gain (↓8%) and terminal body weights (↓12%) were decreased (NS) 

compared to the TP-control; this dose also resulted in decreased (p<0.05) absolute SV and 

LABC weights compared to the TP-control; relative weight was decreased (p<0.05) for SV 

(↓26%). Testicular weights were not affected at this dose. No effects of treatment were noted at 

10 mg/kg of linuron. 

Hershberger Assay 

In a published Hershberger study (Kang et al. 2004), groups of six castrated S-D male rats (4-

weeks old) were administered linuron (99% purity) in corn oil via oral gavage (dose volume 4 

mL/kg) at 0, 25, 50 or 100 mg/kg/day for 10 consecutive days along with TP via s.c. injection 

(dose volume 0.5 mL/kg) at 0.4 mg/kg. The positive agonist control group received only TP 

injections, while the test groups were also orally dosed with flutamide at 1, 5, 10, or 20 

mg/kg/day as a positive antagonist control, or linuron. One day after final dosing, rats were 

euthanized. Blood was collected from the abdominal aorta and serum was prepared immediately 

for measurement of testosterone and LH concentrations. The AST (VP, SV with coagulating 

glands, LABC, CG, and GP), plus the liver, adrenals, and kidneys were excised and weighed. 

Administration of TP produced increases (p<0.05) in all collected AST weights, an increase 

(p<0.05) in serum testosterone, and a decrease (p<0.05) in serum LH compared to the vehicle 

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control. Dosing with flutamide inhibited (p<0.05) the TP-induced re-growth of all AST in a 

dose-dependent manner. Weights decreased (p<0.05) for SV, VP, and LABC starting at the 1.0 

mg/kg dose, and for CG and GP starting at the 5.0 mg/kg dose. Dosing with flutamide also 

caused an increase (p<0.05) in serum LH levels compared to the TP control, but no changes in 

serum testosterone were observed. 

Following administration of linuron, body weights and liver and kidney weights were unaffected, 

but adrenal weights were increased (↑14%; p<0.05) at 100 mg/kg/day. Linuron inhibited 

(p<0.05) TP-induced increases in the relative weights of the AST in a dose-dependent manner. 

At 50 mg/kg, weights were decreased (p<0.05) for SV (↓28%), VP (↓25%) and CG (↓26%). At 

100 mg/kg, weights were decreased (p<0.05) for SV (↓47%), VP (↓38%), CG (↓39%), LABC 

(↓35%) and GP (↓20%). No significant differences were observed for serum testosterone or LH 

concentrations in linuron-treated rats. 

Hershberger Assay 

In a published, multi-component study by Lambright et al. (2000), the potential anti-androgenic 

effects of linuron (purity 99.8%) in vivo were investigated using a Hershberger assay. At PND 

27, groups of 5-7 castrated Sprague Dawley rats were treated for seven consecutive days with 

50 μg/day testosterone propionate (TP) via s.c injection, TP plus 100 mg/kg/day linuron in corn 

oil via oral gavage, or TP and corn oil alone. On the day after the last treatment, males were 

anesthetized with CO2 and body weight was recorded. Rats were euthanized by decapitation; 

body weight and weights of the SV plus coagulating gland (with and without fluid), VP, LABC, 

liver, kidney, adrenal, and pituitary were recorded. 

Administration of linuron reduced (p<0.05) body weight gains (↓16%), and the weights of SV 

(↓44%), VP (↓37%) and LABC (↓22%). Adrenal weights were increased (p<0.05) by 39%. 

Hershberger Assays 

In a published study by Moon et al. (2009) to validate the OECD Phase 3 Hershberger bioassay, 

immature PND 35 male Sprague-Dawley (Crl:CD (SD)) rats were castrated on PND 44, allowed 

to recover for 8 days, then randomized into separate experimental groups (6 rats/group) to obtain 

similar mean body weights between groups. Separate experiments were conducted to investigate 

androgen agonists and antagonists, but linuron was only included in the antagonistic test. The 

test substances (purity 99%) were formulated in corn oil for dosing, and the rats were dosed daily 

for 10 consecutive days via s.c. injection for TP and via oral gavage for the remaining 

compounds; dosing volumes were 0.5 mL/kg for s.c. injection and 5 mL/kg for oral 

administration. A vehicle control group received only corn oil, and all other groups received 

daily subcutaneous injections of TP at 0.4 mg/kg either alone or with another test compound. 

The androgen antagonist study included the following dose groups: TP only (positive agonist 

control); FLUT at 3 mg/kg/day (positive antagonist control); NP at 160 mg/kg/day and DNP at 

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10 mg/kg/day (negative controls); and linuron (purity not reported) at 10 or 100 mg/kg/day. 

Approximately 24 hours after the last dose, rats were anaesthetized and sacrificed by 

exsanguination. The following AST were excised and weighed: VP, SV with coagulating gland, 

LABC, CG, and GP. Liver, kidneys, and adrenal glands were also collected and weighed. 

No significant changes in body, liver, adrenal, or kidney weights were observed for any of the 

treatment groups. For the TP control, AST weights were increased (p<0.05) compared to those 

from the vehicle control. When compared to the TP-stimulated organs, FLUT inhibited (p<0.05) 

TP-induced increases in the weights (relative to body) of all collected AST (VP, SV, LABC, CG, 

and GP). For the negative reference compounds, decreases in SV and LABC relative weights 

were observed for NP, and the relative weight of VP was reduced with DNP. The study authors 

considered these to be false positive results, because no weight changes were observed in rats 

given NP or DNP in the related agonistic test. 

At 10 and 100 mg/kg, linuron inhibited (p<0.05) TP-induced increases in the relative weights of 

the five AST. Compared to the TP-control, linuron at 10 and 100 mg/kg decreased relative 

weights of VP (↓40% and ↓68%, respectively), SVCG (↓48% and ↓71%), LABC (↓28% 

and↓50%), CG (↓32% and ↓55%) and GP (↓12% and ↓19%). At the high dose, linuron 

demonstrated stronger antagonistic activities than were expected by the investigators based on 

previous Phase 2 validation studies. 

In another published study by Moon et al. (2010) to validate the stimulated weanling male assay 

as part of the Hershberger assay, Sprague-Dawley Crl:CD (SD) rats were used for testing in all 

stages. Pregnant dams were allowed to deliver pups naturally, and litters were culled to 10 pups 

and weaned at PND 20 for use in the study; for the remaining stages, immature rats (PND 20) 

were obtained. Rats were randomized by body weight on PND 22 into separate experimental 

groups (6 rats/group) to obtain similar mean body weights between groups. Stage 1 testing was 

conducted to determine the appropriate level of TP to use as a stimulatory dose in subsequent 

anti-androgen studies. In Stage 2, the selected dose of TP was administered with different levels 

of FLUT to determine the optimum dose of FLUT as the reference anti-androgen. Linuron was 

used in Stage 3 testing to assess the capability of the stimulated weanling model to detect a 

known androgen agonist and antagonists. In Stage 4, linuron (99% purity) and other reference 

compounds were blind tested. 

The test substances were formulated in corn oil. Groups were dosed daily for 10 consecutive 

days via s.c. injection for TP and via oral gavage for the remaining compounds; dosing volumes 

were 3 mL/kg for s.c. injection and 5 mL/kg for oral administration. A vehicle control group 

(corn oil only) was included with each stage testing; for Stage 3 and 4 testing, all other groups 

received daily s.c. injections of TP at 1.0 mg/kg. Test groups for Stage 3 included: TP only 

(positive agonist control); FLUT at 3 mg/kg/day (positive antagonist control); and linuron at 10, 

30, or 100 mg/kg/day. Test groups for Stage 4 included: TP only; FLU at 3 mg/kg/day; negative 

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controls of NP at 160 mg/kg/day and DNP at 10 mg/kg/day; and linuron at 10 and 100 

mg/kg/day. Approximately 24 hours after the last dose, rats were anaesthetized and sacrificed by 

exsanguination. The following four AST were excised and weighed: VP, SV with coagulating 

glands, LABC, and CG; testes and epididymides were also weighed. GP was not evaluated as the 

weanling males did not achieve preputial separation by the time of necropsy. In addition, liver, 

paired kidneys and adrenal glands were collected and weighed. 

In the Stage 3 testing, TP alone significantly (p<0.05) increased AST and epididymides weights 

compared to the vehicle control; testes weights were significantly decreased. When compared to 

the relative weights (relative to body) of the TP-stimulated organs, FLUT inhibited (p<0.05) TPinduced increases in the relative weights of all AST and the epididymides, and increased 

(p<0.05) testes weights. 

Results of Stage 3 testing indicate that, compared to the TP-stimulated only group, linuron at 100 

mg/kg decreased (p<0.05) the relative weights of all four AST (VP ↓22%, SV ↓25%, LABC 

↓21%, CG ↓26%) and the epididymides (↓17%). No significant changes were observed for AST 

weights at 10 or 30 mg/kg, and no changes were observed for testes weights at 10, 30, or 100 

mg/kg linuron. 

In the Stage 4 testing, TP alone increased (p<0.05) AST and epididymides weights compared to 

those from the corn oil-treated controls; testes weights were decreased (p<0.05). When compared 

to the relative weights of the TP-stimulated organs, FLUT inhibited (p<0.05) TP-induced 

increases in the relative weights of all collected AST and the epididymides, and increased 

(p<0.05) testes weights. No significant changes in the AST or testes/epididymides weights 

resulted from dosing with NP or DNP compared with the results from the TP-stimulated rats. 

Results of Stage 4 testing indicate that linuron at 100 mg/kg significantly (p<0.05) inhibited TPinduced increases in the relative weights of the four AST. Percent differences from linuron 

dosing compared to the TP-stimulated only organ weights were ↓23%, ↓48%, ↓27%, and ↓34%, 

respectively, for the VP, SVCG, LABC, and CG. Testes weights were not significantly changed, 

but the epididymides weights were significantly decreased (↓21%) by linuron at 100 mg/kg. At 

10 mg/kg, linuron did not significantly affect the AST and/or testes/epididymides weights. 

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Hershberger Assay 

In a published study by Tinwell et al. (MRID 2007) to validate the use of the intact, stimulated 

weanling male rat for the Hershberger assay, groups of six immature Sprague-Dawley (Crl:CD) 

male rats (PND 20) were acclimatized and assigned to treatment groups. The test substances 

were suspended by homogenization in tocopherol-stripped corn oil and administered by oral 

gavage (dose volume 5 mL/kg); TP was administered by s.c. injection (dose volume 2.0 mL/kg). 

Rats were dosed daily for 10 consecutive days with TP at 1.0 mg/kg/day, and co-administered an 

oral dose of linuron (99% purity) at 3, 10, 30, or 100 mg/kg/day, or the positive control antiandrogen FLUT at 3.0 mg/kg/day. A vehicle control group received only corn oil, and a TP 

control group received only TP injections and an oral dose of corn oil. Rats were sacrificed 24 

hours after the last treatment by exsanguination under anesthesia. At necropsy, the following 

tissues were removed and weighed: liver, kidneys, adrenals, testes, epididymides, and AST (CG, 

LABC, SV with coagulating glands, dorsolateral prostate (DP), and VP). Selected tissues were 

fixed, and histopathological assessments were conducted on the testes, SV, and DP from vehicle 

control, TP-stimulated, TP + 100 mg/kg/day linuron, and TP + FLUT groups. The SV and DP 

were evaluated for glandular growth (glandular size, presence and size of a lumen, presence of 

intraluminal secretions, and epithelial maturation); the testes were examined for impaired tubular 

growth (reduced tubular diameter and cellular depletion of the germ cells) and apoptosis (germ 

cells with shrunken eosinophilic cytoplasm and pyknotic or absent nuclei). In addition, VP from 

the same groups used for histological analyses were extracted, and the extracted proteins were 

subjected to 2-dimensional gel electrophoresis. The resulting protein spots of interest were 

analyzed by mass spectrometry or Western blot analysis for L-amino oxidase. 

For the TP control, AST weights (SV, LABC, CG, and epididymides) were increased (p<0.01) 

compared to those from the vehicle control; the testes weights decreased (p<0.05). The study 

authors noted that the significant increases in the liver and kidneys of the TP control group were 

not consistent with the other experiment (DDE testing). When compared to the relative weights 

of the TP-stimulated organs, FLUT inhibited (p<0.01) TP-induced increases in the relative 

weights of the collected AST (DP, VP, SV, LABC, and CG); a decrease (p<0.05) in the 

epididymides weight was observed with FLUT but no changes in testes weights were detected. 

The study conclusions noted that a large interanimal variability was recorded for the testicular 

weights of the anti-androgen-treated stimulated weanlings, causing any testes weight increases 

not to be considered significant. 

At 100 mg/kg, linuron caused significant decreases (p<0.05) in the following accessory sex 

tissue weights: epididymides (↓33%), CG (↓39%), SV (↓41%), and DP (↓32%). Additionally at 

this dose, linuron caused a 20% decrease (p<0.01) in terminal body weight and a 22% decrease 

(p<0.01) in kidney weight. 

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Histopathological examinations revealed that 100 mg/kg linuron had only a slight effect on the 

morphology of the SV and DP compared to TP-treated tissues, slightly reducing the mean 

severity grades for SV (2.5 linuron vs. 3.1 TP) and DP (1.7 linuron vs. 2.4 TP). In the testes, 

linuron increased the mean severity grade for tubular degeneration (2.5 linuron vs. 1.4 TP) and 

for an increased occurrence of apoptotic germ cells (1.3 linuron vs. 0.7 TP). 

Western blot analysis for L-amino oxidase indicated that the primary antibody cross-reacted with 

proteins in at least three distinct, neighboring areas of the gel. It was noted that the primary 

antibody was generated from snake venom, and that the observed cross-reactivity could have 

been a result of non-specific antibody binding, or from the presence of several isoforms of 

L-amino oxidase. Seven reactive spots were analyzed by mass spectroscopy, four of which were 

identified as L-amino oxidase. Further, one spot was confirmed as an isoform of L-amino oxidase 

previously identified in the scientific literature. Linuron treatment at 100 mg/kg resulted in a 

marginal increase (↑42%) in the intensity of the previously identified isoform, compared to an 

approximately 7-fold increase induced by FLU treatment. Of the three other spots, linuron 

treatment had no effect on the modulation of one, and appeared to downregulate another, similar 

to TP treatment. The third spot could not be evaluated, as it was not present on all of the gels. 

