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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

# &

Health Effects Divison

# ::~:E£'~:::e 

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

Environmental

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

THR O UG H: Je ss Rowland

## ~ S

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

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

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

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals
Chemical Name PC Code TXR Number 2,4-D 030001 0057151 Abamectin 122804 0057152 Acephate 103301 0057153
Acetone 044101 0057154 Atrazine 080803 0057155 Benfluralin 084301 0057156 Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159 Carbofuran 090601 0057160 Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164
DCPA 078701 0057165 Diazinon 057801 0057166 Dichlobenil 027401 0057167 Dimethoate 035001 0057168
EPTC 041401 0057169 Esfenvalerate 109303 0057170 Ethoprop 041101 0057171 Fenbutatin-Oxide 104601 0057172 Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178 Linuron 035506 0057179 Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182 Metolachlor 108801 0057183 Metribuzin 101101 0057184 MGK-264 057001 0057185 Myclobutanil 128857 0057186 Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142 PCNB 056502 0057138 Permethrin 109701 0057149 Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141 Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140 Simazine 080807 0057136 Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147 Triadimefon 109901 0057139 Trifluralin 036101 0057135

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

CHEMICAL: METRIBUZIN

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYMETRIBUZIN 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..................................................13

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................16

IV. Committee’s Assessment of Weight of Evidence..........................................20

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................20

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

2. OSRI...............................................................................21

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

C. Androgen Pathway.........................................................................23

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

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

V. EDSP Tier 2 Testing Recommendations....................................................24

VI. References.......................................................................................26

LIST OF TABLES: Table 1. Tier 1 Assays for Metribuzin...............................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for Metribuzin ..................................10
Table 3. Androgenic/Anti-Androge nic Pathway for Metribuzin ................................14
Table 4. Thyroid Pathway for Metribuzin .........................................................18

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................29
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................39

APPENDIX 3. References Not Utilized in the Metribuzin WoE Analysis...........................49METRIBUZIN 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 enerationMETRIBUZIN 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 ConcentrationMETRIBUZIN 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 Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)METRIBUZIN 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-EvidenceMETRIBUZIN FINAL

Page 1 of 49 Executive Summary

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

In determining whether a pestic ide chemical interacts with E, A or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa, and sexes we re considered. Additi onally, the conditions un der which effects occur were considered, in part icular, whether or not endocrine-rela ted responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On August 27, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Offi ce of Science Coordination and Policy (OSCP) conducted a weight-of-evidence analysis (WoE) of the potential interactio n of metribuzin with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in
vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited
OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For the androgen pathway, metri buzin was negative in the in vitro estrogen receptor (ER) binding and ER transactivation a ssay (ERTA) and was equivocal in the aromatase assay. It was positive in the steroidogenesis assay where ther e were dose-dependent increases in estradiol production at metribuzin concentrations of 10

− 5 and 10

− 4 M, respectively. In the in vivo Tier 1 assays, metribuzin showed no evidence for potential estrogen-related effects in the uterotrophic and female pubertal assays. Additionally, there we re no treatment-related estrogen-related effects observed in females in the fish short-term reprod uction assay (FSTRA). Furthermore, there were no estrogen-related effects noted in any of the mammalian or wildlife Part 158 studies.

For the androgen pathway, metri buzin was negative in the in vitro Tier 1 AR binding assay and the steroidogenesis assay. In the in vivo Tier 1 assays, there was no evidence for androgenic or anti-androgenic effects in the Hers hberger and the male pubertal assa ys. While a slight increase
in the incidence of advanced maturation was observed in the gonadal histopathology for male fish in the FSTRA, there were no other tr eatment-related androgen effects observed. Additionally, there were no androge n-related effects noted in a ny of the mammalian or wildlife
Part 158 studies.METRIBUZIN FINAL

Page 2 of 49 There was convincing evidence of potential inter action with the thyroid pathway. In the pubertal assays, at the low dose (60 mg/kg/day), serum T4 levels were decreased and serum thyroid stimulating hormone (TSH) levels were increased in males. At the high dose (120 mg/kg/day), serum T4 levels were decreased in both sexes, serum TSH levels were increased in males, thyroid weights were increased in both sexes. Males at this dose exhibited enlarged thyroids and minimal to slight follicular cell hypertrophy, co lloid alteration, and an increased number of mitoses. The Tier 1 amphibian metamorphosis assay (AMA) requirement was satisfied by other scientifically relevant information (OSRI).

Alterations in thyroid hormone levels, thyroid weights as well as histopathological changes of the thyroid glands were seen in the ma mmalian Part 158 studies. In a rat chronic toxicity/carcinogenicity study, dos e-dependent increases in ab solute thyroid weights and significant increases in serum T 4 and decreases in serum T 3 were observed, as well as thyroid follicular cell hyperplasia in males. Finally, in the rabbit subchronic dermal toxicity study,

serum T 3 levels were decreased in males and T 4 levels were increased in both sexes without concomitant changes in thyroi d weight or histopathology.

In the Tier 1 pubertal assays, metribuzin at the low dose (60 mg/kg/day) decreased serum T4
levels and increased serum TSH levels in male s. At the high dose (120 mg/kg/day), metribuzin
caused a decrease in T4 level and an increase in serum TSH levels (males); increased thyroid weights (both sexes); enlarged thyroids (males); and induced histopathologi cal lesions (males). In the Part 158 mammalian chronic toxicity/c arcinogenicity study, metri buzin at 13.8 mg/kg/day caused significant increases in serum T4 and decreases in serum T3 levels, dose-dependent increases in absolute thyroid weights a nd thyroid follicular cell hyperplasia.

For metribuzin, the point of depa rture (POD) of 1.3 mg/kg/ is ba sed on increased thyroid weights and thyroid follicular cell hyperplasia seen at 13.8 mg/kg/day (LOAEL) in a chronic toxicity/carcinogenicity study in ra ts. This POD is used for deri ving the chronic Reference Dose (RfD) for dietary risk assessmen t and calculating Margins of Exposure (MOEs) for non-dietary
risk assessment.

In the EDSP Tier 1 pubertal assays and the OSRI , there was evidence for potential interaction with the thyroid pathway in studies conducted with adult animals, but no such data exists for its effects in the young animals. Since the POD a nd the toxicity endpoint are based on thyroid toxicity data obtained from adult animals, there is a concern that the POD may not be protective of potential thyroid to xicity in the young.

Consequently, a special thyroid as say in pregnant animals, fetuse s, postnatal animals, and adult
animals is recommended. This special study uses a mechanisti c approach to generate specific data on the thyroid ( i.e. , the primary target of metribuzin ) to protect the developing nervous system from thyroid hormone disrupting chemicals. The specific purpose of this special study is to generate data to establish a POD ( i.e. , NOAELs and LOAELs or benchmark doses) that mayMETRIBUZIN FINAL

Page 3 of 49 be used for human health risk assessment. The POD from this special study would address the concern for the potential ability of metribuzin to disrupt thyroid function in pregnant females and
in the fetus and newborn.

Currently, a definitive chronic endpoint for fish (s urrogates for aquatic-phase amphibians) is not available, as the available data indicated adve rse effects at all concen trations tested (NOAEC value is less than 3.0 mg a.i./L, based on fish early life-stage study). Additional chronic toxicity data for fish has been requeste d under registration review for metr ibuzin. However, as potential effects on the thyroid pathway were indicated, to add to the understand ing of potential thyroid effects, the EDSP Tier 2 Larval Amphibia n growth and Development Assay (LAGDA) is recommended since there is no chronic aquatic amphibian toxicity data available.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for metribuzin. In both spec ies, no effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from metribuzin exposure. Relative to the EDSP, ad ditional testing is not recommended.METRIBUZIN FINAL

Page 4 of 49 I. Introduction

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

In determining whether a pestic ide chemical interacts with E, A or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa, and sexes we re considered. Additi onally, the conditions un der which effects occur were considered, in part icular, whether or not endocrine-rela ted responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On August 27, 2014, 2014, the Tier 1 Assay We ight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of metribuzin with the E, A or T hormone pa thways. 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).

Metribuzin has a vapor pressure of 4.35 × 10 -7 mm Hg at 20°C with a water solubility of 1.2 mg/L. Metribuzin has an octanol/water partition coefficient of 1.7 (Log K ow ). Based on available data, the primary routes of degradation of me tribuzin are microbial metabolism and photolytic degradation on soil. Metribuzin is very stable to abiotic hydrolysis and relatively stable to both aerobic and anaerobic soil metabolism (T 1/2 's of 106 and 112 days, respectively).

The available information considered in determin ing the potential interaction of metribuzin 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. These 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.METRIBUZIN FINAL

Page 5 of 49 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 submitted as OSRI by the Test Order recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on Metribuzin (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and 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 tocharacterize 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.METRIBUZIN FINAL

Page 6 of 49 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 consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finally, under what conditions where the responses occur including consideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemicMETRIBUZIN FINAL

Page 7 of 49 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 gene rally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity 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 ., organ weights, blood chemistry). The circumstances for which a dose/concentration was considered overtly toxic for a particular study are described in Section
IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/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 th ese three main questions is fundamental to the WoE process and in determining whether addi tional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of ke y studies; 3) description of inc onsistent or conflicting data; 4) overall strength of evidence supporting a conclusi on; and, 5) what, if a ny, additional data are needed and why. Assessing the need for additiona l data is based on a case-by-case analysis
which took all available toxi city data into account.METRIBUZIN FINAL

Page 8 of 49 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 biol ogically relevant with regard to treatment-relate d 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 th e presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not re ported 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 metribuzin are shown below
in Table 1.

Table 1: Tier 1 Screening Assays for Metribuzin.

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48617902)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48617903)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48617905)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48617906)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48617901)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48617907)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48617904)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48672203)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48672203)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48672202)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied by OSRI (Based on thyroid effects in Part 158 mammalian toxicity studies)METRIBUZIN FINAL

Page 9 of 49 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

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

. The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-relevant fi ndings from data evaluated as OSRI. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment.

Metribuzin was classified as not interactive (negative) in the Tier 1 in vitro estrogen receptor (ER) binding assay, and as negative in the ER α transcriptional activation assay (ERTA). However, metribuzin was equivocal in the arom atase assay, and positive in the steroidogenesis assay. Aromatase activity was re duced to 63% of control at 10

− 3 M. Since the average lowest portion of the inhibition curve wa s between 50-75% of control activ ity, metribuzin was classified as equivocal in this assay. In the steroidogene sis assay, dose-dependent increases in estradiol production of 1.56- and 2.20-fold were observe d at metribuzin concentrations of 10

− 5 and 10

− 4 M, respectively.

