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 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

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## ~~~ 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: METOLACHLOR

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS


### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYMETOLACHLOR 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..................................................14

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................18

IV. Committee’s Assessment of Weight of Evidence..........................................21

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................21

1. Tier 1 in vivo Assays.............................................................21

2. OSRI...............................................................................22

B. Estrogen Pathway...........................................................................23

C. Androgen Pathway.........................................................................24

D. Thyroid Pathway...........................................................................24

E. Conclusions.................................................................................24

V. EDSP Tier 2 Testing Recommendations....................................................25

VI. References.......................................................................................27

LIST OF TABLES: Table 1. Tier 1 Assays for Metolachlor..............................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Metolachlor...................................11
Table 3. Androgenic/Anti-Androgenic Pathway for Metolachlor ..............................15
Table 4. Thyroid Pathway for Metolachlor .........................................................19

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................31
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................43

APPENDIX 3. References Not Utilized in the Metolachlor WoE Analysis...........................59METOLACHLOR 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 enerationMETOLACHLOR FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
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 ConcentrationMETOLACHLOR FINAL

v Abbreviation Terminology

MTD Maximum Tolerated Dose
N Ne g ative
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 Tie r 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)METOLACHLOR FINAL

vi Abbreviation Terminology

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

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-EvidenceMETOLACHLOR FINAL

Page 1 of 61 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, 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 metolachlor interacts withE, A or T hormone path ways, the number and type of effects induced, the magn itude and pattern of responses observed across stud ies, taxa and
sexes were considered. Additiona lly, the conditions under which effe cts occur were considered, in particular, whether or not endocri ne-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On July 30, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) an alysis of the potential interaction of metolachlor 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).

Metolachlor (CGA-24705) is a racemi c (1:1) mixture of enantiomers (four stereoisomers). This
document included data from metolachlor and S -metolachlor (an isomer-enriched formula).

For the estrogen pathway, there was no convincing evidence of potential interaction with the estrogen pathway. Metalochlor was negative in the in vitro estrogen receptor (ER) binding and the ER transactivation assay (E RTA), gave an equivocal response in the aromatase assay (reduction in activity observ ed (down to 42% at 10

− 3 M), but potential issue with protein (receptor) denaturation and/or compound solubility) and induced a marginal increase (1.2- to 1.3- fold) in estradiol production in the steroidogenesis assay. No tr eatment-related estrogenic effects
were seen in the in vivo uterotrophic or female pubertal assays. In the fish short-term reproduction assay (FSTRA), the only effects se en, which were at the high concentration, occurred in the presence of overt toxicity. There were no trea tment-related effects on female reproductive parameters in the existing metol achlor Part 158 mammalian studies. While there was a 6% increase in cracked eggs (as eggs not cracked/eggs laid) in one Part 158 avian study, there were no complementary effects in this study, the other avian re production study, or the
other in vivo studies.METOLACHLOR FINAL

Page 2 of 61 Metolachlor was positive for androgen receptor (AR) binding but did not alter testosterone production in the in vitro steroidogenesis assay. In th e Hershberger assay (dosed subcutaneously), there was evidence for an an ti-androgenic at the high dose (500 mg/kg/day). However, in the Tier 1 male pubertal assay and Part 158 mammalian studies in which animals were dosed orally, no androgen-rela ted effects were seen. In th e FSTRA, the only effect in the absence of overt toxicity was a reported significant decrease in fat pad score in the mid-treatment level (0.48 mg a.i./L). Overall, there is no c onvincing evidence for a pot ential interaction with the androgen pathway.

There is some evidence for potential interacti on of metolachlor with the thyroid pathway in mammals based on effects on thyroi d histopathology in both sexes in the pubertal assays. These effects are further supported by alterations in T4 levels in females and thyroid stimulating hormone (TSH) levels in males. In the amphi bian metamorphosis assay (AMA), however, the results do not suggest a potential for thyroid in teraction as there were no effects on thyroid histopathology or deve lopmental stage.

In the EDSP Tier 1 pubertal assays there was evidence for potentia l interaction wi th the thyroid pathway in studies conducted with adult animals, but no such da ta exists for potential thyroid toxicity in the young animals.

For mammals, for metolachlor, the current points of departure (POD) for human health risk assessment is 9.7 mg/kg/day is based on decr eased body weight gain in female dog at 32.7 mg/kg/day (LOAEL) in a chroni c toxicity study. This POD is us ed for deriving the chronic Reference Dose (RfD) for dietary exposure. A second POD of 50 mg/kg/day is based on increased incidence of clinical signs, decreased body weight/body weight gain, food consumption and food efficiency seen in mate rnal animals at 500 mg/kg/day (LOAEL) in a developmental toxicity study in rats. This POD is used for deriving the Margins of Exposure (MOEs) for non-dietary risk assessments. The PODs used for overall risk assessments are approximately 5-30-fold lower than the dose ( 300 mg/kg/day) that i nduced histopathological lesions of the thyroid gland in the female pubertal assay.

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. Howe ver in the case of metolachlo r, although no data exists for potential thyroid toxicity in the young, a comp arative thyroid assay (C TA) is not recommended since: 1) the PODs are lower than the dose that caused thyroid lesions; 2) there was no evidence of thyroid toxicity in th e data base; 3) in subchronic and chro nic toxicity studies in dogs, rats and mice, decreased body weight and body weight gain were the most commonly observed effects; 5) the CTA would be tested at higher doses and would not be expected to yield a POD lowerMETOLACHLOR FINAL

Page 3 of 61 than the level currently used for risk asse ssment; and 6) the chronic RfD and MOEs are protective of the thyroid effects seen in the fe male pubertal assay. Theref ore, additional studies to investigate thyroid toxicity in the young are not required.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not
recommended for metolachlor since additional tes ting is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

For wildlife, as there was no convincing evidence for potenti al interaction with estrogen, androgen pathway in fish or thyroid pathway in amphibian s, no additional testing is
recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard
duck are available for metolachlor. In malla rd ducks no reproductive effects were observed, whereas in bobwhite quail an effect on a reprodu ctive parameter was observed. The type of data
obtained from Part 158 avian re production studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from metolachlor exposure. Relative to the EDSP, additional testing is not recommended.METOLACHLOR FINAL

Page 4 of 61 I. Introduction

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, 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 metolachlor interact s with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pa ttern of responses observed across studies, taxa a nd sexes were considered. Additi onally, the condi tions under which effects occur were considered, in particular , whether or not endoc rine-related responses occurred at dose(s) that also resulted in ge neral systemic toxicity or overt toxicity.

On July 30, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Pr ograms (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction of metolachlor with the E, A or T hormone pathways. The
T1WoERC conclusions from the WoE evaluation in this repo rt are presented by pathway (E, A and then T) beginning w ith 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 CF R Part 158 and literature).

Metolachlor (CGA-24705) is a race mic (1:1) mixture of enantiomers (four stereoisomers); the ( S )- enantiomer (CGA 77102, or α -metolachlor) is the active pe sticidal component and the ( R )- enantiomer (CGA 77101) is inactive. In 1997, an isomer-enriched formula called S -metolachlor (88% S -isomer and 12% R -isomer was registered.

Metolachlor has a vapor pressure of 3.14 x 10 -5 mm Hg at 25°C. The repor ted water solubility is 530 mg/L at 20°C with an octanol/w ater partition coefficient (log K ow) of 3.13 to 3.4. The vapor phase is susceptible to photodegr adation, and the particulate phase is susceptible to wet and dry deposition. Metolachlor is also susceptible to direct photolysis and aquatic biodegradation, but minimal biodegradation in soil. Metolachlo r has an estimated whole body bioconcentration factor in fish ranging from <1 to 69.

The available information considered in determin ing the potential interaction of metolachlor with the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxi city studies and other pu blished articles. TheseMETOLACHLOR FINAL

Page 5 of 61 data are summarized in Sections III.A through I II.C. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recomme ndations are presented in Section V.

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 believe d to be relevant to one or more of the Tier 1 assays were requested to be submitted as OSRI by the Test Order recipients and/or the public. This included studies published in the open litera ture and/or data submitted to support pesticide registration (e.g., Part 158 guideli ne studies). However, the Te st Order recipient elected to conduct all eleven Tier 1 screening assays, so no OSRI were submitted in lieu of the Tier 1 assays. The Agency has also conducted a more recen t search of available sc ientific literature for any additional relevant inform ation. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/ studies considered but not uti lized 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 hormonal pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 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 st eps 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.METOLACHLOR FINAL

Page 6 of 61 As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 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 concordance or consistency (complementarity) 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, or 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 rele vant, 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 systemicMETOLACHLOR FINAL

Page 7 of 61 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 FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, 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). For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 st udies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g., lethargy) were also considered when evaluating overt toxicity, es pecially 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 addressing the three main components 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 availa ble toxicity data into account.METOLACHLOR FINAL

Page 8 of 61 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 a substance has the potential to interact with the E, A or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not reported in the table for E, A or T.

A. EDSP Tier 1 Screening Assays

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

.

Table 1. Tier 1 Screening Assays for Metolachlor

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied (MRID 48617807)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OSCPP 890.1300; OECD 455 Requirement Satisfied (MRID 48617808)

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied (MRID 48617806)

Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; OECD 456 Requirement Satisfied (MRID 48617809)

Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied (MRID 48617805)

Uterotrophic Assay (Rat) OSCPP 890.1600; OECD 440 Requirement Satisfied (MRID 48617801)

Hershberger Assay (Rat) OSCPP 890.1400; OECD 441 Requirement Satisfied (MRID 48617802)

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied (MRID 48617804)

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied (MRID 48617803)

Fish Short-term Reproduction Assay OSCPP 890.1350; OECD 229 Requirement Satisfied (MRID 48617811)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; OECD 231 Requirement Satisfied (MRID 48617810)METOLACHLOR FINAL

Page 9 of 61 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for metolachlor to interact w ith the estrogen pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This
table also includes HPG-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.

Metolachlor was negative (or not interactive) in the Tier 1 in vitro estrogen receptor (ER) binding assay and the ER α transcriptional activation assay. Me tolachlor was also negative in an
ERTA considered as OSRI that was conducted in Chinese hamster ovary (CHO) cells (Kojima et
al ., 2004). In the Tier 1 aromatase assay, while enzyme activity was reduced to 42% at the highest tested concentration (10

− 3 M), the data (Hill slope) indicate a potential issue with protein (receptor) denaturation and/or co mpound solubility, and as such, th e results were classified as equivocal. In the Tier 1 steroi dogenesis assay increases (1.2- to 1.3-fold) in estradiol production were observed at the two highest concentrations, 10 and 100 μM.

Metolachlor was negative in the in vivo uterotrophic assay. In the fe male pubertal rat assay, there were no differences in body weight at VO, or final body weights, in the treated gr oups relative to control. Adjusted age at VO was advanced (appr oximately two days) at the low dose, but not at the high dose. Although a statistical ly significant decrease in per centage of animals cycling was noted at the high dose (93.3%) when compared to controls (100%), this findings is not considered treatment-related since this represents only 1/15 animals. There was a decrease in the number of regularly cycling females, but there was no dose-response. In the FSTRA, there was a 98% decrease in fecundity in th e high-concentration group (4.7 mg a.i./L), but no effect on fertility. Female plasma vitellogenin (VTG) wa s decreased by 77% in the high-concentration along with increased incidences of oocyte atre sia, granulosa cell phagocytosis, and decreased early and late vitellogenic oocytes . However, these effects at th e high concentration occurred in conjunction with overt toxicity, ba sed on decreased survival (71%) a nd clinical signs of toxicity ( i.e., erratic swimming, loss of equilibr ium and lack of feeding).

In the mammalian Part 158 toxi city studies, a non- statistically significant (NS) and non-dose responsive increase in pituitar y weights with no co rroborating pituitary histopathological findings in all dose groups in the chronic dog stu dy was noted. No other estrogen-related effects
were observed in the Part 158 mammalian studies.

In the Part 158 avian reproduction studies no treatment-related repr oductive effects were observed in the study with mallard duck up to 80 0 mg/kg-diet. In a study with bobwhite quail,
there was a treatment-related 6% increase in eggs cracked/eggs laid at the high dose (1000METOLACHLOR FINAL

Page 10 of 61 mg/kg-diet); no other treatment-rela ted reproductive effects or signs of parental overt or systemic toxicity were observed.

In a Part 158 fathead minnow early-life cycle st udy, body length was decreased (significance not reported) ≥ 0.220 mg/L, and decreases in we t weight were observed at ≥ 0.110 mg/L and in dry weight at ≥ 0.056 mg/L. In another fish early-life cycle study with sheepshead minnow, there was a decreasing trend in length and weight at concentrations ≥ 2.2 mg/L, and increased mortality

at ≥ 4.1 mg/L.METOLACHLORFINALPage 11 of 61Table 2. Estrogenic/Anti-Estrogenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Metolachlor1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity 2\ Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48617807)NERTA(MRID 48617808)NAromatase(MRID 48617805)ESteroidogenesis(MRID 48617809)PUterotrophic(MRID 48617801)NFemale Pubertal Rat(MRID 48617804)NNNNNNLW:↑22%KW:↑10%↑GGT(H)NFSTRA(MRID 48617811)NENNNNNX(H)4OSRIERTA(Kojimaet al., 2004)N3-Month Subchronictoxicity (Rat; MRID43928923)NENNNEX(H)X(MH,H)3-Month Subchronictoxicity (Rat; MRID44775401, 44775402)NENNNENX(H)METOLACHLORFINALPage 12 of 61Table 2. Estrogenic/Anti-Estrogenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Metolachlor1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity 2\ Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 33-Month Subchronictoxicity (Dog; (MRID43928922)NENNNEX(MH, H)N6-Month Subchronictoxicity (Dog; MRID00032174)NENNNX(H)N21-day Dermal(Rabbit; MRID41833101)NNNENNNDevelopmentaltoxicity (Rat; MRID43928925)NNX(MH, H)NDevelopmentaltoxicity (Rat; MRID00151941)NRNX(H)X(H)Developmentaltoxicity (Rabbit;MRID 43928924)NRNX(H)NDevelopmentaltoxicity (Rabbit;MRID 00041283)NRNX(H)NTwo-generationreproduction (Rat;MRID 00080897)NENENNENX(H)NChronic toxicity/carcinogenicity (Rat;MRID 00129377)NENNNEN NMETOLACHLORFINALPage 13 of 61Table 2. Estrogenic/Anti-Estrogenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Metolachlor1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity 2\ Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Carcinogenicity(Mouse; MRID,00117597)NNNNRNX(H)Chronic toxicity (Dog;MRIDs 40980701,41164501, 42218601,42218602)NENNNN NAvian Reproduction(Duck; MRID44015602)NN NAvian Reproduction(Quail; MRID46508901)P5(H)N N1.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatmentArrows (↓or↑) indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated. LW= Liver weight; KW= Kidney weight; GGT= gamma-glutamyl transferase2. 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. Fordetails see Section IV. A4. Erratic swimming, loss of equilibrium, or lethargic swimming, and increased mortality (survival↓29%) at the high concentration (4.7 mg/L).5. Increases in cracked eggs atthe high test concentration.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedE EquivocalMETOLACHLOR FINAL

Page 14 of 61 2. Effects on Androgen Pathway

The potential for metolachlor to interact with the androgen path way is summarized in Table 3. The various targets of the androgen pathway across the relevant Ti er 1 assays are delineated so as to facilitate determination of potential for a ndrogenic, anti-androgenic or HPG axis effects.
This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

Metolachlor was a positive binder in the Tier 1 in vitro androgen receptor (AR) binding assay. Metolachlor reduced competitive binding to the AR to approximately 50% at 10

− 3 M. However, metolachlor was negative for AR agonism and antagonism in an OSRI AR transcriptional activation (ARTA) assay conduc ted in CHO cells (Kojima et al ., 2004). Metolachlor did not alter testosterone production in the Tier 1 in vitro steroidogenesis assay.