Hershberger Assay 

In a validation study (OECD, 2009), a 10-day Hershberger assay was conducted with linuron 

using intact weanling rats of different strains (Alpk, Sprague-Dawley and Crl:CD). The program 

consisted of three phases: Phase 1, in which optimum TP doses were determined (Phase 1A) for 

co-administration with anti-androgens, which were then co-administered with the anti-androgen 

FLUT (Phase 1B), to select optimum TP and FLUT reference doses (1.0 and 3.0 mg/kg/day, 

respectively); Phase 2, in which the capability of the stimulated weanling rat model to detect an 

androgen agonist (trenbolone acetate; TREN) and linuron (a known weak androgen antagonists) 

was assessed; and Phase 3, in which the participating laboratories evaluated the capability of the 

stimulated weanling version to blindly identify positive and negative substances using the coded 

compounds including linuron. 

A model protocol was used, in which six male weanling rats/dose group were dosed for ten 

consecutive days beginning on PND 21-24 through PND 30-34. TP was administered by s.c. 

injection (dose volume 1.0 mL/kg), and all other test substances were administered by oral 

gavage (dose volume 5.0 mL/kg). Twenty-four h after the final dose, the animals were weighed 

and euthanized. The following AST were collected at necropsy and weighed: VP; SV with 

coagulating glands; LABC; and CG. Collection of additional reproductive (paired testes and 

paired epididymides) and systemic (liver, paired kidneys, and paired adrenal glands) tissues, and 

measurements of serum levels of luteinizing hormone, testosterone, T4, and T3 were performed 

on an optional basis. 

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For linuron in Phase 2, three performing laboratories (Laboratories A, B, and C) participated in 

generating the data. Linuron (purity not reported) was administered at dose levels of 3 

(Laboratory A only), 10, 30, or 100 mg/kg/day, with TP co-administered at 1.0 mg/kg/day. 

Additionally, FLUT was administered at 3.0 mg/kg/day with TP to a separate group; a vehicle 

control group was included as a negative control (Laboratories B and C only). Terminal body 

weights were recorded, and organ weights were determined for the VP, SV, LABC, CG, testes, 

epididymides, liver, adrenals, and kidneys. 

In Phase 3, a pilot study was first conducted by one performing laboratory that included dosing 

with 100 mg/kg/day linuron + 1.0 mg/kg/day TP as described in the model protocol. Terminal 

body weight, and organ weights for liver, testes, epididymides, VP, SV, LABC, and CG were 

recorded at necropsy. Following successful completion of the pilot study, all six performing 

laboratories participated in the final validation tests. Unidentified coded samples were provided 

for linuron (10 and 100 mg/kg/day), DNP (10 mg/kg/day), NP (160 mg/kg/day), and FLUT (3.0 

mg/kg/day); TP (reference androgen) was not coded. A vehicle control was included by 

Laboratories B and D only. Animals were dosed according to the model protocol, and the 

following weights were recorded at necropsy: terminal body weight; VP; SV; LABC; CG; testes; 

epididymides; liver; adrenals; and kidneys. 

In the Phase 2 tests, the TP positive control increased weights of the VP, SV, LABC, CG, and 

epididymides, and decreased testes weights vs. the vehicle control. In comparison with the TP 

positive control, terminal body weights, and testes, liver, adrenal, and kidney weights were 

unaffected by administration of linuron at any dose. In addition, administration of linuron at 3, 

10, or 30 mg/kg/day with TP at 1.0 mg/kg/day did not significantly affect organ weights of any 

of the collected accessory sex tissues. Dosing at 100 mg/kg/day linuron with 1.0 mg/kg/day TP 

caused decreased (p<0.05) weights in the following tissues vs. the TP positive control: VP (↓24-

27%; 2 laboratories); SVCG (↓26-29%; 2 laboratories); LABC (↓20-23%; 3 laboratories); CG 

(↓28%; 1 laboratory); and epididymides (↓19%; 1 laboratory). FLUT with TP caused decreases 

(p<0.05) of greater magnitude in these tissues, along with an increase (p<0.05) in testes weights. 

Interlaboratory comparisons demonstrated that for all organ weights (except testes), a significant 

interlaboratory effect was observed; however, the majority (predominantly the vast majority) of 

the variability was explained by the terms of the model. 

In Phase 3 testing, the TP positive control increased weights of the VP, SVCG, LABC, CG, and 

epididymides, and decreased testes weights vs. the vehicle control (results from two laboratories 

only). The FLU + TP inhibitory positive control significantly reduced mean organ weights of 

VP, SVCG, LABC, CG, epididymides (all laboratories), and testes (three laboratories) vs. the 

positive control. No significant effects were observed for the negative chemical DNP, and for 

NP, one laboratory detected a statistically significant reduction in testes weights and one 

laboratory detected a marginal insignificant change in LABC weight vs. the positive control. 

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For linuron, the pilot study indicated reductions in the 100 mg/kg/day linuron + TP group in 

mean absolute weights of testes, SV, and LABC (all p<0.01), and in CG (p<0.05) vs. the TP only 

group. In final validation testing, terminal body weights, and liver, adrenal, and kidney weights 

were unaffected by administration of linuron at either dose, and 10 mg/kg/day linuron + TP did 

not cause changes (p<0.05) in weights of any of the collected accessory sex tissues. Dosing at 

100 mg/kg/day linuron + TP caused decreased (p<0.05) weights in the following tissues: VP 

(↓23%; one laboratory); SV (↓32-48%; 4 laboratories); LABC (↓20-27%; 3 laboratories); CG 

(↓34%; 1 laboratory); testes (↓24-32%; 2 laboratories); and epididymides (↓21-27%; 3 

laboratories). The interlaboratory comparisons demonstrated that for all organ weights, a 

significant laboratory effect was observed. 

Testosterone Implant Study 

In a published, multi-component study by Lambright et al. (2000), the potential anti-androgenic 

effects of linuron (purity 99.8%) in vivo were investigated using two assays evaluating the results 

of dosing on androgen-dependent tissues and the rate of androgen-dependent gene transcription 

in testosterone-implanted castrated rats. PND 95 castrated male Sprague Dawley rats were 

implanted with testosterone-filled silastic implants, or an empty implant. It was stated that the 

implant would restore serum testosterone to a physiological level of approximately 1.5 ng/ml. 

Four days after surgery, rats were treated with 100 mg/kg/day linuron in corn oil (n=7), 100 

mg/kg/day flutamide in corn oil (n=5), or corn oil control (n=7). An additional group of rats 

(n=8) given empty implants was included. Rats were treated for either seven days (for effects on 

organ weights) or four days (for effects on steady-state levels of TRMP2 and C3 mRNA from the 

ventral prostate). Analyses of TRPM2 and C3 gene mRNA were conducted because previous 

studies have shown changes in steady-state levels of these genes are indicative of chemically 

induced alterations in the rate of androgen-regulated genes transcriptions. 

Following seven days of dosing, males were necropsied, and body, VP, SV, epididymis, liver 

and LABC were weighed, and serum was collected and analyzed for levels of testosterone. 

Linuron caused decreases (p<0.05) in body weights (↓9%) and body weight gains (↓86%), and in 

the weights of epididymis (↓17%), VP (↓33%), and LABC (↓24%). Administration of flutamide 

caused a comparable decrease in LABC weight as linuron, but a greater magnitude of decreases 

in the weights of the epididymis and VP, in addition to reduced weight of the seminal vesicles. 

Following four days of dosing, males were necropsied as before and in addition, VP was 

collected, homogenized, and total RNA was isolated and quantified by northern blot analysis. 

Linuron caused no significant changes in body weight, serum testosterone or any sex accessory 

organ weights. Linuron did, however, cause an increase in TRPM2 and a decrease in C3 mRNA 

levels. The ratio of C3 to TRPM2, was decreased (p<0.05) by flutamide and linuron. The 

TRPM2 increase produced by linuron was significant, but the decrease in C3 was not. 

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Male Pubertal Assay 

In the USEPA (2007c) validation report, linuron was included as a test compound in the multichemical study investigating the sensitivity of the protocol for the male pubertal assay. Each 

chemical was tested at two dose levels selected in an attempt to approximate the MTD (dose 

causing approximately 10% decrease in body weight) and one-half the MTD. Linuron (99-99.5% 

purity) was administered to weanling male Sprague-Dawley rats at 50 or 100 mg/kg/day in corn 

oil in an experimental block that also included propylthiouracil, ketoconazole, phenobarbital, and 

a vehicle control. The test chemicals were administered in corn oil (2.5-5.0 mL/kg) by oral 

gavage from PND 23-53 to 15 males/dose level. The endpoints were: growth (body weight); age 

at PPS; levels of serum testosterone, T4, and TSH; weights of reproductive organs, including SV, 

VP, DP, LABC, epididymis, and testes; histology of epididymis, testis, thyroid, and kidney; and 

weights of thyroid, liver, kidney, adrenal, and pituitary. 

Based on delayed acquisition of PPS, linuron delayed puberty in male rats at 50 and 100 

mg/kg/day dose levels. Both doses of linuron significantly (level of significance not reported) 

decreased weights in all AST (including SV, VP, DLP, LABC, and epididymis), except the 

testes, in which only the high dose effected a significant decrease. In addition, pituitary weight 

decreased significantly at both doses, and liver and kidney weights decreased significantly at the 

high dose. Levels of T4 were reduced at the high dose, while levels of TSH were reduced at only 

the low dose. No treatment-related histopathological changes were observed in the testes, 

epididymis, or thyroid. The authors noted that the body weight decrease of 16% observed at the 

high dose suggested that MTD may have been breached, but that the 7% decrease at the low dose 

indicated that there was no interference from body weight effects on the interaction of linuron 

with the endocrine system. 

15-Day Adult Rat Screening Assay 

In a published study by O’Connor et al. (2002), groups of 15 intact PND 70 male SpragueDawley (Crl:CD®(SD)IGS BR) rats were administered linuron (purity not reported) in 0.25% 

methylcellulose (5.0 mL/kg) by daily gavage at dose levels of 0, 25, 50, 100, or 150 mg/kg/day 

for 15 days. All rats were administered the final dose of test compound approximately 2 h prior 

to euthanasia, and necropsy was performed across the treatment groups in order to control for 

potential variation due to “time-of-day” effects. At necropsy, blood was collected from the 

descending vena cava to obtain serum for hormonal analyses. Serum concentrations of 

testosterone, E2, DHT, LH, FSH, prolactin (PRL), T3, T4, and TSH were measured using 

commercially available RIA kits. The liver, testes, epididymides, prostate, seminal vesicles, and 

AST (prostate, SV with fluid and coagulating glands) were weighed (paired organs together), 

and relative (to body weight) organ weights were calculated. Following fixation, the thyroid 

gland was weighed, and final dissection was performed under a dissecting microscope by one 

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individual. After weighing, the epididymides and testes were fixed and examined 

microscopically. Mean final body weights and organ weights were analyzed statistically. 

Linuron decreased epididymis, prostate, and AST unit weight at the highest dosage (↓9%, ↓19%, 

and ↓11%, respectively) compared to the control, but absolute testis and relative seminal vesicle 

weights were not affected. Microscopically, low incidences of minimal spermatid retention were 

present in the testes of rats in the 100 and 150 mg/kg/day groups (2/15 and 4/15, respectively), 

but there were no compound-related microscopic changes in the epididymides at the dose levels 

tested. Linuron increased serum E2 concentrations in a dose-dependent manner, with statistically 

significantly increases at 50, 100, and 150 mg/kg/day (↑50%, ↑65%, and ↑80%, respectively). 

Serum testosterone and PRL concentrations were significantly decreased at 150 mg/kg/day 

(↓40% and ↓92%, respectively). Serum DHT concentrations were decreased at 100 and 150 

mg/kg/day (↓30% and ↓73%, respectively). Serum FSH and LH concentrations were not 

affected. Linuron administration caused a statistically significant decrease in serum T3

concentrations at 50, 100, and 150 mg/kg/day (↓16%, ↓16%, and ↓22%, respectively). Serum T4

concentrations were decreased in a dose-dependent manner and were significantly decreased at 

all dosages (↓28%, ↓43%, ↓57%, and ↓70% of control at 25, 50, 100, and 150 mg/kg/day, 

respectively). Relative thyroid weights and serum TSH concentrations were not affected, and 

there were no compound-related microscopic changes in the thyroid gland. 

15-Day Adult Rat Screening Assay 

In an interlaboratory validation study of the 15-day adult intact male rat assay (USEPA, 2007d;

also referenced as Lech et al., 2006), linuron (97.7% purity) in aqueous 0.25% (w/v) 

methylcellulose was administered by oral gavage to 15 intact adult male Crl:CD(SD) rats/group 

at 50, 100, and 150 mg/kg/day for 15 days. The rats were at least 70 days old at treatment. 

Animals were sacrificed by decapitation on Day 15 at 2 to 3 hours after administration of the last 

dose. Trunk blood was collected prior to necropsy. Determination of serum concentrations of 

testosterone, LH, TSH, T4, T3, FSH, E2, PRL, and DHT were conducted sequentially. The liver, 

testes, epididymides (paired), prostate (whole), SV (with fluid and coagulating gland), and 

thyroid were weighed. The testes were weighed together and the AST (entire prostate and SV 

with fluid and coagulating gland combined) weight was determined. The right and left testis, 

right and left epididymis and the thyroid were individually identified and examined 

histopathologically. 

No mortality was observed in the linuron-treated groups, and no treatment-related histological 

findings were noted. Decreased motor activity, ataxia, lost righting reflex, unresponsive to touch, 

lacrimation, chromorhinorrhea, chromodacryorrhea, limited use of limbs, cold to touch, and 

dehydration were observed in one or more rats in the 100 and/or 150 mg/kg/day dose groups. In 

addition, impaired righting reflex, comatose, ptosis, perinasal substance, hunched posture, low 

carriage, excess salivation, perioral substance, bradypnea and scant feces were observed in one 

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or more rats at 150 mg/kg/day. Decreases (p<0.05) in overall (Day 1-15) food consumption 

values (↓18-36%), overall body weight gain (↓73-122%), and terminal body weight (↓12-20%) 

were noted in all treated groups. Testes and thyroid weights were unaffected by treatment. 

Absolute liver weights were decreased (p<0.05) by 12-15% in all treated groups, but in a nondose-dependent manner. Absolute AST weights were decreased (p<0.05) by 12-28% in all 

treated groups. Absolute prostate weights were decreased (p<0.05) by 16-26% and absolute 

epididymides were decreased (p<0.05) by 8-14%, both at 100 and 150 mg/kg/day. Absolute SV 

weights were decreased (p<0.05) by 29% at 150 mg/kg/day. The serum concentrations of the 

following hormones were affected (p<0.05) in all treated dose groups: testosterone (↓51-67%), 

T4 (↓34-67%), T3 (↓16-21%), E2 (↑31-49%), and PRL (↓85-94%). DHT was decreased (p≤0.05) 

by 39% at 150 mg/kg/day. LH was decreased (p≤0.05) by 18-19% at 50 and 100 mg/kg/day, and 

was decreased (not statistically significant) by 14% at 150 mg/kg/day. 