In the in vivo Tier 1 assays, metribuzin was negati ve for estrogen-related effects in the uterotrophic and female pubertal assays. Overall, there were no effects observed in the FSTRA that were attributed to exposure to metribuzi n. An increase in prevalence and severity for incidence of egg debris in the ovaries was obser ved with increasing concentration as well as an increase in the incident of oocyte atresia in the highest treatment, but th e pathologist’s report did not consider any finding to be tr eatment-related. Additionally, it is noted in the FSTRA that the mean fecundity rate in the controls was less than the guideline criteria, however, spawning in the control occurred almost daily and the results of the study we re considered adequate for evaluating potential endocrine-related effects.

No estrogen-related effects were noted in any of the Part 158 mammalian studies. There were no
treatment-related effects observed in the Part 158 avian reproduction toxi city studies performed with mallard duck or bobwhite quail up to 420 mg /kg-diet. In a Part 158 early life-stage study with rainbow trout, larval leng th was significantly decreased at all test concentrations ( ≥ 3.0 mg/L) and body weight was reduced at ≥ 11.7 mg/L; survival was also reduced at 48 mg/L.METRIBUZINFINALPage 10 of 49Table 2: Estrogenic/Anti-Estrogenic Pathway for MetribuzinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metribuzin1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48617902)NERTA(MRID 48617903)NAromatase(MRID 48617901)ESteroidogenesis(MRID 48617906)PUterotrophic(MRID 48617907)NP4(L, M, H)NFemale Pubertal Rat(MRID 48672203)NNNNNNP5(L, H)NFSTRA(MRID 48672202)NENNNNNNOSRIDevelopmentalToxicity (Rat; MRIDs00163802, 41551801)N6X(M, H)X(L, M, H)DevelopmentalToxicity (Rabbit;MRIDs 41249201,41835501)NX(H)X(H)Two-GenerationReproduction Toxicity(Rat; MRID 40838401)NE NE N NE NNX(M, H)X(H)METRIBUZINFINALPage 11 of 49Table 2: Estrogenic/Anti-Estrogenic Pathway for MetribuzinLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metribuzin1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormones Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility (Frt)/ Fecundity (Fcd) Vitellogenin Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic Toxicity/Carcinogenicity(Rat; MRID 42672501)NE N N NEX(M, H)X(H)Carcinogenicity(Mouse; MRID00087795)NE N N NEX(H)NChronic Toxicity(Dog; MRID00061260)NE N N NEX(H)X(H)Subchronic Toxicity(Dog; MRID45621001)N N N NX(H)X(H)Subchronic DermalToxicity (Rabbit;MRID 43970701)NE N N NEX(H)NAvian ReproductionToxicity (Duck; MRID43860501)NN NAvian ReproductionToxicity (Quail; MRID43926601)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 indicatesthat is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated.2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X 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. AMETRIBUZINFINALPage 12 of 494. Body weight gains were decreased (p<0.01) by 33, 40, and 46% at the low (10 mg/kg/day), mid (40 mg/kg/day), and high (120 mg/kg/day) doses,respectively.5. Increased salivation was observed in 6/15 rats at the low dose (60 mg/kg/day) and in 7/15 rats at the high dose (120 mg/kg/day) on several occasionsthroughout the study. Absolute, adjusted, and relative liver weights were increased (p≤0.01) by 21-22% at the high dose; relative liver weights wereincreased (p≤0.05) by 10% at the low dose. Enlarged liver was observed in 5/15 rats at the high dose and 1/15 rats at the low dose, and there wereincreased numbers of mitoses in 4/5 livers at the high dose, and in the single liver at the low dose.6. Although no estrogen-related effects were observed inthe study, all three doses showed overt toxicity.P Positive findingsN Negative findings (in vitro)/No effect(in vivo)E Equivocal responseNE Not examinedMETRIBUZIN FINAL

Page 13 of 49 2. Effects on Androgen Pathway

The potential for metribuzin to interact w ith the androgen pathway 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 androgenic, anti-androgenic, or HPG axis effects.
This table also includes HPG-re levant findings from data evaluated as OSRI. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

Metribuzin was classified as negative in the in vitro Tier 1 AR binding assay and the steroidogenesis assay. In the in vivo Tier 1 assays, there were no androgen-related effects in the Hershberger assay and the male pubertal assa y. In the FSTRA, there was a reported higher
incidence of advanced maturation in the testes at the highest treatment group; however, there were no other androgen-related e ffects observed in the FSTRA.

There were no androgenic or anti -androgenic effects noted in a ny of the Part 158 mammalian or
wildlife studies.METRIBUZINFINALPage 14 of 49Table 3: Androgenic/Anti-Androgenic Pathway for MetribuzinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Metribuzin1Study Type/ LiteratureCitation AR Binding Steroidogenesis & Gene Expression 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 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48617905)NSteroidogenesis(MRID 48617906)NHershberger(MRID 48617904)NENN NMale Pubertal Rat(MRID 48672203)NNNNNNNNP4(L, H)NFSTRA(MRID 48672202)NENP5NNNNOSRITwo-GenerationReproduction Toxicity(Rat; MRID40838401)NE N NE N NE NE NX(M, H)NChronic Toxicity/Carcinogenicity(Rat; MRID42672501)N N NE N NEX(M, H)NCarcinogenicity(Mouse; MRID00087795)N N NE NE NEN NChronic Toxicity(Dog; MRID00061260)N N NE NE NEX(H)X(H)Subchronic Toxicity(Dog; MRID45621001)N N N N NX(H)X(H)METRIBUZINFINALPage 15 of 49Table 3: Androgenic/Anti-Androgenic Pathway for MetribuzinLines of Evidence Indicating Potential Interaction withthe Androgenic/Anti-Androgenic Pathway for Metribuzin1Study Type/ LiteratureCitation AR Binding Steroidogenesis & Gene Expression 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 2 Overt Toxicity Observed 3Subchronic DermalToxicity (Rabbit;MRID 43970701)N N NE N NEX(H)NAvian ReproductionToxicity (Duck; MRID43860501)NN NAvian ReproductionToxicity (Quail;MRID 43926601)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 indicatesthat is parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsalprostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP).2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an Xin this 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. At the high dose (120 mg/kg/day), increased salivation was observed in all rats, and one animal in this group had reduced motor activity and laboredrespiration on Day 13 and another rat had ocular discharge on Days 20-25. Absolute, adjusted (for body weight on PND 21), andrelative liver weightswere increased (p≤0.01) by 21-23%. Serum total protein was increased (p≤0.01) by 5%. At necropsy, enlarged liver was observed in 11/15 rats. Therewere increased numbers of mitoses in 7/11examined livers, as well as minimal centrilobular hepatocellular hypertrophy (4/11 rats). At 60 mg/kg/day,increased salivation was observed on several occasions throughout the study in 5/15rats. Relative liver weights were increased (p≤0.01) by 12%, andenlarged liver was observed in 1/15 rats.5. Higher incidence of advanced maturation inthe testes at the highest treatment groupP Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedMETRIBUZIN FINAL

Page 16 of 49 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 female and male rat pubertal assays, and the amphibian metamorphosis assay (frog).

The potential for metribuzin 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 HPG-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 Tier 1 male and female pubertal assays , 120 mg/kg/day metribuz in treatment caused increases in thyroid weight ( ↑ 45-46% in males; ↑ 15% in females) that were associated with an enlarged thyroid (6/15 males) at necropsy. Thyroid histopatho logy showed minimal to slight follicular cell hypertrophy, colloid alteration, and an increased number of mitoses (2/15 males).
Serum T 4 levels were decreased by 43-72% in th e 60 and 120 mg/kg/day males and by 44% in the 120 mg/kg/day females, while serum TSH le vels were significantly increased by 109 and 236% in the 60 and 120 mg/kg/day in males, respec tively. In females, serum TSH levels were non-significantly decreased by 29 and 33% at 60 and 120 mg/kg/day, respectively.

The requirement for the AMA was satisfied by OSRI based on the thyroid-related findings in the
Part 158 mammalian studies as discussed below.

In a rat developmental toxicity study, while effects in the thyroi d glands were observed (increase in thyroid weight and changes in T4 levels), they were seen in the presence of overt toxicity (decreased maternal body weight and food c onsumption). At 200 mg/kg/day metribuzin treatment caused increases in thyroid weight of 57% and 33% on gestation days (GD) 16 and 21, respectively. These thyroid we ight increases were associated with decreases in serum T 4 levels
of 49% and 84% in dams at 70 and 200 mg/kg/day, respectively, on GD 16.

In a rat chronic toxicity/ carcinogenicity study, serum T 3 and T 4 levels were measured at 3, 6, 12, 18 and 24 months. Significant increases in serum T 4 (10 -57%) and decreases in serum T 3 (10 – 30%) were observed at all dose levels in male s and females treated either one or two years beginning at 91 days, and continuing until study term inations at 2 years. However, the changes
in T 4 and T 3 were not consistently dose related at the various time in tervals. There were dosedependent increases in absolute thyroid weights in males after one year and in both sexes after two years. At one year, absolute thyroid weig hts were increased (p<0.05) in males at the mid (13.8 mg/kg/day; ↑ 21%) and high (42.2 mg/kg/day; ↑ 32%) dose. At two years, absolute thyroid weights were increased in the high dose males ( ↑ 24%) and in the mid and high dose femalesMETRIBUZIN FINAL

Page 17 of 49 ( ↑ 18% and ↑ 13%, respectively). Thyroid follicular ce ll hyperplasia (minimal, bilateral, diffuse) was seen in males at the mid (40%) and high (5 5%) dose at one year and at the high (76%) dose at termination (two years). These changes were characterized as an increase in diameter of the centrally located thyroid gland follicle and an increase in follicular epithelial cell height. Statistically significant increases in serum choles terol levels were noted in the mid- and highdose males and females throughout most of the intervals examined.

In a dog chronic toxicity study, metribuzin treatme nt at the high dose (ca. 55 mg/kg/day) caused thyroid weight increases of 38% in a surviving ma le and 20% in a surviving female. There were no corresponding changes in thyroid histology or serum T 4 and T 3 levels. However, the high dose greatly exceeded the maximum tolerated dose , with 3/4 males and 3/4 females dying within two years of treatment.

Finally, in the rabbit subchronic dermal toxicity study, serum T 3 levels were decreased (p<0.05) by 23% in the high-dose males (1000 mg/kg/day), and T 4 levels were increased (p<0.05) in the high-dose males ( ↑ 33%) and females ( ↑ 53%). However, there were no concomitant changes in thyroid weight or histopathology.METRIBUZIN FINAL

Page 18 of 49 Table 4. Thyroid Pathway for Metribuzin.

