UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

SUBJECT: EDSP Weight of Evidence Conclusions on the Tier 1 Screening Assays for the
List 1 Chemicals

PC Code: See table, Attachment A Decision No.: NA Petition No.: NA
Risk Assess ment T y pe: NA
TXR No.: See table

, Attachment A
MRIDNo.:NA DP Barcode: NA Registration No.: NA Regulatory Action: NA
Case No.: NA
CAS No.: NA
40CFR: NA

FROM: Greg Akerman 

# &

Health Effects Divison

# ::~:E£'~:::e 

# f] g:ams d f 'v--

Environmental

# Fat**~~ fec** **~~ ~n** **~** Office of Pesticide Program

THR O UG H: Je ss Rowland

## ~ S

## ~~~ Deputy Divi s ion Director
Health Effects Division
Office of Pe s ticide Programs

TO: Jolene Trujillo Chemical Review Manager
Pe s ticide Re-Evaluation Division Office of Pesticide Programs Ver .A pr. 2010

EPA has completed its Weight of Evidence (WoE) assessment eval u ating results of the
Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals.
The WoE documents for the 52 chemicals are li sted in Attac hm ent A a l ong with the chemical
and report identifiers

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals

Chemical Name PC Code TXR Number 2,4-D 030001 0057151
Abamectin 122804 0057152 Acephate 103301 0057153
Acetone 044101 0057154
Atrazine 080803 0057155
Benfluralin 084301 0057156
Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159
Carbofuran 090601 0057160
Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164
DCPA 078701 0057165
Diazinon 057801 0057166
Dichlobenil 027401 0057167
Dimethoate 035001 0057168
EPTC 041401 0057169
Esfenvalerate 109303 0057170 Ethoprop 041101 0057171
Fenbutatin-Oxide 104601 0057172
Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178
Linuron 035506 0057179
Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182
Metolachlor 108801 0057183
Metribuzin 101101 0057184
MGK-264 057001 0057185 Myclobutanil 128857 0057186
Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142
PCNB 056502 0057138
Permethrin 109701 0057149
Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141
Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140
Simazine 080807 0057136
Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147
Triadimefon 109901 0057139
Trifluralin 036101 0057135

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL
INTERACTION WITH THE ESTROG EN ANDROGEN OR THYROID

PATHWAYS

CHEMICAL: LINURON

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYLINURON FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary..........................................................................................1

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

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

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

B. Other Scientifically Rele vant Information (OSRI)......................................5

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................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-Est rogenic Pathway for Linuron........................................11
Table 3. Androgenic/Anti-Andr ogenic 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................................89LINURON FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance
AOP Adverse Outcome Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r CFR Code of Federal Regulations

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g eneration
F2 Second filial g enerationLINURON FINAL

iv Abbreviation Terminology

Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

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

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

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

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated Concentration
MTD Maximum Tolerated Dose
N Ne g ativeLINURON FINAL

v Abbreviation Terminology

NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating HormoneLINURON FINAL

vi Abbreviation Terminology

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

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

Page 1 of 93 Executive Summary

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

In determining whether 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 conditi ons under which effects occur were considered, in particular, whether or not th e dose(s) at which endocrine rela ted 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 pathwa ys. 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 wi ldlife studies (40 CFR Pa rt 158 and literature).

For the estrogen pathway, although positive results were reported in the li terature for ER-related

responses in vitro , the responses are not supported by the Ti er 1 assays (negative ER binding and uterotrophic assays) or other literature in vitro assays evaluating ER tr ansactivation assays in CHO cells, yeast ER-reporter assays or ER-resp onsive proliferation assay. Linuron showed an equivocal response for aromatase inhibition in vitro . In the female pubertal assay, decreased ovarian weight was observed, however there wa s 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 toxicit y, there were alterations in fecu ndity and fertility at 0.92 mg/L, however, these effects are limited in their diagnos tic 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 OS RI, plasma vitellogenin concentrations were significantly reduced in females; however, no other effects were obs erved in this study. Reductions in egg production (fec undity) were noted in two Part 158 avian reproduction studies, but again, these findings are limited in their ability to identify poten tial estrogen-related interactions; decreases in eggshell thickness was observed in the malla rd reproduction study, and decreased female body weight and food consumpti on was also observed at this concentration.LINURON FINAL

Page 2 of 93 Overall, while some in vitro assays did indicate potential interaction with the estrogen pathway, these findings were not s upported by the results of in vivo mammalian and wildlife studies.

For the androgen pathway, there is consistent complementarity and re dundancy of evidence across the Tier 1 and OSRI to suggest th at 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 test es. The findings of multiple Hershberger assays, male pubertal assays, and reproductive deve lopmental toxicity studies in rats, as well as assays measuring the inhibition of androgen-i nduced 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 wei ghts and histopathology in the testes and epididymides.

There is convincing evidence of potential intera ction of linuron with the thyroid pathway. Although no thyroid histopat hology effects were observed in any of the Tier 1 or OSRI studies,
decreases in serum T3 and/or T4 levels we re observed across multiple studies including the EDSP Tier 1 female pubertal assa y, a 12-week female rat thyroi d toxicity study, and two 15-day adult male rat screening assays considered as OSRI. Additionally, linuron showed weak agonist activity to the T 3 receptor in two in vitro transcriptional activation as says 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 availa ble data suggests that linuron does not interact with the estrogen pathway. For the androgen pathwa y, 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 ho rmone 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 me t- 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 chroni c dietary risk assessmen t 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-rela ted 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 endpo ints of concern are base d on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young.LINURON FINAL

Page 3 of 93 Therefore, a special thyroid a ssay in pregnant animals, pos tnatal, 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 prot ect 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) th at may be used for human health risk assessment. The POD from this special study woul d address the concern fo r the potential ability of linuron to disrupt thyroid f unction in pregnant females a nd 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 obser ved 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 concen tration tested (0.097 mg a.i./L). Given the potential for androgen-related e ffects in fish, the EDSP Tier 2 Medaka Extended One Generation Reproduction Test (MEGORT) using Ja panese 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 satis fied by the OSRI that demonstrated potential interaction with the thyr oid pathway. Therefore, the EDSP Ti er 2 Larval Amphibian Growth and Development Assay (LAGDA) is recommended to a dd to the understanding of potential adverse effects in amphibians.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for linuron. In both speci es, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potenti al reproductive effects to birds from linuron exposure. Relative to the EDSP, ad ditional testing is not recommended.LINURON FINAL

Page 4 of 93 I. Introduction

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

In determining whether 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 conditi ons under which effects occur were considered, in particular, whether or not th e dose(s) at which endocrine rela ted 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 we ight-of-evidence (WoE) analysis of the potential interaction of linuron with the E, A or T hormone path ways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Linuron, a phenylurea herbicide has an octanol/water partition (log K ow ) of 3.00 and a water solubility of 81 mg/L at 20°C. In the envir onment, linuron is expected to be persistent, moderately mobile and semi-volatile.

The available information considered in determin ing the potential interacti on 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 stud ies 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 (U SEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recommendations ar e presented in Section V.LINURON FINAL

Page 5 of 93 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 cons idered relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Orde r 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 re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam (EDRT) on linuron (USEPA, 2010). Since then, the Agency has also conducted a more recent sear ch of available scientif ic literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

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

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

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

Page 6 of 93 designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

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

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the 2013 FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening proce ss. In October 2013, the SAP stated that , “ InLINURON FINAL

Page 7 of 93 summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All effects in endocrine se nsitive 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 wa s generally defined in accordance with EPA’s current approach as used by OPP in reviewi ng 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these an alyses, the effects that EPA considered to be potential evidence of overt toxicity included, but were not limited to: mo rtality; clinical signs such as tremors, ataxia and abnormal swimmi ng (fish and aquatic-pha se amphibians); and body weight decreases of ≥ 10% in mammals. Additionally, ot her effects including morphological ( e.g ., organ weights/histopat hology), 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/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

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

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

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

Page 8 of 93 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)

ER α 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)LINURON FINAL

Page 9 of 93 B. Effects on Hypothalamic-P ituitary-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 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

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 ve ry weak inducer of transcription (EC 50 of 1.45 ×

10

− 5 M compared to 1.55 × 10

− 11 M for estradiol). In additio n, 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 st udy considered as OSRI, linuron was negative
for ER α and ER β 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, l inuron 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 determin ed to be equivocal for aromatase inhibition. The test order requirement for steroidoge nesis assay was satisfied by OSRI (Wilson et al ., 2009); however, estradiol production wa s not evaluated in the study

.
Linuron did not increase uterine weig hts 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 wa s 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 conc entration (9.1 mg/L), including changes in gonadal stag ing, 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 concentrati on (0.92 mg/L) in the absence of overt toxicity. In a FSTRA considered as OSRI with fath ead minnow (Marlatt et al ., 2013), plasma vitellogenin concentrations were significan tly 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 developmenta l rat study, increased post-im plantation loss and number of resorptions (in litters with resorptions) was observed at the highest dose (approximately 50LINURON FINAL

Page 10 of 93 mg/kg/day). In a developmental rabbit study, spon taneous abortion, decreases in the number of live fetuses per litter, and decreased mean fetal body weights we re 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-genera tion 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 toxi city (premating body wei ghts 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 toxi city study, decreases were noted in egg production (30%) and eggshell thickness (7.5% ) at the highest dietary con centration (300 mg/kg diet); possible effects on embryo viability (viable embr yos/eggs set but not statistically significant) were also reported at this concentration. Decr eased 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 rati o of hatchlings that survived to Day 14. In a sheepshead minnow Part 158 early lif e 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.LINURONFINALPage 11 of 93Table 2. Estrogenic/Anti-Estrogenic Pathway for LinuronLines 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) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48617402)NERTARequirement satisfied by OSRI (Gordonet al., 2004; see below)Aromatase(MRID 48617401)ESteroidogenesisRequirement satisfied by OSRI (Wilsonet al.,2009)4Uterotrophic(MRID 48617403)NP5(H)X6(H)Female Pubertal Rat(MRID 48685501)NEN↓12%(H)N↓15%,↓22%(L,H)N NX(L, H)NFSTRA(MRID 48671701)NENNNFrt:↓5%Fcd:↓50%(M)NX7(H)OSRIERTA(Gordonet al., 2004)PERTA (Kojimaet al.,2004)NLINURONFINALPage 12 of 93Table 2. Estrogenic/Anti-Estrogenic Pathway for LinuronLines 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) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Yeast ER assay andFrog OvulatoryResponse (Ortonetal., 2009)PPCell Proliferation(Vinggaardet al.,1999)NFSTRA (Marlattetal., 2013)NENNNNP(L, H)NDevelopmentalToxicity (Rat; MRID00018167)P(H)X(H)NDevelopmentalToxicity (Rabbit;MRIDs 00153867and 40437201)NX(H)X(H)Two-GenerationReproductionToxicity (Rat; MRID48046002)N N N N NENX(H)X(H)Two-GenerationReproductionToxicity (Rat;MRIDs 41463401and 41864701)NE NE NE NE NENX(M, H)X(H)Chronic Toxicity(Dog; MRID40952601)NE N N NE NEX(M, H)NLINURONFINALPage 13 of 93Table 2. Estrogenic/Anti-Estrogenic Pathway for LinuronLines 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) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Carcinogenicity(Mouse; MRID00124195)N N N N NEX(H)X(H)Chronic Toxicity/Carcinogenicity (Rat;MRIDs 00029679,00029680, and00164117)NE NE N N NEX(M, H)X(H)Avian ReproductionToxicity (MallardDuck; MRID42541802)P8(H)NN9(H)Avian ReproductionToxicity (BobwhiteQuail; MRID42541801)P10(H)N NFish Life-cycleToxicity(Fathead minnow;MRID 49396901)NN1.Key to responses: Low treatment (L), Medium-low treatment (ML), Medium treatment (M), Medium-high treatment (MH), High treatment (H).Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in thisassay. 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 anX in this column. For details see Section IV. A3.The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4.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 dayLINURONFINALPage 14 of 936.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 toxicityincluding lethargy and loss of equilibrium in fish at the 9.1mg/L.8.Egg production, and egg shell thickness (significant 7.5% decrease) at 300 ppm9. There were treatment-related decreases inadult 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 responseP Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedLINURON 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 androgeni c, anti-androgenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

The requirements for the Tier 1 AR binding, st eroidogenesis, Hershberger and male pubertal assays were satisfied by OSRI. In the OSRI AR binding assays using rat prostate cytosol,
linuron had an IC 50 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 IC 50 of approximately 20 μM (Lambright et al . 2000). Additionally, linuron was found to have similar

IC 50 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 st udies (ARTA) (Lambright et al ., 2000; Freyberger et al ., 2010; and McIntyre et al ., 2000), linuron antagonized AR transcri ptional 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 5 α -reductase, linuron inhibited 5 α -reductase activity in human prostate homogenates with an IC 50 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 androge nic/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 an ti-androgenic activity as evidenced by consistent reductions in wei ghts of the accessory sex tissues (AST) and epididymides. Overt toxicity was no ted in only two studies (Freyberger et al ., 2009; and Tinwell

et al ., 2007), where terminal body weight were de creased by 12% and 20% at the high dose (100 mg/kg/day in both studies). A dditionally, in a testosterone implant study (Lambright et al

., 2000), linuron administered at 100 mg/kg/day decr eased weights of epididymides and AST and increased expression of the TRMP2 gene, an an drogen-inhibited gene which reportedly peaks within five days of castrati on or anti-androgen treatment.