In the Tier 1 Hershberger assay, the androgenic portion of the assa y was negative. In the antiandrogenic portion of the assay, th e weights for ventral prostate and LABC were decreased by 25 and 22%, respectively, at the high dose, a nd LABC weights also were decreased ( ↓ 17%) in the mid-dose group (300 mg/kg/day). Terminal body we ight decreases in the high-dose metolachlor group (500 mg/kg/day) were 10% and 8% in the androgenic and anti-androgenic portions of the assay, respectively, with concomitant decreases in body weight gains (45 a nd 33%, respectively), demonstrating that the 500 mg/kg/da y dose approached a toxic dose.

In the male pubertal rat assay, no treatment-rela ted changes in body weights, body weight gains, or age and weight at PPS occurred in either treatment group (400 and 80 0 mg/kg/day). Liver weights were increased (p<0.05) by 15 and 20%, and kidney weight s were increased (p<0.05) by 11% each, in both dose groups, respec tively. Mean testosterone c oncentration was decreased in a dose-related manner ( ↓ 45 and 69%), but the observed difference may be an artifact due to unusually high testosterone concentrations (>8.00 ng/mL) in three animals in the control group. No histopathological lesions were observed in the testes or epididymides.

In the FSTRA, there was a significant 63% decr ease in male tubercle score in the highconcentration (4.7 mg a.i./L), and reported signi ficant decreases in fat pad scores in the mid- (0.48 mg a.i./L) and high-concentration groups. There was a marginally significant (p=0.057) decrease of 29% in male GSI at the high concentration along with changes in gonadal histopathology including an incr ease in diploid spermatogonia in germinal epithelium. Overall survival was decreased 29% in the high-concen tration group, and the surviving fish exhibited signs of clinical toxicity (err atic swimming, loss of equilibrium, lethargic swimming, and curved spines).

There were no androgen-related e ffects observed in the Part 1 58 mammalian or avian studies.METOLACHLORFINALPage 15 of 61Table 3. Androgenic/Anti-Androgenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Metolachlor1Study Type/Literature Citation AR Binding AR Activation Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding (MRID48617806)PSteroidogenesis(MRID 48617809)NHershberger (MRID48617802)NEVP↓25%LABC:22% (H)N N4Male Pubertal Rat(MRID 48617803)NNNNNNNNLW:↑15, 20%KW:↑11, 11%(L, H)NFSTRA(MRID 48617811)NENNFat pad:↓20% (M)NNX(H)5METOLACHLORFINALPage 16 of 61Table 3. Androgenic/Anti-Androgenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Metolachlor1Study Type/Literature Citation AR Binding AR Activation Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3OSRIARTA(Kojimaet al., 2004)N3-Month Subchronictoxicity (Rat; MRID43928923)N N NE N NE NEX(H)X(MH, H)3-Month Subchronictoxicity (Rat; MRID44775401,44775402)N N N N NE NEN N3-Month Subchronictoxicity (Dog; MRID43928922)N N NE N NE NEX(MH, H)N6-Month Subchronictoxicity (Dog; MRID00032174)N N NE NE N NEX(H)X(M, H)21-day Dermal(Rabbit; MRID41833101)N NE N NE N NX(H)NTwo-generationreproduction (Rat;MRID 00080897)N N N N NE NENX(H)NChronic toxicity/carcinogenicity (Rat;MRID 00129377)NNNENENENEN NCarcinogenicity(Mouse; MRID00117597)NNRNRNRNRNX(H)NMETOLACHLORFINALPage 17 of 61Table 3. Androgenic/Anti-Androgenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Metolachlor1Study Type/Literature Citation AR Binding AR Activation Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic toxicity(Dog; MRIDs40980701,41164501,42218601, and42218602)NN N N NNEX(M, H)NAvian Reproduction(Duck; MRID44015602)NN NAvian Reproduction(Quail; MRID46508901)NN N1. 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 this parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. Abbreviationsfor androgen sensitive tissues: Seminal vesicles (SV), Ventralprostate (VP), Dorsal prostate(DP), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). BW= Body weight; BWG= Body weight gain;LW= Liver weight; KW= Kidney weight2. 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. Fordetails see Section IV. A4. Body weight decreased 8% in the antagonist portion of the assay at the high dose (500 mg/kg/day; s.c. injection) with a decrease in body weight gain of33%.5. Erratic swimming, loss of equilibrium, or lethargic swimming, and increased mortality (survival↓29%) at the high concentration (4.7 mg/L).P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedMETOLACHLOR FINAL

Page 18 of 61 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 metolachlor to interact with thyroid regulation is 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.

In the female pubertal rat assay with metolach lor, evidence of changes in thyroid function included increased ( ↑ 25%) total thyroxine (T 4 ) in the high-dose (600 mg/kg/day) females with no effect on thyroid stimulating hormone (TSH) and slight changes in the thyroid histopathology. In the male pubertal rats seru m TSH levels were increased ( ↑ 54 and 134%) in both dose groups (400 and 800 mg/kg/day) and sli ght changes in the thyroid hist opathology. There no changes in
serum T4 levels noted at these doses. There were no changes in thyroid wei ghts in either sex.

In the AMA, metolachlor treatment decrease d Day 7 wet weight by 19%, Day 7 snout-vent length (SVL) by 7% and Day 21 normalized hind-limb length (HLL) by 23% at the high concentration (8.4 mg a.i./L), relative to the ne gative control. However, no treatment-related effects on thyroid histopathology or developmental stage were noted.

The only effect observed in the mammalian Part 158 toxicity studies that was related to the thyroid pathway (HPT axis) wa s a non-statistically significan t (NS) and non-dose responsive increase in pituitary weights in a ll dose groups in the chronic dog study.

In a published, non-Guideline study, Jin et al. , 2011 reported effects on thyroid-related gene transcription in juvenile and a dult Japanese medaka fish. The findings showed treatment-related effects on transcription were ge nder-, tissue-, and developmenta l-stage-specific. Effects on males occurred in the presence of estrogen onl y. The study did not evaluate other endocrinerelated endpoints and there is uncer tainty in what impact these alterations in gene transcription would have on higher-levels of biological organization. A study using spotted marsh frogs ( Limnodynastes tasmaniensis ) reported no effects on developmental stage, body length or sex ratio in tadpoles exposed up to 10 μg/L metolach lor; however, as there were no measurements on thyroid or thyroid hormones, the study is limited in its ability to inform about potential thyroidrelated interactions (Spolyarich et al . 2010).METOLACHLOR FINAL

Page 19 of 61 Table 4. Thyroid Pathway for Metolachlor

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Metolachlor 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, BL,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assay

Female Pubertal Rat (MRID 48617804) N P (H) ↑ 25% (H) N N LW: ↑ 22%
KW: ↑ 10% ↑ GGT

(H) N

Male Pubertal Rat (MRID 48617803) N P (L, H) N ↑ 54, 134% (L, H) N LW: ↑ 15, 20%

KW: ↑ 11, 11%

(L, H) N

AMA (Frog; MRID 48617810) N Day 21
HLL: ↓ 23%

(H) Day 7
BW: ↓ 19% SVL: ↓ 7% (H) N 4

OSRI

3-Month Subchronic toxicity (Rat; MRID 43928923) NE N NE X (MH, H) X (MH, H)

3-Month Subchronic toxicity (Rat; MRID 44775401, 44775402) NE N NE N X (H)

3-Month Subchronic toxicity (Dog; MRID 43928922) N N NE X (MH, H) N

6-Month Subchronic toxicity (Dog; MRID 00032174) N N N X (H) X (M, H)

21-day Dermal toxicity (Rabbit; MRID 41833101) NE NE N X (H) N

Two-generation reproduction (Rat; MRID 00080897) N N NE X (H) N

Chronic toxicity/ Carcinogenicity (Rat; MRID 00129377) NE N NE X ( H) N

Carcinogenicity (Mouse; MRID 00117597) NR NR NR X (H) X (H)METOLACHLOR FINAL

Page 20 of 61 Table 4. Thyroid Pathway for Metolachlor

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Metolachlor 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, BL,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

Chronic toxicity (Dog; MRIDs 40980701, 41164501, 42218601, and 42218602) N N N X (L, M, H) N

1. Key to responses: L=Low treatment, ML=Medium- low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the re sponse. A shaded cell indicates that this parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated. LW= liver weight; KW= kidney weight; GGT= gamma-glutamyl transferase 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 4. While clinical signs of toxicity (lethargy or surfacing) were reported until day 8, at the high concentration, these clinical signs of toxicity did not persist throughout the exposure period. There were also 4 mortalities (out of 80) observed at this treatment level during the 21 day exposure period. P Positive findings N Negative findings NE Not examined NR Not reportedMETOLACHLOR FINAL

Page 21 of 61 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 metolachlor to interact with 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 cons istency, coherence and in terrelationships among the measured endpoints within and across studi es. The available findings from standard toxicology studies on the substance may contribute to the WoE evalua tion in helping elucidate 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 that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tier 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l 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).

1. Tier 1 in vivo Assays

All six in vivo assays in the EDSP Tier 1 battery were availab le for metolachlor. In the Hershberger and uterotrophic assays, all animal s survived until schedu led termination, and noMETOLACHLOR FINAL

Page 22 of 61 test substance-related clinical fi ndings were noted in any of the animals. In the female pubertal rat assay, one control animal was sacrificed in extremis; all other rats surv ived until scheduled sacrifice. No treatment-related clinical findings were noted during the da ily examinations at any dose. In the male pubertal rat assay, one high-dose male was found dead (not treatment related)
and one control animal was sacrificed in extremis; all other rats survived until scheduled sacrifice. Clinical findings in males include d salivation prior to dosing, and clear material around the mouth at approximately 1 hour post-dos e in both treatment groups. Clear material also was observed around the ventral neck in high- dose animals. Moderate increases in liver and kidney weights (approximately ↑ 10-20%) were also noted in hi gh-dose females and low- and high-dose males.

In the FSTRA, overall survival was decreased ( ↓ 29%) in the high-concen tration group (4.7 mg a.i./L), and the surviving fish showed signs of erratic swimming, loss of equilibrium, lethargic swimming, and curved spines. In addition, excess feed was observed in the tanks. In the AMA, while clinical signs of toxicity (lethargy or surfacing) were reported until day 8 at the high concentration (8.4 mg a.i./L), these clinical signs of toxicity did not persist throughout the exposure period. There were also 4 mortalities (out of 80) observe d at this treatment level during the 21 day exposure period. The decrease in body weight and SVL at day 7 at the high concentration (but not at day 21) supports initial signs of stress that did not continue throughout the remaining exposure period. Additionally in the AMA, crooked tails were observed in 27 to 57 tadpoles (out of 80 exposed) in the control and all tr eatment groups, but did not appear to be
treatment-related.

2. OSRI

There were no mortalities or clinical signs in a subchronic oral toxici ty study in rats with S-metolachlor. Treatment-related systemic t oxicity was observed in the medium-high and highdose groups as decreases in body weight, body weight gain, and food consumption and efficiency. Body weights were decreased in medium-high ( ↓ 10% at Week 10) and high-dose males ( ↓ 11-13% from Week 1), respectively, an d were decreased in medium-high ( ↓ 10% from Week 11) and high-dose females ( ↓ 12-17% from Week 8), respectively. There were also no mortalities or clinical signs in a subchronic oral toxicity study in rats wi th metolachlor. There was no evidence of any treatment -related effect in male rats, but decreased body weights were observed in the high-dose females ( ↓ 10-11%) from Week 9.

There were no mortalities in a subchronic oral toxici ty study in dogs with S-metolachlor. Clinical signs were limited to incidences of few or no stools in the medium-high and high-dose dogs. There were also no mortali ties or clinical signs in a subchr onic oral toxicity study in dogs with metolachlor. Mean body weight gain wa s decreased (NS) during Weeks 0-13 and 0-26 in the high-dose males ( ↓ 55-63%) and females ( ↓ 44-50%), and mean Week 26 body weights were decreased in the medium- and high-dose males by 11 and 14%, respectively.METOLACHLOR FINAL

Page 23 of 61 Some systemic maternal toxicity was observed in a developmental toxicity study in rats with Smetolachlor. All females in the mid-high and hi gh-dose groups and 9/24 females in the mediumlow dose group exhibited discomfort after daily dose administration which consisted of pushing their heads through the bedding for approximate ly one hour. In addition, body weight gains were decreased in the mid-high and high dose group during dosing a nd over the entire study. In an earlier developmental study in rats with meto lachlor, four dams in the high-dose group died on GDs 7, 8, and 10 (n=2). Clinical signs of toxi city were observed in these animals one or more days prior to death. Clinical signs, observed primarily in the high-dose dams, included excess salivation (25/25), urine-staine d abdominal fur (19/25), excess lacrimation (8/25), and clonic/tonic (6/25) or clonic (5/25) convulsions. One death in the high-dose group due to anorexia and body weight loss was considered tr eatment-related in a developmental toxicity study in rabbits with S-metolachlor. Maternal t oxicity, consisting of litt le, none, or soft stools was observed in the medium- and high-dose animals. Mortalities due to anorexia were observed in an earlier developmental toxic ity study in rabbits with metolachlor. One doe in each the lowand high-dose groups died on study due to persis tent anorexia. Two rabbits aborted; one abortion occurred in a high-dose doe with persistent anorexia. There was a treatment-related increase in the incidence of persistent anorex ia in the does in the high-dose group. There was a treatment-related decrease in body weight gain in the high-dose group during the dosing period ( ↓ 500%) and the combined dosi ng and post-dosing periods ( ↓ 133%).

High-dose females had an increased overall mortality rate due to early deaths during the first few weeks of treatment in a carcinogenicity study in mice with metolachlor. Although the deaths
were attributed to a viral inf ection, the potential contribution of the test material cannot be
dismissed.

No treatment-related mortalities or clinical signs of toxicity were observed in the 21-day dermal toxicity study in rabbits , the two-generation reproduction study in rats, the chronic oral toxicity studies in rats or dogs or th e avian reproduction studies in ma llard ducks and bobwhite quail.

In a fathead minnow early-life cycle study, leng th was decreased (significance not reported) ≥ 0.220 mg/L, and decreases in wet weight were observed at ≥ 0.110 mg/L and in dry weight at

≥ 0.056 mg/L. In another fish early-life cycle study with sheepshead minnow, there was a decreasing trend in length and weight at concentrations ≥ 2.2 mg/L, and increased mortality at

≥ 4.1 mg/L.

B. Estrogen Pathway

There was no convincing evidence of potentia l interaction with the estrogen pathway. Metalochlor was negative in the in vitro estrogen receptor (ER) binding and the ER transactivation assay (ERTA), gave an equivocal response in the aromat ase assay (reduction in activity observed (down to 42% at 10

− 3 M), but potential issue with protein (receptor)METOLACHLOR FINAL

Page 24 of 61 denaturation and/or compound solubility), and induced a marginal increase (1.2- to 1.3-fold) in estradiol production in the steroidogenesis assay. No treatme nt-related estrogenic effects were
seen in the in vivo uterotrophic or female pubertal assays . In the fish short-term reproduction assay (FSTRA), the only effects seen, which were at the high concentration, occurred in the presence of overt toxicity. There were no treatment-related effects on female reproductive parameters in the existing metolachlor Part 158 mammalian studies. While there was a 6% increase in cracked eggs (as e ggs not cracked/eggs la id) in one Part 158 avian study, there were no complementary effects in this study, the other avian reproducti on study, or the other in vivo
studies.