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

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth (BW, SVL)Systemic ToxicityObsserved2Overt ToxicityObserved3

EDSP Tier 1 Assay

Male Pubertal Rat (MRID 48672203) ↑ 45-46% (H) P 4 (H) ↓ 43%, ↓ 72% (L, H) ↑ 109%, ↑ 236% (L, H) N P 5 (L, H) N

Female Pubertal Rat (MRID 48672203) ↑ 15% (H) N ↓ 44% (H) N N P 6 (L, H) N

AMA Assay satisfied by OSRI (MRIDs 00163802, 41249201, 40838401, 42672501, 00087795, 00061260, 45621001, and 43970701; see below)

OSRI Developmental Toxicity (Rat; MRIDs 00163802, 41551801) N 7 NE NE NE NE X (M, H) X (L, M,

H)
Developmental Toxicity (Rabbit; MRIDs 41249201, 41835501) NE NE NE NE NE X (H) X (H)

Two-Generation Reproduction Toxicity (Rat; MRID 40838401) NE NE NE NE NE X (M, H) X (H)

Chronic Toxicity/ Carcinogenicity (Rat; MRID 42672501) 7 P P 8 (M, H) ♂ P 9 (L, M,

H) NE NE X (M, H) X (M,
H ♀ )
Carcinogenicity (Mouse; MRID 00087795) NE N NE NE NE X (H) N

Chronic Toxicity (Dog; MRID 00061260) N N N NE NE X (H) X (H)
Subchronic Toxicity (Dog; MRID 45621001) N N NE NE N X (H) X (H)

Subchronic Dermal Toxicity (Rabbit; MRID 43970701) N N P NE NE X (H) N

1. Key to responses: L=Low treatment, ML=Mediu m-low treatment, M=Medium treatment, MH=Medium high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwise indicated.METRIBUZIN FINAL

Page 19 of 49 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. Enlarged thyroid was observed in 6/15 high dose (120 mg/kg/day) rats. Minimal to slight follicular cell hypertrophy and colloid alteration were observed in the thyroid glands , and an increased number of mitoses was noted in 2/15 males. 5. At the low dose (60 mg/kg/day), increased salivation wa s observed on several occasions throughout the study in 5/15 rats. Relative liver weights were increased (p ≤ 0.01) by 12%, and enlarged liver was observed in 1/15 rats. At the high dose (120 mg/kg/day), increased salivation was observed in all rats on one or several occasions throughout the study. In addition, one animal in this group had reduced motor activity and labored respiration on Day 13. Absolute, adjusted, and relative liver weights were increased (p ≤ 0.01) by 21-23%, and total protein was increased (p ≤ 0.01) by 5%. Enlarged liver was observed in 11/15 high-dose rats, with an increased numbers of mitoses in 7/11 examined livers, as well as minimal centrilobular hepatocellular hypertrophy (4/11 rats). 6. At the low dose (60 mg/kg/day), increased salivation was observed in 6/15 rats on se veral occasions throughout the study. Relative liver weights were increased (p ≤ 0.05) by 10%. An enlarged liver was observed in 1/15 rats and increased numbers of mitoses were observed in this liver. At the high dose (120 mg/kg/day), increased salivation was observed in 7/15 animals on one or several occasions throughout the study. Abso lute, adjusted, and relative liver weights were increased (p ≤ 0.01) by 21-22%. Enlarged liver was observed in 5/15 rats in this group; there were increased numbers of mitoses in 4/5 examined livers. Serum alanine aminotransferase was increased (p ≤ 0.05) in this group. 7. Although thyroid effects were observed in the mid and high dose in this study, all three doses showed overt toxicity. 8. At the mid (300 ppm) and high (900 ppm) doses, increased thyroid size was observed grossly in the males. Thyroid follicular cell hyperplasia (minimal, bilateral, diffuse) was seen in males in the mid (40%) and high (55%) dose groups at 1 year and in the high (76%) dose group at termination (2 years). These changes were characterized as an increase in diameter of the centrally located thyroid gland follicle and an increase in follicular epithelial cell height. 9. Serum T 3 and T 4 were measured at 3, 6, 12, 18 and 24 months. Beginning at 91 days, statistically significant decreases in serum levels of T 4 and significant decreases in T 3 were observed at all dose levels in males and females treated either one or two years. P Positive findings N Negative findings NE Not examined NA Not availableMETRIBUZIN FINAL

Page 20 of 49 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 metribuzin 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 co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

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

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 and OSRI
studies are described below. 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).METRIBUZIN FINAL

Page 21 of 49 1. Tier 1 in vivo Assays

In the female pubertal rat assay, systemic toxici ty was evident at both the low (60 mg/kg/day) and high (120 mg/kg/day) doses, ba sed on clinical signs of increas ed salivation in 6/15 low-dose rats and 7/15 high-dose rats on several occasi ons throughout the study. Additionally, absolute, adjusted, and relative liver weights were increased (p ≤ 0.01) by 21-22% at the high dose, and relative liver weights were increased (p ≤ 0.05) by 10% at the low dose. At necropsy, enlarged liver was observed in 5/15 rats at the high dose and 1/15 rats at the low dose, and there were
increased numbers of mitoses in 4/5 livers at the high dose.

In the male pubertal rat assay, systemic toxicity was also evident at both the low (60 mg/kg/day) and high (120 mg/kg/day) doses ba sed on clinical signs of increas ed salivation in 5/15 low-dose rats on several occasions throughout the study in all high-dose ra ts. Additionally, one high dose rat had reduced motor activity and labored respiration. At the lo w dose, relative liver weights were increased (p ≤ 0.01) by 12%, and enlarged liver was obser ved in 1/15 rats. At the high dose, absolute, adjusted and relative liver weights were increased (p ≤ 0.01) by 21-23%, serum total protein was increased (p ≤ 0.01) by 5% and enlarged liver was obs erved in 11/15 rats. In addition, there were increased numbers of mitoses in 7/11 examined livers, as well as minimal centrilobular hepatocellula r hypertrophy (4/11 rats).

In the uterotrophic assay, body weight gains we re decreased (p<0.01) by 33, 40, and 46% at the low (10 mg/kg/day), mid (40 mg/kg/day) a nd high (120 mg/kg/day) doses, respectively.

No overt toxicity was not observed in the FSTRA or the Hershberger assays.

2. OSRI

In a prenatal developmental toxic ity study in rats), maternal body weight gains were decreased in all treated groups (25, 70, and 200 mg/kg/day) by 37-56% during dosing and by 10-22% during gestation (or 17-38% when corrected for gravid uterine weights). Food consumption was also decreased in all treated groups during dosing. Additionally, fetal body weights (male, female, and combined) were decreased (p<0.05) by 8-16% at the mid (70 mg/kg/day) and high (200 mg/kg/day) doses.

In a developmental toxicity study in rabbits, high dose (85 mg/kg/day) metribuzin treatment resulted in decreased body weight gain during dosing ( ↓ 58%) and decreased food consumption and food efficiency during GD 7-19. Additionally, in the fetuses, irregular spinous process of the scapula was noted in 0 controls vs. 5 tr eated in the high (85 mg/kg/day) dose group.

In the two-generation reproduction toxicity study in rats, body weight gains were decreased (p<0.05) by 9-11% in the hi gh dose (750 ppm) in P and F 1 males during treatment, and by 22-
35% in the P and F 1 females during premating. Food consumption was also decreased (p<0.05) in these animals during the same periods (males ↓ 8-9%, females ↓ 7-10%). During gestation,METRIBUZIN FINAL

Page 22 of 49 body weight gains were decreased (p<0.05) by 11- 14% at the mid (150 ppm) and high doses for

F 1 females, but in a non-dose-dependent manner. In the offspring, lower (p<0.05) median pup body weights on PND 21 and body weight gains fr om PND 0-21 were noted in the high dose F 1 pups. The F 2 pups showed a similar pattern with decreased (p<0.05) median body weights on PND 14 and 21 and body weight gains from PND 0-21 in both the mid (150 ppm) and high dose

groups.

In a chronic toxicity/carcinoge nicity study in rats, absolute body weights were decreased (p<0.05) in the 900 ppm males ( ↓ 3-7%), and in the 300 and 900 ppm females ( ↓ 3-13%) from Week 13 through Week 104. Cumulative body weight gains were decreased at 300 and 900 ppm
as follows: Week 13 – ↓ 7-17% in males, ↓ 12-30% in females; Week 52 – ↓ 4-11% in males, ↓ 14- 26% in females; and Week 104 – ↓ 8-19% in males, ↓ 12-26% in females. Decreases in body weight gain exceeded the decreases in food c onsumption at the 300 and 900 ppm dose levels, supporting a conclusion of test ar ticle related toxicity. At W eek 52, terminal body weights were decreased (p<0.05) by 9% in the 900 ppm fema les; at Week 104, terminal body weights were decreased (p<0.05) by 10% in the 900 ppm males and by 6% and 11% in the 300 and 900 ppm females, respectively. Absolute liver we ights were also increased in the 300 ( ↑ 8%) and 900 ( ↑ 12%) males at Week 52, but were similar to c ontrols at Week 104. Absolute kidney weights were increased (p<0.05) by 13% at 900 ppm in both sexes at Week 104.

In a carcinogenicity study in mice, absolute liv er weights were increased (p<0.05) by 47% in the high dose (3200 ppm) females and absolute kidn ey weights were increased (p<0.05) by 13%.

In a chronic toxicity study in dogs , overt and systemic toxicity were evident. Mortality was high in the high dose (1500 ppm) test group, with two male and two female dogs dying during the first year of treatment. Two additional deaths (one male and one female) occurred in the high dose group after 2 years. After 2 weeks of feeding, the high dose group appeared weakened with dull and bristly coats; feed was frequently re fused. This group showed slightly reduced food intake and gained significantly less weight than the controls, but after approximately 65 weeks only one male and one female survived, and these animals showed normal weight gain. Furthermore, significant differences in the hema tological, and liver and kidney function tests were observed at the high dose. The surviving high dose male also had increased relative liver and pancreas weight.

In a subchronic toxicity study in dogs, overt and systemic toxicity were also evident. A single high dose (1200 ppm) male and female were sacr ificed by Day 59, due to marked body weight decreases (-2.3 and -1.4 kg). Both animals had severely reduced food consumption beginning on Day 43 for the male and Day 52 for the female. Bo th dogs were thin and pale with pale yellow gingiva, with substantial evidence of anemia, and th e female was considered to have an unthrifty appearance. There were increases (at one or more intervals) in ALP, AS T, ALT, GGT, TBILI,METRIBUZIN FINAL

Page 23 of 49 and bile acids, and very low UDP-GT levels. Microscopic lesions included: bone marrow hypoplasia and hyperplasia; myocardial degenera tion and hemorrhage; inflammation of liver, kidney and small intestine; unusual extramedulla ry hematopoiesis in liver and spleen; unusual pigmentation in the liver; pronounced involuti on of the thymus; and renal hemorrhage. Additionally, in the males, findings indicative of slight anemia we re evident at the high dose at
termination. Discoloration of the liver was not iced in two males, one of which was moribund sacrificed (vs 0 controls). Excluding the moribund sacrifices, congestion and pigmentation was observed (1/4 treated vs 0/4 contro ls). In the females, pigmentation was observed in the liver in the high dose (1/4 treate d vs 0/4 controls).