It was concluded that the test was able to identify linuron as an endocrine disrupting chemical. 

Consistent with the mode of action for a weak androgen receptor antagonist, linuron produced 

the expected decreases in absolute epididymal weights and absolute ASG weights, but only at 

doses producing a greater than 15% change in terminal body weights. Consistent with these 

organ weight changes, decreased serum testosterone, DHT, and LH concentrations, and 

increased serum estradiol concentrations were observed. The effects on thyroid weights are more 

difficult to interpret given that relative thyroid weights were statistically increased only at 50 

mg/kg/day and the effects did not follow a dose-response relationship. 

Developmental Toxicity (Rat; OCSPP 870.3700) 

In a Guideline developmental toxicity study (MRID 00018167), 27 presumed pregnant Crl:CD 

rats/dose group were administered linuron (97% purity) in the diet at doses of 0, 50, 125, or 625 

ppm (equivalent to 0, 5.0, 12.1, and 49.8 mg/kg/day) during GD 6-15. The day evidence of 

mating was found was designated GD 1. Maternal body weights and food consumption were 

recorded on GD 6, 10, 16, and 21. On GD 21, all surviving dams were sacrificed and all fetuses 

were weighed and examined for external malformations/variations. Crown-rump length was 

measured on each fetus. Approximately one-half of the fetuses in each litter were fixed in 

Bouin’s solution for visceral examination and the remaining one-half were processed for skeletal 

examination. 

All animals survived to scheduled termination without the appearance of any treatment-related 

clinical signs of toxicity. Gross necropsy was unremarkable. Body weights of the 625 ppm group 

were decreased (p<0.05) on GD 10 (↓6%), 16 (↓11%), and 21 (↓7%). Food consumption of this 

group was also decreased (p<0.05) during GD 6-10 (↓29%) and GD 10-16 (↓24%). 

The LOAEL for maternal toxicity is 625 ppm (49.8 mg/kg/day), based on decreased body 

weights and food consumption. The NOAEL is 125 ppm (12.1 mg/kg/day). 

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No dose- or treatment-related effects were observed on fetal sex ratios, numbers of corpora 

lutea/dam, implantations/dam, live or dead fetuses/dam, fetal body weights, or crown-rump 

length. Post-implantation loss was increased at 625 ppm (14.0% treated vs. 5.8% controls), as 

was the number of resorptions per litter with resorption was 2.1 treated vs. 1.6 controls). One 625 

ppm dam had a total litter loss. No treatment-related external or visceral malformations or 

variations were noted. In the 625 ppm group, bipartite thoracic vetebral centra was observed in 7 

fetuses from 7 litters and unapposed sternebrae were observed in 3 fetuses from 3 litters. These 

anomalies were not found in the control group and were considered indicative of developmental 

delays. 

The LOAEL for developmental toxicity is 625 ppm (49.8 mg/kg/day) based on increases in postimplantation loss and in litter/fetal resorptions. The NOAEL is 125 ppm (12.1 mg/kg/day). 

Developmental Toxicity (Rabbit; OCSPP 870.3700) 

In a Guideline developmental toxicity study (MRID 00153867 and 40437201), 25 presumed 

pregnant New Zealand white rabbits/group were administered linuron (96.2% purity) in 0.5% 

hydroxypropylmethylcellulose by gavage at dose levels of 0, 5, 25, or 100 mg/kg/day on GD 7-

19. On GD 29, all surviving does were sacrificed and the livers weighed. All fetuses were 

weighed and examined for external and visceral malformations/variations including free-hand 

sectioning of the brain. All fetuses were eviscerated and processed for skeletal examination. 

No dose- or treatment-related clinical signs of toxicity, maternal deaths, or necropsy findings 

were observed in any group. Absolute body weights of the 100 mg/kg/day group were decreased 

(p<0.05) by 7% on GD 19. During dosing, body weight losses (p<0.05) were observed in the 100 

mg/kg/day group for the interval GD 13-16 (−0.01 kg treated vs. +0.08 kg controls) and GD 16-

20 (−0.12 kg treated vs. +0.02 kg controls). Body weight gains rebounded (p<0.05) during the 

post-treatment interval (GD 20-29). Food consumption by the 100 mg/kg/day group was 

decreased (p<0.05) during GD 13-16 (↓54%) and GD 16-20 (↓62%). Additionally at this dose, 

absolute liver weights were increased by 129% (p<0.05). 

The LOAEL for maternal toxicity is 100 mg/kg/day, based on reduced body weight gain. The 

NOAEL is 25 mg/kg/day. 

At 100 mg/kg/day, five does aborted between GD 20-25 compared with one control doe on GD 

22. Slight (NS) decreases in the mean number of live fetuses/litter (6.1 treated vs. 6.8 controls) 

and mean fetal body weight (41.99 g treated vs. 45.83 g controls) were observed. No treatmentrelated external or visceral fetal malformations/variations were noted. Skull alterations 

(irregularly shaped fontanelle, hole in parietals, parietals contain intraparietals, and unossified) 

were observed in 19 fetuses (6 litters; p<0.05), compared to 1 fetus (1 litter) for controls. 

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The LOAEL for developmental toxicity is 100 mg/kg/day, based on alterations of the bones of 

the skull. The NOAEL is 25 mg/kg/day. 

Reproductive Development in Male Rats 

In a published study, Hotchkiss et al. (2004) investigated the effects of linuron (100% purity) on 

the developmental reproduction in male rats. Pregnant Sprague-Dawley rats (12/treatment group) 

were treated via oral gavage with corn oil (vehicle), linuron (75 mg/kg/day), butyl benzyl 

phthalate (BBP; 500 mg/kg/day), or linuron and BBP (75 and 500 mg/kg/day, respectively) 

during GD 14-18. On GD 18, dams (6 dams/treatment group) were euthanized for the fetal 

hormone study. The fetuses were removed via cesarean section, anesthetized, killed on ice, and 

fetal sex was determined using a dissecting scope. From each male fetus, the testes were 

removed for measurement of ex vivo testosterone production (n = 16-24/treatment), progesterone 

production (n = 19-22/treatment), and testicular testosterone concentration (n = 15-20/treatment). 

The remaining carcass was extracted for testosterone concentration (n = 35-51/treatment). The 

female fetuses from four (out of the six) litters/treatment group were retained to measure wholebody testosterone concentration. Hormone production (ng per testis per 3 hours) and 

concentration (ng) were determined using radioimmunoassay (RIA), and these data were 

presented as litter means. 

In the developmental study, the remaining dams (6/treatment group) were allowed to deliver 

naturally. On PND 2, the pups were sexed externally, and body weights and AGD were 

measured. On PND 13, the male offspring were weighed again and examined for areolae/nipples 

(dark areas lacking hair found in the area of developing nipple bud). On PND 28-30, the 

offspring were weaned, and the males were weighed and housed with 2 or 3 siblings. At 3 

months, the males were killed, and the ventral surface was shaved. The AGD was measured, and 

these animals were examined externally for the numbers of nipples and individual external 

malformations of genitalia (e.g., cleft prepuce, cleft phallus, hypospadia, exposed os penis, 

vaginal pouch, and incomplete preputial separation). Internal examinations were conducted for 

epididymal agenesis, testicular atrophy, undescended testes, ectopic testes, fluid-filled testes, 

prostate agenesis, and seminal vesicle agenesis. The adrenals, kidneys, and liver were weighed, 

in addition to reproductive organs (including the glans penis, ventral prostate, seminal vesicles, 

whole epididymis, right cauda, right caput and corpus, testes, and LABC). 

In the fetal hormone study, testicular testosterone production was decreased (p<0.05) by 34% in 

the linuron-treated group compared to controls. Similar decreases (p<0.05) were observed in 

testicular testosterone concentration (↓35%), whole-body testosterone concentration (↓48%), and 

progesterone production (↓43%) in these animals compared to controls. It was noted that these 

decreases were not due to general fetotoxicity, as fetal viability and body weights in the treated 

groups were comparable to controls at necropsy on GD 18. 

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In the developmental study, maternal body weight gain in the linuron-treated group was 

decreased (p<0.05) by 67% compared to controls. Pup mortality (post-implantation through PND 

2) was higher in the linuron group (2.00 treated vs. 0.83 controls), and resulting litter size was 

decreased in these animals (12.3 treated vs. 14.0 controls). Pup body weights were decreased 

(p<0.05) by 13% in the males and by 9% in the females. When analyzed with or without body 

weight as a covariate, AGD in the linuron-treated males was decreased (p<0.05) by 25% in the 

pups and by 10% in the adults. The numbers of areolae in the pups and nipples in the adult males 

were higher (p<0.05) in the linuron-treated group (approximately 2) compared to controls (0). 

No external malformations of the genitalia were noted in any group. The only external anomaly 

in the linuron group was incomplete preputial separation (24.2% treated vs 0% controls). The 

authors stated that, although not considered a normal condition, incomplete preputial separation 

was not categorized as a malformation because it is occasionally found in historical controls. 

Internal malformations in the linuron-treated males included adhesions/agenesis of the ventral 

prostate (9.3%), agenesis/small lobes in the seminal vesicles (12.5%), agenesis of the epididymis 

(62.5%), and undescended-ectopic or fluid-filled testes (62.5%). None of these malformations 

were found in the controls. Reproductive organ weights in the linuron-treated group were 

decreased compared to the control group as follows: glans penis (↓8%), ventral prostate (↓27%; 

p<0.05), seminal vesicles (↓8%), testes (↓17%), whole epididymis (↓28%; p<0.05), cauda 

epididymis (↓30%; p<0.05), caput + corpus epididymis (↓15%), and LABC (↓11%; p<0.05). 

Reproductive Development in Male Rats 

In a multi-component study by McIntyre et al. (2000), the effects of linuron exposure in utero

on androgen-dependent reproductive development in the male rat were characterized. For this in 

vivo study, time-mated 8- to 10- week old, nulliparous Crl:CD(SD)BR rats were obtained on GD 

0 (day that sperm was found in the vagina of the mated female) and assigned to treatment groups. 

Eleven dams/dose level were dosed with linuron (99% purity) in corn oil by daily oral gavage 

(dose volume 2 mL/kg) at dose levels of 0, 12.5, 25, or 50 mg/kg/day during GD 12-21. 

Individual dams and offspring were housed together until PND 21, after which the dams were 

euthanized by decapitation, and the offspring were housed up to four/cage by sex and treatment. 

Following PPS (PND 55), all male littermates were transferred to wire cages (2-3/cage) until 

necropsy. Dams were examined daily for clinical signs of toxicity, body weights were recorded 

daily during dosing and weekly during lactation, and food consumption was monitored weekly 

throughout dosing and lactation. Pups were counted and examined for signs of clinical toxicity 

on the day of delivery (PND 1), then sexed by measuring AGD; pups with an AGD less than 

17.5 mm were considered female. Pup litter weights, by sex and litter, and individual pup body 

weights after weaning were recorded weekly. On PND 13, male pups were examined for the 

presence of areolae and/or nipples. An animal was considered a responder if it had one or more 

retained areolae or nipples. Beginning on PND 38, male pups were examined daily for 

malformation of the external genitalia, testicular descent, and PPS; PPS was complete by PND 

55. Females were examined daily for VO beginning on PND 28. When VO was acquired, 

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vaginal lavages were taken for the next 2 weeks to determine time to first estrus and for the 3 

weeks preceding euthanization on PND 85 to determine estrus cyclicity. Following 

euthanization, body and organ weights (liver, kidneys, adrenals, uterus, and ovaries) and number 

of implantation sites were recorded for the dams. Sexually mature male offspring (PND 95-105) 

were euthanized by decapitation, terminal body weights were recorded, and the trunk blood was 

collected for analysis of LH and testosterone. The external genitalia, including the scrotum, 

prepuce, and penis were visually inspected, and the following reproductive organs were 

examined and weighed: testes, epididymides, vas deferens, prostate (ventral and dorsolateral), 

seminal vesicles, and coagulating glands (with fluid). In addition, the following organs were 

collected and weighed: liver, kidneys, adrenals, and levator ani/bulbocavernosa muscle. Tissues 

were fixed in Bouin’s fixative (testes and epididymides) or 10% neutral buffered formalin, and 

stained with hematoxylin and eosin for microscopic examination. Female offspring were 

euthanized by CO2 asphyxiation on PND 85. 

Administration of linuron to pregnant dams during GD 12-21 did not result in signs of toxicity, 

or significant changes in body weight or food consumption during treatment or lactation; dam 

organ weights (kidney, liver, uterus, ovaries, and adrenals) were not affected by treatment. 

Treatment of pregnant dams with linuron (at any dose) did not significantly affect the number of 

implantation sites, live pups born/litter, sex ratio, AGD in either sex; male pup weight at PND 7, 

21, or 100, PPS, or serum testosterone or LH levels (data not reported); or female pup weight, 

weight gain, day of VO, time to first estrus, or estrus cyclicity. No malformations in the external 

genitalia or changes in testicular descent were observed in male offspring at any dose. The 

proportion of pups born alive and surviving to weaning was not affected at 12.5 and 25 mg/kg. In 

utero exposure to linuron at 50 mg/kg decreased (p<0.05) the proportion of pups surviving to 

weaning if the two litters that were immediately cannibalized are included, but had no significant 

effect on the proportion of pups surviving to PND 21 if these litters are removed. 

In utero exposure to linuron resulted in the following changes in male offspring: dose-responsive 

increase in areola/nipple retention; incidence of hypoplastic testes and hypoplastic epididymides; 

and partial agenesis of the epididymides. Following in utero exposure, male offspring exhibited 

1.0, 1.6, and 3.7 areolae/nipples/rat at 12.5, 25, and 50 mg/kg linuron, with a significant increase 

(p<0.05) at 50 mg/kg. Hypoplastic testes were observed in 3.5%, 11.6%, and 11.4% of the rats at 

12.5, 25, and 50 mg/kg linuron. Grossly abnormal testes in the 25 and 50 mg/kg groups 

displayed variable degeneration of the seminiferous epithelium characterized as seminiferous 

tubular degeneration. Similar lesions were observed at both levels; however, the lesions in the 50 

mg/kg group were more severe, with most tubules in the affected testes having complete lack of 

germinal epithelium. Dilation of the rete testis and interstitial edema were also frequently 

observed in lesioned testes in the 25 and 50 mg/kg groups. Occurrence of these changes in the 

testes ranged 2.9-10.1% in the 25 mg/kg group and 4.5-9.1% in the 50 mg/kg group. Linuron 

also induced lesions in the epididymis. Hypoplastic epididymides were observed in 1.7%, 11.6%, 

and 4.5% of rats at the 12.5, 25, and 50 mg/kg doses, and epididymal malformations were 

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observed in 6.8% of 50 mg/kg rats. The testicular and epididymal lesions arose in males from 

different litters and across the linuron exposure groups, confirming that these results were 

biologically relevant. Epididymal weights, excluding grossly malformed epididymides, were 

slightly, but not significantly, decreased in the 25 and 50 mg/kg groups. Agenesis of the vas 

deferens was observed in 4.5% of the rats in the 50 mg/kg group. A decrease (p<0.05) in weight 

was also observed in the DP (↓11%) and kidneys (↓9%) of the 50 mg/kg group. 