C. Androgen Pathway

Metolachlor was positive for AR binding but did not alter testosterone production in the in vitro steroidogenesis assay. In the Hershberger assay (d osed subcutaneously), there was evidence for an anti-androgenic at the high dose (500 mg/kg/day) . However, in the Tier 1 male pubertal assay
and Part 158 mammalian studies in which animal s were dosed orally, no androgen-related effects were seen. In the FSTRA, the only effect in the absence of overt toxicity was a reported significant decrease in fat pad scor e in the mid-treatment level (0.48 mg a.i./L). Overall, there is no convincing evidence for a potential in teraction with the androgen pathway.

D. Thyroid Pathway

There is some evidence for potential interacti on of metolachlor with the thyroid pathway in mammals based on effects on thyroi d histopathology in both sexes in the pubertal assays. These effects are further supported by alte rations in T4 levels in females and TSH levels in males. In the AMA, while a decrease in HLL was observe d at Day 21 at the hi gh test concentration, clinical signs of toxicity (related to swimming) and decr eased body weight and SVL were
observed in this concentration dur ing the beginning phases of the study. Overall, the results to not suggest a potential for thyroi d interaction as there were no effects on thyroid histopathology or developmental stage.

E. Conclusions

Based on the available data, ther e is no convincing evidence for pot ential interact ion with the estrogen or androgen pathways in mammals or wildlife. For the thyroid pathway, there is evidence for potential interaction in mamma ls, but no convincing evidence for potential
interaction in wildlife.METOLACHLOR FINAL

Page 25 of 61 V. EDSP Tier 2 Testing Recommendations

In the EDSP Tier 1 pubertal assays, there was evid ence for potential intera ction with the thyroid pathway in studies conducted with adult animals, but no such da ta exists for potential thyroid toxicity in the young animals.

For metolachlor, the current points of departure (POD) of 9.7 mg/kg/day used for human health risk assessment is based on decreased body we ight gain in female dog at 32.7 mg/kg/day (LOAEL) in a chronic toxicity study. This POD is used for deriving the chronic Reference Dose (RfD) for dietary exposure. A second POD of 50 mg/kg/day is based on increased incidence of clinical signs, decreased body weight/body weight gain, food c onsumption and food efficiency seen in maternal animals at 500 mg/kg/day (LOAEL) in a developmental toxicity study in rats. This POD is used for deriving the Margin s of Exposure (MOEs) for non-dietary risk
assessments. The PODs used for overall risk asse ssments are approximately 5-30-fold lower than the dose (300 mg/kg/day) that induced histopath ological lesions of the thyroid gland in the female pubertal assay.

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. Howe ver in the case of metolachlo r, although no data exists for potential thyroid toxicity in the young, a comp arative thyroid assay (C TA) is not recommended since: 1) the PODs are lower than the dose that caused thyroid lesions; 2) there was no evidence of thyroid toxicity in th e data base; 3) in subchronic and chro nic toxicity studies in dogs, rats and mice, decreased body weight and body weight gain were the most comm only observed effects; 5) the CTA would be tested at higher doses a nd would not be expected to yield a POD lower than the level currently used for risk asse ssment; and 6) the chronic RfD and MOEs are protective of the thyroid effects seen in the fe male pubertal assay. Theref ore, additional studies to investigate thyroid toxicity in the young are not required.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not
recommended for metolachlor since additional tes ting is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

For wildlife, as there was no convincing evidence for potenti al interaction with estrogen, androgen pathway in fish or thyroid pathway in amphibian s, no additional testing is
recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard
duck are available for metolachlor. In malla rd ducks no reproductive effects were observed, whereas in bobwhite quail an effect on a reprodu ctive parameter was observed. The type of data
obtained from Part 158 avian re production studies (OCSPP 850.2300) are considered sufficientMETOLACHLOR FINAL

Page 26 of 61 for evaluating potential reproductive effects to birds from metolachlor exposure. Relative to the EDSP, additional testing is not recommended.METOLACHLOR FINAL

Page 27 of 61 VI. References

Chang, J.C.F. (1995) CGA-77102 Technical 13-week oral toxicity in rats. Unpublished study conducted by Ciba-Geigy Corp, Cr op Protection Division, Environmen tal Health Center for Ciba Crop Protection, Ciba-Geigy Corp. Laborator y Study No. F-00191. February 21, 1995. (MRID 43928923)

Chang, J.C.F. (1995) CGA-77102 Technical Final report 90-day oral toxicity in dogs. Unpublished study conducted by Ciba-Geigy Corp , Crop Protection Division, Environmental
Health Center for Ciba Cr op Protection, Ciba-Geigy Corp. Laboratory Study No. F-00193. June 14, 1995. (MRID 43928922)

Estes, F.L. (1980) 6-Month Chronic Oral T oxicity Study in Beagle Dogs. Unpublished study conducted by International Research and De velopment Corporation (IRDC), Mattawan, MI., Study Number 382-054, May 21, 1980. (MRID 00032174)

Fankhauser, H. (1999) CGA-24705 Final Report. 3-month oral toxicity study in rats (administration in food). Unpublished study conducted by Novartis Crop Protection AG, Toxicology, 4332 Stein, Switzerland. Study ID # 971143, Novartis #1162-98. January 25, 1999. (MRID 44775401)

Fankhauser, H. (1999) CGA-771 02 Final Report. 3-month oral toxicity study in rats (administration in food). Unpublished study conducted by Novartis Crop Protection AG, Toxicology, 4332 Stein, Switzerland. Study ID # 971144, Novartis #1161-98. January 25, 1999. (MRID 44775402)

Gilles, P.A. and Giknis, M.L.A. (1995) A te ratology study of CGA-77102 Technical in New Zealand White rabbits. Unpublished study condu cted by Ciba-Geigy Corp, Crop Protection Division, Environmental Health Center. La boratory Study No. F00192. April 27, 1995. (MRID 43928924)

Hazellette, J.R. and Arthur, A. T. (1989) Meto lachlor Technical, 13/52-Week Oral Toxicity Study in Dogs (MIN 862253). Unpublishe d study conducted by the Division of Toxicology/Pathology, CIBA-GEIGY, Surmnit, NJ and Methpath Laboratories, Rockville, MD, Study Number 862253, January 23, 1989. (MRIDs 40980701, 41164501, 42218601, and 42218602)

Jin, Y., Chen, R., Wang, L., Liu, J., Yang, Y., Z hou, C., Liu, W., and Fu, Z. (2011) Effects of metolachlor on transcription of thyroid system-related genes in juvenile and adult Japanese medaka ( Oryzias latipes ). Gen Comp Endocrinol. 170(3):487-493.METOLACHLOR FINAL

Page 28 of 61 Khalil, S. (1995) CGA-77102 Rat oral teratology. Ciba-Geigy Ltd., Basle, Switzerland for Ciba Crop Protection, Unpublished study conducted by Ciba-Geigy Corp. Laboratory Study No. 941058. August 21, 1995. (MRID 43928925)

Kojima, H., Katsura, E., Takeuchi, S., Niiyami, K., and Kobayashi, K. (2004) Screening for estrogen and androgen receptor activi ties in 200 pesticides by in vitr o reporter gene assays using Chinese hamster ovary cells. Environ. Health Perspect . 112 (5): 524-531.

Lightkep, G.E. (1980) Teratogenic Potential of CGA-24705 in New Zealand White Rabbits Segment II Evaluation. Unpublished study conducted by Argus Research Laboratories, Inc., Horsham, P A. Argus Project 203-001, July 16, 1980. (MRID 00041283)

Lochry, E.A. (1985) Embryo/Fetal Toxicity a nd Teratogenic Potential Study of CGA-24705 (FL841697) Administered Orally via Gavage to Cr l:COBS®CD®(SD) BR Presumed Pregnant Rats. Unpublished study conducted by Argus Research Laboratories, Inc., Horsham, P A. Argus Project 203- 004, August 6, 1985. (MRID 00151941)

Mastrocco, F., Huber, K., Schiavo, D.M., Hazele tte, J.R. and Green, J.D. (1987) 21-Day Dermal Toxicity Study in Rabbits. Unpublished st udy conducted by the Research Department, Pharmaceuticals Division, Ciba-Geigy Cor poration. Study Number 862012, November 16, 1987. (MRID 41833101)

Page, J.G. (1981) Two-Generation Reproduction Study in Albino Rats with Metolachlor. Unpublished study conducted by Technical. Toxi genics, Decatur, IL. Study Number 450-0272, August 31, 1981. (MRID 00080897)

Palmer, S.J., Schneider, S.Z., Kendall, T.Z., and Krueger, H.O. (2012) Metolachlor: Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances. Unpublished study performed by Wildlife Intern ational, Ltd., Easton, MD. La boratory report No. 568A-103. Study sponsored by Metolachlor Task Force, Stilw ell, KS. Study completed January 24, 2012. (MRID 48617810)

Palmer, S.J., Martin, K.H., Kendall, T.Z., and Krueger, H.O. (2012) Metolachlor: Fish Shortterm Reproduction Assay with the Fathead Minnow, ( Pimephales promelas ). Unpublished study performed by Wildlife Intern ational, Ltd., Easton, Maryla nd. Lab Study No.: 568A-102. Study sponsored by Metolachlor Task Force, Stilw ell, Kansas. Study completed January 23, 2012. (MRID 48617811)

Sloter, E. D. (2012) A Hershberg er Assay of Metolachlor Admi nistered Subcutaneously in Peripubertal Orchidoepididymectomized Rats . Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laborat ory project ID: WIL-786010, January 25, 2012. (MRID 48617802)METOLACHLOR FINAL

Page 29 of 61 Sloter, E.D. (2012) Pubertal De velopment and Thyroid Function in Intact Juvenile/Peripubertal Female Rats Following Oral Administration of Metolachlor. Unpublis hed study conducted by WIL Research Laboratories, LLC, Ashland, OH, Laboratory Project ID: WIL-786003, January 25, 2012. (MRID 48617804)

Sloter, E.D. (2012) Pubertal De velopment and Thyroid Function in Intact Juvenile/Peripubertal Male Rats Following Oral Administration of Metolachlor. Unpublished study conducted by WIL Research Laboratories, LLC, Ashland Ohio. WIL 786004, January 25, 2012. (MRID 48617803)

Sloter, E.D. (2012) A Ut erotrophic Assay of Metolachlor Administered Subcutaneously in Ovariectomized Rats. Unpublished study conduc ted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Study No.: WIL-786001, January 3, 2012. (MRID 48617801)

Sousa, J.V. (2012) S-Metolachlor (CGA-77102): Earl y Life-Stage Toxicity Test with Fathead Minnow ( Pimephales promelas ). Unpublished study performed by Springborn Laboratories, Inc., Wareham, MA. Lab Study No. 178 1.6576. Study spons ored by Novartis Crop Protection, Inc., Greensboro, NC. Study completed November 30, 1999. (MRID 44995903)

Taliaferro, L.C., Taliaferro, M.C., and Miller, V.C. (1996) Metolachlor: The reproductive toxicity test of CGA-24705 in Mallard ducks (Anas platyrhynchos). Unpublished study conducted by EBA, Inc., Snow Camp, NC. Spon sored by Ciba Geigy Corporation, Greensboro, NC. Report ID: 029409. (MRID 44015602)

Temple, D.L., et al. (2005) Metolachlor technical: A re production study with the Northern Bobwhite. Unpublished study perf ormed by Wildlife Internationa l Ltd., Easton, MD. Laboratory Project No. 568-102. Study sponsored by the Metola chlor Task Force, Chemical Consultants Intemational, Stilwell, KS. (MRID 46508901)

Thomas, J.A. (2012) In Vitro Rat Prostate Androgen Receptor Competitive Binding Assay. Unpublished study conducted by WIL Research La boratories, LLC, Ashland, OH. Laboratory Study No.: WIL-786007, January 4, 2012. (MRID 48617806)

Thomas, J.A. (2012) In vitro inhibition assay with human reco mbinant aromatase. Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report No.: WIL-786005, January 4, 2012. (MRID 48617805)

Thomas, J.A. (2012) In Vitro Rat Uterine Estrogen Receptor Competitive Binding Assay. Unpublished study conducted by WIL Research La boratories, LLC, Ashland, OH. Laboratory Study No.: WIL-786006, January 4, 2012. (MRID 48617807)

Tisdel, M. (1982) Carcinogenicity Study with Metolachlor in Albino Mice. Unpublished study conducted by Hazleton-Raltech, Inc., Madison, WI. Study Number 79020, August 13, 1982. (MRID 00117597)METOLACHLOR FINAL

Page 30 of 61 Tisdel, M. (1983) Two-year Chr onic Oral Toxicity and Oncogeni city Study with Metolach1or in Albino Rats. Unpublished study conducted by Ha zleton-Raltech, Inc., Madison, WI. Study Number 80030, May 2, 1983. (MRID 00129377)

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental 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.

Wagner, H. (2012) Metolachlor: H295R ster oidogenesis assay. Unpub lished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9179V-100537STER, January 26, 2012. (MRID 48617809)

Ward, S. (1980). Effects of Metolachlor (Dual) on Survival, Growth, and Development of Sheepshead Minnows ( Cyprinodon variegatus ). Unpublished study conducted by EG&G, Bionomics Marine Research Laboratory, Pens acola, Florida. Study No. BP-80-5-80. Submitted by Ciba-Geigy Corporation, Green sboro, NC. (MRID No. 43044602)

Willoughby, J.A. (2012) Metolachlor: estrogen recep tor transcriptional ac tivation (human cell line (HeLa-9903)). Unpublished study conducted by CeeTox, Inc ., Kalamazoo, MI. Laboratory Report No.: 9179V-100537ERTA, January 25, 2012. (MRID 48617808).METOLACHLOR FINAL

Page 31 of 61 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

The 21-day assay (MRID 48617810) of meto lachlor (97.3% purity) on amphibian metamorphosis of Xenopus laevis (African clawed frog) wa s conducted under flow-through conditions. Amphibian larvae at developmental Nieuwkoop-Faber (NF) Stage 51 (80/control and treatment group; 20/replicate) were exposed to no minal concentrations of 0 (negative control), 0.09, 0.9, and 9.0 mg a.i./L. Mean-measured con centrations were <0.0500 (<LOQ; negative control), 0.075, 0.79, and 8.4 mg a.i./L. The test syst em was maintained at 22 ± 1 °C and a pH of

8.1 to 8.3.

On Day 21, mortality was 3.3 and 6.6% in the mid and high treatment groups, respectively. Crooked tails were observed in 27 to 57 tadpoles in the negative control and all treatment groups

Metolachlor significantly redu ced (p<0.05) Day 7 wet weight by 19% and Day 7 snout-vent length by 7% at the high treatment group rela tive to the negative control. Day 21 normalized (for snout-vent length) hind leg length (HLL) was also significan tly reduced (p<0.05) by 23% at the high treatment level relative to the negative control. Metolachlor e xposure had no effect on the NF developmental stage in all treatment groups relative to the negative control on Days 7 and 21; no asynchronous development was observed. Da y 21 normalized HLL at the high treatment level was significantly reduced (p<0.05) by 23% relative to the nega tive control group.

Thyroid gland histopathology obs ervations included thyroid gl and hypertrophy, thyroid gland atrophy, and/or follicular cell hypertrophy in the ne gative control and all treatment groups.

Analysis of Day 21 wet weight, SVL, HLL, a nd normalized HLL excluded late stage tadpoles ( i.e. , NF stage =61-63); there was no pattern in the development of late stage individuals across treatment groups, with the proportion of late stag e tadpoles ranging from 2 to 6% of all tadpoles tested in the negative contro l and all treatment groups.