In a subchronic dermal toxicity study in rabbits , serum cholesterol levels were increased in the high dose (1000 mg/kg/day) males ( ↑ 31%) and females ( ↑ 32%). Additionally liver chemistry analyses demonstrated increased (p<0.05) N -demethylase activity and cytochrome P-450 in the high dose males. There was a slight decrease in high dose male absolu te and relative adrenal weights, and a decrease in high dose female absolute and relative spleen weights.

No systemic or overt toxicity was observed in the two avian reproduc tion studies at dietary concentrations up to 420 ppm. In an early lif e-stage study with rainbow trout, larval length was significantly decreased at all test concentrations ( ≥ 3.0 mg/L) and body weight was reduced at

≥ 11.7 mg/L; survival was also reduced at 48 mg/L.

B. Estrogen Pathway

Metribuzin was negative in the in vitro ER binding and ERTA and was equivocal in the aromatase assay. It was positive in the steroidog enesis assay where there were dose-dependent increases in estradiol production at metribuzin concentrations of 10

− 5 and 10

− 4 M, respectively. In
the in vivo Tier 1 assays, metribuzin showed no eviden ce for potential estrogen-related effects in the uterotrophic and female pubertal assays. A dditionally, there were no treatment-related estrogen-related effects observe d in females in the FSTRA. Furthermore, there were no estrogen-related effects noted in any of th e mammalian or wildlife Part 158 studies.

C. Androgen Pathway

Metribuzin was negative in the in vitro Tier 1 AR binding assay a nd the steroidogenesis assay.
In the in vivo Tier 1 assays, there was no evidence for androgenic or anti-androgenic effects in the Hershberger and the male pubertal assays. While a slight increase in the incidence of advanced maturation was observed in the gonadal histopathology for male fish in the FSTRA, there were no other treatment-related androgen effects observed. Additionally, there were no androgen-related effects noted in any of th e mammalian or wildlife Part 158 studies.METRIBUZIN FINAL

Page 24 of 49 D. Thyroid Pathway

There was convincing evidence of potential inter action with the thyroid pathway. In the pubertal assays, at the low dose (60 mg/kg/day), serum T4 levels were decreased and serum TSH levels were increased in males. At the high dose (120 mg /kg/day), serum T4 levels were decreased in both sexes, serum TSH levels were increased in males and thyroid weights were increased in
both sexes. Males at this dose e xhibited enlarged thyroi ds and minimal to slight follicular cell hypertrophy, colloid alteration, and an increa sed number of mitoses. The Tier 1 AMA requirement was satisfied by OSRI.

Alterations in thyroid hormone levels, thyroid weights as well as histopathological changes of the thyroid glands were seen in the ma mmalian Part 158 studies. In a rat chronic toxicity/carcinogenicity study, dos e-dependent increases in ab solute thyroid weights and significant increases in serum T 4 and decreases in serum T 3 were observed, as well as thyroid follicular cell hyperplasia in males. Finally, in the rabbit subchronic dermal toxicity study,

serum T 3 levels were decreased in males and T 4 levels were increased in both sexes without concomitant changes in thyroi d weight or histopathology.

E. Conclusions

There was no convincing evidence for potential interaction with the estrogen or androgen pathways. The available data i ndicate that metribuzin potential ly interacts with the thyroid pathway.

V. EDSP Tier 2 Testing Recommendations

For metribuzin, the point of departure (POD) of 1.3 mg/kg/day used for human health risk assessment is based on increased thyroid weights and thyroid follicular cell hyperplasia seen at 13.8 mg/kg/day (LOAEL) in a chroni c toxicity/carcinogenicity study in rats. This POD is used for deriving the chronic Reference Dose (RfD ) for dietary risk assessment and calculating Margins of Exposure (MOEs) for non-dietary risk assessment.

In the EDSP Tier 1 pubertal assays and the OSRI , there was evidence for potential interaction with the thyroid pathway in studies conducted with adult animals, but no such data exists for its effects in the young animals. Since the POD a nd the toxicity endpoint are based on thyroid toxicity data obtained from adult animals, there is a concern that the POD may not be protective of potential thyroid to xicity in the young.

Consequently, a special thyroid assay in pregnant animals, fetuses and postnatal animals, and adult animals is recommended. This special study uses a mechanistic approach to generate specific data on the thyroid ( i.e. , the primary target of the chemical of interest) to protect the developing nervous system from thyroid hormone disrupting chemicals. The specific purpose ofMETRIBUZIN FINAL

Page 25 of 49 this special study is to genera te data to establish a POD ( i.e. , NOAELs and LOAELs or benchmark doses) that may be used for human hea lth risk assessment. The POD from this special study would address the concern for the potential ability of metribuzin to disrupt thyroid function in pregnant females and in the fetus and newborn.

Currently, a definitive chronic endpoint for fish (s urrogates for aquatic-phase amphibians) is not available, as the available data indicated adve rse effects at all concen trations tested (NOAEC value is less than 3.0 mg a.i./L, based on fish early life-stage study). Additional chronic toxicity data for fish has been requeste d under registration review for metr ibuzin. However, as potential effects on the thyroid pathway were indicated, to add to the understand ing of potential thyroid effects, the EDSP Tier 2 Larval Amphibia n growth and Development Assay (LAGDA) is recommended since there is no chronic aquatic amphibian toxicity data available.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for metribuzin. In both spec ies, no effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from metribuzin exposure. Relative to the EDSP, additional testing is not recommended.METRIBUZIN FINAL

Page 26 of 49 VI. References

Blanck, M (2011) Metribuzin: assessment of pubertal development and thyroid function in juvenile/peripubertal male and female rats. Bayer S.A.S., Sophia Antipolis Cedex, France. Laboratory Study No. SA 10341, December 1, 2011. MRID 48672203. Unpublished.

Blanck, O (2011) Metribuzin: evaluation in the in vitro (HeLA-9903) estrogen receptor transcriptional activation assay. Bayer S.A.S ., Sophia Antipolis Cedex, France. Laboratory Study No. SA 11157, October 21, 2011. MRID 48617903. Unpublished.

Braune, C, and Bruns, E (2012) Short-term reproduction assay with the fathead minnow ( Pimephales promelas ) exposed to metribuzin technical under flow-through conditions. Bayer CropScience AG, Monheim, Germany. Labor atory Report ID. EBSEY003, March 9, 2012. MRID 48672202. Unpublished.

Christenson, W, and Wahle, B ( 1993) Technical grade metribuzin (Sencor): a combined chronic toxicity/oncogenicity feeding toxicity study in the rat. Miles Inc., Agriculture Division, Toxicology, Stillwell, KS. Laboratory Study No. 88-271-BM, completion date not provided. MRID 42672501. Unpublished.

Clemens, G, and Hartnagel, R (1989) Teratol ogy study in the rabbit w ith Sencor technical (metribuzin). Miles, Inc., Toxicology Depa rtment, Elkhart, IN. Laboratory Study No. MTD 0100, August 2, 1989. MRID 41249201. Unpublished.

Clemens, G.R. and Hartnagel, R.E. (1991) . Supplemental submission to MRID 41249201: a teratology study in the rabbit w ith Sencor technical (metribuzin): Miles, Inc., Toxicology Department, Elkhart, IN. Laboratory Study No. MTD 0100, August 2, 1989. MRID 41835501. Unpublished.

Flucke, W, and Hartmann, E ( 1989) DIC 1468 technical grade (com mon name: metribuzin): subacute dermal toxicity study in rabbits. Bayer AG, Department of Toxicology, Wuppertal, West Germany. Laboratory Study No. T3029453, July 18, 1989. MRID 43970701. Unpublished.

Gagliano, G, and Bowers, L (1992) Chronic toxic ity of SENCOR techni cal to the waterflea ( Daphnia magna ) under flow-through conditions. Miles In corporated, Stilwell, KS. Laboratory Report No. 103248, completion date not provided. MRID 42447802. Unpublished.

Gagliano, G, and Bowers, L (1993) Chronic toxic ity of SENCOR technical to the waterflea ( Daphnia magna ) under flow-through conditions. Miles In corporated, Stilwell, KS. Laboratory Report No. 105023, completion date not pr ovided. MRID 42731201. Unpublished.METRIBUZIN FINAL

Page 27 of 49 Hancock, G (1995a) Effect of technical SENCOR on mallard reproduction. Bayer Corporation, Agriculture Division, Stillwell, KS. Labor atory Report No. 106983, October 6, 1995. MRID 43860501. Unpublished.

Hancock, G (1995b) Effect of technical SENC OR on northern bobwhite reproduction. Bayer Corporation, Agriculture Divi sion, Stillwell, KS. Laborator y Report No. 106984, February 1, 1996. MRID 43926601. Unpublished.

Hayes, R (1981) Metribuzin® (Sencor): oncogen icity study in mice. Mobay Chemical Corp., Kansas City, Mo. Laboratory Study No. CDL:24 6397-A, completion date not provided. MRID 00087795. Unpublished.

Jones, R, and Hastings, T (2002) Technical grade metribuzin (DIC 1468): a subchronic toxicity feeding study in the Beagle dog. Bayer Cor poration, Stilwell, KS. Laboratory Study No. 00- S76-BK, January 16, 2002. MRID 45621001. Unpublished.

Kowalski, R, Clemens, G, Bare, J, et al . (1986) A teratology study with Sencor technical (metribuzin) in the rat. Miles Laboratorie s Inc., Toxicology Depa rtment, Elkhart, IN. Laboratory Study No. 91330, October 3, 1986. MRID 00163802. Unpublished.

Kowalski, R, Clemens, G, Bare, J, et al . (1990) Supplemental submission to EPA Accession No. 265336: a teratology study with Sencor technical (m etribuzin) in the rat. Miles Laboratories Inc., Toxicology Department, Elkhart, IN. Laboratory Study No. 37; 91330-1, completion date not provided. MRID 41551801. Unpublished.

Loser, E, and Mirea, D (1974) Bay 94 337: chro nic toxicity studies on dogs (two-year feeding experiment). Bayer AG, Department of Toxi cology, Wuppertal, West Germany. Laboratory Report No. 4887 & 41814, September 24, 1974. MRID 00061260. Unpublished.