In a study that was part of the validation effo rt for the male pubertal assay (USEPA, 2007c), linuron at 50 or 100 mg/kg/day decreased test es, epididymides, pituitary, and accessory sex organ weights, and delayed PPS, a lthough overt toxicity (body weights ↓ 16%) was observed at 100 mg/kg/day. Similarly in two 15-day a dult rat screening assays (O’Connor et al ., 2002; and USEPA, 2007d), linuron decrease d testosterone levels, caused alterations in gonadal pathology,LINURON FINAL

Page 16 of 93 and decreased epididymides and accessory sex orga n weights. However, in these studies, overt toxicity ( ↓≥ 10% body weight) was observed at the hi gh 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 presen ce of overt toxicity ( ↓ maternal body wt). Alterations in testes and epididymides histopathology ( ≥ 12.5 mg/kg/day) and increase in ni pple 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), th ere were histopathological changes in the testes and epididymis; it is noted in this study that dos ing 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, decr eased anogenital distance (AGD), increased areolae/nipple retent ion. 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 an ti-androgenic effects were noted at doses up to 53 mg/kg/day; how ever, decreases in body weights (>10%) and food consumption, and increases in spleen weights we re 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 le sions in the testes and epididymides, concurrent wi th 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, epid idymides, 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/carcinoge nicity 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, de creased body weight ( ↓ 11%) was observed at the high dose

Pastoor et al . (1986a) exam ined the effects of linuron on Leydig cell hyperplasia in aged male rats. A dose level of 31 mg/kg/day ca used discoloration of the testes and an increase in testicular adenomas and testicular hyperplasia; however, overt toxicity was not ed in these animals (decreased body weights >10%).

In the Tier 1 FSTRA, at the high test concentrat ion (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 (decreasedLINURON FINAL

Page 17 of 93survival and abnormal swimming as well as decreases in male growth parameters). In a nonGuideline FSTRA by Marlatt et al . (2013), no androgen-specific e ffects 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 pr oduction 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-indu ced spiggin production. Similar effects (reduction of siggin production) were reported in the additiona l open literature studies [(Katsiadaki et al . (2006); Pottinger et al . (2013)], however, purity information for th e test substance (i.e., linuron) was not provided in those studies, and ther efore, these results are not pres ented in the table below. In a Part 158 reproductive toxicity study with the ma llard 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.LINURONFINALPage 18 of 93Table 3. Androgenic/Anti-Androgenic Pathway for LinuronLines 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR BindingAssay satisfied by OSRI (Cooket al.1993, Lambrightet al.2000, and USEPA 2007a; see below)SteroidogenesisAssay satisfied by OSRI (Wilsonet al., 2009; see below)HershbergerAssay satisfied by OSRI (Ashbyet al., 2004, Freybergeret al.,2007, Freybergeret al.,2009, Kanget al.,2004, Lambrightet al.,2000, Moonet al.,2009, Moonet al.,2010, and Tinwellet al.,2007; see below)Male Pubertal RatAssay satisfied by OSRI (USEPA 2007c and USEPA 2007d; see below)FSTRA(MRID 48671701)NENNNFrt:↓5%(M)NNEX(H)4OSRIAR Binding(Cooket al.1993)PAR Binding(Lambrightet al.2000)PAR Binding(USEPA 2007a)PAR Binding(Wilsonet al., 2004)PLINURONFINALPage 19 of 93Table 3. Androgenic/Anti-Androgenic Pathway for LinuronLines 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3AR Binding(Wilsonet al., 2007)PARTA (Lambrightetal., 2000)PARTA (Freybergeretal., 2010)PARTA (McIntyreetal., 2000)PFetal Testosterone(Wilsonet al.,2009)PHershberger(Ashbyet al., 2004)↑31%(H)↓19%(H)CG↓13-29%(M, H);SV↓25%,PR↓34%(H)X(M, H)NHershberger(Freybergeret al.,2007)CG↓29%,LABC↓43%,SV↓54%,VP↓50%(H)X(H)NHershberger(Freybergeret al.,2009)NNNX(H)X(H)LINURONFINALPage 20 of 93Table 3. Androgenic/Anti-Androgenic Pathway for LinuronLines 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Hershberger(Kanget al.,2004)NSV↓28-47%,VP↓25-38%,CG↓26-39%(M, H);LABC↓35%,GP↓20%(H)X(H)NHershberger(Lambrightet al.,2000)SV↓44%VP↓37%,LABC↓22%X NHershberger(Moonet al.,2009)VP↓40-68%,SV↓48-71%,LABC↓28-50%,CG↓32-55%,GP↓12-19%(L, H)NNHershberger – Stage3 testing (Moonetal.,2010)N↓17%(H)VP↓22%,SV↓25%,LABC↓21%,CG↓26%(H)NNHershberger – Stage4 testing (Moonetal.,2010)N↓21%(H)VP↓23%,SV↓48%,LABC↓27%,CG↓34%(H)NNLINURONFINALPage 21 of 93Table 3. Androgenic/Anti-Androgenic Pathway for LinuronLines 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Hershberger(Tinwellet al.,2007)NNNX(H)X(H)Hershberger –Phase 2 (OECD,2009)N↓19%(H)VP↓24-27%,SV↓26-29%,LABC↓20-23%,CG↓28%(H)NNHershberger Phase 3(OECD, 2009)↓24-32%(H)↓21-27%(H)VP↓23%,SV↓32-48%,LABC↓20-27%,CG↓34%(H)NNTestosterone Implant(Lambrightet al.,2000)N↓17%VP↓33%,LABC↓24%XNMale Pubertal(USEPA 2007c)NNN↓(L)N↓(L)↓SV,↓VP,↓DP,↓LABC(L)Delayed(L)X(L, H)X(H)15-Day AdultScreening Assay(O’Connoret al.,2002)↓40% (H)NP(MH, H)↓9%(H)NPR↓19%, AST↓11%(H)X(ML,MH, H)NReproductive/Development in MaleRats (McIntyreet al.,2000)N NP(L, M, H)NP(L,M, H)DP↓11%(H)NN NLINURONFINALPage 22 of 93Table 3. Androgenic/Anti-Androgenic Pathway for LinuronLines 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Reproductive/Development in MaleRats (Lambrightet al.,2000)PPX4NReproductive/Development in MaleRats (Wolfet al.,1999)↓22% P↓29-35%P NRSV↓12% (NS),VP↓35%,LABC↓16%,GP↓16%;AGD↓30%X NTwo-GenerationReproductionToxicity (Rat; MRID48046002)NNNNNNNX(H)X(H)Two-GenerationReproductionToxicity (Rat;MRIDs 41463401and 41864701)NNNENNENENX(M)X(H)Multi-GenerationReproductionToxicity in Male Rats(Wolfet al., 1999)N↓20%(H)N↓18%(H)NSV↓14%,CaudaEpididymides↓7% (H)P(H)↑2.8 d(H)NNChronic Toxicity(Dog; MRID40952601)NNNENNEX(M, H)NLINURONFINALPage 23 of 93Table 3. Androgenic/Anti-Androgenic Pathway for LinuronLines 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Carcinogenicity(Mouse; MRID00124195)NNNNNX(H)X(H)Chronic Toxicity/Carcinogenicity (Rat;MRIDs 00029679,00029680, and00164117)↑21%(M,H)NNEP(M )NX(M, H)X (H)Leydig CellHyperplasia in AgedMale Rats (Pastoor,1986a)NPNNNXBiological andHistopathologicalEffects in Rats(Pastoor, 1986b)PNPFSTRA (Marlattetal., 2013)NNNNNAndrogenic/AntiAndrogenic Activityin Fish (Jollyet al.,2009)PAvian ReproductionToxicity (MallardDuck; MRID42541802)NNN6(H)LINURONFINALPage 24 of 93Table 3. Androgenic/Anti-Androgenic Pathway for LinuronLines 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Avian ReproductionToxicity (BobwhiteQuail; MRID42541801)NN NFish Life-cycleToxicity (Fatheadminnow; MRID49396901)NN1. Key to responses: L=Low treatment, ML=Medium-low treatment,M=Medium treatment, MH=Medium high treatment, H=High treatment.Arrows (↓or↑)indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight areabsolute unless otherwise indicated. Abbreviations for androgensensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate(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 OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. A4. Survival was decreased and there were clinical signs of toxicity including lethargy and loss of equilibrium in fish at the 9.1 mg/L. Additionally, decreases inmale 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 bodyweight gains6. There were treatment-related decreasesin adult body weight (NS, male and female)P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedLINURON FINAL

Page 25 of 93 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal female and male rat assays, and the AMA (frog).

The potential for linuron to interact w ith thyroid regulation are summarized in Table 4. The various targets of the th yroid 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 ar e not reported in the ta ble and not considered further in the WOE assessment.

Of the Tier 1 assays utilized for assessing thyr oid 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 T 4 and pituitary weight were observed, but no corr esponding 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 T 4 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 ove rt toxicity. Dose-dependent decreases in both T 3 and T 4 levels were observed in the 15-day adult rat screen ing study (O’Connor et al ., 2002), with no changes in TSH levels or t hyroid histopathology or weights; the effects in the high dose occurred in the presen ce of overt toxicity ( ↓ 11% body weight). Dose-dependent decreases in T 3 and T 4 levels were also observed in a t hyroid 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 stu dy. None of the above
studies showed treatment-relate d changes in thyroid weight or histopathology, and changes in pituitary weight were only noted in the female pube rtal assay and the validation trial of the male pubertal assay.

In an in vitro transcriptional activat ion assays (Hoffman et al ., 2009,) using HepG2 cells
transfected with a luciferase -based reporter gene and a T 3 -responsive element, linuron was reported to have weak agonist activity to the T 3 receptor.

In the Part 158 mammalian long-term toxicity st udies conducted with in mice, rats and dogs, no treatment-related effects were observed on thyr oid weight or histopat hology, or on pituitary weight or histopathology.LINURON FINAL

Page 26 of 93 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 re sponse. A shaded cell indicates that is parameter is not routinely evaluated or is not ap plicable 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 Linuron 1

Study Type/ Literature CitationTR ActivationThyroidWeightThyroid: GrossandHistopathologySerum T3/T4LevelsSerum TSHLevelsPituitaryWeightDevelopmentalstage (± orasynchronous,HLL)Growth (BW,BL, SVL)SystemicToxicity2Overt Toxicity3
EDSP Tier 1 Assay

Female Pubertal Rat (MRID 48685501) N N T 4 ↓ 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 ↓ T 3 ↓ T 4 (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 ↓ T 3 (ML, MH,)

↓ T 4 (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)LINURON FINAL

Page 27 of 93 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of linuron to interact w ith endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, 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 evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP evaluated scientific i ssues associated with weight of evidence evaluation of the resu lts of the Tire 1 assays stated that “In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

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 belo w. Generally, one parameter (i .e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).LINURON FINAL

Page 28 of 93 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 fema le pubertal assays and generally consisted of reductions in body weight and body weight gains, along with cha nges in clinical chemistry parameters and decreased kidney weight ( ↓ 7%) in the pubertal study. No mortal ity 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%, respec tively, at 9.1 mg/L. In addition, lethargy and loss of equilibrium in fish at the 9.1 mg/L leve l indicated that this c oncentration was overtly
toxic.

2. OSRI

Signs of both systemic and overt t oxicity were noted in many of the in vivo OSRI studies, with decreases in body weight and body weight ga ins being the most common finding in many studies. In the mammalian toxicity studies, over all signs of toxicity were generally most pronounced at doses exceeding 50 mg/kg/day.

In the nine reports detailing the various Hershbe rger 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 increas ed 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 th e 15-day adult rat scre ening 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 th e decreases exceeding 10% at doses of ≥ 50 mg/kg/day. In addition, liver weights were increased in one as say and decreased in th e other 15-day assay.LINURON FINAL

Page 29 of 93 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 di et 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 th e fourth study. Systemic a nd 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 t oxicity/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 ra t (27.1/48.3 mg/kg/day [M/F]) studies based on decrea ses in body weight and body weight gain. In addition, all three
chronic studies showed adverse hemato logical changes at the highest doses.