Androgen Receptor (AR) Bindi ng Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48617806), ventral prostate cytosol from previously castrated (23-24 hours prior to collection) Sprague Dawley rats, was used as the source of AR to conduct saturation and competiti ve binding experiments. The saturation binding experiment was conducted to demonstrate that the AR in the rat pros tate was present in reasonable numbers and was functioning with appropr iate affinity for the radio-labeled reference androgen ([ 3 H]-R1881) prior to conducting the co mpetitive binding experiments. The competitive binding assay measured the bi nding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 10 to 10

− 3 M) of metolachlor (97.3% purity, Lot# 20081115). Ethanol was used as the solvent vehicl e at a final assay concentration of 2.6%. AMETOLACHLOR FINAL

Page 32 of 61 total of three runs were performed, and each run included dexamethasone as a weak positive control, and R1881 as the ligand reference standard.

In the saturation binding experime nt, the maximum binding capacity (B max ) was 14.1 fmol/100

μ g protein and the disso ciation constant (K d ) was 0.83 nM. The K d and B max values were within the range reported in the EPA validation progr am. Confidence in these numbers is high according to the goodness of fit (R 2 = 0.985); however, only one run was performed. The specific binding reached a plateau, and non-specific binding (NSB) was ≤ 8.8% of total binding The Scatchard plot for the saturation assay was not linear for the entire [ 3 H]-R1881 concentration range tested; however, it was lin ear over the range from 0.5 to 10 nM, which encompasses the ligand concentration used in the competition binding experiment.

In the competitive binding experiment, the estimated mean log IC 50 s for R1881 (

− 8.8 M) and the weak positive control, dexamethasone (

− 4.2 M), were within the expe cted ranges. The mean relative binding affinity (RBA) for the weak positive control was 0.0026%. The solvent control responses indicated no drift in the study assay. All performance criteria were generally met.

In each run, there were no a pparent reduction in average [ 3 H]-R1881 binding at metolachlor
concentrations from 10

− 10 to 10

− 4 M (87.7-106.8%), but average bi nding of the radiolabeled ligand declined to 48.96-50.99% at 10

− 3 M. The average binding was approximately 50% in Runs 2 and 3 (50.94 and 50.99%), the average binding curve for metolachlor across all three runs indicates that ~50% displacement (com petitive binding to the AR) occurred at 10

− 3 M. The average log IC 50 and RBA for metolachlor were

− 2.994 M and 0.00016%, respectively.

Based on the results from the three runs, metolach lor is classified as a Binder in the Androgen Receptor Binding Assay.

Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP19) assay (MRID 48617805) , metolachlor (97.3% a.i., Lot # 20081115) was incubated with human recombinant ar omatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in 1% ethanol at log concentrations from 10

− 10 to
10

− 3 M for 15 minutes to assess the potential of metolachlor to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Th ree runs were conducted and each run included a full activity control, a bac kground activity control, a pos itive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4-hydroxyandrostenedione ; 4-OH ASDN), and the test chemical series (10

− 10 to 10

− 3 M) with three repetitions per concentration.METOLACHLOR FINAL

Page 33 of 61 Aromatase activity in the fu ll activity controls ranged from 0.2747 to 0.4910 nmol·mgprotein

− 1 ·min

− 1 for the 3 test runs, with a mean and standard deviation of 0.3924±0.0224 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background controls ranged from 0.23 to 1.17% and averaged 0.49% of the fu ll activity controls. Th e responses of the full activity controls and background controls were acceptable for each run.

For the positive control substance (4-OH ASDN) aromatase activity was within the recommended ranges for the top and bottom of the curve, Hill slope, log IC 50 and %CV for replicates of each concentration within runs. The estimated log IC 50 averaged

− 7.3 M and the Hill slope averaged

− 0.93.

For metolachlor, aromatase activity averaged 87.86±6.2% of the full ac tivity control at the
lowest tested concentration of 10

− 10 M and 41.63±3.0% of the full activ ity control at the highest
tested concentration of 10

− 3 M. Aromatase activity at the se cond highest tested concentration (10

− 4 M) averaged 84.53±2.9% of the full activity cont rol. For Runs 1 and 3, the report indicated that the log IC 50 values were "ambiguous" but values of

− 3.79 and

− 3.81 M, respectively, as well as Hill slope values of –8.05 and –8.31, re spectively, were reported. A log IC 50 value and Hill slope were not determined for Run 2 as the concentration response curve did not fit the 4- parameter model.

Although >50% inhibition was observed at the highest test concentration (10

− 3 M) in all three runs, the Hill slope (-8.4) for the average res ponse curve was steeper than -2. The unusually steep slope may indicate an issue with protein denaturation or solubility at this test concentration. Therefore, metolachlor is classified as Equivoc al for aromatase inhibition in this assay. Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding a ssay (MRID 48617807) for metolachlor (97.3% purity, Lot # 20081115), uterine cytosol from previously ovariectomized Sprague Dawley rats was used
as the source of ER to conduct saturation bindi ng and competitive binding experiments. The saturation binding experiment was conducted to de monstrate that the ER in the rat uterine cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the radiolabeled reference estrogen prior to conducting the competit ive 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 increasi ng concentrations (logarithmic increase from 10

− 10 to
10

− 3 M) of metolachlor. Ethanol was used as the solvent vehicle at a final concentration of 2%. A total of 5 runs were performed, and each run included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17- β -estradiol as the natural ligand
reference material.

In the saturation binding experiments, the mean maximum binding capacity (B max ) was 31.7 fmol/100 μg protein and the m ean dissociation constant (K d ) was 0.075 nM. The B max value wasMETOLACHLOR FINAL

Page 34 of 61 within the recommended range and demonstrated that the estrogen receptor was present in a reasonable concentration. The Scatchard plots fo r the three Saturation assays were linear. The specific binding appeared to reach a plateau, an d NSB was generally less than 20% of total binding (1.97 to 21.51%).

For the competitive binding experiment, the estimated mean log IC 50 was

− 8.675 M for 17 β

estradiol and

− 5.254 M for the weak positive control. The mean RBA was 0.045% for the weak positive control. Confidence in these numbers is high due to the small variation. All performance criteria were generally met; however , parameters for 19-noret hindrone were outside of the tolerance limits for a weak positive contro l in Run 5, and a technical error occurred with the negative control, octy ltriethoxysilane, in Run 2.

Metolachlor had no significant e ffect on specific binding of the [ 3 H]-ligand to the ER in runs 2-5; therefore, a log IC 50 and RBA could not be calculated fo r metolachlor (speci fic binding of [ 3 H]-

17 β -estradiol was ≥ 72.28% in Run 1).

Based on the results of the five runs, metolachlor is classified as Not Interactive in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48617808), hER α -HeLa-9903
cells cultured in vitro were exposed to metolachlo r (97.3% a.i., Lot #20081115) at logarithmically increasing concentrations from 10

− 11 to 10

− 4 M in DMSO (0.1%) for approximately 24 hours in two independent runs . The experiments were performed using 96- well plates and each metolachlor concentration was tested in 6 wells/plate in each run. Cells were exposed to the test agent for 24±2 hours at 37±2°C to induce report er (luciferase) gene products. Luciferase expression in response to ac tivation of the estrogen receptor was measured upon using a proprietary luciferase assay

Metolachlor was tested up to a cytotoxic le vel, which was observed at concentrations ≥ 10

− 4 M.
The mean RPC Max was 3.3% for the first run and 0.6% for the second, and the associated PC Max was 10

− 7 M for both.

In the main assays, the responsiveness of th e cells to the very weak positive control 17 α

methyltestosterone was lower than the expected values, indicating a decrea sed sensitivity of the assay to very weak agonists. Although the conditi ons of this assay were not optimal to detect very weak activity, metolachlor responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to
reach a maximum of 36.5-46.3% PC. Metolachlo r was only able to reach a maximum of 0.6- 3.3% PC when tested up to the highest concentr ation possible based on cytotoxicity. BecauseMETOLACHLOR FINAL

Page 35 of 61 the RPC Max < PC 10 in both assay runs, metolachlor was considered negative for estrogen receptor transcriptional activation in this test system.

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

The 21-day short-term reproduction assay (M RID 48617811) of metolachlor with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions. Adult fish (16 spawning groups, 2 males and 4 females in each group; 5.5 months old) were exposed to metolachlor (97.3% purity) at nominal concentr ations of 0 (negative control), 0.050, 0.50, and 5.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.0300, negative control), 0.046, 0.48, and 4.7 mg a.i./L. The test syst em was maintained at 24.1 to 25.2 o C and a pH of 7.0 to 8.2.

Overall survival was significantly reduced (p<0.05) by 66.7% for males, 73.3% for females, and 70.8% for the combined sexes at the high treatme nt level compared to the negative control. Additionally, male and female fish in the high tr eatment level showed signs of erratic swimming, loss of equilibrium, lethargic swimming, and cu rved spine; excess feed was observed in high treatment tanks. There were no significant differe nces (p>0.05) for male or female growth parameters (weight and length); however, ther e was a non-statistically significant decrease (p>0.05) of 23% in male body weight at the high treatment compared to the negative control.

Mean spawning frequency and fecundity in the ne gative control were at least every 4 days and 28 eggs/female/day (range 20.2-33.9), respectively; fertilization success in the negative control was 97.9%. There was a significant re duction (p<0.05) in fecundity of 98% at the high treatment level relative to the negative control; only one replicate in the high tr eatment level produced eggs, and there was 100% fertiliza tion success in that replicate.

Female vitellogenin (VTG) levels were sign ificantly reduced (p<0.05) by 77% at the high treatment level relative to the negative control. Male VTG levels were not significantly different (p>0.05) from the negative control. There we re no significant differe nces in female gonadosomatic index (GSI) compared to the negative control, but there was a marginally significant increase (p=0.057; Mann-Whitney) of 29% in male GSI at the high treatment level compared to the negative control. There was a significant re duction (p<0.05, Mann-Whitn ey) in male tubercle score at the high treatment level (median tubercle score: 5) compared to the negative control (median tubercle score: 22). No tubercles were observed in female fish. The study authors also reported a statistically-significant decrease (p<0. 05; Jonckheere-Terpstra trend test) in mean male fatpad scores in the mid and high treatm ent groups compared to the negative control. Sex
steroids were not measured.

Gonadal histopathologic changes in females in cluded increased oocyte atresia related to increases in granulosa cell hypertrophy and hyperplasia a nd increased granulosa cell phagocytosis as well as decreased early and late vitellogenic oocyt es and ovary size at the high
treatment level. In males of th e high treatment group, germinal epithelium showed an increasedMETOLACHLOR FINAL

Page 36 of 61 presence of diploid spermatogonia with a decreased presence of spermatocysts containing spermatocytes and spermatids. One in six males in the high treatment group showed moderate
alterations in these ratios and could not be st aged under normal criteria. These treatment-related changes appeared to result from an inability of spermatogonia in th e testes of affected fish to enter into spermatogenesis.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger Assay (MRID 48617802) screen ing for androgenic activity, metolachlor (97.3% a.i., Lot #20081115) in corn oil was administer ed daily via subcutaneous (s.c.) injection to groups of six 54- to 56-day old, castrated ma le Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 500 mg/kg/day. An andr ogenic positive control group consisted of six castrated rats exposed to 0.2 mg/kg/day of test osterone propionate (T P) by s.c. injection.

To screen for potential anti-a ndrogenic activity, metolachlor ( 97.3% a.i.) in corn oil was administered daily via s.c. injection to groups of six 54- to 56-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 500 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.2 mg/kg/day by s.c. injection. The anti-androgenic positive control group consisted of six cas trated rats exposed to 0.2 mg/ kg/day TP (s.c. injection) and 3 mg/kg/day flutamide (FT, via or al gavage). TP alone was used as the anti-androgenic negative
control.

For both components of the assay, body weights were determined da ily. The animals were dosed for 10 consecutive days and terminated a pproximately 24 hours after the final dose administration. At necropsy, the five androgen- dependent tissues, adrenal glands, kidney, and liver were collected and weighed. One animal in the 100 mg/kg/day group in the androgen agonist assay was inadvertently given FT on the la st day of dose administration; therefore, this animal was removed from the study.

One animal in the 300 mg/kg/day group in th e anti-androgen assay was found dead on Day 10, within one hour of dosing; no macroscopic findi ngs were noted at necropsy. All other rats
survived until scheduled sacrifice. No animal s exhibited any dose-rela ted clinical signs of toxicity and there were no treatmen t-related gross pat hological findings.

In the androgen agonist assay, overall body weight gains were decreased compared to controls by 18% (not significant [NS]) and 45% (p <0.01) in the 300 and 500 mg/kg/day groups, respectively. Body weights were also decr eased on Days 6-11 in the 500 mg/kg/day group; however, the decreases were not statistically signif icant. Body weights in the 100 and 300 mg/kg/day dose groups and the positive control group were comparable to controls throughout the duration of the assay. Th ere were no treatment related e ffects in accessory sex organs,METOLACHLOR FINAL

Page 37 of 61 adrenal glands, kidneys, or liver for any meto lachlor treatment group. Animals in the positive control group had increased (p<0.01) accessory se x organ weights as follows: 327% in seminal vesicles; 381% in ventral prostate; 132% in levator ani-bulbocavernosus (LABC); 322% in Cowper’s gland; and 53% in glans penis. Ad renal glands, kidney, and liver weights in the positive control group were comparable to the vehicle control.

Percent CVs for the vehicle control and the 500 mg /kg/day treatment groups were compared to the performance criteria in the Guidelines. All %CV values were less than the maximum permissible values, with the ex ception of the %CV for ventral prostate for the 500 mg/kg/day group (CV = 48.7%; maximum permissible CV = 45%).

In the anti-androgen assay, overa ll body weight gains were d ecreased by 16% (NS) and 33% (p<0.01) in the 300 and 500 mg/kg/day groups, compar ed to controls. Body weights were also decreased on Days 6-11 in the 500 mg/kg/da y group; however, the decreases were not statistically significant. B ody weights in the 100 and 300 mg/kg/day dose groups and the positive control group were comparable to controls throughout the duration of the assay. At 500 mg/kg/day, the weights for ventral prostate and LABC were decreased (p<0.05) by 25% and 22%, respectively. LABC weights were also de creased (p<0.05) in the 300 mg/kg/day group ( ↓ 17%). There were no treatment related effects in seminal vesicles, Cowper’s glands, glans penis, adrenal glands, kidneys, or liver for any metolachlor group. Animals dosed with TP + FT (positive control) had decreas ed (p<0.01) accessory sex organ weights as follows: 74% in seminal vesicles; 74% in ventra l prostate; 58% in LABC; 75% in Cowper’s gland; and 33% in glans penis. Adrenal glands, kidney, and liv er weights in the positive control group were comparable to the vehicle control. The perf ormance criteria indicated that this assay was performing as expected.

The high dose of metolach lor selected for this study (500 mg /kg/day) is appropriate because it induced anti-androgenic effects but did not cause significant toxicit y. In addition, reductions in body weight grains of 16-18% (NS) at 300 mg /kg/day and 33-45% (p<0.01) at 500 mg/kg/day indicate that the doses selected were adequate.

Metolachlor was negative for androgenic activity in the Hershberger assay as no statistically significant increases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist assay. Metolachlor was positive for potential anti-androgenic activity in this Hershberger assay as statistically significant decreases in organ weights were seen in two or more of the androgen sensitive tissues in the androg en antagonist assay.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48617804), 15 Crl:CD(SD) Sprague-Dawley rats/dose group were treated daily via oral ga vage (5 mL/kg) with metolach lor technical (97.3% a.i., LotMETOLACHLOR FINAL

Page 38 of 61 #20081115) in corn oil at doses of 0, 300 or 600 mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening da ily beginning on PND 22, a nd weight at day of attainment was recorded. Following sacrifi ce on PND 42, total thyroxi ne (T4) and thyroid stimulating hormone (TSH) levels were an alyzed using an electrochemiluminescent immunoassay (T4) and a magnetic [ 125 I]rTSH gamma counter immunoassay (TSH). Kidneys, liver, adrenal glands, thyroid, pituitary, and ova ry weights were recorded, and microscopic examination of the thyroid, kidneys, ova ry and uterus were performed.