Porter, M. Jasty, and Hartnage l, R (1988) A two-generation re production study in rats with Sencor technical (metribuzin). Miles, Inc., Toxicology Department, El khart, IN. Laboratory Project ID 98295, September 23, 1988. MRID 40838401. Unpublished.

Rouquie, D. (2011). Evaluation of Metribuzin in the Androgen Receptor Binding Assay. Bayer SAS, Sophia Antipolis Cedex, France. La boratory Study No.: SA 11191, November 30, 2011. MRID 48617905. Unpublished.

Rouquie, D. (2011). Evaluation of Metribuzin in the Estrogen Receptor Binding Assay. Bayer SAS, Sophia Antipolis Cedex, France. La boratory Study No.: SA 11213, December 2, 2011. MRID 48617902. Unpublished.METRIBUZIN FINAL

Page 28 of 49 Sheet, L and Hall, T. (2012). Weight of evidence to evaluate the potential fo r metribuzin to affect estrogen, androgen or thyroid sy stems. Bayer CropScience, Res earch Triangle Park,NC. Report
Number USO267. MRID No. 48672201

Tinwell, H. (2011) Evaluation of metribuzin in the aromatase assay. Bayer S.A.S., Bayer CropScience, Sophia Antipolis Cedex, France. Laboratory Report No.: SA 11062, November 10, 2011. MRID 48617901. Unpublished.

Tinwell, H. (2011) Evaluation of metribuzin in the H295R steroidogenesis assay. Bayer S.A.S., Bayer CropScience, Sophia Antipolis Cedex, France. Laboratory Study No.: SA 11268, December 09, 2011. MRID 48617906. Unpublished.

Totis, M. (2011) Metribuzin evaluation in th e Hershberger bioassay. Bayer S.A.S., Sophia Antipolis Cedex, France. Laboratory St udy No.: SA 10342, November 10, 2011. MRID 48617904. Unpublished.

Totis, M. (2011). Metribuzin Evaluation in the Immature Rat Uterotrophic Assay. Bayer SAS, France. Laboratory report number SA 10345, November 10, 2011. MRID 48617907. Unpublished.

USEPA (2010) METRIBUZIN - Report of the Endoc rine Disruptor Review Team- Test Order#: EDSP-101101-127. November 10, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, D.C.

USEPA (2011) Endocrine Disrupt or Screening Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention Science Coor dination and Policy. U.S. Environmental Protection Agency, Washington, DC.

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

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

Page 29 of 49 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100)

Requirement satisfied by OSRI (MRIDs 00163802, 41249201, 40838401, 42672501, 00087795, 00061260, 45621001, and 43970701); see Appendix 2 for findings.

Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48617905), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and a competitive binding experiment. The sa turation binding experiment was conducted to demonstrate that the AR in the rat prostate cy tosol was present in reasonable numbers and was functioning with appropriate affi nity for the radio-labeled refe rence androgen (R1881) prior to routinely conducting AR competitive binding e xperiments. The competitive binding assay was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (10

− 10 to 10

− 3 M) of metribuzin (94.4%, Batch# K285067). No precipitation of test substance was observed at any concentration and DMSO was used as the solvent vehicle at a final assay concentration of ≤ 3.3%. Three independent runs were performed,
and each run included dexamethasone as a w eak positive control and R1881 as the ligand
reference standard.

In the saturation binding experiment , the mean maximum binding capacity (B max ) was 14 fmol/100 μg protein and the m ean dissociation constant (K d ) was 1.01 nM. The K d obtained from Run #2 (0.59 nM) was below recommende d values for saturation binding, but the K d s from
the other two runs were with in the recommended range (0.685 to 1.57 nM), and the Scatchard plots for the three Saturation assays were ge nerally linear. The specific binding reached a plateau, and non-specific binding (NSB) wa s <20% of total binding (4.6-12.6%).

In the competitive binding experiment, specific [ 3 H]-R1881 binding was >75% at all
concentrations of metr ibuzin tested in all three runs. An IC 50 and relative binding affinity (RBA)
could not be calculated for metribuzin.

The estimated average log IC 50 s for R1881 and dexamethasone were

− 9.1 M and

− 4.4 M, respectively. Compared to the reference ligand, the mean RBA for dexamethasone was 0.0021%. No precipitation of the test compound was visually observed at ≤ 10

− 3 M. With the exception of the low K d from run 2, all performance criteria were met.

Based on the results from the three runs, metri buzin is classified as a Non-Binder in the Androgen Receptor Binding Assay.METRIBUZIN FINAL

Page 30 of 49 Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48617901), Metribuzin (94.4% a.i., batch # AEF055208-01-13) was incubated with human recombin ant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in dimethyl sulfoxi de (DMSO) at increasing logarithmic concentrations from 10 –10 M to 10

− 3 M for 15 minutes to assess the potential of metribuzin 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 metribuzin. Tritiated water was quantified using liquid scintillation counting (LSC ). Three runs were conducted and each run included a full activity c ontrol, a background activity control, a positive control series (10

− 10 to
10

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

− 10 to 10

− 3 M) with three repetiti ons per concentration.

For the positive control (4-OH ASDN), ar omatase activity averaged 0.356±0.0526 nmol/mgprotein/min at the lowest tested concentration (10

− 10 M) and 0.0028±0.0005 nmol/mgprotein/min at the highest tested concentration (10

− 5 M). These results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC 50 and %CV. For 4-OH ASDN, the estimated log IC 50 averaged

− 7.23 M, and the Hill slope was

− 0.79. Activity in the background controls ranged 2.9 to 3.9% and averaged 3.4% of the fu ll activity control.

For metribuzin, aromatase activity averaged 0.3 18±0.057 nmol/mg-protein/min at the lowest test concentration (10

− 10 M) and 0.217±0.029 nmol/mg-protein/ min at the highest tested concentration (10

− 3 M). As a percent of control, ar omatase activity was 91.4-103.3% at 10

− 10 to
10

− 4 M metribuzin, but was 63% at 10

− 3 M. Although the data for metribuzin did not fit the 4-parameter regression model, the average lowest portion of the inhibition curve across runs was between 50-75% activity, indica ting an equivocal response.

Based on the data from the average response curve, metribuzin is classified as Equivocal for aromatase inhibition in this assay.

Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding assa y (MRID 48617902), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct saturation and competitive binding experiments. A saturation binding experiment wa s conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriateMETRIBUZIN FINAL

Page 31 of 49 affinity for the radiolabeled reference estrogen prior to conducting ER competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presen ce of increasing concentrations (10

− 10 to 10

− 3 M) of metribuzin (94.4%; Batch # K285067) . DMSO was used as the vehicle at a
final concentration of <2%. A total of three independent 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.

For the saturation binding experiment, the approp riate protein concentration for the saturation binding assay was determined to be 0.5 mg/mL as it bound 25-35% of the [ 3 H]-17 β -estradiol.
The mean K d for [ 3 H]-17 β -estradiol was 0.070 nM, and the estimated B max was 0.034 nM for the prepared rat uterine cytosol.

For the competitive binding experiment, the da ta for metribuzin did not fit the non-linear regression model and percen t binding was >75% at all concentrations tested ( ≤ 10

− 3 M) in all three runs. Therefore, a mean log IC 50 and relative binding affi nity (RBA) could not be
calculated for metribuzin.

The estimated mean log IC 50 for 17 β -estradiol and 19-norethindrone was

− 8.8 M and

− 5.0 M, respectively, and the mean RBA for 19-noret hindrone was 0.017% compared to the natural ligand. Several performance criteria were not met for 17 β -estradiol: the Hill slope was outside the recommended range (

− 2.0); the bottom % binding (2.1 to 2.5%) was just above the recommended range; and the top % binding (93 %) was just below the recommended range. However, the responses for the weak positive control (19-norethindrone ) and negative control (octyltriethoxysilane ) were as expected.

Metribuzin was tested over a c oncentration range that fully defi ned the top of the curve and the maximum percent binding was within approximate ly 25 percentage points of the lowest
concentration of the estradiol standa rd. Each of the three runs was classified as not interactive. The lowest average percent radiolabeled estr adiol binding to the ER in the presence of
metribuzin at ≤ 10

− 3 M was >75%.

Based on the results of the three runs, metribuzin is classified as Not Interactive in the Estrogen Binding Assay.

ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48617903), hER α -HeLa-9903
cells cultured in vitro were exposed to metribuzin (94.4% a.i. Batch # K285067) at logarithmically increasing concentrations from 10

− 10 M to 10

− 4 M in DMSO (0.1%) for 24 hours,METRIBUZIN FINAL

Page 32 of 49 in two independent runs conducted on separate days. The experiments were performed using 96- well plates and each metribuzin c oncentration was tested in triplicate (3 wells /plate). Cells were exposed to the test agent for 23±1 h to induce re porter (luciferase) gene products. Luciferase expression in response to activation of the es trogen receptor by metribuzin was measured upon addition of a luciferase substrate and detection with a luminometer with acceptable sensitivity.

The laboratory proficiency assays using the required reference co mpounds demonstrated that the assay was functioning properly. Th e only deviation was a weak positive response in one run to corticosterone (negative contro l compound), otherwise the perfor mance criteria were met for
17 β -estradiol and corticosterone. For 17 α -estradiol (weak agonist), the Log PC 10 was lower than the expected range in the first assay, as was the Hill Slope. For 17 α -methyltestosterone (very weak agonist), the Log PC 50 was lower than the expected range in run 2, and the Log PC 10 was below the excepted range in both runs. These results suggest increased sensitivity of the assay.

Metribuzin was tested up to the limit of solubility, 10

− 4 M. The mean RPC Max for metribuzin was 0.7% for the first run and 14.1% for the second run, and the associated PC Max was 10

− 4 M
for the first run and 10

− 6 M for the second run. Although the RPC Max ≥ PC 10 in run 2 (14.1%), this high value was due to only one of the three samples for that run, and the response was not dose-dependent. Therefore, methomyl was c onsidered negative for estrogen receptor transcriptional activation in this test system.

Fish Short Term Reproducti on Assay (FSTRA; OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID 48672202) of metribuzin with fathead minnows (Pimephales promelas) was conducted under flow-through conditions. Adult fish (16 spawning groups; 2 males and 4 females in each gr oup; ca. 6.5 months old) were exposed to metribuzin (94.4% purity) at nominal concentr ations of 0 (negative control), 0.200, 1.00, and 5.00 mg a.i./L; mean-measured concentrations were <LOQ (<0.02; negative control), 0.199, 1.03, and 5.40 mg a.i./L. The test system was ma intained at 25.2 to 26.0oC and a pH of 6.7 to
7.3.