Reproductive Development in Male Rats 

In a multi-component study, Lambright et al. (2000) investigated the transgenerational effects 

of linuron on the histology of the testis and epididymis in rats. Sprague-Dawley rats were dosed 

with linuron (100% purity) at 100 mg/kg/day. This dosage level was selected because it was 

minimally toxic or nontoxic, produced minor effects on body weight gains, and this dose was 

known to cause malformations in male rat offspring when administered during sexual 

differentiation. Dams in the control group and the positive control group (dibutyl phthalate) were 

dosed from GD 14 to LD 3. Dams in the linuron group were not dosed after GD 18 due to a 

reduction in maternal weight gain. At 5 to 6 months of age male rat offspring were necropsied, 

and organ weights were collected from 1-2 males randomly selected from each litter. Tissues 

were routinely fixed, stained, and examined for microscopic lesions (graded as minimal, mild, 

moderately severe, or severe/high). 

In the testes of the linuron group, 8/13 males displayed seminiferous tubules devoid of active 

spermatogenesis, compared to 0 controls. In the epididymis, rats displayed a variety of lesions 

including diffuse epithelial hypertrophy (50% with mild to moderate severity) and hypospermia 

(58% moderate to severe). The results in the linuron treated group were nearly identical to the 

positive control. 

Reproductive Development in Male Rats 

In a multi-component study, Wolf et al. (1999) conducted an in vivo assay to determine the 

transgenerational effects of linuron. Sprague-Dawley dams were administered linuron (100% 

purity) in corn oil at 0 (10 dams) or 100 (8 dams) mg/kg/day during GD 14-18. Typically, pup 

body weight and AGD were measured on PND 2 and during PND 10-13, and the offspring were 

examined for areolae with and without a nipple bud. A dissecting scope was used to determine 

the presence of a bud with the areola. At PND 28, male offspring were weaned and housed in 

groups of two or three with litter mates, and examined for PPS beginning on PND 35. After 

sexual maturation, the males were examined for external malformations. At approximately 5 

months of age, males were killed via decapitation within 15 seconds after removal from the 

home cage in a separate room during the dark phase of the animal’s daily light cycle. The ventral 

surface of each male was shaved and examined externally for abnormalities (including number 

and location of retained nipples, cleft phallus, vaginal pouch, and hypospadias). The animals 

were examined internally for: ectopic or atrophic testes; agenesis of the gubernaculum, 

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epididymis, sex accessory glands, and ventral prostate; epididymal granulomas; hydronephrosis; 

and enlarged bladder with stones. Weight measurements included the body, pituitary, adrenal, 

kidney, liver, ventral prostate, seminal vesicle (with coagulating gland and fluid), testis, and 

epididymis. Samples of the testes, epididymis, and accessory sex tissues were taken for 

microscopic evaluation. 

All dams survived until scheduled termination. Maternal body weight gain was decreased at 100 

mg/kg/day during the treatment period. However, dams showed recovery during the posttreatment period (LD 18-22), with weight gain in the treated group approaching controls (35 g 

treated vs 44 g controls). Pup weights in the treated group were 20% lower (p<0.0001) than 

controls at birth, and the adjusted (for body weight by analysis of covariance) AGD in the male 

offspring was decreased (p<0.005) by 30%. The incidence of areolae (with and without nipples) 

was increased from 0% in the controls to 44% in the 100 mg/kg/day male pups (mean number of 

aerolae and retained nipples was 2.1 ± 0.7). At necropsy, one out of the 13 males treated with 

linuron had epispadias (partial hypospadias with the urethral opening half way down the 

phallus). Numerous epididymal and testicular malformations were found in the linuron-treated 

group, with 56% of the males displaying agenesis or atrophy of one or both organs compared to 

0% controls. Malformations of the epididymis included agenesis of the caput and/or corpus 

epididymides. Some testes were atrophic, fluid-filled, and flaccid. However, testicular agenesis 

was not observed in the treated males. Decreases in androgen-dependent tissue weights were 

observed in the linuron-treated male offspring, including the seminal vesicles (↓12%), ventral 

prostate (↓35%), epididymides (↓29%), cauda epididymis (↓35%), testes (↓22%), glans penis 

(↓16%), and levator ani/bulbocavernosus (LABC) muscle (↓16%). Epididymal weights that 

displayed agenesis and testes weights with severe atrophy were excluded from these analyses. 

With the exception of the seminal vesicles, these decreases in organ weights were statistically 

significant (p<0.03). 

A previously developed pseudohermaphroditism index (PHI) was used to describe the degree of 

demasculinization/feminization based on a suite of androgen-dependent parameters. Two PHI 

scores were calculated, one for dihydrotestosterone (DHT)-dependent parameters (AGD, aerolae, 

retained nipples, ventral prostate weight, hypospadias, and vaginal pouches) and the other for 

testosterone-dependent variables (weights and abnormalities of the seminal vesicles, 

epididymides, and testes). Offspring of animals treated with linuron were approximately 30% 

“feminized” for DHT-dependent parameters and 40% feminized for testosterone-dependent 

parameters. 

Reproductive toxicity in Male Rats 

In a published, multi-component study, Cook et al. (1993) examined the effects of linuron on 

sexually immature and mature male rats. Groups of 10 male CD rats/dose were administered 

linuron (97% purity) in methocel vehicle by daily oral gavage (dose volume 5.0 mL/kg) at dose 

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levels of 0 or 200 mg/kg/day for 14 consecutive days. Sexually immature rats were dosed from 

PND 32-45; sexually mature rats were dosed from PND 93-107. Rats were euthanized the 

morning after the last dose on Study Day 15. All rats were weighed daily and these weights were 

used to calculate the daily dose. Because linuron is known to reduce body weight gain, a pair-fed 

control group was included in order to control for the effects of reduced body weight on organ 

weights and hormonal levels. All rats were euthanized by exsanguination under anesthesia. 

Blood was collected from the descending vena cava, and serum was prepared for 

radioimmunoassay analysis to determine testosterone, E2, and luteinizing hormone (LH) 

concentrations. Testes, epididymides, accessory sex organ unit (prostate, seminal vesicles, and 

coagulating glands), prostate, seminal vesicles with fluid removed, and coagulating glands were 

weighed at necropsy and relative organ weights (percentage of body weight) were calculated. 

In the sexually immature rats treated with linuron, decreased final body weights (↓27%) and 

body weight gains (↓46%) were noted compared to the control. As expected, daily food 

consumption for the pair-fed control group was similar to that for the linuron-treated rats. Final 

body weights and overall body weight gains were similar between the pair-fed control and the 

linuron-treated rats, suggesting that the decreased body weight was primarily due to reduced 

food consumption. The following relative organ weights were decreased (p<0.05) in the linurontreated rats compared to the pair-fed controls: epididymides (↓21%), accessory sex organ unit 

(↓42%), prostate (↓28%), and seminal vesicles (↓46%). Except for the epididymides, these 

relative organ weights were also decreased when compared to those of the control. Serum 

testosterone, estradiol, and LH levels in the linuron-treated rats were similar to the controls and 

the pair-fed controls. 

In the sexually mature rats treated with linuron, final body weights were decreased by 18% and a 

body weight loss of −10.2 g was observed compared to the controls. Even though daily food 

consumption for the pair-fed control group was similar to the linuron-treated rats, the decreases 

in body weights and body weight loss of the linuron-treated rats were greater in magnitude than 

those of the pair-fed control. These data suggest that the decreased body weight in the mature 

linuron-treated rats is not solely due to reduced food consumption. The following relative organ 

weights were decreased in the linuron-treated rats when compared to those in the pair-fed 

control: accessory sex organ unit (↓17%) and prostate (↓19%). These relative organ weights were 

also significantly decreased when compared to those in the control. Serum testosterone, estradiol, 

and LH levels were unchanged in the linuron-treated rats when compared to those in the control; 

however, serum estradiol (↑27%) and LH (↑23%) levels were significantly elevated in the 

linuron-treated rats when compared to those in the pair-fed control. 

Two-Generation Reproduction Toxicity (Rat; OCSPP 870.3800)

In a Guideline two-generation reproduction toxicity study (MRID 48046002), 25 Wistar 

rats/sex/dose group were administered linuron (94.7% purity) continuously in the diet at dietary 

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levels of 0, 25, 125, or 625 ppm (equivalent to 0/0, 1.9/2.1, 9.8/10.6, and 52.8/55.8 mg/kg/day in 

males/females) for two consecutive generations. The P generation animals were exposed to the 

test diets beginning at approximately 7 weeks of age, for 10 weeks prior to mating to produce the 

F1 litters. F1 offspring selected to be parents of the next generation (25/sex/dose group) were fed 

the same test diet concentrations as their parents for 18 weeks prior to mating to produce the F2

generation. The F2 offspring were terminated after weaning. 

All P and F1 generation parents survived to scheduled sacrifice. There were no remarkable 

clinical observations during the study or gross findings at necropsy in the P or F1 generation 

males or females. 

At 625 ppm, body weights (↓8-13% P males; ↓9-14% P females; ↓19-33% F1 males; ↓16-27% F1

females) and overall body weight gains were decreased (p<0.05) throughout the pre-mating 

period in both sexes from both generations. Additionally in the females from both generations, 

body weights (↓14-18% P females; ↓16-20% F1 females) and overall body weight gains were 

decreased (p<0.05) during gestation and lactation. Absolute food consumption was decreased 

(p<0.05) generally throughout pre-mating in both sexes and both generations, and absolute food 

consumption was decreased (p<0.05) throughout gestation and lactation in the P and F1 females. 

No treatment-related changes in absolute or relative weights were seen in the testes, 

epididymides, seminal vesicle, prostate, ovaries, uterus, thyroid, adrenal or pituitary glands of the 

P and F1 parental animals and F1 and F2 offspring. In addition, no treatment-related 

histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, 

coagulating glands, ovaries, uterus, vagina, cervix, thyroid, adrenal or pituitary glands of the P 

and F1 parents. 

Absolute and relative (to body and brain) spleen weights were increased (p<0.01) by 34-73% at 

625 ppm in the P and F1 males and females. In the spleen, increased incidences of hemosiderosis 

were found in the 625 ppm P males (24/25 treated vs. 4/25 controls) and females (19/25 treated 

vs. 2/25 controls), and F1 males (23/25 treated vs. 0/25 controls) and females (20/25 treated vs. 

3/25 controls). Additionally in the spleen, increased hematopoiesis was found at 625 ppm in the 

P males (22/25 treated vs. 4/25 controls) and females (18/25 treated vs. 0/25 controls) and F1

males (20/25 treated vs. 2/25 controls) and females (19/25 treated vs. 0/25 controls). 

Slight to moderate lymphoid atrophy was noted in the thymus in the females of the P (10/25 

treated vs. 2/25 controls) and F1 (12/25 treated vs. 5/25 controls) generations. 

The LOAEL for parental toxicity is 625 ppm (equivalent to 53/56 mg/kg/day in males/females) 

based on decreased body weights, body weight gains, and food consumption and effects on the 

spleen (increased weight, hemosiderosis, and hematopoiesis) and thymus. The NOAEL is 125 

ppm (equivalent to 10/11 mg/kg/day in males/females). 

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No treatment-related effects on the mating, fertility, or gestation indices were observed in either 

generation. There were no effects of treatment on gestation duration in either generation. 

At 625 ppm, the pre-coital interval was slightly increased compared to controls in both 

generations. In both the P and F1 generations, the number of implantations was reduced, and 

post-implantation loss was increased. Total litter loss was observed in three P dams and five F1

dams at this dose. The combined effect of decreased number of implantations and increased postimplantation loss resulted in a decreased litter size on PND 0. Furthermore, a greater proportion 

of the pups born at this dose were stillborn, resulting in a decreased live birth index.

The LOAEL for reproductive toxicity is 625 ppm (equivalent to 53/56 mg/kg/day in 

males/females) based on decreased implantation, increased post-implantation loss, and decreased 

live birth index. The NOAEL is 125 ppm (equivalent to 10/11 mg/kg/day in males/females). 

No clinical signs of toxicity were observed in the pups of either generation from birth to PND 21. 

Pup sex ratio on PND 0 was not affected by treatment in either generation. There were no 

treatment-related effects on organ weights, gross pathology, or histopathology. 

In both the F1 and F2 litters at 625 ppm, mean litter size continued to be decreased throughout the 

post-natal period, resulting in a decreased viability index in both generations and a decreased 

lactation index in the F2 litter. Overall (PND 1-21) pup body weight gains at this dose were 

decreased by 26% in both generations compared to controls. 

The LOAEL for offspring toxicity is 625 ppm (equivalent to 53/56 mg/kg/day in males/females) 

based on decreased litter size, pup body weights and viability and lactation indices. The NOAEL 

is 125 ppm (equivalent to 10/11 mg/kg/day in males/females). 

Two-Generation Reproduction Toxicity (Rat; OCSPP 870.3800)

In another Guideline two-generation reproduction toxicity study (MRIDs 41463401 and 

41864701), groups of 30 male and 30 female Crl:CD BR rats were administered linuron (96.2% 

purity) in the diet at concentrations of 0, 12.5, 100, or 625 ppm (equivalent to 0/0, 0.736/0.917, 

5.83/7.33, and 35.5/45.1 mg/kg/day in males/females) for two generations. One litter was 

produced by each generation. P and F1 parental animals were treated for 72 or 75 days, 

respectively, prior to mating and continuously throughout mating, gestation, and lactation, and 

until necropsy. 

No treatment-related clinical signs of toxicity or mortalities were observed in the adult animals 

of either generation. Body weights, body weight gains, and food consumption were decreased 

(p<0.05) beginning on Day 7 for the 625 ppm P animals and throughout premating for the 100 

and 625 ppm F1 animals. At the end of premating, body weights of the 625 ppm P males and 

females were decreased 19% and 14%, respectively, with overall body weight gains decreased 

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by 41% and 45%, respectively. For the 625 ppm F1 males and females, final premating body 

weights were decreased 24% and 25%, respectively, with body weight gains decreased 23% in 

both sexes. Mean daily food consumption levels for the 625 ppm groups during the premating 

interval were decreased 15-20%. Absolute body weights of the 100 ppm P males were 

occasionally decreased (p<0.05), with premating weight gains and food consumption 12% and 

6% (p<0.05), respectively, less than controls. For the 100 ppm F1 males and females, final 

premating body weights, body weight gains, and food consumption were decreased 6-8%. For 

the 100 and 625 ppm dams of both generations, decreased body weights during gestation and 

lactation were considered a continuation of premating effects. 