One animal in the control group was sacrificed in extremis on PND 25 due to impairment of the
left forelimb. All other rats survived until scheduled sacrifice. No treatment-related clinical findings were noted during the daily examinations at any dose level. There were no significant differences in body weight at va ginal opening or final body weights in the trea ted groups relative to controls. Adjusted age at VO was significantl y earlier at 300 mg/kg/day (p<0.05) but not at 600 mg/kg/day. One female in the control group failed to attain vaginal opening. There were no statistically significant differences in mean age at first vaginal estrous or mean cycle length. The cycle status at necropsy was si milar among groups. Serum TSH wa s not affected by treatment.

Unadjusted, adjusted and relative liver wei ghts were increased at 600 mg/kg/day (p<0.05, ↑ 20- 22%); only relative liver we ight was significantly increa sed at 300 mg/kg/day (p<0.05, ↑ 9%). Unadjusted, adjusted and relative kidney we ights were increased at 600 mg/kg/day (p<0.05, ↑ 8- 10%). No other effects on organ weights were reported. The percent of females cycling (93.3%) and regularly cycling (77.8%) were lower at 600 mg/kg/day (p<0.05) than the controls (100% for both). Percent regularly cyc ling was also reduced at 300 mg /kg/day (p<0.05, 53.8%). There were dose-related increases in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, respectively; the increa se was significant (p<0.05) at 600 mg/kg/day only. Mean globulin levels were significantly higher in the 300 mg/kg/day (p<0.05, ↑ 7%) and 600 mg/kg/day (p<0.05, ↑ 7%) groups; there were corre sponding reductions in A/ G ratios of 8% and 12%, respectively, with only the high dos e being significant (p <0.05). GGT was significantly elevated at 600 mg/kg/day (p<0.05, ↑ 700%).

There was an increased incidence of Grade 2 follicular cell height at 600 (11/14) mg/kg/day relative to control (4/14); also, one animal at the high dose was observed with Grade 3. There
was an increased incidence of Grade 4 colloid ar ea at 600 mg/kg/day (2/14, 4/15 and 11/14 in the control, 300 and 600 mg/kg/day respectively). Th e differences in follicular cell height and colloid area were significant at 600 mg/kg/day (p=0.005). Th e ovaries appeared normal histopathologically. There were no microscopic findings in the uter us related to treatment with the test material. Renal tubular cysts were in creased in the 300 and 600 mg/kg/day groups (1/14, 2/15 and 4/15 in the 300 and 600 mg/ kg/day groups, respectively).METOLACHLOR FINAL

Page 39 of 61 No effects were observed on body weights or body weight gain in the study. Increased liver weights accompanied by elevated GGT levels an d increased kidney weights demonstrate organ toxicity as a result of trea tment. However, histopathology was not conducted on the liver and microscopic findings in the kidney were minimal, so the degree of toxicity in these organs is unclear. Age at vaginal openi ng was not affected by treatment. There was a dose-related
increase in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, respectively; the increase was significant (p<0.05) at 600 mg/kg/day only. Serum TSH was unaffected by treatment. The dosing levels tested in this assay are adequate based on systemic toxicity (increased liver and kidney weights) at the high dose.

Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48617803), 15 Crl:CD(SD) Sprague-Dawley rats/dose group were treated daily via oral gavage with me tolachlor technical (97.3% a.i., Lot #20081115) in
corn oil at doses of 0, 400 or 800 mg/kg/day from pos t-natal day (PND) 23 to 53. Animals were examined for preputial separation (PPS) daily beginning on PND 30 and weight at day of attainment was recorded. Following sacrifi ce on PND 53, blood was taken for total thyroxine (T 4 ), testosterone, thyroid stimulating hormone (T SH) and clinical chemistry analysis. Although the hormones were reportedly analyzed by ra dioimmunoassay (RIA) or chemiluminescence, the specific details of the methods were not provided. W eights were recorded for kidneys, liver, adrenal glands, thyroid, pituitary, epididymides, ve ntral prostate, dorsal prostate, seminal vesicle, levator ani plus bulbocavernosus (LABC) muscles and testes, and microscopic examinations were performed on the thyroid, kidn eys, testis and epididymides.

One male in the 800 mg/kg/day group (Ani mal No. 79812-07) was found dead on PND 40; no significant clinical or macroscopic findings were observed in this animal. One male in the control group (Animal N o. 79823-03) was sacrificed in extremis on PND 47 following clinical findings of a swollen right forelimb resulting in imp aired use; gross examination of this animal at necropsy revealed a punctured esoph agus probably due to a dosing error. All other rats survived until scheduled sacrifice. Clinical findings included salivation prior to dosing and clear material around the mouth approximately 1 hour post-dosing in the 400 and 800 mg/kg/day dose groups. At 800 mg/kg/day, clear material was also observed around the ventral neck.

No treatment-related changes in body weights, body weight gains, or ag e and weight at PPS
occurred in either treatment group. Liver weights were in creased (p<0.05) by 15-20% and kidney weights were increased (p<0.05) by 11% each at 400 and 800 mg/kg/day, respectively. A significant trend for increased thyroid weights was observed, although pairwise comparisons were not significant; the increasing trend in th yroid weights did not correlate with higher TSH levels or microscopic findings. Although a sign ificant trend for decreas ing right epididymis weights was observed, it was not si gnificant by pairwise comparis on and was not observed in the left epididymis.METOLACHLOR FINAL

Page 40 of 61 TSH levels were increased (p<0.05) by 54 a nd 134% at 400 and 800 mg/kg/day, respectively. There was no corresponding effect on T 4 levels, and the increas ed TSH levels did not consistently correlate with thyr oid gland weight or histopathol ogical findings on an individual
animal basis.

Mean testosterone levels were d ecreased in a dose-related manner ( ↓ 45%, NS and ↓ 69%, p<0.05) in the 400 and 800 mg/kg/day group s, respectively. The control group had three animals with unusually high testosterone levels (>8.00 ng/mL), and the control mean was above the performance criteria maximum (3.96 ng/mL) when th ese animals are included in the analysis. When these animals were excluded from the analysis, the mean value for the control group was 2.27 ng/mL, which is similar to the values fo r the treated groups (2.29 and 1.35 ng/mL) although a statistical analysis was not c onducted using the adjusted mean.

At the scheduled euthanasia, no treatment-related findings were noted. A s light treatment-related effect on thyroid morphology is considered possi ble based the larger number of animals with follicular height Grade 1 and colloid area Grade 5 in the control group relative to the 400 and 800 mg/kg/day animals; this is consistent with a higher number of treated animals with follicular height and colloid area scores of 3. These findings are consistent with higher mean thyroid weights and elevated TSH levels in the treated animals; how ever, these endpoints were not
correlated when evaluated on an individual anim al basis. Additionally, increased follicular height was not necessarily correlated with decreas ed colloid area in individual animals. There
were no treatment-related findings in the testes, epididymides or kidneys.

The high dose tested was considered to be adeq uate based on the results of a study in which a metolachlor dose of 1000 mg/kg/day (route of ad ministration not reporte d) resulted in overt toxicity (all of the males found d ead, euthanized in extremis or terminated early). In addition, organ toxicity was observed in the current study with both liver and kidney weights significantly increased at 400 and 800 mg/kg/day.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48617809), H295R cells cultured in vitro in 24-well plates
were incubated with metolachlor

, (97.3% a.i., Batch # 20081115) at log concentrations from 0.0001 to 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle,
at a final concentration was 0.05%.

Testosterone and estradiol concentrations we re measured by HPLC/MS-MS with positive ion multiple reaction monitoring. Three independent experiments were performed. A Quality Control (QC) plate was run concur rently with each independent r un of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentrationMETOLACHLOR FINAL

Page 41 of 61 levels; positive controls include d the known inhibitor (p rochloraz) and indu cer (forskolin) of
estradiol and testos terone production. The highest suitable concentration of metolachlor in all three runs was 100 μM.

Guideline recommendations were met, includi ng adequate production of testosterone and estradiol, acceptable reproducibili ty (low %CV), and appropriate induction and inhibition with positive controls. Cytotoxicity greater than 20% was not observed.

Statistically significant changes in testosterone production after incubation with metolachlor were not observed, although the mean fold -difference was decreased 17% for 100 μM metolachlor. Statistically significant increases in estradiol production we re observed in two of the three runs at 10 μM, and in all three runs at the 100 μM concentration. The mean folddifferences were approximatel y 1.2- and 1.3-fold, respectively, and showed a concentrationdependent response.

Based on the hormone responses in each of the three independent runs, metolachlor treatment resulted in statistically signifi cant and reproducible in creases in estradiol production, but did not result in a statistically signi ficant and reproducible alteration s in testosterone production.

Uterotrophic Assay; (OCSPP 890.1600)

In an uterotrophic assay (MRID 48617801) conducted to screen for potential estrogenic activity, metolachlor (97.3% a.i., lot # 20081115) in corn oil was administered daily via subcutaneous (s.c.) injection to groups of six ovariectomized Sprague Dawl ey rats at dose levels of 0 (vehicle), 100, 300, or 1000 mg/kg/day on post- natal days (PND) 57-59. A positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) at 0.5 μ g/kg/day by s.c. injection. Body weights were determined daily. All animals were terminated and necropsied on PND 60 approximately 24 hours after the final dose administration to determine wet and blotted uterine weights.

All animals survived until scheduled termination. No test substance-related clinical findings were noted in any of the animals during the study. Body weights were comparable to the controls throughout the duration of the assay, but overall (Days 1-4) body weight gains were decreased (p<0.01) in the 300 mg/kg/day ( ↓ 42%) and 1000 mg/kg/day ( ↓ 53%) metolachlor groups compared to the controls. Body weight ga ins in the positive control (EE) group were also decreased (p<0.01) by 57% compared to the controls.

Uterine weights in the metolachlor treated groups were comparable to the controls. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p<0.05) as expected by 288% and 175%, respectively.METOLACHLOR FINAL

Page 42 of 61 Based on the observed decreases (42% and 53%) in body weight gain at 300 and 1000 mg/kg/day, adequate dose levels were achieved in this study.

No statistically significant changes were seen in uterine weight in this assay. Metolachlor is negative in the uterotrophic assay.METOLACHLOR FINAL

Page 43 of 61 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

ER/AR Transcript ional Activation

In a published, non-Guideline study ( Kojima et al ., 2004) , the potential of 200 chemicals (including metolachlor; purity 95-100%) to interact with two human ER subtypes (hER α and
hER β ) and one human androgen receptor (hAR) in tr ansactivation assays with Chinese hamster ovary cells (CHO-K1 cells) was inve stigated. For detection of hER α or hER β activity, cells were transfected with pcDNAER α or pcDNAER β , pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZ eoSV2AR, pIND-ARE, an d pRL-SV40. Flutolanil was tested over a concentration range from 10

− 8 to 10

− 5 M, and the responses were compared to the activity of 17 β -estradiol (E 2 ; purity >97%) or 5 α -dihydrotestosterone (DHT; purity 95%) for the ER or AR assays, respectively. For meas urement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 or 10

− 10 M DHT was added to the cell cultures along with the test compound, respectively. A gonistic activities were eval uated by the relative activity expressed as 20% relative effective concentration (REC 20 ), the concentration of the compound demonstrating 20% of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activities were evaluated similarly by the relative activity expressed as 20% relative i nhibitory concentration (RIC 20 ), the concentrat ion of the compound showing 20% inhibition of the activity of E 2 or DHT against ER α , ER β , and AR, respectively. Metolachlor did not show agonist or antagonist activity for ER α , ER β , or AR in this study.

90-Day (Subchronic) Oral Toxicity in Rodents (Rat; OSCPP 870.3100)

In a subchronic oral toxicity study in rats ( MRID 43928923 ), S-metolachlor (89.6% purity; 93.7% S-isomer) was administered to Crl: COBS (SD) BR rats (15/sex/group, except for females in the low-, medium-low, and medium-high dose groups; n=10/group) in the diet at nominal concentrations of 0 (acetone-t reated diet), 30, 300, 3000, or 10,000 ppm for 13 weeks. Estimated compound intakes (by standard conversion) were 0, 1.5, 15, 150, and 500 mg/kg/day. Five rats/sex in the control and high-dose groups we re permitted a 28-day reco very period after the 13-week treatment period. Organs that were wei ghed included the liver, kidneys, testes, ovaries,
and adrenals. Gross and microsc opic pathological examinations were conducted on these tissues
as well.

No deaths occurred during the study, and no tr eatment-related effects on appearance, behavior, or physical examinations were noted in any treat ment group. Treatment-related systemic toxicity was observed in the medium-high and high-dos e groups as decreases in body weight, body weight gain, and food consumption and efficien cy. Body weights were decreased (p<0.05 and 0.01) in medium-high ( ↓ 8-10% from Week 2) and high-dose males ( ↓ 11-13% from Week 1), respectively, and were decreased (p<0.05 and 0.01) in medium-high ( ↓ 8-10% from Week 8) and high-dose females ( ↓ 5-17% from Week 1), respectively. B ody weight gains were decreased by 15.7 and 24.3% in males and 18.8 and 40.8% in females in the medium-high and high-doseMETOLACHLOR FINAL

Page 44 of 61 groups, respectively. No apparent treatment-re lated effects were noted in ophthalmological examinations, or in hematology, clinical chemistr y, or urinalysis parameters. Decreased (p<0.05 and 0.01) absolute adrenal weights were noted in high-dose animals (( ↓ 17 and 22%), and kidney weights were increased (p <0.01) in the medium-high and high-dose males ( ↑ 18%), with increases in relative kidney weights in males and females. No gross pathology findings were noted, and the only histopathol ogical finding was an increased incidence of eosinophilic intracytoplasmic inclusion bodies in hi gh-dose males (6/10 vs. 0/10 control).

The systemic toxicity NOAEL is 300 ppm ( 15 mg/kg/day) and the LOAEL is 3000 ppm (150 mg/kg/day) based on decreased body weight, body weight ga in, and food consumption and efficiency in both sexes and incr eased kidney weights in males.

In another subchronic oral toxicity study in rats ( MRIDs 44775401 and 44775402 ), metolachlor (97.7% purity) was administered to Crl:CD (SD) BR rats (20/sex for c ontrol, 10/sex/treated group) in the diet at nominal concentr ations of 0, 30, 300, or 3000 ppm (mean-measured concentrations of 0, 2.00, 20.2, and 210 mg /kg/day for males and 0, 2.32, 23.4, and 259 mg/kg/day for females). Organs that were we ighed included the liver, kidneys, testes, epididymides, ovaries, and adrenals. Gross and microscopic pathological examinations were conducted on these tissues, as well as the prostate, seminal vesicle, uterus, vagina, pituitary, and thyroids.

No treatment-related deaths or clinical signs occurred during th e study (two deaths occurred unrelated to treatment). In addition, there we re no treatment-related effects on ophthalmologic parameters, water consumption, urinalysis, food efficiency, or gross/microscopic pathology in
either males or females. There was no evidence of any treatment-related effect in male rats. Decreased (p<0.05 or 0.01) body weights were observed in the high-dose females ( ↓ 8-11%); decreases (p<0.05) in th e medium-dose group were not consid ered toxicologically relevant ( ↓ 7- 9%). Decreased (p<0.01, ↓ 22%) overall body weight gain was observed in the high-dose females, but decreased (NS) body weight gains in the low- and medium-dose groups displayed no dose response ( ↓ 13 and 12%, respectively). Decrease d (p<0.05 or 0.01) food consumption was reported in all female dose groups during Week 1 ( ↓ 12-14%), and in the low- and mediumdose groups throughout the study (p<0.05, ↓ 10-13%). Overall mean food consumption was decreased (p<0.01) similarly in the lo w-, medium-, and high-dose females ( ↓ 11, 11, and 12%). The toxicological effect of treatment on food consumption is questionable as there was no doseresponsive effect and food e fficiency was not affected.