Overall survival was 100, 96.9, 96.9, and 90.6% in the negative control a nd low, mid, and high treatment groups, respectively; all mortalities in the treatment groups were female (one, one, and three female mortalities in the low, mid, and high treatment groups, respectively). There were no notable observations with regards to behavi or or changes in appearance (specifically color/banding, ovipositor appearan ce, or size of dorsal nape pa d) in the negative control or treatment groups. There were no significant differe nces (p>0.05) in male or female body weight or length compared to the negative control.METRIBUZIN FINAL

Page 33 of 49 In the negative control replicat es, spawning occurred at least ev ery 4 days and mean fecundity was 8.84 eggs/female/day/replicate (range: 6.74-13) . Fertilization success in the negative control averaged 96.9% (range: 95-98.5%). Fecundity wa s not significantly reduced (p>0.05) in any treatment group compared to the negative contro l. There was no significant difference (p>0.05)
in fertilization success between the ne gative control and treatment groups.

Male nuptial tubercle scores were not significantly different (p>0.05) in any treatment group compared to the negative control. None of the surviving females were found to have tubercles. For males and females, gonado-somatic index (GSI) in the treatment groups was not significantly different (p>0.05) from the nega tive control. Plasma vitelloge nin (VTG) was not significantly different (p>0.05) in males or females. For males, it is noted that at th e low test concentration, VTG was increased 195% compared to the control. However, this percent difference is due to one fish for which the VTG value was substantially greater than the control values and this
increase does not reflect an overall increase in the fish exposed at this concentration. Sex steroids were not measured in this study.

In males, there was a higher incidence of advanc ed maturation (stage 3) at the high treatment level compared with the negative control and other treatment groups. The study author reported that while a relationship to e xposure could not be excluded, the high treatment level did have increased female mortality, and therefore, the matu ration could be related to overt toxicity or due to the lower density of females in the replicates. However, the reviewer did not discern a trend
between the males with the advanced maturation and replicates that contained dead females. Overall, no substantial female hi stopathological results that coul d be correlated with exposure to
the test substance were noted.

Hershberger Assay (OCSPP 890.1400)

In a Hershberger Assay (MRID 48617904) screeni ng for androgenic activity, metribuzin (94.4% a.i., batch/lot K285067) in an aqueous soluti on of 0.5% methylcellulose 400 + 0.4% Tween 80 was administered daily via oral gavage to groups of 58-day old, castrated male Sprague Dawley rats (6/group) at dose levels of 0 (vehicle), 40, or 80 mg/kg/day. An androgenic positive control group consisted of six castrated rats exposed to 0.4 mg/kg/day of testoste rone propionate (TP) by subcutaneous (s.c.) injection.

To screen for potential anti-a ndrogenic activity, metribuzin in an aqueous solution of 0.5% methylcellulose 400 + 0.4% Tween 80 was also admi nistered daily via oral gavage to groups of 58-day old, castrated male Sprague Dawley rats (6/group) at dose le vels of 0 (vehicle), 20, 40, or 80 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic pos itive control group consisted of six castrated rats were dosed orally with flutamide (FT) in corn oil at 3 mg/ kg/day for 10 days in conjugation with a daily s.c.METRIBUZIN FINAL

Page 34 of 49 dose of TP (0.4 mg/kg/day). Animals received 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 were collected and weighed.

In the androgen agonist assa y, body weights throughout the study and body weight gains from Days 1 to 10 were similar across all groups. At 80 mg/kg/day, there was an increase in glans penis weight ( ↑ 28%) which was statistically significant (p ≤ 0.01, ANCOVA using terminal body weight as the co-variable). There were no trea tment-related effects on seminal vesicles, ventral prostate, levator ani-bulbocavernosus (LABC), or Cowper’s glands in either dose group. Animals in the TP positive control group had increased (p ≤ 0.01) accessory sex organ weights as follows: 1280% in seminal vesicles; 2200% in ventral prostate; 197 % in LABC; 570% in Cowper’s gland; and 89% in glans penis. Five of the ten possible indivi dual CVs in the control and high dose treatment groups exceeded the guideline performan ce criteria values.

In the anti-androgen assay, body we ights throughout the study and body weight gains from Days 1 to 10 were similar across all groups. There were no treatment-related effects on accessory sex organ weights in either dose group. Animals dosed with TP + FT (positive control) had decreased (p ≤ 0.01) accessory sex organ weights as follo ws: 78% in seminal vesicles; 67% in ventral prostate; 49% in LABC; 61% in Cowper ’s gland; and 27% in glans penis. The performance criteria indicated that this assay was performing as expected.

The high dose used in this study (80 mg/kg/day) was considered ad equate based on data from the dose range-finding study which showed decreases in total body weight gain of 21% (compared to control) at this dose.

Statistically significant organ wei ght changes were not seen in tw o or more of the five androgen sensitive issue weights. Metribuzin was negativ e for androgenicity and anti-androgenicity in the Hershberger assay.

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48672203), 15 Sprague Dawley rats/dose group were treated daily via oral gavage with metribuzin ( 94.4% a.i., Batch/lot# K285067) in aqueous 0.5% methylcellulose 400 + 0.4% Tween 80 (vehicle) at doses of 0 (vehicle), 60, or 120 mg/kg/day from post-natal day (PND) 22 to 42. Animals we re examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment were recorded. Following sacrifice on PND 42, serum thyroxine (T 4 ) and thyroid stimulating ho rmone (TSH) levels wereMETRIBUZIN FINAL

Page 35 of 49 determined using enzyme immunoassay kits. Ad renals, liver, pituitary, thyroid, and urogenital organ weights were recorded and microscopic ex amination of the ovaries , uterus, thyroid, and kidneys were performed. In addition, any livers found to be enlarged at necropsy were subjected to microscopic examination.

There were no effects of treatment on mean age at first vaginal estrus, mean cycle length, percent cycling, or percent regularly cyc ling; on the weights of kidneys, p ituitary, adrenals, ovaries, or uterus (wet or blotted); or on levels of creatinine, blood ur ea nitrogen, total protein, total bilirubin, aspartate aminotransfe rase, or alkaline phosphatase. Th ere were no treatment-related histopathological findings in the thyroid gland, ovarie s, uterus, or kidneys.

At 120 mg/kg/day, increased salivation was obs erved in 7/15 animals on one or several occasions throughout the study. The age (PND 36.47 treated vs. PND 35.00 controls) and body weight ( ↑ 6%) at VO were slightly increased (not si gnificant; NS) compared to the controls. Overall body weight gains were comparable to the control. Absolute, adju sted (for body weight on PND 21), and relative liver weights were increased (p ≤ 0.01) by 21-22% and adjusted thyroid gland weights were increased (p ≤ 0.05) by 15% in the 120 mg/kg/day group compared to controls. At necropsy, enlarged thyroid was obs erved in 1/15 rats a nd enlarged liver was observed in 5/15 rats in this gr oup; there were increased numbers of mitoses in 4/5 examined
livers. Serum T 4 levels were decreased (p ≤ 0.01) by 44% in rats dosed at 120 mg/kg/day; mean TSH levels were increased (NS) by 33% and alanine aminotransferase was increased (p ≤ 0.05) in this group.

At 60 mg/kg/day, increased saliva tion was observed in 6/15 rats on several occasions throughout the study. Final body weight and body weight ga in, and age and body weight at VO in this group were comparable to the control. Relative liver weights were increased (p ≤ 0.05) by 10%; thyroid gland weights were comparab le to the controls at this dose level. At necropsy, enlarged
liver was observed in 1/15 rats and increased numb ers of mitoses were observed in this liver. Mean TSH levels were increased (NS) by 29% at 60 mg/kg/day; however, serum T 4 and alanine aminotransferase levels in this group were comparable to the controls.

The dose levels tested in this assay are adequa te based on the results of a dose range-finding study.

Male Pubertal Assay (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48672203), 15 Sp rague Dawley rats/dose group were treated daily via oral gavage with metribuzin ( 94.4% a.i., Batch/lot# K285067) in aqueous 0.5% methylcellulose 400 + 0.4% Tween 80 (vehicle) at doses of 0 (vehicle), 60, or 120 mg/kg/day from post-natal day (PND) 23 to 53. Animals we re examined for preputial separation (PPS)METRIBUZIN FINAL

Page 36 of 49 daily beginning on PND 30 and age and weight at day of attainment were recorded. Following sacrifice on PND 53, total seru m testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed using enzyme immunoassay kits. Adrenals, liver, pituitary, thyroid, and urogenital organ weights were recorded a nd microscopic examinations of the testes, epididymides, thyroid, and kidneys were performed. In addition, any livers found to be enlarged at necropsy were subjected to microscopic examination.

There were no effects of treatment on final body weights, overall body wei ght gain, or age and body weight at PPS; on the weights of kidneys, pituitary, adrenals, se minal vesicles plus coagulating gland (with or without fluid), ventral prostate, dors olateral prostate, levator anibulbocavernosus muscle complex (LABC), epididymid es, or testes; or on levels of testosterone, creatinine, blood urea nitrogen, total bilirubin, aspartate aminotransferase, alanine aminotransferase, or alkaline phosphatase. There were no treatment-related histopathological findings in the testes, epididymides, or kidneys.

At 120 mg/kg/day, increased sali vation was observed in all rats on one or several occasions throughout the study. In addition, one animal in this group had reduced motor activity and labored respiration on Day 13 and another rat had ocular discharge on Days 20-25. All rats in this group achieved PPS before study terminatio n. Absolute, adjusted (for body weight on PND 21), and relative liver we ights were increased (p ≤ 0.01) by 21-23% and absolute and adjusted thyroid weights were increased (p ≤ 0.01) by 45-46% compared to controls. Serum T 4 levels were decreased (p ≤ 0.01) by 72% in rats dosed at 120 mg/kg/da y; mean TSH levels were increased (p ≤ 0.01) by 236% and total protein was increased (p ≤ 0.01) by 5% in this group. At necropsy, enlarged thyroid was observed in 6/15 rats a nd enlarged liver was obs erved in 11/15 rats. Minimal to slight follicular cell hypertrophy and colloid alteration were observed in the thyroid glands; an increased num ber of mitoses was noted in 2/15 males in this group. There were
increased numbers of mitoses in 7/11 examined livers in the 120 mg/kg/day group, as well as minimal centrilobular hepatocellula r hypertrophy (4/11 rats).