No treatment-related lesions were noted at necropsy of the P males or females. In the 625 ppm F1

males, gross lesions included testes reduced in size (9/30), abnormally large (3/30), and soft 

(5/30), and small epididymides (8/30), and unspecified deformities of the epididymides (5/30); 

these were noted as significant incidences, but control rates were not provided in the original 

DER. Microscopically, increased (p<0.05) incidences of testicular and epididymal lesions were 

found in the 625 ppm F1 males as compared with the controls: atrophy (14/30), fibrosis (8/30), 

and hyperplasia (7/30) in the testes; and arteritis (6/30), inflammation (5/30), and oligospermia 

(12/30) in the epididymides. Only one incidence each of atrophy and oligospermia were 

observed in control animals. Absolute testes weights of the 625 ppm F1 males were decreased by 

20% (p<0.05; 80%) compared to the controls. In the 625 ppm F1 females, gross findings 

included cystic ovaries (4/30), dilatation (3/30), and fluid filled uterine horns (2/30), none of 

which were observed in control animals. 

In addition, lesions of the eye were noted in the 625 ppm F1 males and females. An increase 

(p<0.05) was seen in the number of F1 males with any type of corneal or conjunctival change 

(14/30 vs 4/30 controls). The lesions included corneal degeneration/basophilia and conjunctival 

inflammation/basophilia. Degeneration of the lens was observed in 3/30 males and 3/29 females 

compared with only 1/30 control male. However, a clear treatment-related effect in females was 

not considered to be definitive. 

The LOAEL for systemic toxicity is 100 ppm (average premating doses 5.83-9.24 mg/kg/day) 

based on reduced body weight gains in males and females during both generations. The NOAEL 

is 12.5 ppm (average premating doses 0.736-1.12 mg/kg/day). 

No treatment-related adverse effects were found on the reproductive performance of either 

generation. For the control, low-, mid-, and high-dose group, pup viability for lactation days 0-4 

was 99.4, 98.0, 99.8, and 91.7% (p<0.05), respectively, for the F1 generation and 96.8, 92.7, 

99.5, and 76.2% (p<0.05), respectively, for the F2 generation. The mean number of pups per litter 

in the 625 ppm F2 group was less (p<0.05) than the control throughout lactation. The number of 

litters with pups showing clinical signs was increased (p<0.05) in both generations. Body 

weights of the 100 and 625 ppm F1 male and female pups and of the 625 ppm F2 male and 

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female pups were less (p<0.05) than those of the controls throughout lactation. These data were 

not considered for setting the reproductive toxicity NOAEL in the original DER. 

The reproductive toxicity NOAEL was greater than or equal to 625 ppm (average premating 

doses 35.5-63.0 mg/kg/day) and the reproductive toxicity LOAEL was not established. 

Two-Generation Reproduction Toxicity in Rats

In a published, multi-component study, Cook et al. (1993) examined the effects of linuron on 

reproduction of male rats and the resulting male offspring. In this two-generation reproduction 

study, one set of litters was produced in each generation; however, no information was provided 

on the F2 pups. Groups of 30 CD rats/sex were fed diets containing 0, 12.5, 100, or 625 ppm 

linuron (equivalent to 0, 0.736, 5.83, and 35.5 mg/kg/day for the P males and 0, 0.948, 7.77, and 

54.0 mg/kg/day for the F1 males during pre-mating). The P males were treated continuously 

beginning at PND 60, and were euthanized after 122 to 124 days of feeding. The F1 males were 

treated beginning at PND 21, and were euthanized after 147 to 161 days of feeding. At 

termination, blood was collected and serum was prepared for analysis of testosterone, estradiol, 

and LH concentrations by radioimmunoassay. For the P males, testes, epididymides, accessory 

sex organ unit (prostate, seminal vesicles, coagulating glands), prostate, seminal vesicles with 

fluid removed, and coagulating glands were weighed at necropsy and relative organ weights 

(percentage of body weight) were calculated. 

In the P generation, final body weights were decreased in the males at 100 ppm (↓6%) and 

625 ppm (↓19%). Overall body weight gain (Days 0-119) was also decreased in the males at 100 

ppm (↓12%) and 625 ppm (↓37%). There were no decreases in relative organ weights in the P 

males. Serum estradiol (↑55%) and LH (↑75%) levels were significantly increased in the 625 

ppm P males. 

In the F1 generation, final body weights were decreased in the males at 100 ppm (↓6%) and 

625 ppm (↓25%). Overall body weight gain (Days 0-154) was also decreased in the F1 males at 

100 ppm (↓6%) and 625 ppm (↓25%). There were no decreases in relative organ weights in the 

F1 males. Serum estradiol (↑15%) and LH (↑68%) levels were significantly increased in the F1

625 ppm males. 

Multi-Generation Reproduction Toxicity in Rats

In a published, multi-component study, Wolf et al. (1999) characterized the reproductive effects 

of linuron on male rats. The P generation was administered the test material by daily oral gavage 

from weaning, through puberty, young adulthood, mating, and lactation. F1 pups were evaluated 

through mating, but were exposed only via the dam during gestation and lactation. In the current 

study, linuron (100% purity) in corn oil was administered to pregnant LE hooded rats daily via 

oral gavage at dose levels of 0, 10, 20, or 40 mg/kg/day beginning at weaning on PND 21. The 

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corn oil control group included 12 rats/sex, and the remaining treatment groups were comprised 

of 8 rats/sex. In the P females, dosing continued through PND 20, while males were dosed daily 

from weaning until necropsy. Parameters examined in the P generation animals included age at 

sexual maturation, estrous cyclicity, fertility, and reproductive system evaluation at necropsy. 

When the P males were necropsied, serum was collected for hormonal analyses, organs were 

weighed (testes, liver, kidneys, adrenal, seminal vesicles plus coagulating glands with fluids, 

epididymis, pituitary, and terminal body weight), the testes were examined microscopically, and 

caudal and epididymal sperm counts were conducted. When the F1 offspring reached PND 25, 

the P dams were euthanized. The body, liver, kidney, adrenal, pituitary, and ovaries were 

weighed, and the number of implantation sites in the uterus was counted. Litter size and weights 

in the F1 offspring were determined throughout the study. After sexual maturation, F1 offspring 

were randomly selected from the highest dose with sufficient number of surviving animals for 

fertility assessment under continuous mating conditions (16/sex/dose group). F2 pups were 

counted and removed at birth. After determination of the age of sexual maturation, all excess F1 

animals were necropsied and examined for malformations. After termination of the breeding 

phase of the study, the F1 rats were necropsied as were the P animals. 

Linuron did not affect any serum hormone concentration. In the P males at 40 mg/kg/day, 

seminal vesicle weight (with coagulating glands and fluids) was decreased (p<0.05) by 14% 

compared to controls, cauda epididymal weight was 7% lower (p<0.05) than controls, and age at 

PPS increased (PND 45.8 treated vs. PND 43.0 controls; p<0.05). Additionally at this dose, the 

F1 males exposed in utero and during lactation produced a lower (p<0.01) cumulative number of 

pups (65 vs 104 pups) when mated continuously over 12 breeding cycles, and the male offspring 

had decreased (p<0.05) weights of the testes (↓20%) and epididymis (↓18%), and decreased 

(p<0.05) spermatid numbers (↓16%). 

Chronic Toxicity (Dog; OCSPP 870.4100) 

In a Guideline chronic toxicity study (MRID 40952601), groups of 4 beagle dogs/sex were 

administered linuron (96.2% purity) in the diet at concentrations of 0, 10, 25, 125, or 625 ppm 

(equivalent to 0/0, 0.29/0.30, 0.79/0.77, 4.17/3.49, and 18.6/16.1 mg/kg/day in males/females) 

for one year. 

No treatment-related clinical signs of toxicity or mortalities were observed at any dose level. 

Body weights, body weight gains, and food consumption were not affected by treatment. No 

treatment-related ophthalmological lesions or changes in urinalysis parameters were noted and 

gross necropsy was unremarkable. 

RBC counts, hemoglobin, and hematocrit were slightly (NS) decreased throughout the study in 

high-dose males and females as compared with those of the controls. WBC and platelet counts 

were increased (p<0.05) in high-dose females at 3, 6, and 9 months and platelet counts were 

increased (p<0.05) in high-dose males at 3 months. Methemoglobin and sulfhemoglobin levels 

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were increased (p<0.05) in the 625 ppm males and females at all time points as compared with 

those of the controls. In addition, for the 125 ppm groups methemoglobin levels were increased 

(p<0.05) in males and females at 3 and 6 months while sulfhemoglobin levels were (p<0.05) 

increased at 9 months in males and at 3, 9, and 12 months in females. Increased hematopoiesis 

was observed in the bone marrow from 3/4 high-dose males and 4/4 high-dose females, 

compared with none of the control males and only 1/4 control females. 

Cholesterol levels were increased (p<0.05) in the high-dose groups at 3, 6, 9, and 12 months for 

males and 3 months for females. Absolute liver weights were slightly (NS) increased in the 625 

ppm males and relative liver weights were increased (p<0.05) in the 125 and 625 ppm males. No 

effects on liver weights were noted in females. Increases in the incidence and/or severity of 

brown pigment (hemosiderin) deposition in the liver were observed microscopically in high-dose 

males and females. 

No treatment-related changes were seen in absolute or relative weights of testes, ovaries, and 

thyroid glands. In addition, no treatment-related histopathological lesions were seen in the testes, 

epididymides, prostate, ovaries, vagina, mammary glands, thyroid, adrenal or pituitary glands. 

The LOAEL for systemic toxicity is 125 ppm (4.17/3.49 mg/kg/day in males/females) based on 

abnormal hematology findings (increased met- and sulfhemoglobin levels). The NOAEL is 25 

ppm (0.79/0.77 mg/kg/day in males/females). 

Carcinogenicity (Mouse; OCSPP 870.4200) 

In a Guideline oncogenicity study (MRID 00124195), groups of 80 CD®-1 mice/sex were 

administered linuron (97.0% purity) in the diet at concentrations of 0, 50, 150, or 1500 ppm 

(equivalent to 0/0, 8/12, 23/35, and 261/455 mg/kg/day for males/females) for 24 months. 

No significant treatment-related effects were seen in clinical signs or survival. Body weights 

were consistently lower (p<0.05) in males and females at 1500 ppm than in the control groups 

throughout the study. At 52 weeks, the group mean body weights and weight gains of high-dose 

males were 8% and 15% lower than the controls, respectively, and high-dose females were 

decreased by 11% and 21%. At 104 weeks, the body weights and weight gains of high-dose 

males were 10% and 20% less than the controls, and in high-dose females were 8% and 14% less 

than the controls, respectively. The overall food intake for high-dose males was decreased by 

about 14% and by 10% for high-dose females compared to the controls. Food efficiency for the 

2-year study was not significantly affected in treated animals. 

Increases of 9-18% in group mean erythrocyte cell volume and mean cell hemoglobin were seen 

in males and females after 6 months of treatment at 1500 ppm. Erythrocyte counts were 

decreased by 9% and 14% in high-dose males and females, respectively, compared to the 

controls at 6 months. These hematology values returned to near control levels later in the study. 

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An increased (p<0.05) incidence of hemosiderin deposits in the spleens of both sexes at 1500 

ppm is suggestive of a compensated hemolytic anemia in high-dose animals. Methemoglobin 

levels were increased in high-dose females by 117% compared to the control group, and were 

increased in high-dose males by 613% compared to the 50 ppm group (the male control value 

was not available). Differential white cell counts were within normal parameters for both sexes 

at all doses. 

The absolute and relative (to body) liver weights were increased by 20% and 24%, respectively 

in high-dose males and by 65% in high dose females compared to the controls. Microscopic 

evidence of liver toxicity at 1500 ppm included increased incidences of focal hepatocellular 

cytoplasmic alteration, focal centrilobular peliosis, and centrilobular hepatocytomegaly in both 

sexes, and increased incidence of hepatocellular cytoplasmic vacuolation in females. 

No treatment-related changes in absolute or relative weights were seen in the testes, 

epididymides, ovaries, uterus, cervix, thyroid, adrenal or pituitary glands. In addition, no 

treatment-related histopathological lesions were seen in the testes, epididymides, prostate, 

ovaries, uterus, vagina, cervix, mammary, thyroid, adrenal or pituitary glands. 

The LOAEL for systemic toxicity is 1500 ppm (equivalent to 261/455 mg/kg/day in 

males/females), based on microscopic liver changes, methemoglobinemia, and decreased body 

weight and weight gain in both sexes. The NOAEL is 150 ppm (23/35 mg/kg/day in 

males/females). 

Treatment of up to 104 weeks with 1500 ppm linuron resulted in an increase in the incidence of 

hepatocellular adenomas (25% treated vs. 6% controls; p<0.05) in female mice under the 

conditions of this study. Dosing was considered adequate based on the liver changes, 

methemoglobinemia, and decreased body weights. 

Chronic Toxicity/Carcinogenicity (Rat; OCSPP 870.4300) 

In a Guideline chronic toxicity/oncogenicity study (MRIDs 00029679 and 00029680), groups of 

70 ChR-CD1 rats/sex/dose were administered linuron (96.9-97.2% purity) in the diet at 

concentrations of 0, 50, 125, or 625 ppm (equivalent to 0/0, 2.09/3.13, 5.11/7.75, and 27.1/48.3 

mg/kg/day in males/females) for up to 2 years. Additional groups of 10 rats/sex/dose were 

administered the same diets for 12 months for interim evaluation. All clinical pathology data 

were reanalyzed (MRID 00164117) due to inappropriate statistical methods used in the original 

study report. 

Linuron had no effect on mortality at any dose, and there were no treatment-related clinical signs 

reported. Absolute body weights of the 625 ppm males were decreased to 88-91% of controls 

during Weeks 1-19 with decreased body weight gains during Weeks 0-13 and 52-104, resulting 

in decreased overall body weight gains (89% of controls). Absolute body weights of the 625 ppm 

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females were decreased throughout the study, with the magnitude of the decrease generally 

increasing throughout the course of the study. The body weight gains of this group for the entire 

study were 57% of controls. Body weight gains by the 125 ppm males was decreased to 64% of 

controls during Week 52-76, with a body weight loss during Week 76-104 being increased to 

148% of controls. Body weight gains by the 125 ppm females were decreased to 75% of controls 

during Week 52-76. There were no toxicologically significant treatment-related effects on food 

consumption; however, food efficiency values of the 625 ppm females were decreased to 74-

88% of controls during weeks 1-4 and to 58% of controls for the entire study period. 