In the two guideline avian reproduction studi es, transient decreases were observed in body weight of adult ducks during the first week of e xposure. No other overt toxicity was noted in either study at dietary co ncentration 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 wa s tested at concentrations from 0.206 to 2.93 mg/L. Survival was decreased at the two highest con centrations (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.LINURON FINAL

Page 30 of 93 B. Estrogen Pathway

Although positive results were reported in th e 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 transactiva tion assays in CHO cells, yeast ERreporter assays or ER-responsive proliferation assay. Linuron show ed an equivocal response for
aromatase inhibition in vitro . In the female pubertal assa y, decreased ovarian weight was observed, however there was no corroborating micr oscopic findings. In addition, pituitary weights were also decreased in the study. Ther e were no estrogen-related effects noted in the
mammalian Part 158 studies. In the Tier 1 FST RA (with fathead minnow), in the absence of overt toxicity, there were alte rations in fecundity and ferti lity at 0.92 mg/L, however, these effects are limited in their diagnostic specificity fo r estrogen-related alterati ons in of themselves;
no effects were observed in a Part 158 fath ead minnow life-cycle study up to 0.097 mg/L. In another FSTRA considered as OSRI, plasma v itellogenin concentrations were significantly reduced in females; however, no other effects we re 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 malla rd reproduction study, and decreased female body weight and food consumption was al so observed at this concentra tion. 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 redundanc y 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 mu ltiple 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 histopatholo gy in the testes and epididymides.

D. Thyroid Pathway

There is convincing evidence of potential intera ction of linuron with the thyroid pathway. Although no thyroid histopat hology effects were observed in any of the Tier 1 or OSRI studies,
decreases in serum T3 and/or T4 levels we re observed across multiple studies including the EDSP Tier 1 female pubertal assa y, a 12-week fem ale rat thyroi d toxicity study, and two 15-dayLINURON FINAL

Page 31 of 93 adult male rat screening assays considered as OSRI. Additionally, linuron showed weak agonist activity to the T 3 receptor in two in vitro transcriptional activation as says 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, linur on 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 interac tion with the estrogen pathway in mammals. There was convincing evidence for poten tial interaction with the andr ogen 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 me t- 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 chroni c dietary risk assessmen t 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-rela ted 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 endpo ints of concern are base d on data obtained from adult animals, there usually would be a concern that the POD may not be protective of potential thyroid toxicity in the young.

Therefore, a special thyroid a ssay in pregnant animals, pos tnatal, 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 prot ect 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) th at may be used for human health risk assessment. The POD from this special study woul d address the concern fo r the potential ability of linuron to disrupt thyroid f unction in pregnant females a nd 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 effectsLINURON FINAL

Page 32 of 93 observed), anti-androgenic effects were obser ved 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 concen tration tested (0.097 mg a.i./L). Given the potential for androgen-related e ffects in fish, the EDSP Tier 2 Medaka Extended One Generation Reproduction Test (MEGORT) using Ja panese 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 satis fied by the OSRI that demonstrated potential interaction with the thyr oid pathway. Therefore, the EDSP Ti er 2 Larval Amphibian Growth and Development Assay (LAGDA) is recommended to a dd to the understanding of potential adverse effects in amphibians.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for linuron. In both speci es, effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potenti al reproductive effects to birds from linuron exposure. Relative to the EDSP, ad ditional testing is not recommended.LINURON FINAL

Page 33 of 93 VI. References

Beavers, J, Foster, J, and Jaber, M (1992) Li nuron: a one-generation reproduction study with the northern bobwhite ( Colinus virginianus ). Wildlife International Ltd., Easton, MD. Sponsored by E.I. duPont de Nemours and Company, Ne wark, DE. Laboratory Project No. 112-271, completion date not provided. Unpublished. (MRID 42541801)

Beavers, J, Foster, J, and Jaber, M (1992) Li nuron: 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 th yroid function assay of linuron administered orally in intact juve nile/peripubertal female rats. WIL Research Laboratories, LLC, Ashland, OH. Laboratory Project ID WIL-851 004, 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 fr om two-year rat study with INZ326. Central Research and Development Depart ment, 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 numb er 100-80 [MRID 00029680], with no new laboratory or registrant report or study number provided.) Au gust 13, 1986. Unpublished. (MRID 00164117)

Coder, P (2011) A uterotrophic as say of linuron administ ered orally in ovariectomized rats. WIL Research Laboratories, LLC, Ashland, OH. Laboratory Study ID: WIL-851003, December 8, 2011. Unpublished. (MRID 48617403)

Culik, R (1979) Teratogenicity study of 3-(3,4- dichlorophenyl)-1-methoxy-1- methyl-urea in rats. E.I. du Pont de Nemours and Company, Haske ll Laboratory for Toxicology and Industrial Medicine, Newark, DE. La boratory Report No. 33-79, Ma y 8, 1979. Unpublished. (MRID 00018167)

Everett, R (1980) Two-year feeding study-ChR-CD rats. Haskell Laboratory for Toxicology and Industrial Medicine, Central Research and Deve lopment Department, E.I. du Pont de Nemours and Company, Newark, DE. Pathology Report No. 1-80 (data from Haskell Laboratory Report number 100-80 [MRID 00029680]), Februa ry 4, 1980. Unpublished. (MRID 00029679)LINURON FINAL

Page 34 of 93 Gordon, J, Chu, A, Chu, M, et al . (2004) Detection of estroge n receptor endocrine disruptor potency of commonly used organochlorine pe sticides using the LUMI-CELLTM bioassay. Organohalogen Compounds, Dioxin 66 : 169-174.

Hoberman, A, and Staples, R (1985) Developmen tal toxicity study of IN Z-326 administered via gavage to New Zealand white rabbits. Argus Research Laboratories, Inc., Horsham, PA. Laboratory Study No. 104-009, September 16, 1985. Unpublished. (MRIDs 00153867 and 40437201)

Kaplan, A, Smith, L, Koniecki, W, et. al . (1980) Long-term feeding st udy in rats with 3-(3,4- dichlorophenyl)-l-methoxy-l-methylurea (Lorox' , linuron, INZ-326). Haskell Laboratory for Toxicology and Industrial Medici ne, Central Research and Development Department, E.I. du Pont de Nemours and Company, Newark, DE. Laboratory Report No. 100-80, completion date not provided. Unpublished. (MRID 00029680)

Lech, J (2006) Interlaboratory va lidation of the 15-day adult int act male rat assay with linuron and phenobarbital. Charles River Laboratories Preclinical Services, Hors ham, PA. Sponsored by Battelle, Columbus, Ohio. Laboratory Report No. RTP00004, May 2, 2006. Unpublished. Note: This study was also referenced as U.S. EPA, 2007d (MRID#48321201) below.

Malley, L (1988) Chronic toxicity study with INZ326-118: one-ye ar feeding study in dogs. E.I. du Pont de Nemours and Company, Haskell Labo ratory for Toxicology a nd Industrial Medicine, Newark, DE. Laboratory ID No. 181-88, October 28, 1988. Unpublished. (MRID 40952601)

Marlatt, V, Lo, B, Ornostay, A, et al . (2013) The effects of the urea-based herbicide linuron on reproductive endpoints in the fathead minnow ( Pimephales promelas ). Comp. Biochem. Physiol.
C. Toxicol. Pharmacol. 157 : 24-32.

Moon, H-J, Kang, T, Kim, T, et al . (2009) OECD validation of phase 3 Hershberger assay in Korea using surgically castrated male rats with coded chemicals. J. Appl. Toxicol. 29 : 350-355. (MRID 48321205)

Mullin, L (1990) Reproductive and fertility eff ects with IN Z326-118 (linuron) multi-generation reproduction study in rats. E.I. du Pont de Nemours and Company, Haskell Laboratory for Toxicology and Industrial Me dicine, Newark, DE. Laborat ory 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 Me dicine, Newark, DE. Laboratory Study No. not provided, April 3, 1991. Unpublished. (MRID 41864701)LINURON FINAL

Page 35 of 93 Orton, F, Rosivatz, E, Scholze, M, et al . (2011) Widely used pestic ides 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-Z 326-198 to embryo and larval rainbow trout, Onorhynchus mykiss . Haskell Laboratory for Toxi cology and Industrial Medicine, E.I. du Pont de Nemours and Company, Newar k, 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 andr ogen-primed female three-spined sticklebacks - prediction of combined effects by co ncentration addition. Aquat Toxicol . 140-141: 145-156.

Rickard, R (1981) Long-term feeding st udy with 3-(3,4-Dichlorophenyl)-1-methoxy-1- methylurea (Lorox ® ; Linuron: INZ-326) in mice. Hask ell Laboratory for Toxicology and Industrial Medicine, E.I. du Pont de Nemour s and Co., Newark, DE. Laboratory Project No. 3336, April 1981. Unpublished. (MRID 00124195)

Suter, P, Vogel, W, and Wilson, J (1988) Tw o-generation reproduction study in the rat. RCC AG, Itingen, Switzerland. Laboratory Pr oject No. 071504, November 18, 1988. Unpublished. (MRID 48046002)

USEPA (1995) Reregistration Eligibility D ecision (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 thyr oid 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 Pr otection Agency. August 27, 2007. MRID 48321201. Note: This study was also referenced as Lech, 2006 above.

USEPA (2010) 

### Linuron - report of the Endocrine Disr uptor Review Team – Test Order #:

EDSP-035506-122. Dece mber 16, 2010. Office of Chemical Safe ty and Pollution Prevention, 

### U.S. Environmental Protection Agency, Washington, DC.LINURON FINAL

Page 36 of 93 USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention Science Coordina tion and Policy, U.S. Environmental Protection Agency, Washington, DC.

USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

Vinggaard, A, Breinholt, V, and Larsen, J (1999) Sc reening of selected pe sticides 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 - Labor atory Proficiency Data for ERTA assays and Saturation Binding Data for AR and ER Bi nding 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 recept or 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)LINURON FINAL

Page 37 of 93 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) Bindi ng 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 tritiate d androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]-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 con centration of chemical. Tritiated water was quantified using liquid scintillation counting (L SC). Three independent runs were conducted,
and each run included a full activ ity 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 fu ll activity controls ranged from 0.494 to 0.726 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a m ean 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 activ ity 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 ra nges for the top of the curve, bottom curve, Hill slope, log IC 50 , and coefficient of varia tion for replicates of each concentration within runs. Also, the coefficients of variations (%CVs) fo r replicates of each
concentration of linuron within a run were generally within the 15% guideline. For 4-OH ASDN, the estimated log IC 50 averaged –7.27 M, and the slope was –1.00.LINURON FINAL

Page 38 of 93 For linuron, aromatase activity av eraged 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 stra ight line (14% inhibition at 10 –4 M and essentially no inhibition was observed at other doses). The aver age 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 cu rve, linuron is classifi ed as Equivocal for aromatase inhibition in this assay.

Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48617402), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiment s. 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 appropr iate 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]-17 β -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 con centration of 2%. The assay included 19-norethindrone as a weak positive control, oc tyltriethoxysilane as a negative control, and 17 β

estradiol as the natural ligand reference material.

Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e performing laboratory were submitted following a request by the Agency. The K d for [ 3 H]-17 β -estradiol was 0.303±0.090 nM, and the
estimated B max was 33.59±0.70 fmol/100 μg protein. The K d 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 bi nding of radiolabeled estradiol to ER at concentrations of ≤ 10

− 4 M in all runs. A mean log IC 50 was not calculated for linuron as 50% binding displacement was not achieved in any run. The mean log IC 50 was

− 8.9 M for the natural ligand, 17 β -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.LINURON FINAL

Page 39 of 93 Based on the results of the three runs, linuron is classified as Not Interactive in the Estrogen Receptor Binding Assay.

ER α 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 (M RID 48671701) of linuron w ith 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 concentrati ons 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 25 o C and a pH of 7.0 to 7.6.

Overall mean survival values were 100% for bot h males and females in the negative control, mid, and low treatment groups; 2 males and 1 fe male 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 da rk coloration and partia l/complete loss of equilibrium in males and females of the hi gh 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/replic ate (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%, respectivel y, at the mid treatment level.

Male vitellogenin (VTG) and gonadal-somatic i ndex (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 compar ed to the negative control.
Tubercles were not observed in female fish.LINURON FINAL

Page 40 of 93 Although not analyzed statis tically, in male fish, there was an increase in incidence and severity of increased proportion of sperma togonia at the high treatment le vel compared to the negative control. In female fish, there were changes in gonadal staging, decreased yolk synthesis, and increased oocyte atresia of predominantly pre-vi tellogenic 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 linur on technical (99.4% a.i ., Lot # 1012A3876) in corn
oil at doses of 0, 50 or 100 mg/kg/day from post-na tal day (PND) 22 to 42 or 43. Animals were examined for vaginal opening (VO) daily begi nning on PND 22, and the age and weight at day
of attainment of VO was recorded. Followi ng sacrifice on PND 42 or 43, blood was collected for clinical chemistry analyses, including total thyroxine (T 4 ) and thyroid stimulating hormone (TSH), which were analyzed using an electrochemiluminescent immunoassay or radioimmunoassay, respectively. Kidney, liver, adrenal gland, t hyroid, 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-dos e or at the daily examinations (~24 hours post-dose).