At 60 mg/kg/day, increased sali vation was observed on several occasions throughout the study in 5/15 rats. All but one rat in this group achieved PPS before study termination. Relative liver weights were increased (p ≤ 0.01) by 12%; thyroid gland weights we re comparable to the controls
at this dose level. Serum T 4 levels were decreased (p ≤ 0.01) by 43% and mean TSH levels were increased (p ≤ 0.01) by 109% in rats dosed at 60 mg/kg/day; total protein levels in this group were comparable to controls. At necropsy, enlarged liver was observed in 1/15 rats.

The high dose of metribuz in selected for this study (120 mg /kg/day) was adequate based on the increased liver weights observed at this dose level.METRIBUZIN FINAL

Page 37 of 49 Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48617906), H295R cells cultured in vitro in 24-well plates
were incubated with metribuzin (94.4% purity, Lot # AE F 055208-01-13) at serial log
concentrations of 10

− 10 to 10

− 4 M for 48 hours in triplicate in thr ee independent tests. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentration of 0.1%.

Testosterone and estradiol levels were measured by using EIA. The hormone assay interference
test indicated no interference in the measurement of testosterone and estradiol concentrations in the presence of metri buzin. A Quality Control (QC) plate was run concurrently with each independent run of the test chem ical plate to demonstrate that the assay responded properly to positive control agents at two concentration le vels. The positive controls included a known inhibitor (prochloraz) and a know n inducer (forskolin) of estrad iol and testosterone production.

Precipitation of the test compound, and cytotoxic ity, were not observed at any concentration tested ( ≤ 10

− 4 M). Guideline acceptability recommendati ons and requirements were generally met, including lack of cytotoxicity, adequate produc tion of testosterone and estradiol, acceptable reproducibility (low coefficients of variation, %CV), and appropriate i nduction and inhibition with positive controls. Exceptions included that th e induction of testosterone on the QC plates in all assay runs (1.50- to 1.86-fold) was below the ≥ 2-fold Guideline criteria, and the inter-plate % CV of the solvent control (SC) for estradiol (33%) in the QC plates exceeded the Guideline criteria ( ≤ 30%).

No significant effects of metribuzin on testoste rone production (0.81- to 0.97-fold change) were
noted between 10

− 10 and 10

− 4 M. No effects of metribuzin we re noted on estradiol production at
concentrations of 10

− 10 to 10

− 6 M; however, dose-dependent increases of 1.56- and 2.20-fold
were observed for estradiol at metribuzin concentrations of 10

− 5 and 10

− 4 M, respectively.

Based on hormone responses in each of the inde pendent runs, metribuzin treatment resulted in statistically significant and repr oducible increases in estradiol production, but did not result in statistically significant and reproducible alterations in testosterone production.

Uterotrophic Assay (OCSPP 890.1600)

In an Uterotrophic Assay (MRID 48617907) con ducted to screen for potential estrogenic activity, metribuzin (94.4%, Batch # K285067) in 0.5% methylcellulose 400 + 0.4% Tween 80 was administered daily via oral gavage to groups of 6 immature female, Sprague-Dawley rats at dose levels of 0 (vehicle), 10, 40, or 120 mg/kg/day on post-natal days (PND) 19-21. A positive control group was treated with 17 α -ethynyl estradiol (EE) in corn oi l by gavage at a dose level ofMETRIBUZIN FINAL

Page 38 of 49 1 μg/kg/day. All animals were terminated and necropsied on PND 22 approximately 24 hours
after the final dose to determine wet and blotted ut erine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for any dose groups. Body weights in the metribuzin treated groups were comparable to the controls throughout the study; however, the b ody weight gains for Days 1-3 were decreased in all metribuzin treated groups. The body wei ght gains were decreased (p<0.01) by 33, 40, and 46% at 10, 40, and 120 mg/kg/day, respectively. Uterine weights in th e metribuzin treated groups were comparable to the controls. Abso lute wet and blotted uterus weights for the EE group were increased (p<0.01) by 109% and 110%, respectively, as expected.

The doses tested were determined to be ade quate based on the dose-dependent decreases in body weight gains (33-46%).

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

Page 39 of 49 APPENDIX 2: Other Scientifical ly Relevant Information (OSRI)

Developmental Toxicity (Rat; OCSPP 870.3700)

In a prenatal developm ental toxicity study ( MRIDs 00163802 and 41551801 ), groups of 33 pregnant Sprague-Dawley (Crl:CD BR) rats were administered metribuzin (92.6% purity) in aqueous 0.5% (v/v) Emulphor by oral gavage at dose levels of 0, 25, 70, or 200 mg/kg/day during gestation days (GD) 6- 15. Five dams/group were sacrificed on GD 16 for measurement of serum triiodothyronine (T 3 ) and T 4 , and the thyroids were rem oved, weighed, and fixed. The remaining dams (28/group) were sacrificed on GD 20. Blood was collected for maternal T 3 and

T 4 measurements and the thyroids were collected . The abdominal and thoracic organs of all dams were examined grossly. For dams s acrificed on GD 20, the abdomen was opened, the ovaries removed, and corpora lutea were count ed. The gravid uterus was then removed, weighed, and the fetuses were collected by caesar ean section. Implantation and resorption sites
were also determined. The fetuses were examin ed for viability, sexed, and weighed. Individual placentas were also weighed. All fetuses were exam ined for gross external anomalies. One half of the fetuses were examined internally for abdom inal and thoracic organ anomalies. The fetuses were then fixed for examination of the eyes and cranium. The remaining fetuses were fixed in alcohol, eviscerated, and proce ssed for skeletal examination.

All animals survived to scheduled euthanasia. Hypoactivity and ptosis were noted in most treated animals; ataxia was observed in all mid and high dose animals. Body weight gains were decreased in all treated gr oups by 37-56% during dosing, by 10 -22% during gestation, and by 17-38% during gestation when corrected for gravid uterine weights. Food consumption was also decreased in all treated groups during dosing. Gr avid uterine weights we re decreased by 4-13%
with no difference in litter size. This observati on parallels a decrease in fetal body weights.

Thyroid weights were increased (p<0.05) by ↑ 57% on GD 16 and by ↑ 33% on GD 20 at the high dose. On GD 16, T 4 levels were decreased by 49% and 84% at the mid and high dose, respectively; however, on GD 20, T 4 levels were increased by 26% and 48% at the mid and high dose, respectively. The levels of T 3 were reduced on GD 16 and 20 at the high dose, but differences did not attain significance (wide variability was apparent).

No treatment-related changes were seen in preg nancy rate, number of corpora lutea, mean number of implantations/litter, post implantation loss, early or late resorptions, number of live fetuses/litter, fetal sex ratio, or so ft tissue abnormalities at any dose.

Fetal body weights (male, female, and combined) were decreased (p<0.05) by 8-16% at the mid and high dose; female fetal weights were de creased (p<0.05) by 5% at the low dose. Furthermore, reduced or unossified skull bones, ribs, vertebrae, sternebrae, pelvic bones, and appendages were observed in fetuses from the high-dose group. There was a delay in
ossification of several skelet al elements for the high-dose group, which was statisticallyMETRIBUZIN FINAL

Page 40 of 49 significant for both the fetal and li tter incidence. The delay in ossification corresponds to the reduction in fetal weight for th e high-dose group and should be c onsidered further evidence of developmental toxicity.

The maternal LOAEL is 25 mg/kg/day, base d on decreased body weight gains and food consumption. The maternal NOAEL was not observed. The developmental LOAEL is 200 mg/kg/day, based on decreased fe tal body weights and reduced ossification. The developmental NOAEL is 70 mg/kg/day.

Developmental Toxicity (Rabbit; OCSPP 870.3700)

In a developmental toxicity study ( MRIDs 41249201, 1989; and 41835501, 1991 ), groups of 17 pregnant American Dutch rabbits were administ ered metribuzin (92.7% purity) in aqueous 0.5% (w/v) carboxymethylcellulose + 0.4% (w/v) Tween -80 by oral gavage at dose levels of 0 (vehicle), 10, 30, or 85 mg/kg/day during GD 6-18. On GD 28, all surviving does were sacrificed, and the abdomen was opened, ovaries were excised, corpora lutea counted, and pregnancy determined. The uterus was remove d intact and weighed, then opened, and fetuses and resorptions removed, and each implant note d. Abdominal and thoracic viscera were examined in maternal rabbits and any gross anatomical changes recorded. E ach fetus was examined for viability, then blotted dry, wei ghed, and subjected to a complete external examination. Following external examination, fetu ses were individually id entified and subjected to a complete internal examina tion of the abdominal and thoracic viscera. Skin was removed from the head for purposes of examining the eyes , and a transverse sect ion was made through the skull posterior to the coronal suture through the cerebrum. Following internal examination, fetuses were eviscerated, fixed, skinned, and processed for skeletal examination.

During the dosing period, one rabbit each from the 10, 30, and 85 mg/kg dose groups died on GD 27, 17, and 16, respectively. The deaths did not appear to-be related to treatment with the test article, but apparently th e result of either dosing trau ma or systemic infection.

Administration of metribuzin to pregnant rabbits resulted in ma ternal toxicity at 85 mg/kg, as evidenced by decreased body weight gain during dosing ( ↓ 58%), and decreased food consumption and food efficiency during GD 7-19. There were no apparent test article related gross pathological changes in mate rnal rabbits at any dose level. No treatment-related changes were seen in pregnancy rate, number of corpora lutea, mean number of implantations/litter, post implantation loss, resorptions/dam, number of live fetuses/litter, or fetal sex ratio at any dose.

Nonspecific external changes unrel ated to treatment were observed in all fetuses with external abnormalities, including control fetuses. These in cluded umbilical hernia (1 control fetus), and missing phalanges (2 fetuses from 85 mg/kg/day dose group). No treatment related visceral abnormalities were observed in any fetus from an y dose group. Skeletal abnormalities in fetuses were determined within the laboratory historical incidence of the skeletal abnormalities and wereMETRIBUZIN FINAL

Page 41 of 49 not significant by litter incidence, with the exception of irregular spinous process of the scapula (0 controls vs. 5 treated) in the 85 mg/kg dose group.

The maternal LOAEL is 30 mg/kg/day, based on decreased body weight gains on days 18-28. The maternal NOAEL is 10 mg/kg/day. The de velopmental LOAEL is 85 mg/kg/day, based on increased incidence of irregular spinous proce ss of the scapula. The developmental NOAEL is 30 mg/kg/day.