Changes in absolute and relative organ weights were limited to decreased liver weight in the low- and medium-dose females ( ↓ 11 and 12%, respectively), and in creased relative liver (highdose females) and kidney weights (all treated fe males). These effects were not considered toxicologically relevant due to the small magn itude and the lack of accompanying histopathology changes. The LOAEL for female rats is 259 mg/kg/day based on decreased body weight andMETOLACHLOR FINAL

Page 45 of 61 body weight gain; the NOAEL for females is 23.4 mg/ kg/day). The LOAEL for male rats is not established ( ≥ 210 mg/kg/day), and the NOAEL for >210 mg/kg/day.

3-Month (Subchronic) Oral Toxicity in Non-Rodents (Dog; OCSPP 870.3150)

In a subchronic oral toxicity study in dogs ( MRID 43928922 ), S-metolachlor (95.4% S-isomer) was administered to Beagle dogs (4/sex/group) in the diet at nominal concentrations of 0 (acetone-treated diet), 300, 500, 1000, or 2000 ppm for 13 weeks. Due to palatability issues at the highest dose, high-dose dogs were administer ed the test compound by capsule (700 mg/day) starting at Week 3, and the study was extended fo r three weeks to achieve capsule dosing for 14 weeks (high dose) and test diet exposure for 16 weeks (lower doses). Mean-measured compound intakes were 0, 9.0, 15.1, 31.1, and 62 mg/kg/ day for males and 0, 10, 17.2, 31.5, and 74 mg/kg/day for females. Organs that were weighed included the liver, kidneys, testes, ovaries, thyroid, and adrenals. Gross and microscopi c pathological examinations were conducted on these tissues, as well as the pituitary, mammary gland, epidid ymides, prostate, vagina, and

uterus.

No animals died during the study, and clinical si gns were limited to incidences of few or no stools in the medium-high and high-dose dogs. No significant treatment -related effects were noted on body weight, although body wei ght gains were decreased (NS;

− 0.48 and

− 0.43 kg, respectively) in the medium-high and high- dose males during Week 1, and were negative (p<0.01;

− 0.98 kg) in the high-dose females during W eek 1. Food consumption was decreased in the medium-high dose males and high-dose males and females during Weeks 1 and 2, but recovered during Weeks 13-16. No treatment-rela ted effects were noted for ophthalmological examinations, in hematology, clinical chemistry, or urinalysis parameters, or for organ weights or gross and microscopic pathology.

The systemic toxicity NOAEL is ≥ 2000 ppm (62 and 74 mg/kg/day in males and females, respectively) and the LOAEL is >2000 ppm based on a lack of biologically-relevant findings at doses up to 2000 ppm (700 mg/day) excep t for the palatability issue.

6-Month (Subchronic) Oral Toxicity in Non-Rodents (Dog; OCSPP 870.3150)

In a subchronic oral toxicity study in dogs ( MRID 00032174 ), metolachlor (purity not reported?) was administered in the diet to Beagle dogs (8/sex/group for cont rol and high dose groups; 6/sex/group for low- and mid-dose groups) at nominal concentrations of 0, 100, 300, or 1000 ppm (mean-measured doses of 0, 2.92, 9.71 and 29.61 mg/kg/day, respectively, in males, and 0, 2.97, 8.77, and 29.42 mg/kg/day, respectively, in female s) for six months. Organs that were weighed included the liver, kidneys , testes, ovaries, pituitary, thyr oid, and adrenals. Gross and microscopic pathological examinations were co nducted on these tissues, as well as the mammary gland, prostate, and uterus.METOLACHLOR FINAL

Page 46 of 61 There were no deaths or clinical signs of toxicity. Mean body weight gain was decreased (NS) during Weeks 0-13 and 0-26 in the high-dose males ( ↓ 55-63%) and females ( ↓ 44-50%), and mean Week 26 body weights were decreased in the medium- and high-dose males by 11 and 14%, respectively. Mean overall food consumption was not affected in the high-dose males, but was slightly decreased ( ↓ 9%) in the high-dose females. Findi ngs of decreased activated partial thromboplastin time and increased alkaline phosp hatase concentrations were not considered toxicologically significant and we re not related to the endocrine system. No treatment-related effects were noted in the opthalmological examin ations and urinalysis investigations. No treatment-related effects were noted in organ weights, gross pathology, or histopathological examinations. The LOAEL is 1000 ppm ( 29.61/29.42 mg/kg/day in males and females, respectively) based on decreased body wei ght gain. The NOAEL is 300 ppm (9.7l/8.77 mg/kg/day in males and females, respectively).

Subchronic Dermal Toxicity in Non-Rodents (Rabbit; OCSPP 870.3200)

In a 21-day dermal toxicity study ( MRID 41833101 ), metolachlor (96.4% purity) was applied to
the shaved skin of New Zealand White rabbits (5/sex/dose group) at nom inal doses of 0, 10, 100, or 1000 mg/kg/day, 6 hours/day, over a 21-day peri od. Organs that were weighed included the liver, kidneys, adrenals, pituitary, ovaries, testes, prostate, epidi dymides, and uterus. Gross and microscopic pathological examinations were conducted on the liver and kidneys.

All animals survived the treatment. There were no treatment-related e ffects on clinical signs, body weight/body weight gain, food consumpti on, ophthalmoscopic examinations, hematology, or necropsy examinations. Significant increases in total bilirubi n were observed in the mediumand high-dose females ( ↑ 68 and 72%, respectively), but thes e increases were not considered toxicologically significant. Absolute liver weight was increased (p<0.05, ↑ 64%) in the high-dose males, but there no accompanying laboratory or necropsy findings. There was evidence of skin
irritation in all treated groups. Very slight erythema and dry skin were observed in all treated animals; one female in the low-dose group had fissuring. Fissuring increased in the mediumdose group (n=2/sex) and one male had moderate er ythema. Fissuring and wr inkling of the skin were noted in all high-dose animals. Histopath ological examination showed hyperkeratosis in all animals (except low-dose females, n=3), with do se-dependent parakeratosis, congestion of the dermis, edema, and subacute lymphocytic infilt ration reported in some or all of the treated animals. The systemic LOAEL is not estab lished, and the NOAEL is 1000 mg/kg/day (HDT). The dermal irritation LOAEL is 10 mg/kg/day (L DT) based on very slight erythema, dry skin, and fissuring (one animal). The NOAEL is not established.

Prenatal Development in Rodents (Rat; OCSPP 870.3700)

In a developmental toxicity study in rats ( MRID 43928925 ), S-metolachlor (95.6% S-isomer) was administered by gavage to mated female Tif: RAI f (SPF) rats (24/ group) at doses of 0 (0.5% carboxymethylcellulose), 5, 50, 500, or 1000 mg/kg/day on GDs 6 to 15, inclusive. TheMETOLACHLOR FINAL

Page 47 of 61 rats were sacrificed at GD 21 of gestation a nd subjected to gross necr opsy. All fetuses were weighed, sexed, and examined for gross external alterations. One-half of the fetuses in each
litter were examined for visceral soft tissue alterations by the Wilson technique. The remaining
fetuses in each litter were examined for skeletal alterations.

No maternal toxicity was observed in this st udy. All females in the medium-high and high-dose groups, and 9/24 females in the medium-low dos e group exhibited discomfort after daily dose administration ( e.g. , pushing head through bedding for appr oximately one hour). Decreased (p<0.01) body weights were observed in the high-dose dams on GDs 15 ( ↓ 7%) and 21 ( ↓ 8%), and decreases (p<0.01) in body wei ght gains of 19 and 28%, respec tively, were observed in the medium-high and high-dose dams during GDs 6-16. Body weight gains in the higher dose groups were not decreased during GDs 16-21, and ove rall body weight gains were affected to a lesser degree (p<0.05 and 0.01) with decreases of 12 and 19%, resp ectively. Treatment-related decreases in food consumption were also not ed during dose administration (GDs 6-11 and 11- 16); no differences were observed for GDs 16-21. There were no tr eatment-related findings at

necropsy.

Reproductive (cesarean section) pa rameters were generally not affected by treatment with metolachlor; there were no abortions or early deliv eries. A decrease in litter size was noted at the high dose (12.8 vs. 15.0 control; live fetuses/dam), but was not significant due to increased variability. No treatment-relate d effects were noted during exte rnal, visceral, or skeletal
examinations. The maternal LOAEL is 500 mg/kg/day, based on decreased body weight, body weight gain, and food consumption and efficien cy, and the maternal NOAEL is 50 mg/kg/day. The developmental LOAEL is not determined (>1000 mg/kg/day), and the developmental
NOAEL is ≥ 1000 mg/kg/day.

In an earlier developmental study in rats ( MRID 00151941 ), metolachlor (96.4% purity) in 0.5% aqueous carboxymethylcellulose was administered by gavage to Crl: COBS (SD) BR rats (25/group) at doses of 0, 30, 100, 300, or 1000 mg/kg/ day on GDs 6 to 15, inclusive. The rats were sacrificed at GD 20 of gestation and s ubjected to gross necropsy. All fetuses were weighed, sexed, and examined for gross external alterations. One-half of the fetuses in each
litter were examined for visceral soft tissue alterations by the Wilson technique. The remaining
fetuses in each litter were examined for skeletal alterations.

Four dams in the high-dose group died on study on GDs 7, 8, and 10 (n=2); clinical signs of toxicity were observed in these animals one or more days prior to death. Treatment-related increases (p<0.01) in clinical signs in the high-dose dams in cluded excess salivation (25/25), urine-stained abdominal fur (19/25), excess lacrima tion (8/25), and clonic/tonic (6/25) or clonic (5/25) convulsions. Excess salivation was al so observed in the medium-high dose animals (16/25). Because the increased salivation may re sult from gastric irritation, this finding is not considered toxicologically relevant. Body wei ght gain was decreased in the high-dose group forMETOLACHLOR FINAL

Page 48 of 61 the dosing period (p<0.05, ↓ 17%) as well as for the entire gestation period (p<0.01, ↓ 12%) and the dosing plus post-dosing periods combined (p<0.05, ↓ 12%). Treatment-related decreases in food consumption were also noted for these same periods (NS, ↓ 3-5%). Gross pathological findings at necropsy were not reported.

Reproductive (cesarean section) pa rameters were generally not affected by treatment with metolachlor; there were no abortions or early deliveries. Non-significant decreases in the number of implantations/dam (14.6 vs. 15.8 contro l) and litter size (13.8 vs. 15.2 control) were noted at the high dose. Mean fetal weight was also decreased (p<0.05, ↓ 4%) in the high-dose group. No treatment-related effects were noted during external, vis ceral, or skeletal
examinations. The maternal LOAEL is 1000 mg/kg/day, based on deaths and increased incidences of clinical signs, and the matern al NOAEL is 300 mg/kg/day. The developmental LOAEL also is 1000 mg/kg/day based on reductions in the number of implantations per dam and the resulting decreased litter size, as well as the decrease in mean fetal weight. The developmental NOAEL is 300 mg/kg/day.

Prenatal Development in No n-Rodents (Rabbit; OCSPP 870.3700)

In a developmental toxicity study in rabbits ( MRID 43928924 ), S-metolachlor (89.6% purity; 93.7% S-isomer) was administered to artifici ally-inseminated, female New Zealand White rabbits (19/dose group) by gavage at doses of 0 (3% corn starch with 0.5% Tween 80 suspension), 20, 100, or 500 mg/kg/day from GDs 7-19, inclusive. On GD 29, all surviving does were sacrificed, and all fetuses were weighed, sexed, and external examinations were conducted. Each fetus was weighed, examined for gross ex ternal observations, sacrificed, examined for visceral abnormalities, then eviscerated, stained with Alizarin Red S and examined for skeletal
abnormalities.

One high-dose rabbit died after anorexia and bod y weight loss, and was considered treatment
related. One death in the lo w-dose group was related to abor ting, and a medium-dose doe was sacrificed after breaking a hindlimb. Maternal to xicity, consisting of li ttle/none/soft stools was observed in the medium- and high-dose animals (14/ 19 and 19/19, respectively vs. 6/19 control). Decreased (p<0.05) body weight was obser ved in the high-dose animals on GD 19 ( ↓ 8%) with a slight decrease on GD 29 (NS; ↓ 3%). Body weight gain was de creased during the dosing period (GDs 7-19) at the high dose ( ↓ 121%), with a compensatory increase during GDs 19-29 ( ↑ 254%); the overall decrease in body weight gain was 29 % in the high-dose grou p. Decreases in food consumption and efficiency were also noted in the high-dose group, primarily during the treatment period. There were no trea tment-related findings at necropsy.

Reproductive (cesarean section) pa rameters were generally not affected by treatment with Smetolachlor. Two does aborted during the st udy (one each in the low- and medium-dose groups), but a relation to treatmen t was not established. No treatment-related effects were noted during external, visceral, or sk eletal examinations. The ma ternal LOAEL is 100 mg/kg/day,METOLACHLOR FINAL

Page 49 of 61 based on clinical signs, and the maternal NOA EL is 20 mg/kg/day. The developmental LOAEL is not determined (>500 mg/kg/day) , and the developmental NOAEL is ≥ 500 mg/kg/day.

In an earlier developmental toxicity study in rabbits ( MRID 00041283 ), metolachlor (95.4%purity) in 0.75% aqueous hydroxymethylcellulose was administered by gavage to assumedpregnant New Zealand White ra bbits (16/group) from GDs 6-18, inclusive, at doses of 0, 36, 120, or 360 mg/kg/day. The animals were sacrif iced on GD 30 and the fetuses were weighed, sexed, and examined for evidence of developmenta l effects. Each fetus was weighed, examined for gross external observations, sacrificed, examined for visceral abnor malities excluding the brain, then eviscerated, stained with Alizarin Re d S, and examined for skeletal abnormalities.

One doe in each the low- and high-dose groups died on GDs 24 and 29, respectively; the cause of death in both animals was attributed to pers istent anorexia. Two rabbits aborted, one in the medium-dose group (GD 25) and a second doe with persistent anorexia in the high-dose group (GD 17). One rabbit in each group (control and tr eatment) reportedly delivered prior to GD 30.
There was a treatment-rela ted increase in the incide nce of persistent anorexia in the does in the high-dose group; anorexia was defined as consump tion of less than one-h alf of the daily food allotment. Food consumption data were not pr ovided to support this finding. There was a treatment-related decrease in body weight gain in the high-dose group for the dosing period (p<0.0l, ↓ 500%) and the combined dosing and post-dosing periods (NS, ↓ 133%). There was no treatment-related increase in gross pathological findings in maternal an imals at necropsy. Reproductive (cesarean section) para meters were not affected by treatment with metolachlor, and no treatment-related external, visceral, or sk eletal developmental effects were observed.

The maternal toxicity LOAEL is 360 mg/kg/day based on an increased incidence of clinical observations (persistent anorexia) and decr eased body weight gain; the NOAEL is 120 mg/kg/day. The developmental toxicity LOAEL is not established (>360 mg/kg/day), and the
NOAEL is ≥ 360 mg/kg/day.