There were no significant diffe rences in mean age at VO, body weight at VO, or final body weights in the treated groups rela tive to controls. There were no treatment-related effects on the weights of liver, adrenals, uterus (wet or blotted), orthyroid. 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 regular ly cycling. No treatment-related microscopic
lesions were observed on the th yroid, ovary, uterus, or kidney. Serum TSH was not affected by

treatment.LINURON FINAL

Page 41 of 93 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 wa s 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 kidn ey 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 cont rols. At 50 mg/kg/day, the unadjusted, adjusted, and relative pi tuitary weights were decreased (p ≤ 0.05) by 13-15%.

There was a dose-dependent decrease (p ≤ 0.05) in serum T 4 levels of 27% and 48% at 50 and 100 mg/kg/day, respectively. However, there was no asso ciated 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 bilir ubin was increased (p ≤ 0.05) by 33%, and total protein was decreased (p ≤ 0.05) by 5% compared to the cont rols. 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 adeq uate based on the results of a dose range-finding study showing body weight loss at 100 mg/kg/day.

Male Pubertal A ssay; (OCSPP 890.1500)

Requirement satisfied by OSRI (USE PA 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 potenti al 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 17 α

LINURON FINAL

Page 42 of 93 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 ap proximately 24 hours after the final dose on PND 75 to determine liver, and wet a nd blotted uterine weights.

All animals survived until sche duled 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 ga ins were decreased (p<0.05) for the 30, 60 and 120 mg/kg/day groups by 66, 117, and 192%, re spectively. Uterine and liver weights in the linuron-treated groups were comparable to th e controls. Absolute wet and blotted uterus weights for the positive EE group were increase d (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.LINURON FINAL

Page 43 of 93 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of linuron with the endoc rine system are summarized below. Given the breadth of toxicity data availa ble on linuron, the only Part 158 st udies 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-dich lorophenylurea; 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 wa s prepared using tissue from rats castrated 24 h prior to harvest. The cytoso l prep was incubated with [ 3 H]-testosterone in the absence or presence of increasing concentra tions of the unlabeled ligand. From these data, the concentration of ligand which produced 50% displacement (IC 50 ) of [ 3 H]-testosterone from the androgen receptor was calculated.

The calculated IC 50 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 IC 50 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 domai n using a rat prostate competitive binding assay and a COS whole cell hAR binding assay.

For the rat prostate cytosol assay, ventral prosta te tissue was obtained fr om 90-day old rats 24 h after castration. In four separate re plicates, 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. Nonsp ecific binding was assessed with 100-fold molar excess of unlabelled R1881. Following incubation, ligand-bound receptor was separated from unbound, and quantified by liquid scin tillation counting. The EC 50 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,LINURON FINAL

Page 44 of 93 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 ra dioactivity of the lysate was determined by liquid scintillation counting. The a ssay was replicated a to tal of four times. Linuron inhibited R1881 AR binding by about 25% at 5 μM (p<0.05) with an EC 50 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 in cluded in the competitive binding assays conducted by the lead laboratory and in the ma in interlaboratory validation study. In the competitive binding assays using the two-parameter model, the mean IC 50 for the standard curve

was 9.42 x 10

− 10 M. Linuron was analyzed in dupl icate at concentrations of 10

− 8 to 10

− 3 M. The

mean IC 50 for linuron was 80,302 ± 14,938.3 nM (

− 4.10 log M), and the mean IC 50 for R1881 was 0.99 nM (

− 9.00 log M), for an RBA of 0.001%. The R 2 value for the individual curve fit

was 0.997.

In the interlaboratory validation studies, th ree independent runs we re conducted by each laboratory with a standard, a weak positive cont rol (dexamethasone), and each test chemical; each concentration was analyzed in triplicate. Li nuron 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 IC 50 for the standard curve was

− 8.624 log M, and the mean log IC 50 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 IC 50 values from the four laboratories ranged

− 4.05 to

− 3.85 log M, mean R 2 values ranged 0.68-0.97, and log RBAs ranged

− 4.79 to

− 4.22. Using the four-parameter model, mean log IC 50 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 IC 50 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 linur on from each of two laboratories either did not converge or produced a poor f it to the standard curve (R 2 value of 0.15); howev er, the other two
runs in both laboratories had R 2 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 investig ated. COS-1 African green monkey kidney cells were transiently transfected w ith a vector containing either fathead minnow AR or hAR for comparison. Twenty-four h post-transfecti on, the cells were incubated with [ 3 H]-R1881 in the presence of increasing concentrations of linuron. Linuron was found to have an IC 50 of approximately 42 μM (

− 4.38 log M) in this study.LINURON FINAL

Page 45 of 93 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 huma ns was investigated. COS-1 African green monkey kidney cells were tr ansiently transfected with vectors containing either fathead minnow AR, rainbow trout AR, or hAR. Twenty-four h post- transfection, the cells were incubated with [ 3 H]-R1881 in the presence of increas ing concentrations of linuron.

Linuron was found to have log EC 50 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 tota l of four times. Linuron inhibited DHT-induced transcriptional activation at doses as low as 10 μM, which was also the EC 50 value. It was not
clear whether the data came from the exposure which occurre d 24 or 48 hours after transfection.

For the MDA-MB-45J-KB2 cell hAR transcrip tional activation assay, MDA-MB-453-KB2 cells, which contain endogenous hAR, stably transfect ed 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-i nduced luciferase expression at 5 μ M and above with an

EC 50 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 stab ly transfected with hAR and a luciferase gene under the control of mouse mammary tumor viru s [MMTV]) was validated for its ability to detect androgen agonists and antagonists. Se veral known androgen agoni sts and antagonists, including linuron (99.7% purity), were tested. Linur on was shown to be a w eak antagonist in the assay when tested with 0.2 nM methyldihydrotes tosterone 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 concentra tion of 10 μM was too low to co nsistently produce a sufficiently complete concentration response curve suitable for robust EC 50 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 aLINURON FINAL

Page 46 of 93 reporter gene assay. Human hepatoma HepG2 cells were transfected with three plasmids: 1) human AR; 2) an androgen-respon sive MMTV-luciferase (luc) re porter 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 concentrati ons listed. The cells were incuba ted 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 transcriptio nal activity induced by DHT in a dose-dependent manner, with a K d 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 lu ciferase reporter gene plasmid (pGudLuc7ere) containing the estrogen responsive element (ERE ) and luciferase reporter gene to create the BG1Luc4E2 cell line (LUMI-CELL TM 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 e xpression. 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 (con centrations 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 17 β -estradiol after subtracti on of background radioactivity. Each compound was tested at least three times on three different sets of plates, and the EC 50 value was determined.

The bioassay yielded an EC 50 value of 1.45 x 10

− 5 M for linuron, with a relative induction factor
of 1.07 x 10

− 6 compared to 17 β -estradiol (1.55×10 -11 M).

Receptor Transcriptional Activation

In a published study by Kojima et al . (2004) , 200 pesticides (incl uding linuron; purity 95-100%) were evaluated for their potenti al to interact with human ER α , ER β and AR using Chinese hamster ovary (CHO-K1) cells transfected with th e appropriate plasmid. It was reported that the chemicals were tested at non-cytotoxic c oncentrations, but no data were provided.LINURON FINAL

Page 47 of 93 Linuron was negative for ER α , ER β , and AR transcriptional activa tion in this test system. No data were presented for negative findings. Li nuron did exhibit weak anti-androgenic activity,
with a RIC 20 (relative inhibitory concentration to 20% of the activity invoked by 10

− 10 M dihydrotestosterone; DHT) of 2.0 × 10

− 4 M and a relative lucifera se 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 a nd androgen agonist/anta gonist activities using recombinant yeast assays (YES and YAS), and a frog ( Xenopus laevis ) ovulatory response assay. The yeast assays used E 2 or testosterone as agonists. Fo r 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 th e pesticides. Medium concentrat ions of estrogen, testosterone, and progesterone were also measured. Linuron wa s 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-estr ogenic activity at c oncentrations of 1.9-31.3 μM in the YES assay, and anti-androgenic activity at concentra tions of 0.98-62.5 μM in the YAS assay. In the ovulation assay, 62.5 μM linuron e xposure caused an increase (p<0.05) in progesterone ( ↑ 70%), a slight decrease (NS) in testosterone ( ↓ 12%) and E 2 ( ↓ 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 hER α 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 ce ll proliferation assay and from 0.2 to 500 μM in the YES. Linuron was reported to be negative in both assays (data on ly reported for MCF-7 assay results).LINURON FINAL

Page 48 of 93 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 ge ne expression were investigated. Time-mated pregnant Sprague-Dawley rats we re dosed with linuron (99.8% purit y) in corn oil by daily oral gavage (dose volume 2.5 mL/kg) at dose le vels 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 CO 2 and exsanguinated. Fetal te stes 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 peptid e 3 (insl3) along with several enzymes in the steroidogenic pathway (cyp17a, StAR, and c yp11a) 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-Daw ley dams were euthanized and necropsied on GD 18, and the testes were collect ed from all male fetuses and incubated as previously described. In the first experime nt, 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) ur ea (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 di fferences between groups in maternal survival, maternal body weight, litter loss, or li tter size. While all the dams gained weight, body weight gains were generally dose-dependently decreased (not signi ficant [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 si gnificantly decreased in all treated groups. However, when evalua ted on a litter basis, ex vivo testosterone production was significantly reduced (p<0.01) only at 50 and 75 mg/kg ( ↓≈ 25%). Progesterone produ ction 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 ex periment, indicating that linuron can directly inhibit testosterone production in the absence of cytotoxicity. Ketoconazole resulted in significantly decreased (p<0.0001) testosterone production, demonstr ating the expected response to the positive control in both experiments.LINURON FINAL

Page 49 of 93 Thyroid Receptor Transcriptional Activation Assays

In a published study by Hoffman et al. ( 2009 ) linuron (purity 97%) wa s tested for hormone agonism and antagonism in a thyroid hormone re sponsive luciferase-based reporter gene assay. Human hepatoma HepG2 cells were transfected with T 3 -responsive element (TRE) reporter plasmid [p(DR4)2-SV40-Iuc+] and either a TR α 1 or a TR β 1 expression plasmid (pRS-TR α 1 or pRS-TR β 1 ). Test chemicals, including the natural ligand T 3 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 pitu itary hormones. Female Sprague Dawley rats were ovariectomi zed at 3 months of age, we ighed, and randomly assigned to experimental groups (n=12). The test compounds we re administered daily with the food for 12 weeks. The following test groups were established: an ovx control; E 2 at 1.2 mg/kg/day (327 μg/animal/day) as the positive control; and the fi ve endocrine active test substances (including linuron) at low and high doses. Linuron was ad ministered at nomina l doses of 16.3 and 65.5 mg/kg/day (4.4 and 17.7 mg/animal/day).

Prior to the beginning of dosing a nd 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 sacr ificed by decapitation, and blood was collected from the trunk. The following serum parameters were measured: serum lip ids including leptin, triglycerides, and low and high density lipopro teins (LDL and HDL); and pituitary hormones including LH, TSH, T 4 , and T 3 . Serum LH, TSH, leptin, and t hyroid hormones were measured using radioimmunoassay kits, and serum choles terol, HDL, LDL and triglycerides were measured using commercially availabl e 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, E 2 decreased water and f ood 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.