No treatment-related changes were seen in absolute or relative weights of the pituitary and 

adrenal glands. At termination, there were significant (p<0.05) increases in the mean absolute 

and relative testicular weights in the mid (121 and 122%) and high (131 and 143%) dose groups. 

No treatment-related histopathological lesions were seen in prostate, ovaries, mammary, thyroid, 

and adrenal or pituitary glands. 

Histopathology observations consistent with hemolysis were observed at increased incidences in 

the 125 and 625 ppm males and females from the main study and interim sacrifice groups, 

including hemosiderin in Kupffer cells and increased hemosiderosis in bone marrow, spleen, 

and/or mesenteric lymph nodes, and transient decreases in the erythrocyte count, hemoglobin 

concentration, and hematocrit of the high-dose female group were noted at 6 and/or 12 months. 

Male rats of the main study group had increased (p<0.05) incidences of mineralization/calculi in 

the renal pelvis, transitional cell hyperplasia in the renal pelvis, and subacute perivasculitis 

and/or vasculitis in the epididymides at 125 and 625 ppm. Female rats of the main study group 

had increased (p<0.05) incidences of calculi in renal tubules at 125 and 625 ppm and increased 

(p<0.05) incidences of hepatic sinusoidal ectasia and collecting duct ectasia in the kidney at 625 

ppm. The 625 ppm females also had an increased (NS) incidence of transitional cell hyperplasia 

in the renal pelvis compared to controls. Hepatocellular megalocytosis/syncytium formation with 

fibroplasia radiating between hepatic cords, and occasional increased hepatocellular 

intracytoplasmic basophilia was only observed in the main study 625 ppm females (15/68 

animals examined at that site) and may have been related to induction of hepatocellular protein 

synthesis. The significance of this finding is unknown but considered adverse. 

The LOAEL for systemic toxicity is 125 ppm (equivalent to 5.11/7.75 mg/kg/day in 

males/females), based on decreased body weight gains in both sexes, microscopic observations 

consistent with hemolysis (hemosiderin in Kupffer cells and increased hemosiderosis in bone 

marrow, spleen, and/or mesenteric lymph nodes), and increased incidences of microscopic 

changes in the epididymides (perivasculitis/vasculitis) and renal pelvis (transitional cell 

hyperplasia and mineralization/calculi) of males and kidneys (calculi in renal tubules) of 

females. The NOAEL is 50 ppm (equivalent to 2.09/3.13 mg/kg/day in males/females). 

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There was a treatment-related increase in the incidence of testicular interstitial adenomas at the 

125 and 625 ppm treatment levels (5.7, 27.5, and 53.6% for control, mid-, and high-dose males, 

respectively; p<0.01). Common neoplasms, included pituitary adenomas of the pars anterior in 

both male and female rats and mammary fibroadenomas in female rats. Decreased incidences of 

both these tumor types were noted in the high-dose female group. Dosing was considered 

adequate based on the decreases in body weight and body weight gain of high-dose females. 

Leydig Cell Hyperplasia in Aged Male Rats 

In a special study by Pastoor (1986a), linuron (94.5% purity) was administered in the diet to 

aged male Crl:CD(SD)BR rats to investigate the induction of testicular interstitial cell (Leydig 

cell) hyperplasia and adenomas. Two groups of 25 rats each were given 625 ppm linuron 

beginning at 12 or 18 months of age and continuing for 6 and 12 months; terminal sacrifice was 

carried out at 24 months of age. Another group of 25 rats was given basal diet and served as 

controls. Compound intake for both treated groups was 22-23 mg/kg/day for the treatment 

interval. 

Treatment-related clinical signs of toxicity were limited to swollen testes observed in 2, 4, and 5 

rats in the control, 6-, and 12-month groups. A total of 6, 4, and 4 rats, respectively, were found 

dead or were sacrificed in extremis prior to scheduled termination; further details were not 

included. Body weights of both treated groups were reduced (p<0.05) compared with the control 

group within two weeks after the initiation of treatment. Final body weights for the control, 6-, 

and 12-month groups were 848.5, 670.1, and 668.5 g, respectively (additional data not provided). 

Body weight gains during the treatment intervals were less (p<0.05) than the controls with a 

weight loss for animals treated for 6 months (−107.8 g treated vs. +45.6 g controls) and a weight 

gain of 2.1% of the control level for the animals treated for 12 months. Food consumption and 

food efficiency values were slightly reduced [NS] for both treated groups as compared with the 

controls throughout the treatment intervals. 

At necropsy, discoloration of the testes was observed in 2/25, 4/25, and 6/25 rats in the control, 

6- and 12-month groups, respectively. However, the observation did not correlate with either 

interstitial cell adenoma or hyperplasia. For rats sacrificed at study termination, dose-related 

increases in the incidence of testicular adenoma were observed. The incidences in the control, 6-, 

and 12-month groups were 0/19, 2/21, and 6/21 (p<0.05), respectively. In addition, testicular 

hyperplasia was observed in 6/19, 7/21, and 14/21 (p<0.05) rats, respectively. No increased 

incidences of hyperplasia or adenoma in the adrenal cortex, adrenal medulla, or the pituitary 

gland were observed in the treated groups as compared with the control group. 

Biochemical and Histopathological Effects in Rat 

In another special study by Pastoor (1986b), the biochemical and histopathological effects of 

linuron (94.5% purity) under a variety of conditions were examined in Crl:CD® BR rats. The 

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rats utilized for various parts of this study were young adult males approximately 22 days old 

(young), retired breeder males approximately 11 months old (old), and male and female F1b and 

F2b rats from a multigeneration study (MRID 41463401) maintained on diets of 0, 12.5, 100, or 

625 ppm (equivalent to 0/0, 0.75/1.1, 4.1/6.1, and 22/37 mg/kg/day in males/females). 

Additionally, Leydig cell enzyme analyses were conducted in vitro.

The activities of five testicular steroidogenic enzymes were assayed in vitro using horse 

testicular microsomal preparations in the presence of 0, 0.5, 5, 50, 500, or 5000 μM linuron or 

linuron metabolites. Enzyme activities measured included aromatase, 17-20 desmolase 

(desmolase), 3-β-hydroxysteroid dehydrogenase/isomerase (isomerase), 17-hydroxylase 

(hydroxylase), and 17-ketosteroid reductase (reductase). Linuron concentrations of 500 and 

5000 μM resulted in significantly decreased aromatase and desmolase and increased reductase 

activities. At 50 μM the activities of aromatase, desmolase, isomerase, and hydroxylase were 

decreased by 10-20% and reductase was increased by 20% as compared with the controls. 

Effects of linuron metabolites on enzyme activities were highly variable and generally only seen 

at 5000 μM. 

The testosterone metabolic clearance rate was measured in young male rats. Groups of 5 animals 

were administered 0 or 200 mg/kg/day for eight days, castrated, and then infused with 

testosterone at 3 or 6 μg/hour. Blood samples were taken every 30 minutes for 180 minutes after 

the start of infusion. No differences between the treated and control groups were noted at the 

lower infusion rate. At the higher infusion rate, the mean plateau concentrations (60-180 

minutes) in the control and treated rats were 769±344 and 605±67.4 μg/dL, respectively, 

resulting in calculated metabolic clearance rates of 780 and 992 mL/h, respectively. Due to the 

large variability between individual animals it was concluded that linuron does not affect 

testosterone clearance in young, castrated rats. 

Two trials evaluated the response of Leydig cells to LH. In trial 1, the groups consisted of five 

young or five old males treated with 0 or 200 mg/kg/day for three days, and five 0-ppm and five 

625-ppm F2b males which were approximately 11 months old. In trial 2, the groups consisted of 

five young or five old males treated with 0 or 200 mg/kg/day for seven days, and five each of the 

0-, 12.5-, 100-, and 625-ppm F2b males which were approximately 19 months old. Leydig cells 

were isolated from the testes and incubated with up to 1000 ng LH/tube. Results from trial 1 

showed no differences in the response between the treated and control young rats. However in 

old rats, Leydig cells from the treated animals were less responsive than the controls both in 

maximum response and potency. In contrast, Leydig cells from the chronically treated F2b males 

were significantly more responsive to LH stimulation as compared with their controls. In trial 2, 

Leydig cells from treated young and old rats had decreased maximum responses and potency as 

compared with controls, with old rats more affected than young rats. From the chronically 

exposed rats, Ledig cells were moderately responsive from the control and low-dose groups, 

minimally responsive from the mid-dose group, but significantly greater in response from the 

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high-dose group. Taken together, the two trials were reasonably consistent with a preliminary 

conclusion that there are dose- and time-related effects of linuron upon the sensitivity of rat 

Leydig cells to stimulation by LH. 

Limited body weight and food consumption data were included for the F1b and F2b animals. 

High-dose animals had significantly (p ≤ 0.05) reduced body weights as compared to controls 

during days 469-708 for F1b males and females, days 0-533 for F2b males (none remained on test 

after this day), and days 0-709 for F2b females. At the mid dose, body weights were significantly 

(p ≤ 0.05) reduced for the F1b and F2b females on days 540-596 and 0-654, respectively. Food 

consumption was slightly less for the high-dose F2b males (21.8 g vs 28.3 g for controls) during 

days 519-526; additional data were not given. In the mid- and high-dose groups alopecia was 

observed in a slightly greater number of F1b females and a significantly (p ≤ 0.05) greater 

number of F2b females. 

F1b and F2b rats were sacrificed at two years of age for histopathological evaluation of selected 

tissues. The number of high-dose males of both generations with small or discolored testes was 

increased as compared with the controls (stated in text of DER; incidence rates not readable). No 

other gross observations were noted. Microscopically, mid- and high-dose males had increased 

incidences of interstitial cell adenomas and hyperplasia. Combining data from both generations, 

adenomas were observed in 1/19, 0/25, 6/25, and 2/16 animals and hyperplasia was observed in 

2/19, 0/25, 7/25, and 3/19 animals from the control, low-, mid-, and high-dose groups, 

respectively. The lower incidences in the high-dose group were probably due to fewer animals 

available for examination. In females the combined incidence rate for cervical endometrial 

hyperplasia was 0/28, 6/30, 9/29, and 13/29, respectively, and of cervical cystic hyperkeratosis 

was 0/28, 1/30, 1/29, and 7/29, respectively. These lesions in both males and females are 

consistent with findings in a 2-year chronic study. 

Avian Reproduction Toxicity (OCSPP 850.2300) 

In a Guideline avian reproduction toxicity study (MRID 42541802), adult (23-week old) Mallard 

ducks (Anas platyrhynchos) were administered linuron (98.4% purity) in the feed at nominal 

concentrations of 0 (control; acetone and corn oil), 50, 100, or 300 ppm (actual mean-measured 

concentrations 42.7, 87.3, and 262 mg/kg diet) for 18 weeks (eight weeks prior to 

photostimulation and during ten weeks of egg production). Each treatment group was comprised 

of 16 pens each with one male and one female bird; offspring did not receive treated feed. A 

progressive photo period was employed in this study: 8 hours/day through Week 8 and 

17 hours/day from the beginning of Week 9 to sacrifice of adult birds. 

All adult birds were observed for signs of toxicity or abnormal behavior at least once daily, and 

all birds that died during the study and their sacrificed pen mates were necropsied. Individual 

adult body weights were recorded at test initiation and on Weeks 2, 4, 6, and 8, and at 

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termination; food consumption was determined once a week throughout the study. At study 

termination, all surviving birds were sacrificed and necropsied. 

Eggs were collected daily from all pens and stored until incubation. Eggs were removed from 

storage and candled weekly; cracked or abnormal eggs were discarded. One egg was collected 

weekly from each odd or even numbered pen during odd or even weeks, respectively, for shell 

thickness determinations. The average thickness of the dried shell plus membrane was 

determined. All eggs not used for shell thickness measurements were incubated. Eggs were 

candled on Day 14 to determine embryo viability and on Day 21 to determine embryo survival. 

Eggs were placed in a hatcher on Day 24. All hatchlings and unhatched eggs were removed from 

the hatcher on Day 27 or 28, and the average weight of the hatchlings by pen was determined. 

Hatchlings were placed in brooding pens until 14 days old, at which time the average body 

weight by parental pen was determined for the survivors. 

There were no treatment-related mortalities or signs of overt toxicity at any concentration tested. 

There was one incidental mortality in the control group, but necropsies of the dead bird and its 

pen mate showed no treatment related findings. Incidental clinical signs were noted in various 

groups during the study, but these were associated with physical injury and wear and/or 

interaction among pen mates. 

There were no treatment-related effects on body weight, food consumption, reproduction, 

offspring survival, or offspring weight at 50 or 100 ppm. There were treatment-related decreases 

(p<0.05) in adult body weight (NS, male and female) during Week 1, feed consumption during 

Week 1, egg production, and egg shell thickness (significant 7.5% decrease) at 300 ppm. There 

was also a possible effect (not significant) upon the percent viable embryos of eggs set, the 

number of viable embryos, and the number of live embryos at 300 ppm. The reduction in the 

number of eggs laid and the slight reduction in the number of viable embryos resulted in a 

reduction (p<0.05) in the number of hatchlings and 14-day old survivors of maximum set. There 

were no significant differences between the controls and any treatment group in body weights of 

the offspring at hatching or at 14 days of age. 

The NOAEC for mallard ducks exposed to linuron in the diet for 18 weeks was 100 ppm. 

In an another Guideline avian reproduction study (MRID 42541801), 25-week old Bobwhite 

quails (Colinus virginianus) were administered linuron (98.4% purity) in the feed at nominal 

concentrations of 0 (control; acetone and corn oil), 50, 100, or 300 ppm (actual mean-measured 

concentrations 42.7, 87.3, and 262 mg/kg diet) for 21 weeks (seven weeks prior to egg laying 

and during eleven weeks of egg laying). Each treatment group was comprised of 16 pens each 

with one male and one female bird. A progressive photo period was employed in this study: 

8 hours/day through Week 7 and 17 hours/day from the beginning of Week 8 to sacrifice of adult 

birds. 

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All adult birds were observed for signs of toxicity or abnormal behavior at least once daily, and 

all birds that died during the study and their sacrificed pen mates were necropsied. Individual 

adult body weights were recorded at test initiation and on weeks 2, 4, 6, and 8, and at 

termination; food consumption was determined once a week throughout the study. At study 

termination, all surviving birds were sacrificed and necropsied. 

Eggs were collected daily from all pens and stored until incubation. Eggs were removed from 

storage and candled weekly; cracked or abnormal eggs were discarded. One egg was collected 

weekly from each odd or even numbered pen during odd or even weeks, respectively, for shell 

thickness determinations. The average thickness of the dried shell plus membrane was 

determined. All eggs not used for egg shell thickness measurements were incubated. Eggs were 

candled on Day 11 to determine embryo viability and on Day 21 to determine embryo survival. 