Two-Generation Reproduction in Rodents (Rat; OCSPP 370.3800)

In a two-generation reproduction study in rats ( MRID 00080897 ), metolachlor (95.4% purity)
was administered in the diet to two consecuti ve generations of CD albino rats (15 males/30 females) at nominal concentrations of 0, 30, 300, or 1000 ppm (mean-measured intake over 15- week premating period: 0, 2.4, 23.5, and 75.8 mg/kg/day in F 0 males and 0, 2.5, 26.0, and 85.7 mg/kg/day in F 0 females; mean-measured intake ove r 17-week premating period: 0, 2.3, 23.7, and 76.6 mg/kg/day in F 1 males and 0, 2.6, 25.7, and 84.5 mg/kg/day in F 1 females). Animals were observed daily of mortalit y, morbidity and overt signs of toxicity. Food consumption was monitored weekly in parental animals during th e pre-mating periods. Pa rental animals were weighed weekly during pre-mating, and males we re weighed monthly thereafter. Pregnant females were weighed on GD 0, 6, 15 and 20 a nd LD 0, 4, 7, 14 and 21. Pups were weighed individually on LD 4, 7, 14 and 21. Body weights of all animals were also determined atMETOLACHLOR FINAL

Page 50 of 61 sacrifice. Parent males were s acrificed at 157-167 days of age and female were sacrificed at 196-208 days of age. At sacrifice, the testes of all parental males (F 1 and F 0 ) were examined histopathologically for spermatogenesis Organ weights for adult animals were determined for the adrenals, brain, heart, kidneys, liver, testes , and thyroid. Histopat hological examinations were conducted on these organs a nd others, including the testes (with epididymis), prostate, ovaries, uteri, mammary gland, a nd pituitary and thyroid glands.

There were no deaths in the F 0 generation. Two females of the F 1 generation died during thepremating period (one each in the medium- and high-dose groups). One female in the mediumdose group was found dead on GD 19 and a control group female was sacrificed in a moribund condition on LD 1. Based on necropsy examina tions, none of the deaths were considered
treatment-related. There were no treatment-related c linical signs of toxicity in either generation. Body weight, body weight gain, and food c onsumption were unaffected in the F 0 generation. In
the F 1 generation, food consumption was decreased (p<0.05 or 0.01) in females of the high-dose group at Weeks 1, 8, and 15; however, there wa s no effect on body weight/body weight gain. There were no treatment-related effects on orga n weights in parental animals in either generation, including the adrenals, liver, kidneys, brain, testes (with epid idymides) and thyroid gland. There were also no treatment-related hist opathological finding in e ither generation in the ovaries, uterus, mammary glands, testes, prostate, epididymides, thyroid and pituitary glands.
There also was no evidence of a treatment-related effect on any of the reproductive parameters for either generation.

Offspring body weight was decreas ed (p<0.05 or 0.01) in the F 1 litters on LDs 14 and 21 ( ↓ 4- 9%) and in the F 2 litters on LDs 4, 7, 14, and 21 ( ↓ 5-8%). These offspring weight loss findings are considered toxicologically significant. The parental toxicity LOAEL is not established, and the NOAEL is 1000 ppm (75.8/85.7 mg/kg/day in F 0 males and females, respectively; 76.6/84.5 mg/kg/day in F 1 males and females, respectively). Th e reproductive toxicity LOAEL is not established, and the NOAEL is 1000 ppm (75.8/85.7 mg/kg/day in F 0 males and females, respectively; 76.6/84.5 mg/kg/day in F 1 males and females, respectively). The offspring LOAEL is established at 1000 pp m (75.8/85.7 mg/kg/day in F 0 males and females, respectively; 76.6/84.5 mg/kg/day in F 1 males and females, respectively) base d on decreased offspring body weights in the F1 and F2 litters. The NOAEL is 300 ppm (23.5/26.0 mg/kg/day in F 0 males and females, respectively; 23.7/25.7 mg/kg/day in F 1 males and females, respectively).

Chronic Oral Toxicity/Carcinogenici ty in Rodents (Rat; OCSPP 870.4300)

In a chronic oral toxicity/ carcinogenicity study in rats ( MRID 0012937748617809 ), metolachlor (95.3% purity) was administered to Crl:CD (SD) BR albino rats (70/sex/group for control and high-dose groups; 60/sex/group for low- and mid-dos e groups) at nominal concentrations of 0, 30, 300, or 3000 ppm (0, 1.5, 15, or 150 mg/kg/day based on standard conversion) for two years. The additional ten rats/sex/group in the cont rol and high-dose groups were kept on study forMETOLACHLOR FINAL

Page 51 of 61 twelve months; five rats/sex/group were sacrifi ced after twelve months of treatment, and the remaining five/sex/group were fed the negative control diet for four weeks and sacrificed. Organs that were weighed include d the liver, kidneys, heart, spl een, testes, and ovaries. Gross and microscopic pathological examinations were conducted on these tissues and others, including the prostate, uterus, mammary gla nd, pituitary, adrenals, and thyroid glands.

Comparable mortality rates were observed in th e treated and control an imals. There were no treatment-related clinical signs of toxicity, although si gns of sialodacryoadenitis viral infection were noted in approximately half of the study an imals (all groups and sexes). Mean body weight gains were decreased (NS) in the high-dose females ( ↓ 11-17%) from Week 26 to the end of the study, and mean food consumption was slightly decreased (NS, ↓ 4-9%) throughout the study in the high-dose females. Absolute and relative liver weights were increased ( ↑ 7 and 13%, respectively) in the high-dose males, and these increases persisted in the high-dose males after the four-week recovery period. However, the toxicological significa nce of the finding is questionable as there were no accompanying clinical pathology or histological changes. No
treatment-related effects were not ed on other organ weights in ma les, or for organ weights in females, including the testes and ovaries. In addition, no histopathological findings were noted in the kidneys, adrenal gland, ovaries, uterus, mamma ry glands, testes, prostate, or thyroid gland.

The LOAEL is 3000 ppm (estimated 150 mg/kg/ day) for females based on decreased body weight gain and food consumption. The NOAE L is 300 ppm (estimated 15 mg/kg/day) for
females. A LOAEL is not established for males, but the NOAEL is 3000 ppm (estimated 150 mg/kg/day).

In addition, the HED Cancer Peer Review Co mmittee evaluated the ca rcinogenic potential of
metolachlor on several occasions. Administratio n of metolachlor in the diet up to 3000 ppm (150 mg/kg/day) yielded increased incidences of liver adenomas and combined
adenomas/carcinomas in female rats. Males showed a statistical trend (but no pair-wise significance) for liver tumors. Metolachlor is classified as a Group C possible human carcinogen due to liver tumors in rats, w ith risk quantitated with a Ma rgin of Exposure approach.

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200)

In a carcinogenicity study in mice ( MRID 00117597 ), metolachlor (reported to be 95% purity) was administered in the diet to CD-1 mice ( 68/sex/group) at nominal concentrations of 0, 300, 1000, or 3000 ppm (0, 45, 150, or 450 mg/kg/day, base d on standard conversion). Based on the calculated mean intakes in treatment groups at Weeks 26, 52, 78, and 104, the overall meanmeasured intakes were 50, 170, and 526 mg/kg/ day, respectively, in males and 64, 224, and 704 mg/kg/day, respectively, in females. Eight mice/se x/group were sacrificed at 12 and 18 months. Information regarding the organs/tissues select ed for terminal weight s and histopathological examination were not provided.METOLACHLOR FINAL

Page 52 of 61 High-dose females had an increased (p<0.05) overal l mortality rate due to a number of deaths during the first few weeks of treatment (34/52 vs . 24/52 control, at termination). Although the early deaths were attributed to a viral infection, the potential c ontribution of the test material cannot be dismissed. Body weight was decreased (p<0.01 or 0.05) from Week 2 in the high-dose males ( ↓ 5-9%) and decreased (p<0.05) at approximately two-thirds of the time points from Week 32 in high-dose females ( ↓ 5-7%). Body weight gains were c onsistently decreased in the highdose males ( ↓ 12-51%) and females ( ↓ 14-41%). Food consumption was comparable between treated and control groups until Week 90 of treatment, at which time the consumption in the high-dose males was decreased by 10%; occasion al statistical significance was mentioned. There was no effect on food consumption in trea ted females. There were no treatment-related effects on hematology, clinical chemistry, or urin alysis parameters. Organ weights were not affected except for a dose-related decrease (signi ficance not reported) in the absolute and relative weights of the seminal vesicles of high-dose ma les. However, there was no effect on testes weight and no accompanying histologi cal changes were noted in the seminal vesicles; therefore, the toxicological significance of the finding is questionable. Th ere were no treatment-related microscopic changes, and no treatment-related in creases in tumor incide nce in the study.

The LOAEL is 3000 ppm (526/704 mg/kg/day in ma les and females, respectively) based on possible treatment-relate d deaths in females and decrease d body weight/body weight gain in males and females. The NOAEL is 1000 ppm (170/224 mg/kg/day in males and females, respectively).

Chronic Oral Toxicity in Non-Rodents (Dog; OCSPP 870.4100)

.
In a chronic toxicity study in dogs ( MRIDs 40980701, 41164501, 42218601, and 42218602;

1989 ), metolachlor (97% purity) was administered to Beagle dogs (6 /sex/group in the control and high-dose groups and 4/sex/group in the low- and medium-dose groups) in the diet at concentrations of 0, 100, 300, or 1000 ppm for up to 52 weeks (mean-measured doses of 0, 3.5, 9.7, and 32.7 mg/kg/day, respectively, in male s and 0, 3.6, 9.7, and 33.0 mg/kg/day, respectively, in females). Two dogs/sex in the control and high-dose groups were designated as recovery animals and allowed a four-week recovery peri od after the 52-week treatment period. An additional 4 dogs/sex/group were admi nistered the same treatments and sacrificed after Week 13. Organs that were weighed included the liver, kidneys, testes, epididymides, ovaries, pituitary, thyroid, and adrenals. Gross and microscopi c pathological examinations were conducted on these and other tissues, including the mammar y glands, prostate, vagina, and uterus.

There were no treatment-related d eaths or clinical signs of to xicity. Mean body weight gains were decreased (NS) in the high-dose fema les for animals sacrif iced at Week 13 ( ↓ 16%) and at Week 52 ( ↓ 17%). Decreases in food consumption were noted in high-dose males and females, but were not generally associated with decreased body weights. Alkaline phosphatase
concentrations were increased (p<0.05, 0.01, or NS) in the hi gh-dose females throughout theMETOLACHLOR FINAL

Page 53 of 61 study ( ↑ 27-81%); however, the increases were not considered toxicol ogically significant due to the lack of accompanying necropsy findings. Ther e was a treatment-related decreased in kidney weights of males in the low- (NS, ↓ 16%), medium- (p<0.05, ↓ 19%), and high-dose (p<0.01, ↓ 27%) groups, with no treatment effects obser ved on kidneys in females and no accompanying necropsy findings. No other treatment-relate d changes were noted in organ weights although pituitary weights were increased (NS) in both males and females at the low-, mid- and high- dose levels (males, ↑ 31, 56 and 22%; females, ↑ 13, 22, 40%). No treatment-related gross or microscopic pathology changes were noted in an y tissues, including the ova ries, uterus, vagina, mammary gland, testes, epididymides, prostate, adrenals, thyroid or pi tuitary glands.

The LOAEL is 1000 ppm for females (33.0 mg/kg/ day) based on decreased body weight gain, and the NOAEL is 300 ppm (9.7 mg/ kg/day). The LOAEL for males is not established, and the NOAEL for males is 1000 ppm (32.7 mg/kg/day).

Avian Reproduction (OCSPP 850.2300)

In an avian reproduction study ( MRID 44015602 ), 24-week old Mallard ducks ( Anas platyrhynchos ) were administered metolachlor (97.3% purity) in the feed at nominal concentrations of 0 (no solven t required), 50, 200, or 800 ppm (mg/kg) diet for 20 weeks (ten weeks prior to the start of egg production and ten weeks of egg production). Mean dietary doses were not calculated for the study. Each treatment group was comprised of sixteen mating pairs, with one pair per pen. A progressive photo peri od was employed in this study: 7 hours/day through Week 9 and 17 hours/day until the end of the study. Temperature was maintained at 15.6-27.8°C throughout the study and hu midity was not reported. Body weights were measured at test initiation, end of Week 3, during Weeks 7 and 10, and at test termination. Overall weight gain for males and females was determined from initial and final body weights. Food consumption in each replicate was measured bi-weekly throughout the pre-treatment and treatment periods of the adult phase. Individual chick body we ights were recorded on Day 14 after hatching; egg shell thickness was measured every week during egg production (alternating from even and odd numbered pens during even and odd numbered weeks). Adult birds and chicks were observed routinely fo r mortality and clinical signs.

No overt signs of toxicity or treatment-related mo rtalities were reported. There was a significant difference in 14-day survivor weig hts at the low and high concentra tions compared to control. However, because the mean 14-day survivor we ight at the high concentration was increased (p<0.05, ↑ 11%), the decreased (p<0.05, ↓ 9%) weight at the low concentration was not
considered to be treatment related. There we re no treatment-related effects on any of the measured reproductive endpoints. The slight decr ease in the percentage of 14-day-survivors of normal hatchlings at the medium concentration was not considered to be treatment related (93 vs. 97% control) because the percentages of viable em bryos of eggs set and of normal hatchlings ofMETOLACHLOR FINAL

Page 54 of 61 live 3-week embryos were slightly decreased (NS) compared to the control group. Therefore, the NOAEC is established as 800 pprn and the LOAEC is not determined ( ≥ 800 ppm).

In another avian reproduction study ( MRID 46508901 ), 26-week old Bobwhite quail ( Colinus virginianus ) were administered metolachlor (97.2% purity) in the feed at nominal concentrations of 0 (acetone, 0.2% v:w and corn oil, 0.3% v: w), 160, 400, or 1000 mg/kg diet for 20 weeks (ten weeks prior to the start of egg production and te n weeks of egg production). Calculated mean dietary doses were 13.9, 34.6, and 85.9 mg/kg/day, re spectively for the low-, medium-, and highdose groups. Each treatment group was comprised of sixteen mating pairs, with one pair per pen. A progressive photo period was employed in this study: 8 hours/day through Week 7 and 17 hours/day until the end of the study. Temp erature was maintained at 24.1±0.7°C throughout the study and humidity was maintained at 54.9±15.7%. Individual adult body weights were recorded at Week 0, 2, 4, 6, 8, and 20, and body weight changes for the study period were determined. Food consumption in each repl icate was measured weekly throughout the pretreatment and treatment periods of the adult phase. Individual ch ick body weights were recorded after hatching and on Day 14 after hatching; e gg shell thickness was measured every week during egg production (alternating from even and odd numbered pens during even and odd numbered weeks). Adult birds and chicks were observed routinely for mortality and clinical signs.

There was no reported treatment-related effect on adult mortality; there were three incidental deaths during the study in the high -dose group. No overt signs of toxicity were observed due to treatment, and all birds appeared normal thr oughout the study, except for incidental clinical findings. Adult body weight changes and food c onsumption during the treatment period were generally similar in all groups, and there was no evidence of a treatment-related effect. Occasional increases in food consumption were noted in the low-and medium-dose groups, but overall mean food consumption was 17-18 g/bird/day throughout the study. No treatmentrelated findings were observed at necropsy.