E 2 completely prevented the increase in the pa ratibial 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 E 2 produced the lowest serum leptin and cholesterol concentrations, and reduced (p<0.05) LDL and HDL; serum triglyceride concen trations increased. For linuron, serum leptin concentrations were dose-dependently reduced (p<0.05) , and serum cholesterol, LDL, andLINURON FINAL

Page 50 of 93 triglyceride concentrations we re reduced (p<0.05) at 65.5 mg/kg; serum HDL concentrations were increased (p<0.05) at 16.3 mg/kg. For the pi tuitary hormones, serum TSH levels were not significantly affected by a ny of the test substances , although treatment with E 2 showed a tendency for increased TSH. Serum T 4 levels were unaffected by E 2 , but T 3 and LH levels were reduced (p<0.05). Dose-depe ndent reduction of serum T 4 levels was observed for linuron (p<0.05 for the high dose), T 3 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 li nuron may have a direct effect within the thyroid gla nd as serum TSH levels were no t affected, but a dose-dependent
reduction was seen for T 3 and T 4 . The decrease in thyroid horm one 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 He rshberger assay and an intact immature male assay. The castrated rats (Alpk:AP fSD; 4-6 rats/group) used in th e Hershberger bioassay studies
were 6 weeks old. Rats used in the stimulate d 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 w eanlings. Rats were give n 10 consecutive doses of TP, or 10 consecutive doses of the synthetic a ndrogen 17-methyltestosterone (17MT), and a coadministered second oral dose of the chemical under investigation (linuron), or the positive control anti-androgen flutamide. Preliminary wean ling 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 we re euthanized by cervical dislocation under anesthesia. At necropsy, the following tissues were removed and weighed: liver, kidneys, Cowper’s glands (CG), levator ani and bulbocaver nosus muscles (LABC), seminal vesicles (SV) with coagulating glands, and prostate. In additi on, 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) suspen ded 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 test es weight. Linuron increased (p<0.05) liver weights at 30 mg/kg/day and above ( ↑ 2-10%) but these minor increases were notLINURON FINAL

Page 51 of 93 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 potentia l, 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%), we re 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 com pounds. Linuron was not tested for androgenic potential. To test for anti-andr ogenicity, 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, respect ively. One day after the last treatment, the rats were anaesthetized and sacrificed by exsa nguination. The liver and the following AST were collected, trimmed of fat and adjacent tissue, a nd weighed: ventral pros tate (VP), SV, LABC, CG, and GP. The liver and VP were immediately frozen until further an alysis. Gene expression analysis in the prostate was conducted usi ng Taqman® Real-time pol ymerase 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 f ood 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 we re not statistically significant. When compared to TP-control, the high dose of linuron significantly (p<0.01) decreased the weight (rela tive to body weight) of all androgen-sensitive tissues ( ↓ 29-54%) except GP; relative liver weight s 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 wei ghts or organ or tissue weight s. In the androgen-responsive gene expression analysis, the high dose of li nuron and FLUT slight ly 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 Hers hberger bioassay using juvenile intact male (stimulated weaning) rats , male Wistar (Hsd/Cpb:WU) pups at PND 19 were acclimated for three days and randomly assigne d to the control and treatment groups (6 rats/group). For dosing, the test compounds, includ ing 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 antiLINURON FINAL

Page 52 of 93 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 gr oups: 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 dos e levels were expected to
result in a no/low effect level and a clear eff ect level, respectively. One day after the last treatment, the rats were anaes thetized and sacrif iced 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 sa mples were immediately frozen until further analysis.

No mortality was observed through the treatment period. When compared with the vehicle-only control, administration of TP al one resulted in slightly increa sed body weight gain and final body weight (NS). TP treatment increased absolu te weights for the VP, SV, LABC, CG and epididymides, and decreased relative testes weig hts (p<0.01). Liver weights were not affected by treatment. Relative organ and tissue weights we re not reported. The positive control, FLUT, significantly (p<0.01) decr eased all absolute and relative AST weights ( ↓ 32-64%) and increased (p<0.05) testicular weight ( ↑ 45%) compared to the TP-contro l. The negative control NP
decreased absolute but not rela tive 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, clin ical signs (digging and cleani ng 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 re sulted in decreased (p<0.05) absolute SV and LABC weights compared to the TP-control; rela tive weight was decreased (p<0.05) for SV ( ↓ 26%). Testicular weights were not affected at th is 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 lin uron (99% purity) in corn oil via oral gavage (dose volume 4 mL/kg) at 0, 25, 50 or 100 mg/kg/day for 10 cons ecutive days along with TP via s.c. injection (dose volume 0.5 mL/kg) at 0.4 mg/kg. The positiv e 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 fi nal dosing, rats were
euthanized. Blood was collected from the abdomin al aorta and serum was prepared immediately
for measurement of testosterone and LH concen trations. The AST (VP, SV with coagulating glands, LABC, CG, and GP), plus the liver, ad renals, 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 vehicleLINURON FINAL

Page 53 of 93 control. Dosing with flutamide inhibited (p< 0.05) the TP-induced re-gro wth 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 th e 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 linur on, body weights and liver and ki dney 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 we ights of the AST in a dose-dependent manner. At 50 mg/kg, weights were de creased (p<0.05) for SV ( ↓ 28%), VP ( ↓ 25%) and CG ( ↓ 26%). At 100 mg/kg, weights were decr eased (p<0.05) for SV ( ↓ 47%), VP ( ↓ 38%), CG ( ↓ 39%), LABC ( ↓ 35%) and GP ( ↓ 20%). No significant differe nces 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 CO 2 and body weight was recorded. Rats were euthanized by decapitation; body weight and weights of the SV plus coagulat ing 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 separa te 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 follo wing dose groups: TP only (positive agonist control); FLUT at 3 mg/kg/day (positive antagon ist control); NP at 160 mg/kg/day and DNP atLINURON FINAL

Page 54 of 93 10 mg/kg/day (negative controls ); and linuron (purity not repo rted) at 10 or 100 mg/kg/day. Approximately 24 hours after the last dose, ra ts were anaesthetized and sacrificed by exsanguination. The following AST were excised a nd weighed: VP, SV with coagulating gland, LABC, CG, and GP. Liver, kidneys, and adrenal glands were also co llected and weighed.

No significant changes in body, liver, adrenal, or kidney weights were obs erved for any of the treatment groups. For the TP control, AST weight s 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, b ecause 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-i nduced 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 investig ators 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 assa y, Sprague-Dawley Crl:CD (SD) rats were used for testing in all stages. Pregnant dams were allowed to deliver pu ps 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 appropri ate level of TP to use as a stimulatory dose in subsequent anti-androgen studies. In Stage 2, th e selected dose of TP was admini stered 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 Stag e 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 tes ting; for Stage 3 and 4 testing, all other groups received daily s.c. injections of TP at 1.0 m g/kg. Test groups for Stage 3 included: TP only (positive agonist control); FLUT at 3 mg/kg/da y (positive antagonist control); and linuron at 10, 30, or 100 mg/kg/day. Test groups for Stage 4 incl uded: TP only; FLU at 3 mg/kg/day; negativeLINURON FINAL

Page 55 of 93 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 afte r the last dose, rats were an aesthetized and sacrificed by exsanguination. The following four AST were exci sed and weighed: VP, SV with coagulating glands, LABC, and CG; testes and epididymides we re also weighed. GP was not evaluated as the weanling males did not achieve pr eputial 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 orga ns, 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, comp ared to the TP-stimulated only group, linuron at 100 mg/kg decreased (p<0.05) the rela tive weights of all four AST (VP ↓ 22%, SV ↓ 25%, LABC ↓ 21%, CG ↓ 26%) and the epididymides ( ↓ 17%). No significant cha nges 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 colle cted AST and the epididymides, and increased (p<0.05) testes weights. No si gnificant 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 linur on at 100 mg/kg significantly (p<0.05) inhibited TPinduced increases in the relativ e weights of the four AST. Percent differences from linuron dosing compared to the TP-stimul ated 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 th e AST and/or testes/e pididymides weights.LINURON FINAL

Page 56 of 93 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, gr oups 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 administ ered by s.c. injection (dose volume 2.0 mL/kg). Rats were dosed daily for 10 consecutive days w ith TP at 1.0 mg/kg/day, and co-administered an oral dose of linuron (99% purit y) at 3, 10, 30, or 100 mg/kg/day, or the positive control antiandrogen FLUT at 3.0 mg/kg/day. A vehicle cont rol group received only corn oil, and a TP control group received only TP in jections 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, test es, epididymides, and AST (CG, LABC, SV with coagulating glands , dorsolateral prostate (DP), and VP). Selected tissues were fixed, and histopathological assessments were con ducted on the testes, SV, and DP from vehicle control, TP-stimulated, TP + 100 mg/kg/day lin uron, 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 epith elial maturation); the testes were examined for impaired tubular growth (reduced tubular diameter and cellular de pletion 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 we re extracted, and the extracted proteins were subjected to 2-dimensional gel electrophoresis. Th e resulting protein spot s 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 decr eased (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 incr eases 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 interanima l variability was recorded for the testicular weights of the anti-androgen-treat ed stimulated weanlings, causi ng any testes weight increases not to be considered significant.

At 100 mg/kg, linuron caused si gnificant 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.LINURON FINAL

Page 57 of 93 Histopathological examinations revealed that 10 0 mg/kg linuron had only a slight effect on the morphology of the SV and DP compared to TP-t reated tissues, slightly reducing the mean severity grades for SV (2.5 linuron vs. 3.1 TP) a nd DP (1.7 linuron vs. 2.4 TP). In the testes,
linuron increased the mean severity grade for tubular dege neration (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 dis tinct, 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 analy zed 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 sc ientific literature. Linuron trea tment 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 thr ee 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 ev aluated, as it was not pres ent 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-D awley and Crl:CD). The program consisted of three phases: Phase 1, in which opt imum 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 a nd 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) an d linuron (a known weak androgen antagonists) was assessed; and Phase 3, in which the participa ting 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 gr oup were dosed for ten consecutive days beginning on PND 21-24 thro ugh 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 af ter the final dose, the animals were weighed and euthanized. The following AST were collect ed 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 adre nal glands) tissues, and
measurements of serum levels of luteinizing hormone, testosterone, T 4 , and T 3 were performed on an optional basis.LINURON FINAL

Page 58 of 93 For linuron in Phase 2, three perfor ming laboratories (Laboratories A, B, and C) participated in generating the data. Linuron (purity not reporte d) 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 cont rol (Laboratories B and C only). Terminal body weights were recorded, and orga n 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 fo r 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 code d 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 wei ght; VP; SV; LABC ; CG; testes; epididymides; liver; adrenals; and kidneys.

In the Phase 2 tests, the TP positive control in creased weights of the VP, SV, LABC, CG, and epididymides, and decreased testes weights vs. th e 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 do se. 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 follo wing 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 w ith 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 fo r all organ weights (excep t 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 incr eased weights of the VP , SVCG, LABC, CG, and epididymides, and decreased testes weights vs. th e vehicle control (result s from two laboratories only). The FLU + TP inhibitory positive contro l significantly reduced mean organ weights of VP, SVCG, LABC, CG, epididymides (all laboratories), and test es (three laboratories) vs. the positive control. No significant effects were observed for the negative chemical DNP, and for NP, one laboratory detected a statistically si gnificant reduction in te stes weights and one laboratory detected a marginal insignificant chan ge in LABC weight vs. the positive control.LINURON FINAL

Page 59 of 93 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 we ights, and liver, adrenal, and kidney weights were unaffected by administration of linuron at e ither 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 interlaborator y comparisons demonstrated th at 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 tis sues 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 w ith 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 st eady-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 a ndrogen-regulated gene s 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 ma gnitude of decreases in the weights of the epididymis and VP, in addi tion to reduced weight of the seminal vesicles.

Following four days of dosing, males were necr opsied as before and in addition, VP was collected, homogenized, and tota l 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, howev er, cause an increase in TRPM2 and a decrease in C3 mRNA levels. The ratio of C3 to TRPM2, was decr eased (p<0.05) by flutam ide and linuron. The TRPM2 increase produced by linuron was signifi cant, but the decrease in C3 was not.LINURON FINAL

Page 60 of 93 Male Pubertal Assay

In the USEPA (2007c) validation report, linuron was incl uded 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 wei ght) and one-half the MTD. Linuron (99-99.5% purity) was administered to wean ling male Sprague-Dawley rats at 50 or 100 mg/kg/day in corn oil in an experimental block that also include d propylthiouracil, ketoconazole, phenobarbital, and
a vehicle control. The test chemicals were admi nistered 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, T 4 , and TSH; weights of repr oductive organs, including SV, VP, DP, LABC, epididymis, and testes; histology of epididymis, testis, thyroid, and kidney; and weights of thyroid, liver, ki dney, adrenal, and pituitary.

Based on delayed acquisition of PPS, linuron de layed puberty in male rats at 50 and 100 mg/kg/day dose levels. Both doses of linuron si gnificantly (level of si gnificance 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 an d kidney weights decreased significantly at the high dose. Levels of T 4 were reduced at the high dose, while levels of TSH were reduced at only the low dose. No treatment-related histopathol ogical 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 ha ve been breached, but that th e 7% decrease at the low dose
indicated that there was no inte rference from body weight effect s 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 fi nal dose of test compound approximately 2 h prior to euthanasia, and necropsy was performed acro ss the treatment groups in order to control for potential variation due to “time-of-day” eff ects. At necropsy, blood was collected from the descending vena cava to obtain serum for hor monal analyses. Serum concentrations of testosterone, E 2 , DHT, LH, FSH, prolactin (PRL), T 3 , T 4 , and TSH were measured using commercially available RIA kits. The liver, testes , epididymides, prostate, seminal vesicles, and AST (prostate, SV with fluid and coagulating gl ands) were weighed (paired organs together), and relative (to body weight) organ weights we re calculated. Following fixation, the thyroid gland was weighed, and final di ssection was performed under a dissecting microscope by oneLINURON FINAL

Page 61 of 93 individual. After weighing, th e epididymides and testes were fixed and examined microscopically. Mean final body weights and organ weights were analyzed st atistically.