Eggs were place in a hatcher on Day 21. All hatchlings and unhatched eggs were removed from 

the hatcher on Day 25 or 26, and the average weight of the hatchlings by pen was determined. 

Hatchlings were placed in brooding pens until 14 days old, at which time the average body 

weight by parental pen was determined for the survivors. 

There were no treatment-related mortalities or signs of overt toxicity at any concentration tested. 

There were two incidental mortalities in the 100-ppm group, but necropsies of the dead birds and 

their pen mates showed no treatment related findings. Incidental clinical signs (ruffled 

appearance, wing droop, lethargy, and/or ventral head curl) were noted in various treated groups 

during the study, but these were determined unrelated to treatment. 

There were no treatment-related effects on adult body weight at any treatment concentration. 

There were no other significant differences between the controls and any treatment group at any 

body weight interval. There were increases (p<0.05) in food consumption in all treatment groups 

compared to controls. The differences appeared to be due, in part, to low values in the control 

group and it could not be determined if the differences were treatment related. 

There were no apparent treatment related effects on reproduction at 50 or 100 ppm. At 300 ppm, 

there was a slight (NS) reduction in egg production, and a reduction (p<0.05) in the ratio of 

hatchlings that survived to 14 days. There also appeared to be a reduction (p<0.05) in the ratio of 

14-day survivors/eggs set. When compared to controls, there were no apparent or significant 

differences in egg shell thickness or offspring body weights at hatching or at 14 days of age at 

any concentration tested. 

The NOAEC for Bobwhite quails exposed to linuron in the diet for 21 weeks was 100 ppm. 

FSTRA 

In a published study by Marlatt et al. (2013), the mode of action of linuron (99.7%) was 

examined in male and female fathead minnows (Pimephales promelas) using the biomarker 

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VTG, nuptial tubercle formation, GSI, egg hatching, and 7-day egg survival. The expression 

patterns of a number of key genes involved in steroidogenesis in the ovary were also examined, 

and gene clustering was performed to better elucidate the MOA of linuron compared to the 

model androgen DHT and the model estrogen E2. During the pre-exposure period, adult fathead 

minnows were distributed into groups of 2 males and 4 females per tank. Fish (4 

replicates/treatment) were exposed for 21 days to one of the following treatments: water control; 

solvent control (MeOH, 0.01%); E2 (0.1 μg/L); DHT (100 μg/L); linuron (1, 10, or 100 μg/L); 

and a co-treatment of DHT (100 μg/L) and linuron (100 μg/L) under semi-static conditions. The 

concentration of linuron was measured using an ELISA kit. Each test chamber contained two 

spawning substrates (semicircular PVC tiles with no base), and eggs adhering to a tile were 

quantified daily. For each treatment, 10 eggs were cultured in 100 mL of dechlorinated water in 

triplicate, and survival, number of days to hatch, and deformities at hatching were recorded. 

After 21 days, the fish were sacrificed and body weight and length were recorded. Blood was 

collected and centrifuged; the plasma was removed and stored at −80°C for VTG analysis using 

an ELISA kit. The gonadal tissue was removed and weighed to determine the GSI. The head was 

removed and stored in 10% neutral buffered formalin for subsequent tubercle counts. The 

number of tubercles on the snout of each fish was evaluated, and the tubercle size was 

quantitatively ranked as present, enlarged, or pronounced. Testes and ovaries of one male and 

one female per replicate were preserved and examined histopathologically to determine relative 

maturation stage and changes due to treatment. Three sections from the anterior, median, and 

posterior region of a gonad were examined microscopically and severity scores qualitatively 

assigned to pathological findings, based on the relative amount of affected gonad section 

compared to that of the controls. Additionally, RNA was extracted from ovarian tissue and 

processed, genomic DNA was removed, and real-time PCR analysis was performed. 

After 21 days of exposure, no significant differences were observed in GSI, egg production, days 

to hatch, or larva survival or deformity rates between any exposure group and the water control. 

There was no increase in the incidence of pathologies in the ovaries or testes in linuron-, DHT-, 

or mixture-treated fish. The DHT and DHT + linuron co-treatment significantly increased 

tubercle formation in females to approximately the same degree (mean±SE: 17±3 and 21±3, 

respectively) compared to the pooled control treatment groups (p = 0.013 for both comparisons). 

In males, there were no significant changes in tubercle formation between treatments. Treatments 

with linuron had significant effects on plasma VTG concentrations in both males and females, 

and in a sex-specific manner. Pooled control females exhibited plasma VTG levels with a 

median of 491.0 mg/mL and range of median replicate values from 68.1 to 1360.0 mg/mL. The 

DHT treatment caused a significant decrease in plasma VTG levels (median=8.4 mg/mL, range= 

0.9-33.6 mg/mL) in females compared to the controls. A similar significant anti-estrogenic effect 

on basal plasma VTG concentrations was observed in females exposed to linuron at 1 μg/L 

(median=4.6 mg/mL, range=0.7-65.0 mg/mL) and 100 μg/L (median=6.9 mg/mL, range=3.0-

12.8 mg/mL), but not at 10 μg/L (median=236.0 mg/mL, range= 4.2-1662.0 mg/mL) compared 

to the control treatments. The males in the DHT + linuron co-treatment and the 0.1 μg/L E2

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treatment exhibited significant (p<0.05) increases in plasma VTG (median values of replicates 

ranged 27.8-468.9 and 57.6-756.0 mg/mL, respectively) compared to the pooled control. 

Exposure to individual treatments of linuron or DHT did not change plasma VTG levels in 

males. For transcript expression in the ovary, only one transcript, p450ssc, showed significant 

variation across treatments, and the only difference observed was between the control and DHT 

+ linuron co-treatment, where p450ssc was decreased approximately 20-fold. The hierarchical 

clustering of mRNA transcript expression patterns revealed two distinct expression clades. One 

expression clade contained all three linuron treatments and the E2 treatment while the other 

expression clade contained control, DHT, and DHT + linuron. The gene expression patterns also 

showed that genes in the linuron treatments grouped more closely with E2 than DHT. 

Fish Early Life-Stage Toxicity (OCSPP 850.1400)

In a Guideline estuarine fish early life-stage study (MRID 43996502), Sheepshead minnows 

(Cyprinodon variegatus) <24 hours old were exposed to linuron in 0.1 mL/L DMF at meanmeasured concentrations of <0.0500 (LOQ; dilution and solvent control), 0.206, 0.357, 0.766, 

1.55, or 2.93 mg/L in a flow-through system for 36 days (32 days post hatch). The study 

evaluated percent hatch and day of hatch (2 replicates/treatment x 20 embryos/replicate), and 

time to swim-up, growth, and survival (2 replicates/treatment x 15 larvae/replicate). This study 

was considered supplemental as it only included two replicates instead of the required four 

replicates for embryo exposure. 

The post-hatch percent of embryos that produce live fry was 97.5% and 95% for the dilution 

water and solvent controls, respectively. The percentage survival at hatch was calculated by the 

study reviewer as the maximum number of live fish at hatch from Day 1 to Day 4, which differed 

slightly from those reported by the authors (only considered number of live fish on Day 4). 

Several fish in the three highest concentrations were lying motionless on their sides or were 

observed to be smaller than the controls. Percent of survival at hatch were similar to controls 

(92.5-97.5%) with linuron exposure at 0.206, 0.357, and 0.766 mg/L, but percent survival 

decreased to 80-90% and 7.5-12.5% with linuron exposure at 1.55 and 2.93 mg/L, respectively. 

Time to hatch was 3 days for all groups, except the linuron 2.93 mg/L group which was >4 days. 

Survival at 36 days (32 days post-hatch) dropped from 93.3-100% to 26.7-40% with 0.766 mg/L 

linuron and 0% survived with 1.55 and 2.93 mg/L linuron. Total length and wet weight were 

decreased in the 0.766 mg/L treatment group and could not be measured in the two highest 

treatment groups as there were no survivors. The NOAEC and LOAEC for linuron for 

sheepshead minnows were 0.357 and 0.766 mg/L, respectively. 

Another Guideline fish early life-stage study (MRID 42061804) was conducted with rainbow 

trout (Oncorhynchus mykiss). Trout embryos (21 hours post-fertilization) were exposed to 

linuron at 0 (dilution water control), 0.042, 0.11, 0.21, 0.39, 0.79, 1.6, and 3.2 mg a.i./L (meanmeasured) in a flow-through system for 80 days. The study evaluated embryo survival, day of 

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hatch, and swim up (2 replicates/treatment x 40 embryos/replicate), and survival, growth and 

behavioral effects (2 replicates/treatment x 15 larvae/replicate). Egg chambers were inspected 

daily and infertile, damaged, or diseased embryos were removed. The number of live embryos 

was recorded daily. After hatching was complete (Day 35) most alevins remained in the egg cups 

until swim-up was complete (Day 47). The number of fingerlings per replicate was randomly 

thinned to 15 and they were released into the aquarium. The number of live fingerlings was 

determined daily. Length of surviving trout after 47 (sampled at thinning) and 80 days (test 

termination) was determined. Wet weight of surviving fish was determined at test termination. 

Embryo survival at Day 47 (end of swim up) was significantly affected at 3.2 mg/L; NOAEC = 

1.6 mg/L, LOAEC = 3.2 mg/L. Fingerling survival at days 47 and 80 was affected at 

concentrations ≥0.39 mg/L; NOAEC = 0.21 mg/L, LOAEC = 0.39 mg/L. The 47-day LC50 was 

0.46 mg/L (95% CI = 0.39-0.57 mg/L) and the 80-day LC50 was 0.38 mg/L (95% CI = 0.25-1.0 

mg/L). Fish weight at test termination was significantly lower than the control at concentrations 

≥0.21 mg/L; NOAEC = 0.11 mg/L, LOAEC = 0.21 mg/L. Fish length at 47 and 80 days in all 

treatment levels was significantly lower than the control; NOAEC < 0.042 mg/L, LOAEC = 

0.042 mg/L. Toxicant-related behavioral effects were observed at 0.39 mg/L. 

The overall NOAEC was <0.042 mg/L and LOAEC was 0.042 mg/L based on reductions in fish 

length at all exposure levels. 

Fish Life-cycle Reproduction Toxicity (OCSPP 850.1500) 

The toxicity of linuron (99%) to the life cycle of fathead minnows (Pimephales promelas) was 

studied under flow-through conditions (MRID 49396901). The F0 generation was exposed for 

38 weeks (264 days) starting at the embryo stage (<24 hours). The F0 generation was allowed to 

reproduce, and F1 embryos were exposed for 30 days. Nominal concentrations of linuron were: 0 

(negative control), 0.0026, 0.0064, 0.016, 0.040, and 0.10 mg a.i./L. Mean-measured 

concentrations of linuron were: <0.00022-0.00036 (<LOQ, negative control), 0.0025, 0.0066, 

0.016, 0.037, and 0.097 mg a.i./L. The reviewer’s analysis detected a significant reduction 

(Dunnett’s, p<0.05) in Day 264 (Week 38) F0 female length at the mean-measured 0.016 mg 

a.i./L treatment level relative to the negative control. However, no reductions were detected at 

the higher treatment levels by Dunnett’s or Williams tests. Therefore, the reviewer agrees with 

the study author that this reduction was not biologically relevant or treatment-related. There were 

no effects on any of the other endpoints evaluated in this study (i.e., F0 hatching success and 

time-to-hatch; F0 4-week survival and length; F0 8-week survival and length; F0 27-week 

survival, length, and wet weight; F0 38-week survival, length, and wet weight; F0 eggs/spawn, 

spawns/female, and eggs/female; F1 hatching success, time-to-hatch; and F1 survival, length, 

and wet weight). The NOAEC and LOAEC were 0.097 mg a.i./L and >0.097 mg a.i./L, 

respectively). 

Androgenic/Anti-Androgenic Activity in Fish 

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Page 86 of 93 

In a published study by Jolly et al. (2009), the androgenic/anti-androgenic properties of linuron 

in fish were investigated using in vivo and in vitro assays. Exposure to exogenous androgens 

induces production of the glue protein spiggin in female stickleback kidney. In the in vitro

experiments, kidney cell cultures were prepared from batches of female three-spined sticklebacks 

(Gasterosteus aculeatus) exposed to DHT at a concentration of 5 μg/L for 10 days. To determine 

whether any of the test chemicals possessed anti-androgenic activities, DHT was added to the 

culture medium at a concentration of 3 μg/L. The ability of linuron (99% purity) to inhibit 

androgen-induced spiggin expression was determined by adding a range of concentrations of the 

test compounds (10−14 to 10−6 M) to the DHT-treated kidney cells. The agonistic activity of the 

seven chemicals was also assessed by exposing cell cultures at the same range of concentrations 

of the test compounds alone. After 48 hours of exposure, the spiggin content of the cells was 

determined by extraction and analysis using ELISA. In the in vivo experiments, 20 stickleback 

fish/treatment level were placed in glass aquaria and exposed to linuron in saltwater under flowthrough conditions for 21 days. A range of six concentrations was dissolved in methanol 

(≤0.01%), and co-administered with 5 μg/L DHT. The nominal concentrations chosen for linuron 

were: 2, 10, 25, 75, 100, and 250 μg/L. Additionally, each exposure experiment included a 

negative control (water), solvent control (methanol), a positive control (5 μg/L DHT), and a test 

compound control (test compound at the highest concentration tested in the absence of DHT). 

After 21 days of exposure, the fish were sacrificed and frozen in liquid nitrogen. After body 

weight measurement, the kidney was dissected, weighed and stored frozen until spiggin analysis. 

The sex of each fish was verified and only female fish were used for analysis. Spiggin level 

measurements were performed using ELISA and the protein levels were expressed as units of 

spiggin per gram of body weight. The actual concentrations of the test compounds in the water 

were verified from samples collected from each test aquarium on Days 0, 7, 14, and 21. A 

radioimmunoassay (RIA) was used for analysis of DHT and LC-MS was used for linuron. The 

recovery of DHT in all assays ranged from 59.5-102.2% of nominal and recoveries of linuron 

were 30.3-77.3%. 

In the in vitro androgenic experiment, DHT alone induced a statistically significant, 

concentration-dependent increase in spiggin production at all concentrations (≥3 pg/L). No 

significant effect on spiggin cell content was observed in cells treated with linuron at any 

treatment level. In the anti-androgenic experiment, linuron significantly inhibited the stimulatory 

effect of DHT on spiggin production in a concentration-dependent manner. 

In the in vivo androgenic experiment, DHT (positive control) alone induced a significant increase 

in spiggin production, resulting in a spiggin content of 104

-105 units/g body weight, compared to 

the negative and solvent controls (101

-102 units/g bw). No significant increases in spiggin 

production were observed compared to the controls in any test compound (linuron) at up to the 

highest concentrations tested. In the anti-androgenic experiment, linuron induced significant 

decreases in DHT-induced spiggin production at ≥100 μg/L. 