No treatment-related effects were observed on egg production or quality, fertility, embryonic development, hatchability, or survival of hatchlings at the low or medium treatment
concentrations. At the low treatment concentrat ion, there was slight increase (p<0.05) in egg production (percentage of eggs laid of maximum number laid) that also resulted in increases (p<0.05) in hatchlings and 14-day old survivors. Because the increase in egg production at the low concentration was slight, not concentration dependent, and represented an improvement in reproductive performance, the difference was not cons idered to be treatment related. There were
no differences between the control and the medium treatment groups for any of the reproductive parameters measured; however, there was a slight reduction in the percentage of viable embryos of eggs set (86 vs. 91% control) . This decrease was due to data from one pen with only 2/46 eggs set containing viable embryos . The percentage of viable embr yos (91%) was identical to the
control value if the data from this one pen was not used.METOLACHLOR FINAL

Page 55 of 61 Effects were noted in four of the parameters evaluated: eggshell th ickness, Day 14 survivor weight, eggs cracked, and percenta ge eggs not cracked/eggs lai d. Eggshell thinning occurred at all treatment concentrations (p<0.01, 0.22 mm vs. 0.23 mm control). Because mean eggshell thickness was the same (0.22 mm) for all treatment groups, eggshell thinning was considered to be not biologically significant at the low and medium concentra tions (in the absence of other adverse effects ( e.g. , cracking, reduced viabil ity). Eggshell thinning occurred at the high concentration in conjunction with an increase in eggs cracked/e ggs laid (p<0.05, 6 vs. 1%). A decrease (p<0.05) in Day 14 survivor weight was noted at the medium concentration ( ↓ 5%), but this response was not dose dependent.

The highest dose (85.9 mg/kg/day) is the LOAEC for the Bobwhite quail due to an increased incidence in eggs cracked, a nd 34.6 mg/kg/day is the NOAEC.

Fish Early Life-Stage Toxicity (OCSPP 850.1400)

In an early life-stage estuarine/marine fish study ( MRID 43044602, 1980 ), Sheepshead minnow ( Cyprinodon variegatus ) embryos were obtained by stripping eggs from adult fish that were collected wild. Egg production was enhanced by addition of human chorionic gonadotrophin, and a sperm suspension prepared from macerated testes was added to fertilize the eggs. Fish (100 embryos/treatment) were exposed to metolach lor (97.0% purity) at nominal concentrations of 0 [negative and solvent (TEG; 130 mg/L ) controls], 0.62, 1.2, 2.5, 5.0, or 10 mg/L (meanmeasured concentrations were 0.55, 1.0, 2.2, 4.1, a nd 8.6 mg/L). Embryo mortality and time hatched were observed, and survival of juven iles was monitored for 22 days. The photoperiod was 12 hours light/12 hours dark, instead of the recommend 16 hours light/8 hours dark. Standard length and weight were measured at study termination.

The mean-measured concentrations ranged from 82 to 89% of the nominal concentrations. Hatching was not affected at concentrations ≤ 8.6 mg/L. Hatching was >90% complete by Day 7 in all treatments (and ≥ 99% complete by Day 8), with no delay in hatch observed. Several embryonic development abnormalities (not reported) were observed, but were not attributed to metolachlor. The test was terminated at Day 26; it was reportedly because fish were dying as a
result of the artificial salt introduced in th e system, but increased mortality was not evident
across all of the treatments. Mortal ity was significant at concentrations ≥ 4.1 mg/L after Days 8, 15, and 22 post-hatch, with overall mortalities of 40 and 100% in the 4.1 and 8.6 mg/L treatment groups, respectively. Length wa s decreased (p<0.05) in the 4.1 mg/L treatment group (no survivors at the highest concentration). Weight was not significantly a ffected, although a trend in decreasing weight and length was observed at concentrations ≥ 2.2 mg/L. Therefore, the LOAEC is 2.2 mg/L based on decreased wei ght and length, and the NOAEC is 1.0 mg/L.

In an early life-stage freshwater fish study ( MRID 44995903 ), Fathead minnow ( Pimephales promelas ) embryos were obtained from an in-house culture. Fish (120 embryos/treatment, thinned to 80 embryos/treatment) were exposed to S-metolachlor ( 98.6% purity) at nominalMETOLACHLOR FINAL

Page 56 of 61 concentrations of 0 (nega tive control), 31, 63, 130, 250, 500, or 1000 μg/L (mean-measured concentrations were 30, 56, 110, 220, 450, and 870 μg/L ). Embryo mortality and time to hatch were observed, and survival of juveniles was monitored for 22 days. The photoperiod was the recommend 16 hours light/8 hours dark. Standa rd length, and wet and dry weights, were measured at study termination.

The mean measured concentrations ranged from 85 to 97% of the nominal concentrations. No sublethal signs of toxicity were reported. Hatching was not affected at concentrations

≤ 1000 μg/L. The mean percent hatch ranged from 85-90%, and no delay in hatch was observed. The 30-day post-hatch percent survival ranged from 93-100%. Length was decreased (significance not reported) ≥ 220 μg/L. Treatment-related decreases in wet weight were observed

at ≥ 110 μg/L and in dry weight at ≥ 56 μg/L. Therefore, the LOAEC is 56 μg/L based on decreased dry weight, and the NOAEC is 30 μg/L.

Mechanistic Studies: Thyroid System-Related Genes (Fish)

In a published, non-Guideline study ( Jin et al. , 2011 ), the effect of metolachlor treatment on the thyroid system of juvenile a nd adult Japanese medaka fish was determined. In place of plasma/serum hormone determination, transcri ptional analyses of genes regulating the hypothalamic-pituitary-thyroid (HPT) axis were c onducted on RNA isolated from whole juvenile
fish and from the brains and livers of adult fish.

The Japanese medaka fish used in the study were propagated at the study laboratory. Newlyhatched fish were maintained in glass tanks (wit h charcoal-dechlorinated ta p water at a constant temperature) until they reached juve nile stage (1 month) or adult stage (4 months). For exposure testing, male and female juvenile fish were raised in two beakers/sex/dose group (n=4 fish/beaker) containing metolachlo r (98% purity) at nominal concen trations of 0 (control), 1, 10, 100, and 1000 μg/L in 0.1% ethanol in water (v:v). Adult male and female fish were dosed in separate aquariums, two/dose group, containing six fish each; adult fish were dosed at the same nominal concentrations as juvenile fish. All fi sh were exposed to metolachlor for 14 days at ambient temperature (26  1°C); the rearing water and exposure water were changed daily. Fish were anesthetized on ice prior to sample collectio n. Each whole juvenile fish was considered to be one sample, yielding eight sa mples/sex/dose group. The brain and liver were collected from each adult fish, and the tissues from three same sex fish from each exposure tank were combined, yielding four samples of each tissue/sex/dose group.

The effect of estrogen exposure, with or without metolachlor exposure, on juvenile male medaka fish was also examined. Fish were exposed to estrogen at 25, 50, and 100 ng/L with or without
metolachlor at a concentration of 100 μg/L. Two vessels were created for each exposure group, each containing four fish. This study was conduc ted concurrently with the metolachlor study so that the 100 μg/L and 0 μg/L metolachlor-exposure groups serv ed as the positive and negativeMETOLACHLOR FINAL

Page 57 of 61 controls, respectively. Fish were exposed as desc ribed previously, and each whole juvenile fish was considered to be one sample.

Total RNA was isolated from individual juvenile fi sh and from the brains and livers of adult fish;
the RNA was denatured and then a reverse transcri ptase kit was used to synthesize cDNA. The expression of five genes were determined: Rpl- 7 (used as a house-keepin g gene to standardize results), Trh (thyrotropin-releasing ho rmone), Dio2 (deiodinase enzyme), Thr α (thyroid receptor), and Thr β (thyroid receptor).

No fish died during exposure. In female juve nile fish, metolachlor exposure increased (p<0.05) the expression of Trh at 100 μg/L and the expression of Dio2, Thr α , and Thr β at all treatment
concentrations ≥ 10 μ g/L. In male juvenile fish, there we re no differences in the expression of Trh, Dio2, Thr α , and Thr β due to metolachlor treatment. In the brains of female adult medaka, metolachlor exposure at 100 and 1000 μg/L increase d (p<0.05) the expression of all four genes related to the thyroid system, with no effects in male adult medaka. Expression of Dio2 in adult female livers was increased (p<0.05) at 1000 μg/L , with no effects of me tolachlor exposure on the expression of Dio2 in adult male livers or the expression of Thr α and Thr β in adult male or female livers. In juvenile male medaka exposed to metolachlor at 100 μg/L in the presence of estrogen, expression of Dio2 was increased (p<0.05) at the 50 ng/ L concentration, and expression of Dio2, Thr α , and Thr β was increased (p<0.05 or 0.01) at 100 ng/L. No effects of metolachlor exposure in the pr esence of estrogen were obser ved on expression of Trh.

The study authors concluded that exposure to metolachlor (10, 100, and 1000 μg/L for juvenile female medaka, and 100 and 1000 μg/L for adult female medaka) increased transcription of genes related to the thyroid system; transcript ion induced by metolachlor was gender-, tissue-, and developmental stage-specific. Estrogen co -exposure was found to increase transcription of thyroid system genes in male juvenile medaka treated with metolachlor.

Amphibian Development: Spolyar ich et al. 2010 (ECOTOX 153638)

Spotted marsh frog larvae ( Limnodynastes tasmaniensis ), which is indigenous to Australia, were rear in 80-L aquaria filled with dechlorinated tap water with complete water renewal every 14 days. Larvae were fed every 2 nd day with a 1:1 mix of Sera Micron ® and Nutrafin ® Fry Food with increasing food ration as th e animals matured. The larval development assay was based on Xenopus Metamorphosis Assay (XEMA) with expos ure starting at early limb bud emergence (Gosner stage 28) and continui ng until the majority of larv ae were undergoing tail resorption (Gosner stage 42). Each repl icate contained 10 larvae in 10 L of water within 14.5-L glass aquaria fitted with a glass lid to limit evapora tion. Each aquarium was aerated and was on a 12:12 light:dark cycle. Exposure commenced just prior to gonadal differe ntiation (Gosner stage 30). Animals were removed at Gosner stage 44 or 45. Spiking took place after every water renewal after every 3 rd or 4 th day of exposure. DO, temperature and pH were measured daily. Sex ratios initially based on gross gonadal mor phology. Male gonads from Tests 1, 6, 7 (atrazineMETOLACHLOR FINAL

Page 58 of 61 only) and Test 4 (atrazine, metolachlor and thio bencarb mixture) were examined histologically for the presence of oocytes. Atrazine (98% ai), metolachlor (99.5% ai), thiobencarb (99% ai) concentrations were analyzed weekly in the 4-day water following the water renewal. Tadpoles were exposed to metolachlor at nominal concen trations of 0.1, 1 and 10 μg/L. No atrazine,
metolachlor or thiobencarb were detected in c ontrols. Recovery of atrazine was on average 121.5%; average recovery of metolachlor was 90.8% ; average recovery of thiobencarb was 34%. Thiobencarb recovery was 27.2% in a mixtur e with atrazine and 28.1% in a mixture with
atrazine and metolachlor. The authors conclude the exposure of spotted march frog lavae to atrazine (0.1, 1, 3 and 30 μg/L, metolachlor (0.1, 1 and 10 μg/L) and thiobencarb (90, 180, and 360 μg/L) from Gosner stage 28 to 42 under controlled lab conditions had no significant effect on tadpole growth, development and sex ratio. Bina ry mixtures of atraine and thiobencarb as well as ternary mixtures of all three also ha d not significant effect on the developing larvae; abnormal gonad morphology was observed on two occa ssions, both from atrazine at 0.1 μg/L
with one larvae with testicular oovarian follicle bu t this did not exhibit a concentration associated response. In this study, the review er noted that the study used w ild-caught animals; therefore, prior exposure history is unknow n. Also, histology was only c onducted on male gonads. Sex was apparently based on gross gonadal morphology al one and was not verified across all of the test animals with histology.METOLACHLOR FINAL

Page 59 of 61 APPENDIX 3: References Not Utilized in the Metolachlor 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 Metolachlor 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.

Aït-Aïssa, S., Laskowski, S., Laville, N., Porche r, J. M., and Brion, F. (2010) Anti-androgenic activities of environmental pesticides in the MDA-kb2 reporter cell line. Toxicol In Vitro. 24:
1979-1985.

Buccafusco, R.J.; Stiefel, C.; Sullivan, D.M.; et al. (1978) Acute T oxicity of CGA-24705 to Rainbow Trout ( Salmo gairdneri ): Report #BW-78-6-186. (Unpublished study received Jul 13, 1978 under 100-597; prepared by EG&G, Bionomi cs, submitted by Ciba- Geigy Corp., Greensboro, N.C.; CDL:234396-B) (MRID 00018722)METOLACHLOR FINAL

Page 60 of 61 Buccafusco, R.J.; Stiefel, C.; Sullivan, D.M. ( 1978) Acute Toxicity of CGA-24705 to Bluegill (Lepomis macrochirus ): Report #BW- 78-6-181. (Unpublis hed study received Jul 13, 1978 under 100-597; prepared by EG&G, Bionomics, submitt ed by Ciba-Geigy Corp., Greensboro, N.C.; CDL:234396-C) (MRID 00018723)

Fink, R. (1974) Final Report: Eight-Day Dietar y LC50--Mallard Ducks: Project No. 108-111. (Unpublished study received on unknown date under 5G 1553; prepared by Truslow Farms, Inc. in cooperation with Maryland, Dept. of Agri culture, Div. of Inspection and Regulation, submitted by Ciba-Geigy Corp., Green sboro, N.C.; CDL:094220-O) (MRID 00016425)

Fink, R. (1974) Final Report: Eight-Day Diet ary LC50--Bobwhite Quail: Project No. 108-110. (Unpublished study received on unknown date under 5G 1553; prepared by Truslow Farms, Inc. in cooperation with Maryland, Dept. of Agri culture, Div. of Inspection and Regulation, submitted by Ciba-Geigy Corp., Green sboro, N.C.; CDL:094220-P) (MRID 00016426)

Fink, R.; Beavers, J.B.; Brown, R.; et al. (1981) Final Report: Acute Oral LD50--Mallard Duck: Project No. 108-200. (Unpublished study received Mar 31, 1981 under 100-587; prepared by Wildlife International, Ltd. and Washington College, submitted by Ciba-Geigy Corp., Greensboro, N.C.; CDL:244691-B) (MRID 00062465)

Mai, H., Cachot, J., Brune, J., Geffard, O., Belles, A., Budzinski, H., and Morin, B. (2012). Embryotoxic and genotoxic effects of heavy metals and pesticides on early life stages of Pacific oyster ( Crassostrea gigas ). Mar Pollut Bull. 64(12):2663-2670.

Mathias, F. T., Romano, R. M., Sleiman, H. K ., de Oliveira, C. A., and Romano, M. A. (2012). Herbicide metolachlor causes changes in re productive endocrinology of male Wistar rats. ISRN Toxicology . 2012:130846.

Murphy, T. (1987). Dermal Absorption of Metolachlor in Rats. CIBA-GEIGY Corporation, Agricultural Divi sion, Metabolism Department, Gree nsboro, NC and WIL Research Laboratories, Ashland, Ohi o. Laboratory Study No. ABR-87051, August 25, 1987. MRID No.
41833102.

Pereira, S. P., Fernandes, M. A., Martins, J. D., Santos, M. S., Moreno, A. J., Vicente, J. A., Videira, R. A., and Jurado, A. S. (2009). Toxi city assessment of the herbicide metolachlor comparative effects on bacterial and mitochondrial model systems. Toxicol In Vitro . 23(8):1585-

1590.

Reylea, R.A. (2009) A cocktail of contamin ants: how mixtures of pesticides at low concentrations affect aquatic communities. Oecologia : 159, 363-376. DOI 10.1007/s00442-008-

1213-9.METOLACHLOR FINAL

Page 61 of 61 Spolyarich, N., R. Hyne, S. Wilson, C. Palmer and M. Byrne. 2010. Growth, development and sex ratios of Spotted Marsh Frog ( Limnodynastes tasmaniensis ) larvae exposed to atrazine and a herbicide mixture. Chemosphere 78: 807 - 813. (ECOTOX Ref. No. 153638)

Williams, B. K. and Semlitsch, R. D. (2010). Larval responses of three midwestern anurans to chronic, low-dose exposures of four herbicides. Arch Environ Contam Toxicol. 58(3):819-827.