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. Micros copically, low incidences of mi nimal 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 cha nges in the epididymides at the dose levels
tested. Linuron increased serum E 2 concentrations in a dose-depende nt manner, with statistically significantly increases at 50, 100, and 150 mg/kg/day ( ↑ 50%, ↑ 65%, and ↑ 80%, respectively).
Serum testosterone and PRL concentrations we re significantly decreased at 150 mg/kg/day ( ↓ 40% and ↓ 92%, respectively). Serum DHT concentr ations 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 sta tistically significant decrease in serum T 3 concentrations at 50, 100, and 150 mg/kg/day ( ↓ 16%, ↓ 16%, and ↓ 22%, respectively). Serum T 4 concentrations were decreased in a dose-depende nt 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 seru m TSH concentrations were not affected, and there were no compound-related microsc opic 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, T 4 , T 3 , FSH, E 2 , 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 th e 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, atax ia, lost righting reflex, unresponsive to touch, lacrimation, chromorhinorrhea, chromodacryorrh ea, 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 refle x, comatose, ptosis, perinasal substance, hunched posture, low carriage, excess salivation, perior al substance, bradypnea and scant feces were observed in oneLINURON FINAL

Page 62 of 93 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 weight s 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 se rum concentrations of the following hormones were affected (p<0.05) in all treated dose gr oups: testosterone ( ↓ 51-67%),

T 4 ( ↓ 34-67%), T 3 ( ↓ 16-21%), E 2 ( ↑ 31-49%), and PRL ( ↓ 85-94%). DHT was decreased (p ≤ 0.05) by 39% at 150 mg/kg/da y. LH was decreased (p ≤ 0.05) by 18-19% at 50 and 100 mg/kg/day, and was decreased (not statistically si gnificant) 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 anta gonist, 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 test osterone, DHT, and LH concentrations, and
increased serum estradiol concentrations were ob served. 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 fo llow a dose-respons e 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 surviv ing dams were sacrificed and all fetuses were weighed and examined for external ma lformations/variations. Crown-rump length was measured on each fetus. Approximately one-half of the fetuses in each li tter were fixed in
Bouin’s solution for visceral exam ination and the remaining one-hal f were processed for skeletal
examination.

All animals survived to scheduled termination w ithout the appearance of any treatment-related clinical signs of toxicity. Gr oss 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 pp m (49.8 mg/kg/day), based on decreased body weights and food consumption. The NOAEL is 125 ppm (12.1 mg/kg/day).LINURON FINAL

Page 63 of 93 No dose- or treatment-related effects were obs erved on fetal sex ratios, numbers of corpora lutea/dam, implantations/dam, live or dead fe tuses/dam, fetal body weights, or crown-rump length. Post-implantation loss was increased at 625 ppm (14.0% tr eated 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-re lated external or visc eral malformations or
variations were noted. In the 625 ppm group, bipartite thoracic vete bral centra was observed in 7 fetuses from 7 litters and unapposed sternebrae we re observed in 3 fetuses from 3 litters. These anomalies were not found in the control group an d were considered indicative of developmental delays.

The LOAEL for developmental toxicity is 625 pp m (49.8 mg/kg/day) base d on increases in postimplantation loss and in litter/fetal reso rptions. 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 ra bbits/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 fetu ses were eviscerated and processed for skeletal examination.

No dose- or treatment-related c linical signs of toxicity, matern al deaths, or necropsy findings were observed in any group. Absolute body weight s of the 100 mg/kg/day group were decreased (p<0.05) by 7% on GD 19. During dos ing, 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 a borted between GD 20-25 compared with one control doe on GD 22. Slight (NS) decreases in the mean number of live fetuses/litte r (6.1 treated vs. 6.8 controls) and mean fetal body weight (41.99 g treated vs. 4 5.83 g controls) were observed. No treatmentrelated external or visceral fetal malformations/variations were noted. Skull alterations (irregularly shaped fontanelle, ho le in parietals, parietals contai n intraparietals, and unossified) were observed in 19 fetuses (6 litters; p<0.05), compared to 1 fetus (1 litter) for controls.LINURON FINAL

Page 64 of 93 The LOAEL for developmental toxicity is 100 mg/kg/day, based on altera tions 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. Pre gnant 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 a nd BBP (75 and 500 mg/kg/day, respectively) during GD 14-18. On GD 18, dams (6 dams/treatmen t group) were euthanized for the fetal hormone study. The fetuses were removed via cesare an section, anesthetized, killed on ice, and fetal sex was determined using a dissecting scop e. 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 te stosterone concentratio n (n = 15-20/treatment). The remaining carcass was extracted for testoster one 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 pr oduction (ng per test is 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 we ighed 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 we ighed and housed with 2 or 3 siblings. At 3 months, the males were killed, and the ventra l surface was shaved. The AGD was measured, and these animals were examined externally for th e numbers of nipples a nd 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, undesce nded 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, vent ral prostate, seminal vesicles, whole epididymis, right cauda, right caput and corpus, testes, and LABC).

In the fetal hormone study, testic ular testosterone production wa s decreased (p<0.05) by 34% in the linuron-treated group compared to controls. Similar decreases (p<0.05) were observed in
testicular testoster one 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 c ontrols at necropsy on GD 18.LINURON FINAL

Page 65 of 93 In the developmental study, maternal body wei ght 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-trea ted males was decreased (p<0.05) by 25% in the pups and by 10% in the adults. The numbers of areo lae in the pups and nipples in the adult males were higher (p<0.05) in the linur on-treated group (approximately 2) compared to controls (0). No external malformations of the genitalia we re noted in any group. The only external anomaly in the linuron group was incomplete preputial se paration (24.2% treated vs 0% controls). The authors stated that, although not considered a normal condition, incomplete preputial separation was not categorized as a malfor mation because it is occasionally found in historical controls.
Internal malformations in the linuron-treated males included adhesions/a genesis 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 orga n weights in the linuron-treated group were decreased compared to the contro l 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, nulli parous Crl:CD(SD)BR rats were obtained on GD 0 (day that sperm was found in the vagina of th e mated female) and assigned to treatment groups.
Eleven dams/dose level were dosed with linuron (99% purity) in corn o il 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 cons umption 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 r ecorded weekly. On PND 13, m ale pups were examined for the presence of areolae and/or nipples. An animal wa s considered a responder if it had one or more retained areolae or nipples. Beginning on P ND 38, male pups were examined daily for malformation of the external ge nitalia, testicular descent, and PPS; PPS was complete by PND 55. Females were examined daily for VO beginning on PND 28. When VO was acquired,LINURON FINAL

Page 66 of 93 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, kidney s, adrenals, uterus, and ovaries) and number of implantation sites were recorded for the da ms. Sexually mature male offspring (PND 95-105) were euthanized by decapitation, terminal body we ights 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 inspecte d, and the following reproductive organs were examined and weighed: testes, epididymides, vas deferens, prostate (ven tral and dorsolateral), seminal vesicles, and coagulati ng glands (with fluid). In addi tion, the following organs were collected and weighed: liver, kidneys, adrenals , and levator ani/bulbocavernosa muscle. Tissues were fixed in Bouin’s fixative (testes and epid idymides) or 10% neutral buffered formalin, and stained with hematoxylin and eosin for micr oscopic examination. Female offspring were euthanized by CO 2 asphyxiation on PND 85.

Administration of linuron to pre gnant dams during GD 12-21 did not result in signs of toxicity, or significant changes in body wei ght or food consumption during treatment or lactation; dam organ weights (kidney, liver, uter us, 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 leve ls (data not reported); or female pup weight, weight gain, day of VO, time to first estrus, or es trus cyclicity. No malformations in the external genitalia or changes in testicul ar descent were observed in male offspring at any dose. The proportion of pups born alive and surviving to we aning 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 immediatel y cannibalized are include d, 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 reten tion; incidence of hypoplastic test es 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 te stes 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 seminife rous 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. Occurr ence of these changes in the testes ranged 2.9-10.1% in the 25 mg/kg gr oup and 4.5-9.1% in the 50 mg/kg group. Linuron also induced lesions in the ep ididymis. 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 wereLINURON FINAL

Page 67 of 93 observed in 6.8% of 50 mg/kg rats. The testicular and epididymal lesions arose in males from different litters and across the linuron exposur e groups, confirming that these results were biologically relevant. Epididym al 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 th e testis and epididymis in rats . Sprague-Dawley rats were dosed with linuron (100% purity) at 100 mg/kg/day. Th is dosage level was selected because it was minimally toxic or nontoxic, produced minor eff ects 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 linur on group were not dosed after GD 18 due to a reduction in maternal weight gain. At 5 to 6 m onths of age male rat offspring were necropsied, and organ weights were collected from 1-2 males randomly select ed from each litter. Tissues were routinely fixed, stained, and examined for microscopic lesions (gra ded 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 hypert rophy (50% with mild to modera te severity) and hypospermia (58% moderate to severe). The results in the linuron treated group were ne arly 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 a nd 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 decapitat ion within 15 seconds after removal from the home cage in a separate room during the dark phas e of the animal’s daily light cycle. The ventral
surface of each male was shaved and examined externally for abnorma lities (including number and location of retained nipples, cleft phallus , vaginal pouch, and hypospadias). The animals were examined internally for: ectopic or at rophic testes; agenesis of the gubernaculum,LINURON FINAL

Page 68 of 93 epididymis, sex accessory glands, and ventral pr ostate; 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. Ma ternal body weight gain was decreased at 100 mg/kg/day during the treatment period. Howeve r, 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 adjust ed (for body weight by analysis of covariance) AGD in the male offspring was decreased (p<0.005) by 30%. The in cidence 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 n ecropsy, one out of the 13 males treated with linuron had epispadias (partial hypospadias wi th the urethral opening half way down the phallus). Numerous epididymal and testicular ma lformations 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 offs pring, 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 se vere 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 pseudoherm aphroditism index (PHI) was used to describe the degree of
demasculinization/feminization based on a suit e of androgen-dependent parameters. Two PHI scores were calculated, one for dihydrotestoster one (DHT)-dependent parameters (AGD, aerolae, retained nipples, ventral prostate weight, hypos padias, and vaginal pouches) and the other for testosterone-dependent variables (weights and abnormalities of the seminal vesicles, epididymides, and testes). Offspring of animal s treated with linuron were approximately 30% “feminized” for DHT-dependent parameters a nd 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 doseLINURON FINAL

Page 69 of 93 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 ra ts were weighed daily and these weights were used to calculate the daily dose. Because linuron is known to reduce body we ight gain, a pair-fed control group was included in or der 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 ve na cava, and serum was prepared for radioimmunoassay analysis to determine te stosterone, E 2 , and luteinizing hormone (LH) concentrations. Testes, epididymides, accessory sex organ unit (prostate, seminal vesicles, and coagulating glands), prostate, se minal vesicles with fluid remove d, and coagulating glands were weighed at necropsy and relative organ weights (percentage of body wei ght) 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 sim ilar 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, s uggesting that the decreased body wei ght was primarily due to reduced food consumption. The following relative organ we ights 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 linur on-treated rats were similar to the controls and the pair-fed controls.

In the sexually mature rats treated with li nuron, 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 sim ilar to the linuron-treat ed 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 sugge st that the decreased body weight in the mature
linuron-treated rats is not sole ly due to reduced food consumption. The following relative organ weights were decreased in the li nuron-treated rats when compar ed 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 administ ered linuron (94.7% purity) continuou sly in the diet at dietaryLINURON FINAL

Page 70 of 93 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

F 1 litters. F 1 offspring selected to be parents of the next generati on (25/sex/dose group) were fed the same test diet concentrations as their pa rents for 18 weeks prior to mating to produce the F 2 generation. The F 2 offspring were terminated after weaning.

All P and F 1 generation parents survived to scheduled sacrifice. There were no remarkable
clinical observations during the study or gross findi ngs at necropsy in the P or F 1 generation
males or females.

At 625 ppm, body weights ( ↓ 8-13% P males; ↓ 9-14% P females; ↓ 19-33% F 1 males; ↓ 16-27% F 1 females) and overall body weight gains were decreased (p<0.05) throughout the pre-mating period in both sexes from both generations. Addi tionally in the females from both generations, body weights ( ↓ 14-18% P females; ↓ 16-20% F 1 females) and overall body weight gains were decreased (p<0.05) during gesta tion 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 lactat ion in the P and F 1 females.

No treatment-related changes in absolute or relative weights were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, ut erus, 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, epidi dymides, 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 F 1 males and females. In the spleen, in creased 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 F 1 males (23/25 treated vs. 0/25 controls) and females (20/25 treated vs. 3/25 controls). Additionally in the spleen, incr eased hematopoiesis was found at 625 ppm in the P males (22/25 treated vs. 4/25 c ontrols) and females (18/25 treated vs. 0/25 controls) and F 1 males (20/25 treated vs. 2/25 controls) and fe males (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 F 1 (12/25 treated vs. 5/25 controls) generations.

The LOAEL for parental toxicity is 625 ppm (equi valent 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).LINURON FINAL

Page 71 of 93 No treatment-related effects on th e mating, fertility, or gestation indices were observed in either generation. There were no effect s of treatment on gestation duration in either generation.