Page 94 of 101
LINURON FINAL 

Page 87 of 93 

Anti-Androgenic Activity in Fish 

In a published study by Katsiadaki et al. (2006), linuron was screened for anti-androgenic 

activity in female stickleback fish. Exposure to exogenous androgens induces production of the 

glue protein spiggin in female stickleback kidney. The fish were exposed to linuron (purity not 

provided) at 15 or 150 μg/L for three weeks using semi-static exposure conditions (replacement 

of approximately two-thirds of the exposure water every 48 h with fresh water containing 

linuron). The fish were co-exposed to 0.5 μg/L 17α-methyltestosterone (17α-MT) as the 

androgenic agent. At the end of the exposure period, the fish were killed and snap frozen in 

liquid nitrogen. The fish were weighed and their kidneys removed and solubilized. Kidney 

spiggin content was then quantified by ELISA. 

At 150 μg/L, linuron significantly inhibited the androgenic activity of 17α-MT based on spiggin 

induction in the kidneys (data presented graphically). At 15 μg/L, linuron did not inhibit spiggin 

induction by 17α-MT. 

Anti-Androgenic Activity in Fish 

In a published study by Pottinger et al. (2013), the anti-androgenic activity of linuron (≥99% 

purity) and other known anti-androgens was determined using the androgenized female 

stickleback screen with the quantitative assessment of androgen-induced spiggin production after 

exposure for 21 days. Only female stickleback fish were used in the study because low levels of 

endogenous spiggin are required for testing. The female stickleback fish were exposed to test 

chemicals in untreated raw (lake or sea) water under flow-through conditions at 100 mL/min. 

The test solutions were formulated in methanol such that the concentration of methanol in the 

exposure tanks was 0.1%. Water samples (1 L) were collected from each tank at the start of the 

study and at 7, 14, and 21 days after the start of the study for test chemical concentration 

analyses. For the single chemical exposures, female sticklebacks were exposed to 5 μg/L DHT 

with the linuron treatment concentrations. The additional controls used in each test included a 

negative control, containing the highest concentration of the test chemical without DHT, an 

absolute control, containing only water, and a solvent control, containing only methanol (at 

0.1%). The single compound test with linuron was conducted with seven nominal test 

concentrations ranging from 0.25 to 250 μg/L. The single compound data from all studies were 

pooled for data analysis. The mixture study consisted of a series of exposure tanks in which the 

individual chemicals were tested at both the IC50 and IC50/10 along with mixture tanks receiving 

all four anti-androgens at mixture ratios proportional to individual potencies ranging from IC50 to 

IC50/100. The controls used in the mixture study included water-only absolute control, methanolonly solvent control, and DHT-only positive control. At study termination (21 days), fish were 

killed immediately and stored individually at −20°C. The fish kidneys were dissected and stored 

at -20°C until processing for the spiggin ELISA. The DHT concentrations in the water extracts 

were determined using an established radioimmunoassay procedure. The concentrations of test 

Page 95 of 101
LINURON FINAL 

Page 88 of 93 

chemicals in the water extracts were determined by high performance liquid chromatography 

with electro spray ionization and selected ion mass spectrometry. 

In all individual and mixture tests, mortality was ≤1.5% for all control and treatment groups. For 

the single chemical exposure of linuron, the mean measured recovery from the exposure tanks 

was 43.3 ± 4.8%. It was noted that differences between measured concentrations and nominal 

concentrations may occur because of uptake by fish, losses due to degradation, adsorption to 

surfaces, evaporation, photolysis, or hydrolysis. Linuron was the least potent of the four antiandrogens with an IC50 of 172 μg/L and 95% confidence interval of 121-258 μg/L. Both mixture 

tests resulted in concentration-dependent inhibition of spiggin induction in DHT-primed female 

sticklebacks. 

 

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Page 89 of 93 

APPENDIX 3: References Not Utilized in the Linuron WoE Analysis 

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

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

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

as “other scientifically relevant information (OSRI) was provided in the Report of the Endocrine 

Disruptor Review Team (USEPA, 2010). 

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies 

previously submitted to the agency for registration/reregistration, published literature articles 

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

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

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

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

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

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

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

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

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

analysis for Linuron 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. 

Akahori, Y, Nakai, M, Yamasaki, K, et al. (2008) Relationship between the results of in vitro 

receptor binding assay to human estrogen receptor α and in vivo uterotrophic assay: comparative 

study with 65 selected chemicals. Toxicol. In Vitro 22: 225-231. (MRID 48041701) 

Bauer, E, Daxenberger, A, Petri, T, et al. (2001) Characterization of the affinity of different 

anabolic and synthetic hormones to the human androgen receptor, human sex hormone binding 

globulin and to the bovine progestin receptor. APMIS 108 (12): 838-846. (MRID 48041703) 

Clayton, R, Wesley Jr, and Harper, K et al. (1965) Effect of H634 upon reproductive function in 

the rat: Part I F0 (Parent) generation. DuPont de Nemours and Co. and Huntingdon Research 

Centre, England. Laboratory Report No. 1352/65/270, November 5, 1965. 

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Crago, J, and Klaper, R (2012) A mixture of environmentally realistic concentration of a 

phthalate and herbicide reduces testosterone in male fathead minnow (Pimephales promelas) 

through a novel mechanism of action. Aquatic Toxicology: 110-111: 74-83. 

Degitz, S, Holcombe, G, Flynn, K, et al. (2005) Progress towards development of an amphibianbased thyroid screening assay using Xenopus laevis. Organismal and thyroidal responses to the 

model compounds 6-propylthiourqcil, methimazole, and thyroxine. Toxicol. Sci. 87: 353-364. 

(MRID 48041705) 

Diehl, K, and Leist, K (1987) Testing for the effect of linuron - active ingredient technical on the 

Leydig cell system in the male rat; 4-week substance application by gavage. Pharma Research 

Toxicity and Pathology, Hoechst Aktiengesellschaft. Laboratory Study No. 87.0114, March 4, 

1987. 

EURISKED (2010) Summary of results of the EURISKED project. 

http://www.eurisked.org/results.htm Note: address does not work. (MRID 48041706) 

Gordon, J, Chu, A, Chu, M, et al (2003) Validation of the LUMI-CELL© ER recombinant 

bioassay for rapid evaluation of chemicals for potential estrogenic activity. Organohalogen 

Compounds 65: 78-81. 

Hogan, N, Gallant, M, and van den Heuvel, M (2012) Exposure to the pesticide linuron affects 

androgen-dependent gene expression in the three-spined stickleback (Gasterosteus aculeatus). 

Environ. Toxicol. Chem. 31: 1391-1395. 

Hodge, H, and Downs, W (1964) Second reproduction study of rats fed linuron. Department of 

Pharmacology, University of Rochester School of Medicine and Dentistry, Rochester, NY. 

February 26, 1964. (MRID 00018169) 

Hodge, H, Downs, W, Smith, D et al. (1968) Oral toxicity of linuron (3-(3,4-dichlorophenyl)-1-

methoxy-1-methylurea) in rats and dogs. Fd. Cosmet. Toxicol. 6: 171-183. 

Iwasaki, T, Hase, A, and Totsukawa, K (2001) The endocrine-disrupting activity of linuron in 

mice. Environmental Sciences 8: 274-5. (MRID 48041711) 

Jolly, C, Katsiadaki, I, Le Belle, N, et al. (2006) Development of a stickleback kidney cell 

culture assay for the screening of androgenic and anti-androgenic endocrine disrupters. Aquat. 

Toxicol. 79: 158-166. (MRID 48041712) 

Katsiadaki, I, Scott, A, Hurst, M, et al. (2002) Detection of environmental androgens: a novel 

method based on enzyme-linked immunosorbent assay of spiggin, the stickleback (Gasterosteus 

aculeatus) glue protein. Environ. Tox. Chem. 21 (9): 1946-1954. (MRID 48041718) 

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Leist, K (1988) Study on the effect of linuron - substance technical on the reproductive system of 

male rats during 3 months of treatment. May 27, 1988. (MRID 40950201) 

Lo, S, King, I, Alléra, A, et al. (2007) Effects of various pesticides on human 5α-reductase 

activity in prostate and LNCaP cells. Toxicol. in Vitro 21: 502-508. (MRID 48041721) 

Luccio-Camelo, D, and Prins, G (2011) Disruption of androgen receptor signaling in males by 

environmental chemicals. J. Steroid Biochem. Mol. Biol. 127: 74-82. 

Martyniuk, C, Alvarez, S, Lo, B, et al. (2012) Hepatic protein expression networks associated 

with masculinization in the female fathead minnow (Pimephales promelas). J. Proteome Res. 11: 

4147-4161. 

Nishihara, T, Nishikawa, J-I, Kanayama, T, et al. (2000) Estrogenic activities of 517 chemicals 

by yeast two-hybrid assay. J. Health Sci. 46(4): 282-298. (MRIDs 48033010, 48041723) 

OECD (2007) Validation of the amphibian metamorphosis assay as a screen for thyroid-active 

chemicals: Integrated Summary Report. Organization of Economic Cooperation and 

Development. October 16, 2007. 

Opitz, R, Braunbeck, T, Bogi, C, et al. (2005) Description and initial evaluation of a Xenopus

metamorphosis assay for detection of thyroid system disrupting activities of environmental 

compounds. Environ. Toxicol. Chem. 24: 653-664. (MRID 48041725) 

Opitz, R, Hartmann, S, Blank, T, et al. (2006) Evaluation of histological and molecular 

endpoints for enhanced detection of thyroid system disruption in Xenopus laevis tadpole. 

Toxicol. Sci. 90 (2): 337-348. (MRID 48041726) 

Orton, F, Lutz, I, Kloas,W, et al. (2009) Endocrine disrupting effects of herbicides and 

pentachlorophenol: in vitro and in vivo evidence. MRID 48041727. Environ. Sci. Technol. 43 

(6): 2144-50. 

Orton, F, Rosivatz, E, Scholze, M, et al. (2012) Competitive androgen receptor antagonism as a 

factor determining the predictability of cumulative anti-androgenic effects of widely used 

pesticides. Environ. Health Perspect. 120: 1578–1584 

Owens, W, Zeiger, E, Walker, M, et al. (2006) The OECD program to validate the rat 

Hershberger bioassay to screen compounds for in vivo androgen and anti-androgen responses. 

Phase 1: use of a potent agonist and a potent antagonist to test the standardized protocol. 

Environ. Health Perspect. 114: 1259-1265. 

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Owens, W, Gray, L, Zeiger, E, et al. (2007). The OECD program to validate the rat Hershberger 

bioassay to screen compounds for in vivo androgen and antiandrogen responses: Phase 2 dose 

response studies. Environ. Health Perspect. 115 (5): 671-678. 

Pastoor, T (1984) Multi-generation reproduction study in rats with 3-(3,4-dichlorophenyl)-1-

methoxy-1-methylurea (LOROX®, LINURON, INZ-326). Haskell Laboratory for Toxicology 

and Industrial Medicine, E. I. du Pont de Nemours and Company, Newark, DE. Laboratory 

Project No. 4580-001, October 26, 1984. Unpublished. (MRID 00146071) 

Rider, C, Wilson, V, Howdeshell, K, et al. (2009) Cumulative effects of in utero administration 

of mixtures of "anti-androgens" on male rat reproductive development. Toxicol. Pathol. 37: 100-

113. 

Rider, C, Furr, J, Wilson, V, et al. (2010) Cumulative effects of in utero administration of 

mixtures of reproductive toxicants that disrupt common target tissues via diverse mechanisms of 

toxicity. Int. J. Androl. 33: 443-462. 

Rogers, J, and Denison, M (2000) Recombinant cell bioassays for endocrine disruptors: 

development of a stably transfected human ovarian cell line for the detection of estrogenic and 

anti-estrogenic chemicals. In Vitr. Mol. Toxicol. 13: 67-82. (MRID 48041728) 

Schmutzler, C, Gotthardt, I, Hofmann, P, et al. (2007) Endocrine disruptors and the thyroid 

gland - a combined in vitro and in vivo analysis of potential new biomarkers. Environ. Health 

Perspect. 115 (Suppl. 1): 77-83. (MRID 48322304) 

Sherman, H, Hood, D, Barnes, J, Schepers, G, et al. (1961) The toxicity of 

3-(3,4-dicholorphenyl)-l-methoxy-l-methyl urea (INZ 326). Haskell Laboratory Report No. 47-

61. October 19, 1961. 

Shin J, Moon H, Kang I, et al. (2007) OECD validation of the rodent Hershberger assay using 

three reference chemicals: 17α-methyltestosterone, procymidone, and p,p'-DDE. Arch. Toxicol. 

81: 309-318. 

Thienpont, B, Tingaud-Sequeira, A, Prats, E, et al. (2011) Zebrafish eleutheroembryos provide a 

suitable vertebrate model for screening chemicals that impair thyroid hormone synthesis. 

Environ. Sci. Technol. 45: 7525-7532. 

Tietge, J, Holcombe, G, Flynn, K, et al. (2005) Metamorphic inhibition of Xenopus laevis by 

sodium perchlorate: effects on development and thyroid histology. Environ. Toxicol. Chem. 24: 

926-933. (MRID 48041730) 

USEPA (2005a) http://www.epa.gov/endo/pubs/aromatase_prevalidation.pdf. 

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USEPA (2005b) http://www.epa.gov/endo/pubs/edmvs/aromatase_drp_final_3_30_05.pdf. 

USEPA (2007b) Integrated summary report for validation of an estrogen receptor binding assay 

using rat uterine cytosol as source of receptor as a potential screen in the Endocrine Disruptor 

Screening Program Tier 1 battery. October 2007. Office of Science Coordination and Policy, 

Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC.

Vinggaard, A, Hnida, C, Breinholt, V, et al. (2000) Screening of selected pesticides for 

inhibition of Cyp19 aromatase activity in vitro. MRID 48041732. Toxicology in Vitro 14: 227-

234. 

Waller, C, Juma, B, Gray Jr., L, et al. (1996) Three dimensional quantitative structure activity 

relationships for androgen receptor ligands. Toxicol. Appl. Phamacol. 137: 219-227. (MRID 

48041734) 

Wilson, V, Blystone, C, Hotchkiss, A, et al. (2008) Diverse mechanisms of anti-androgen action: 

impact on male rat reproductive tract development. Int. J. Androl. 31: 178-187. (MRID 

48041736) 

Yamasaki, K, Noda, S, Imatanaka, N, et al. (2004) Comparative study of the uterotrophic 

potency of 14 chemicals in a uterotrophic assay and their receptor-binding affinity. Toxicol. Lett.

146: 111-120. (MRID 48041740) 

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