At 625 ppm, the pre-coital interval was sligh tly increased compared to controls in both generations. In both the P and F 1 generations, the number of implantations was reduced, and post-implantation loss was increased. Total litter loss was observed in three P dams and five F 1 dams at this dose. The combined effect of d ecreased number of implantations and increased postimplantation loss resulted in a decreased litter size on PND 0. Furtherm ore, 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 (equivale nt to 10/11 mg/kg/day in males/females).

No clinical signs of toxicity we re observed in the pups of either generation from birth to PND 21. Pup sex ratio on PND 0 was not affected by trea tment in either generation. There were no treatment-related effects on organ wei ghts, gross pathology, or histopathology.

In both the F 1 and F 2 litters at 625 ppm, mean litter size co ntinued to be decreased throughout the post-natal period, resulting in a decreased viabi lity index in both generations and a decreased
lactation index in the F 2 litter. Overall (PND 1-21) pup body we ight gains at this dose were decreased by 26% in both generations compared to controls.

The LOAEL for offspring toxicity is 625 ppm (equi valent to 53/56 mg/kg/day in males/females) based on decreased litter size, pup body weights and viability a nd 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-genera tion reproduction toxicity study ( MRIDs 41463401 and

41864701 ), groups of 30 male and 30 female Crl:C D 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/female s) for two generations. One litter was produced by each generation. P and F 1 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 we re 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 F 1 animals. At the end of premating, body weights of the 625 ppm P males and females were decreased 19% and 14%, respecti vely, with overall body we ight gains decreasedLINURON FINAL

Page 72 of 93 by 41% and 45%, respectively. For the 625 ppm F 1 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 leve ls 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 premati ng weight gains and food consumption 12% and 6% (p<0.05), respectively, less th an controls. For the 100 ppm F 1 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 con tinuation of premating effects.

No treatment-related lesions were noted at necrop sy of the P males or females. In the 625 ppm F 1 males, gross lesions included testes reduced in size (9/30), abnormally large (3/30), and soft (5/30), and small epididymides (8/30), and unspeci fied deformities of the epididymides (5/30); these were noted as significant incidences, but co ntrol 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 F 1 males as compared with the contro ls: atrophy (14/30), fibrosis (8/30), and hyperplasia (7/30) in the test es; and arteritis (6/30), infla mmation (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 F 1 males were decreased by 20% (p<0.05; 80%) compared to the controls. In the 625 ppm F 1 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 F 1 males and females. An increase (p<0.05) was seen in the number of F 1 males with any type of co rneal or conjunctival change (14/30 vs 4/30 controls). The le sions included corneal degenera tion/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 premati ng doses 0.736-1.12 mg/kg/day).

No treatment-related adverse effects were f ound 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 F 1 generation and 96.8, 92.7, 99.5, and 76.2% (p<0.05), respectively, for the F 2 generation. The mean number of pups per litter in the 625 ppm F 2 group was less (p<0.05) than the contro l throughout lactation. The number of litters with pups showing c linical signs was increased (p <0.05) in both generations. Body weights of the 100 and 625 ppm F 1 male and female pups and of the 625 ppm F 2 male andLINURON FINAL

Page 73 of 93 female pups were less (p<0.05) than those of th e controls throughout lact ation. These data were not considered for setting the reproductive toxicity NOAEL in the original DER.

The reproductive toxicity NOAEL was greater th an or equal to 625 ppm (average premating doses 35.5-63.0 mg/kg/day) and the reproductive toxicity L OAEL 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 ge neration; however, no information was provided
on the F 2 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 F 1 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 F 1 males were treated beginning at PND 21, and were euthan ized after 147 to 161 da ys of feeding. At termination, blood was collected and serum was prepar ed for analysis of testosterone, estradiol, and LH concentrations by radioimmunoassay. For th e P males, testes, epididymides, accessory sex organ unit (prostate, seminal vesicles, coagulating glands), pr ostate, seminal vesicles with fluid removed, and coagulating glands were we ighed at necropsy and relative organ weights (percentage of body wei ght) 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 significan tly increased in the 625 ppm P males.

In the F 1 generation, final body weights were de creased in the males at 100 ppm ( ↓ 6%) and 625 ppm ( ↓ 25%). Overall body weight gain (Days 0-154) was also decreased in the F 1 males at 100 ppm ( ↓ 6%) and 625 ppm ( ↓ 25%). There were no decreases in relative organ weights in the

F 1 males. Serum estradiol ( ↑ 15%) and LH ( ↑ 68%) levels were significantly increased in the F 1 625 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 admini stered the test material by daily oral gavage from weaning, through puberty, young adulthood, mating, and lactation. F 1 pups were evaluated through mating, but were exposed only via the dam during gestation and lact ation. In the current study, linuron (100% purity) in corn oil was admi nistered 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. TheLINURON FINAL

Page 74 of 93 corn oil control group included 12 rats/sex, and the remaining treatment groups were comprised of 8 rats/sex. In the P females, dosing conti nued 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 co llected for hormonal analyses, organs were weighed (testes, liver, kidneys, adrenal, seminal vesicles plus coagulati ng glands with fluids, epididymis, pituitary, and terminal body weight), the testes were examined microscopically, and caudal and epididymal sperm counts were conducted. When the F 1 offspring reached PND 25, the P dams were euthanized. The body, liver, ki dney, adrenal, pituitary, and ovaries were weighed, and the number of implantation sites in the uterus was counted. Litter size and weights
in the F 1 offspring were determined throughout the study. After sexual maturation, F 1 offspring were randomly selected from the highest dose w ith sufficient number of surviving animals for fertility assessment under continuous ma ting conditions (16/sex/dose group). F 2 pups were counted and removed at birth. After determinatio n of the age of sexual maturation, all excess F1 animals were necropsied and examined for malf ormations. 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 con centration. In the P males at 40 mg/kg/day, seminal vesicle weight (with coagulating gla nds and fluids) was d ecreased (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

F 1 males exposed in utero and during lactation produced a lower (p<0.01) cumulative number of pups (65 vs 104 pups) when mated continuously ove r 12 breeding cycles, and the male offspring had decreased (p<0.05) we ights 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 we re observed at any dose level. Body weights, body weight gains, and food consum ption were not affected by treatment. No treatment-related ophthalmological lesions or changes in urinalys is parameters were noted and gross necropsy was unremarkable.

RBC counts, hemoglobin, and hematocrit were sl ightly (NS) decrease d throughout the study in high-dose males and females as compared with th ose 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 mont hs. Methemoglobin and sulfhemoglobin levelsLINURON FINAL

Page 75 of 93 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 mont hs 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 increas ed (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 wei ghts 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) depos ition 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 hi stopathological lesions were seen in the testes, epididymides, prostate, ovaries, vagina, mammary glands, thyroid, adrena l 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 di et at concentrations of 0, 50, 150, or 1500 ppm (equivalent to 0/0, 8/12, 23/35, and 261/455 mg/kg/day for male s/females) for 24 months.

No significant treatment-related e ffects 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 contro ls, respectively, and hi gh-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 f ood intake for high-dose males was decreased by about 14% and by 10% for high-dose females compar ed 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 retu rned to near control le vels later in the study.LINURON FINAL

Page 76 of 93 An increased (p<0.05) incidence of hemosideri n 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 nor mal 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 fema les compared to the controls. Microscopic evidence of liver toxicity at 1500 ppm included increased incidences of focal hepatocellular cytoplasmic alteration, focal centrilobular peliosi s, and centrilobular hepatocytomegaly in both sexes, and increased incidence of hepatocellu lar 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 re sulted 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 consider ed adequate based on the liver changes, methemoglobinemia, and decreased body weights.

Chronic Toxicity/Carcinoge nicity (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 (e quivalent 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 year s. Additional groups of 10 rats/sex/dose were
administered the same diets for 12 months for interim evaluation. All c linical pathology data were reanalyzed ( MRID 00164117 ) due to inappropriate statistica l methods used in the original study report.

Linuron had no effect on mortality at any dose, and there were no tr eatment-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 ppmLINURON FINAL

Page 77 of 93 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 ga ins 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 signi ficant (p<0.05) increases in the mean absolute and relative testicular weight s in the mid (121 and 122%) and high (131 and 143%) dose groups. No treatment-related histopatholog ical lesions were seen in prostate, ovaries, mammary, thyroid, and adrenal or pituitary glands.

Histopathology observations consiste nt with hemolysis were observe d at increased incidences in the 125 and 625 ppm males and females from th e main study and interim sacrifice groups, including hemosiderin in Kupffe r cells and increased hemoside rosis in bone marrow, spleen, and/or mesenteric lymph nodes, and transient decreases in the erythr ocyte count, hemoglobin concentration, and hematocrit of the high-dose female group were not ed 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 ce ll hyperplasia in the re nal 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 ectasi a and collecting duct ecta sia in the kidney at 625 ppm. The 625 ppm females also had an increased (NS) incidence of tran sitional cell hyperplasia in the renal pelvis compared to controls. Hepa tocellular megalocytosis/syncytium formation with fibroplasia radiating between hepatic cord s, 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 be en related to induction of hepatocellular protein synthesis. The significance of this findi ng 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 ga ins in both sexes, microscopic observations consistent with hemolysis (hemosiderin in K upffer cells and increased hemosiderosis in bone marrow, spleen, and/or mesent eric lymph nodes), and increase d incidences of microscopic changes in the epididymides (perivasculitis/v asculitis) and renal pelvis (transitional cell hyperplasia and mineralization/calculi) of male s 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).LINURON FINAL

Page 78 of 93 There was a treatment-rela ted increase in the incide nce 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 p ituitary adenomas of the pars anterior in both male and female rats and mammary fibroadenom as in female rats. Decreased incidences of both these tumor types were noted in the hi gh-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 admi nistered in the diet to aged male Crl:CD(SD)BR rats to investigate the i nduction of testicular inte rstitial 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 continui ng for 6 and 12 months; terminal sacrifice was carried out at 24 months of age. Another group of 25 rats was given basa l diet and served as controls. Compound intake for both treated gr oups was 22-23 mg/kg/da y for the treatment
interval.

Treatment-related clinical signs of toxicity were limited to swo llen 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 terminati on; further details were not included. Body weights of both treated groups were reduced (p<0.05) compar ed 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, resp ectively (additional data not provided). Body weight gains during the treatment interval s 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 anim als 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 obs erved 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 hype rplasia. For rats sacrificed at study termination, dose-related
increases in the incidence of test icular 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 ad renal 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 hist opathological effects of linuron (94.5% purity) under a variety of cond itions were examined in Crl:CD® BR rats. TheLINURON FINAL

Page 79 of 93 rats utilized for various part s of this study were young adult males approximately 22 days old (young), retired breeder males approximately 11 months old (old), and male and female F 1b and

F 2b 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 th e presence of 0, 0.5, 5, 50, 500, or 5000 μM linuron or linuron metabolites. Enzyme activities meas ured included aromatase, 17-20 desmolase (desmolase), 3- β -hydroxysteroid dehydr ogenase/isomerase (isomerase), 17-hydroxylase (hydroxylase), and 17-ketosteroid reductase (reductase). Linuron concentrations of 500 and 5000 μM resulted in significantly decreased arom atase and desmolase and increased reductase activities. At 50 μM th e activities of aromatase, desmolas e, isomerase, and hydroxylase were decreased by 10-20% and reducta se 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 m easured in young male rats. Groups of 5 animals were administered 0 or 200 mg/kg/day for ei ght 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 th e treated and control gr oups 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, a nd five 0-ppm and five 625-ppm F 2b males which were approximately 11 months old. In trial 2, the groups consisted of five young or five old males treate d with 0 or 200 mg/kg/day for se ven days, and five each of the 0-, 12.5-, 100-, and 625-ppm F 2b males which were approximat ely 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, Leydi g cells from the chronical ly treated F2b males were significantly more responsive to LH stimulati on 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 theLINURON FINAL

Page 80 of 93 high-dose group. Taken together, the two trials we re reasonably consistent with a preliminary
conclusion that there are dose- and time-relate d 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 F 1b and F 2b animals. High-dose animals had significantly (p ≤ 0.05) reduced body weights as compared to controls during days 469-708 for F 1b males and females, days 0-533 for F 2b males (none remained on test after this day), and days 0-709 for F 2b females. At the mid dose, body weights were significantly (p ≤ 0.05) reduced for the F 1b and F 2b females on days 540-596 and 0-654, respectively. Food consumption was slightly less for the high-dose F 2b 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 F 1b females and a significantly (p ≤ 0.05) greater
number of F 2b females.

F 1b and F 2b rats were sacrificed at two years of age for histopathological ev aluation of selected tissues. The number of high-dose males of both gene rations 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- a nd high-dose males had increased
incidences of interstit ial cell adenomas and hyperplasia. Co mbining data from both generations, adenomas were observed in 1/19, 0/25, 6/25, a nd 2/16 animals and hyperplasia was observed in 2/19, 0/25, 7/25, and 3/19 animals from the c ontrol, 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 combin ed incidence rate for cervical endometrial hyperplasia was 0/28, 6/30, 9/29, and 13/29, respectiv ely, and of cervical cystic hyperkeratosis was 0/28, 1/30, 1/29, and 7/29, resp ectively. 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 pr oduction). 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 toxic ity or abnormal behavior at least once daily, and all birds that died during the study and their sacrificed pen ma tes were necropsied. Individual adult body weights were recorded at test initiation and on Weeks 2, 4, 6, and 8, and atLINURON FINAL

Page 81 of 93 termination; food consumption was determin ed once a week throughout the study. At study termination, all surviving birds we re sacrificed and necropsied.

Eggs were collected daily from all pens and st ored until incubation. Eggs were removed from storage and candled weekly; cracked or abnorma l eggs were discarded. One egg was collected weekly from each odd or even numbered pen duri ng odd or even weeks, respectively, for shell thickness determinations. The average thickne ss of the dried shell plus membrane was determined. All eggs not used for shell thickne ss 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 hatc hlings and unhatched eggs were removed from the hatcher on Day 27 or 28, and the average wei ght of the hatchlings by pen was determined. Hatchlings were placed in br ooding 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 contro l group, but necropsies of the dead bird and its pen mate showed no treatment related findings. Inci dental clinical signs were noted in various groups during the study, but these were associat ed 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 fe male) during Week 1, feed consumption during Week 1, egg production, and egg shell thickness (s ignificant 7.5% decrease) at 300 ppm. There was also a possible effect (not significant) upon the percent viab le 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 sl ight reduction in the number of viable embryos resulted in a reduction (p<0.05) in the number of hatchlings and 14-day old surv ivors of maximum set. There were no significant differences between the cont rols 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 progre ssive 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.LINURON FINAL

Page 82 of 93 All adult birds were observed for signs of toxic ity or abnormal behavior at least once daily, and all birds that died during the study and their sacrificed pen ma tes 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 determin ed once a week throughout the study. At study termination, all surviving birds we re sacrificed and necropsied.

Eggs were collected daily from all pens and st ored until incubation. Eggs were removed from storage and candled weekly; cracked or abnorma l eggs were discarded. One egg was collected weekly from each odd or even numbered pen duri ng odd or even weeks, respectively, for shell thickness determinations. The average thickne ss of the dried shell plus membrane was determined. All eggs not used for egg shell thic kness 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 wei ght of the hatchlings by pen was determined. Hatchlings were placed in br ooding 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 not ed in various treated groups during the study, but these were determ ined unrelated to treatment.

There were no treatment-related effects on adu lt 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 lo w values in the control group and it could not be determined if the differences were treatment related.

There were no apparent treatment related eff ects on reproduction at 50 or 100 ppm. At 300 ppm, there was a slight (NS) reduc tion in egg production, and a reduc tion (p<0.05) in the ratio of hatchlings that survived to 14 da ys. There also appeared to be a reduction (p<0.05) in the ratio of 14-day survivors/eggs set. When compared to c ontrols, there were no apparent or significant differences in egg shell thickness or offspring body weights at hatc hing or at 14 days of age at any concentration tested.

The NOAEC for Bobwhite quails exposed to li nuron in the diet for 21 weeks was 100 ppm.

FSTRA

In a published study by Marlatt et al

. ( 2013 ), the mode of acti on of linuron (99.7%) was examined in male and female fathead minnows ( Pimephales promelas ) using the biomarkerLINURON FINAL

Page 83 of 93 VTG, nuptial tubercle formation, GSI, egg ha tching, and 7-day egg survival. The expression patterns of a number of key genes involved in st eroidogenesis in the ovary were also examined, and gene clustering was performed to better el ucidate the MOA of linuron compared to the model androgen DHT and the model estrogen E 2 . During the pre-exposur e 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%); E 2 (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 plas ma 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 fo rmalin for subsequent tubercle counts. The
number of tubercles on the snout of each fi sh 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 exam ined histopathologically to determine relative maturation stage and changes due to treatment. Th ree sections from the anterior, median, and posterior region of a gonad were examined micr oscopically and severity scores qualitatively assigned to pathological findi ngs, 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 re al-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 patholog ies in the ovaries or testes in linuron-, DHT-,
or mixture-treated fish. The DHT and DHT + linuron co-treat ment 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 tu bercle 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 significan t decrease in plasma VTG levels (median=8.4 mg/mL, range= 0.9-33.6 mg/mL) in females compared to the contro ls. A similar significant anti-estrogenic effect on basal plasma VTG concentrations was observe d 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 E 2LINURON FINAL

Page 84 of 93 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 treatment s 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 diffe rence observed was between the control and DHT + linuron co-treatment, where p450ssc was decreased approximately 20-fold. The hierarchical clustering of mRNA transcript e xpression patterns revealed two distinct expression clades. One expression clade contained all three linuron treatments and the E 2 treatment while the other expression clade contained contro l, DHT, and DHT + linuron. The gene expression patterns also showed that genes in the linuron trea tments grouped more closely with E 2 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 replic ates/treatment x 15 larvae /replicate). This study was considered supplemental as it only include d 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, resp ectively. 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 we re 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, excep t 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-fe rtilization) were exposed to linuron at 0 (dilution wate r 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 ofLINURON FINAL

Page 85 of 93 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 ch ambers were inspected daily and infertile, damaged, or diseased embr yos were removed. The number of live embryos was recorded daily. After hatching was complete (D ay 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 th e aquarium. The number of live fingerlings was determined daily. Length of surviving trout af ter 47 (sampled at thi nning) and 80 days (test termination) was determined. Wet weight of surv iving 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. Fi ngerling 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 LC 50 was 0.46 mg/L (95% CI = 0.39-0.57 mg/L) and the 80-day LC 50 was 0.38 mg/L (95% CI = 0.25-1.0 mg/L). Fish weight at test term ination 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 wa s 0.042 mg/L based on reductions in fish length at all exposure levels.

Fish Life-cycle Reproduc tion 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 da ys. 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 an alysis detected a significant reduction (Dunnett’s, p<0.05) in Day 264 (Week 38) F0 fe male length at the mean-measured 0.016 mg a.i./L treatment level relative to the negative cont rol. However, no reductions were detected at the higher treatment levels by Dunnett’s or William s tests. Therefore, the reviewer agrees with the study author that this reducti on was not biologically relevant or treatment-related. There were no effects on any of the other endpo ints 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 su rvival, length, and wet weight; F0 eggs/spawn, spawns/female, and eggs/female; F1 hatching su ccess, time-to-hatch; and F1 survival, length, and wet weight). The NOAEC and LOAEC we re 0.097 mg a.i./L and >0.097 mg a.i./L, respectively).

Androgenic/Anti-Androgenic Activity in FishLINURON FINAL

Page 86 of 93 In a published study by Jolly et al . (2009) , the androgenic/anti-androge nic properties of linuron in fish were investigated using in vivo and in vitro assays. Exposure to exogenous androgens induces production of the glue protein spi ggin in female stickleback kidney. In the in vitro experiments, kidney cell cultures were prepared from batches of fe male 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 an ti-androgenic activities, DHT was added to the
culture medium at a concentra tion of 3 μg/L. The ability of linuron (99% purity) to inhibit androgen-induced spiggin expression was determin ed 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 e xposure, 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 inglass 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. Th e nominal concentrati ons 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 cont rol (5 μg/L DHT), and a test compound control (test compound at the highest con centration tested in the absence of DHT). After 21 days of exposure, the fish were sacr ificed and frozen in liquid nitrogen. After body weight measurement, the kidney was dissected, we ighed 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 th e protein levels were expressed as units of spiggin per gram of body weight . The actual concentra tions 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 i nduced a statistically significant, concentration-dependent increase in spi ggin production at al l concentrations ( ≥ 3 pg/L). No significant effect on spiggin cell content was obs erved in cells treate d 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 10 4 -10 5 units/g body weight, compared to the negative and solv ent controls (10 1 -10 2 units/g bw). No signifi cant 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 experime nt, linuron induced significant decreases in DHT-induced spiggin production at ≥ 100 μg/L.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 us ing 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 17 α -methyltestosterone (17 α -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 inhi bited the androgenic activity of 17 α -MT based on spiggin induction in the kidneys (data pr esented graphically). At 15 μg/L , linuron did not inhibit spiggin induction by 17 α -MT.

Anti-Androgenic Activity in Fish

In a published study by Pottinger et al . (2013), the anti-androgenic ac tivity of linuron ( ≥ 99% purity) and other known anti-andr ogens was determined using the androgenized female stickleback screen with the quantitative assessm ent 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) wa ter under flow-through c onditions 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, fema le sticklebacks were exposed to 5 μg/L DHT
with the linuron treatment concentrations. The a dditional controls used in each test included a negative control, containing the highest concentration of the te st chemical without DHT, an absolute control, containing only water, and a solvent control, contai ning 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 consiste d of a series of expos ure tanks in which the
individual chemicals were tested at both the IC 50 and IC 50/10 along with mixture tanks receiving all four anti-androgens at mixt ure ratios proportional to i ndividual potencies ranging from IC 50 to
IC 50/100 . The controls used in the mixture study incl uded 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 esta blished radioimmunoassay procedure. The concentrations of testLINURON FINAL

Page 88 of 93 chemicals in the water extracts were determ ined by high performance liquid chromatography with electro spray ionization and selected ion mass spectrometry.

In all individual and mixt ure tests, mortality was ≤ 1.5% for all control a nd 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 be tween measured concentrations and nominal concentrations may occur because of uptake by fish, losses due to degradation, adsorption to surfaces, evaporation, photolysis, or hydrolysis. Li nuron was the least potent of the four antiandrogens with an IC 50 of 172 μg/L and 95% confidence inte rval of 121-258 μg/L. Both mixture
tests resulted in concen tration-dependent inhibition of spiggin induction in DHT-primed female
sticklebacks.LINURON FINAL

Page 89 of 93 APPENDIX 3: References Not Utilized in the Linuron WoE Analysis

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

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

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

Akahori, Y, Nakai, M, Yamasaki, K, et al . (2008) Relationship be tween 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 F 0 (Parent) generation. DuPont de Nem ours and Co. and Huntingdon Research Centre, England. Laboratory Re port No. 1352/65/270, November 5, 1965.LINURON FINAL

Page 90 of 93 Crago, J, and Klaper, R (2012) A mixture of e nvironmentally realisti c concentration of a phthalate and herbicide reduces test osterone 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 eff ect of linuron - active in gredient technical on the Leydig cell system in the male rat; 4-week subs tance application by gavage. Pharma Research Toxicity and Pathology, Hoech st Aktiengesellschaft. La boratory Study No. 87.0114, March 4,

1987.

EURISKED (2010) Summary of results of the EURISKED project. http://www.euriske d.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 evaluati on of chemicals for potential estrogenic activity. Organohalogen Compounds 65 : 78-81.

Hogan, N, Gallant, M, and van den Heuvel, M (2 012) 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 reproducti on 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 screeni ng of androgenic and anti-androg enic endocrine disrupters. Aquat.
Toxicol

. 79 : 158-166. (MRID 48041712)

Katsiadaki, I, Scott, A, Hurst, M, et al . (2002) Detection of envi ronmental 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)LINURON FINAL

Page 91 of 93 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 5 α -reductase activity in prostate and LNCaP cells. Toxicol. in Vitro 21 : 502-508. (MRID 48041721)

Luccio-Camelo, D, and Prins, G (2011) Disrup tion 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 expres sion 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 activit ies of environmental compounds. Environ. Toxicol. Chem. 24 : 653-664. (MRID 48041725)

Opitz, R, Hartmann, S, Blank, T, et al . (2006) Evaluation of hi stological 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 predicta bility of cumulative anti-androg enic 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 com pounds 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.LINURON FINAL

Page 92 of 93 Owens, W, Gray, L, Zeiger, E, et al. (2007). The OECD program to valid ate 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-genera tion reproduction study in rats with 3-(3,4-dichlorophenyl)-1- methoxy-1-methylurea (LOROX®, LINURON, IN Z-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) Cumulativ e 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 co mmon 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 transfec ted human ovarian cell line for th e 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 disr uptors 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: 17 α -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 chemi cals that impair thyroid hormone synthesis.
Environ. Sci. Technol. 45 : 7525-7532.

Tietge, J, Holcombe, G, Flynn, K, et al . (2005) Metam orphic 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/e ndo/pubs/aromatase_prevalidation.pdf.LINURON FINAL

Page 93 of 93 USEPA (2005b) http://www.epa.gov/e ndo/pubs/edmvs/aromatase_drp_final_3_30_05.pdf.

USEPA (2007b) Integrated summary report for valid ation 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 Devel opment, U.S. Environmental Protection Agency, Washington, DC.

Vinggaard, A, Hnida, C, Breinholt, V, et al . (2000) Screening of se lected 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 st udy of the uterotrophic potency of 14 chemicals in a uterotrophic assay and their recepto r-binding affinity. Toxicol. Lett.

146 : 111-120. (MRID 48041740)