Two-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a 2-generation reproduction study in rats ( MRIDs 40838401 ), groups of 30 (Crl:CD SD) BR rats/sex were administered metribuzin (92.6% pu rity) in the diet at dos e levels of 0, 30, 150 or 750 ppm (equivalent to 0, 1.5, 7.5, or 37.5 mg/kg/day) for 2 generati ons. P animals were treated for 10 weeks prior to mating and continued th roughout lactation until lactation day (LD) 21, when they were sacrificed. F 1 pups selected for mating (for F 2 offspring) received the treatment diet for ten weeks prior to mating. The F 1 males were sacrificed when the F 2 pups were born,
and the F 1 females were sacrificed at LD 21. The F 2 pups were culled at PND 4 and the remaining pups were sacrificed on PND 21. Ten randomly selected P and F 1 rats/sex/dose group were bled via cardiac puncture to obtain samples for analysis of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) activities. Furthermore, the vagina, uterus, cervix, ovaries, testes, epididymides, semi nal vesicles, prostate, pituitary, liver and any tissue with significant pathology in the P and F 1 parental animals were removed and saved. The tissues from control and high dose animals along with any low and mid dose tissues with gross lesions were examined histologically. All liver s from low- and mid-dose animals and prostate glands from low- and mi d-dose males of the P generation were examined.

At 750 ppm, body weight gains were decr eased (p<0.05) by 9-11% in the P and F 1 males during treatment, and by 22-35% in the P and F 1 females during premating. Food consumption was also decreased (p<0.05) in these animal s during the same periods (males ↓ 8-9%; females ↓ 7-10%). During gestation, body weight gains were decr eased (p<0.05) by 11-14% in the 150 and 750 F 1 females, but in a non-dose-dependent manner. No other systemic effects were noted.

No effect of the test compound was noted on the measured parameters of reproductive performance (male or female fertility indices, gestation index, gestati on length, lactation index, parturition, or fetal se x ratio) of the P or F 1 adults.

In the offspring, lower (p<0.05) median pup body weights on PND 21 and body weight gains from PND 0-21 were noted in the 750 ppm F 1 pups. The F 2 pups showed a similar pattern with decreased (p<0.05) median body weights on PND 14 and 21 and body weight gains from PND 0- 21 in both the 150 and 750 ppm groups. It was noted that these changes were very small and that no changes in body weight gain were seen until PND 14 when pups began to consume the diet.METRIBUZIN FINAL

Page 42 of 49 No treatment related necropsy findi ngs were noted in the P and F 1 parents, or F 1 and F 2 stillborn
or culled neonates. No treatment related effect s were noted in microscopic examination of the reproductive organs, pituitary, or tissues with gross pathology. Since the investigators noted elevations of GGT activity in F 1 females, livers were examined microscopically and a dose-related increase in hypertr ophy of hepatocytes of the cent rilobular and midzonal regions
were noted with the most relevant increases in high dose males, and mid and high dose females. No other biologically releva nt observations were noted.

The LOAEL for parental toxicity is 150 ppm (e quivalent to 7.5 mg/kg/ day), based on decreased body weight gain and hypertrophy of the hepatocy tes. The NOAEL for parental toxicity is 30 ppm (equivalent to 1.5 mg /kg/day). The LOAEL for offspring toxicity is 150 ppm (equivalent to 7.5 mg/kg/day), based on decreased median bod y weights and body weight gains. The NOAEL for offspring toxicity is 30 ppm (equivalent to 1.5 mg/kg/day). The LOAEL for reproductive toxicity was not observed. The NOAEL for reproduc tive toxicity is 750 ppm (equivalent to 37.5 mg/kg/day).

Chronic Toxicity/Carcinoge nicity (Rat; OCSPP 870.4300)

In a chronic toxicity /carcinogenicity study ( MRID 42672501, 1993 ), groups of 50 Fischer [CDF(F-344)/BR] rats/sex were administered metri buzin (93.0% purity) in the diet at dose levels of 0, 30, 300, or 900 ppm (equivalent to 0/0, 1.3/1.6, 13.8/17.7, and 42.253.6 mg/kg/day in males/females) for 104 weeks. An additional 20 rats/sex/dose level were administered metribuzin as above at 0 or 900 ppm, and an add itional 10 rats/sex/dose le vel were treated at 30 or 300 ppm, both for 52 weeks. All rats sacrificed in extremis or at study termination were killed by carbon dioxide asphyxiation and subjected to gross necropsy. At sacrifice the following organs were weighed: adrenals, brain, heart, kidneys, liver, lu ngs, spleen, testes, and ovaries.

There were no treatment-related effects on mort ality, hematological parameters. Absolute body weights were decreased (p< 0.05) in the 900 ppm males ( ↓ 3-7%), and in the 300 and 900 ppm females ( ↓ 3-13%), beginning at Week 13 and c ontinuing through Week 104. Cumulative body weight gains were decreased at 300 and 900 ppm as follows: Week 13- ↓ 7-17% in males, ↓ 12- 30% in females; Week 52- ↓ 4-11% in males, ↓ 14-26% in females; and Week 104- ↓ 8-19% in males, ↓ 12-26% in females. Decreases in body wei ght gain exceeded th e decreases in food consumption at the 300 and 900 ppm dose levels, s upporting a conclusion of test article related toxicity. At Week 104, an increase in cornea l neovascularization was observed in the 300 and 900 ppm males.

The most biologically relevant change in serum chemistry involved increases in levels of T 4 and
decreases in levels of T 3 . In general, an increase in T 4 levels and a decrease in T 3 levels could be observed at 30 and 300 ppm for male and female ra ts, but this trend did not continue at the 900 ppm dose. In addition, it was difficult to identif y a potential mode of ac tion for metribuzin in this respect, as TSH levels were not measured. Although the changes in T 4 and T 3 occurred atMETRIBUZIN FINAL

Page 43 of 49 the lowest dose level (30 ppm), these were not co nsidered biologically rele vant by the registrant, as other systemic effects were not observed at this dose.

No treatment-related changes were se en in absolute or relative wei ghts of the testes, ovaries, or adrenal glands. At Week 52, terminal body wei ghts were decreased (p<0.05) by 9% in the 900 ppm females; at Week 104, terminal body weight s were decreased (p<0.05) by 10% in the 900 ppm males and by 6% and 11% in the 300 and 900 ppm females, respectively. At Week 52, absolute thyroid weights were increased (p<0.05) in the 300 ( ↑ 21%) and 900 ( ↑ 32%) ppm males; female thyroid weights were unaffected by treatm ent. At Week 104, absolute thyroid weights were increased by 24% in the 900 ppm male s and by 18% and 13% in the 300 and 900 ppm females, respectively. The increases were s upported by microscopic evidence of follicular cell hyperplasia in male rats at 300 a nd 900 ppm. Absolute liver weights were increased (p<0.05) in the 300 ( ↑ 8%) and 900 ( ↑ 12%) males at Week 52, but were si milar to controls at Week 104. Absolute kidney weights were increased (p<0.05) by 13% at 900 ppm in bot h sexes at Week 104.

Observations at gross necropsy included increas ed size of the thyroid, increased incidence of ovarian cysts, exophthalmos, enlarged adrenals, discolored zone in th e liver, and epididymal masses. However, none of these observations achi eved significance, and there did not appear to be any microscopic support for these changes, except for thyroid.

No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, vagina, cervix, clitoral, mammary, preputial, adrenal or pituitary glands. Thyroid follicular cell hyperplasi a (minimal, bilateral, diffuse) were seen in males at 300 (40%) and 900 (55%) ppm at Week 52 and at 900 ppm (76%) at Week 104. This lesion was characterized as an increase in diameter of the centrally located thyroid gland follicle and an increase in follicular epithelial cell height.

There was no evidence of carcinogenicity in the study. The NOAEL is 30 ppm (<1.3/1.6 mg/kg/day, M/F). The LOAEL is 300 ppm (13.8/17.7 mg/kg/da y, M/F) based on decreased body weight gains in the females, increased thyroid weights in male s, and increased liver weights in males and females. Thyroid is considered a target organ for metribuzin in this study.

Carcinogenicity (Mouse; OCSPP 870.4200)

In a mouse carcinogenicity study ( MRID 00087795, 1981 ), groups of 50 CD-1 mice/sex were administered metribuzin (92.9% purity) in the diet at dos e levels of 0, 200, 800, or 3200 ppm (equivalent to 0/0, 25/35, 111/139, and 438/567 mg/ kg/day in males/females) for 24 months.

There were no treatment-related effects observe d on mortality, body weights, or gross pathology.

It was stated that the hemoglobi n and hematocrit values were be low the expected range for the 3200 ppm female mice, and concluded that these re sults were treatment-related. Absolute liver weights were increased (p<0.05) by 47% in the 3200 ppm females; absolute kidney weights wereMETRIBUZIN FINAL

Page 44 of 49 also increased (p<0.05) by 13% in these animals. No treatment-related changes in absolute weights were seen in the test es, ovaries or adrenal glands.

Mice sacrificed at the re mainder or at termination of the study were reported to have gross lesions which were varied and most frequently found in the liver and lung. Histological diagnoses of non-neoplastic lesi ons included glandular hyperplasi a and chronic inflammation of the gastric mucosa, and amyloidosis. It was stated that these lesions were spontaneous in nature, and were not compound or dose related. No trea tment-related histopat hological lesions were seen in the testes, seminal vesicles, prostate, ovaries, uterus, cervix, mammary, thyroid, adrenal or pituitary glands.

There was no evidence of carcinogenicity in the study. The NOAEL is 800 ppm (111/139 mg/kg/day, M/F), and the LOAEL is 3200 ppm (438/567 mg/kg/day, M/F) based on increased liver weights and decreased hematological parameters.

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

In a chronic toxicity study ( MRID 00061260 ), groups of four beagle dogs/sex were administered metribuzin (99.5% purity) in the diet at dose le vels of 0, 25, 100, or 1500 ppm (equivalent to 0/0, 0.82/0.84, 3.44/3.56 and 55.65/55.30 mg/kg/day in males/females). After 24 months, all surviving dogs were euthanized by exsanguination. The inner organs were grossly examined and the weights of the thyroid gland, heart, lung, liver, spleen, pancreas , kidneys, adrenal glands and gonads were measured. Histopath ological examinations were made on the following tissues: brain, pituitary gland, eyes, thyr oid gland, salivary glands, cervi cal lymph nodes, tonsils, thymus, heart, aorta, lung, diaphragm, liver, gall bl adder, spleen, pancreas, esophagus, stomach, intestines, kidneys, adrenal glands, urinary bladder, testicles/ovaries, prostate, uterus, skeletal muscle, sciatic nerve, bone marrow, femur, and mammary gland (females).

After 2 weeks of feeding, the 1500 ppm group animal s appeared weakened with dull and bristly coats; feed was frequently refused. Mortality was high in the 1500 ppm test group (2 male and 2 female deaths), while only one other death occu rred (in the 25 ppm test gr oup) after 1 year. An additional 2 deaths (1 male and 1 female) occu rred in the 1500 ppm group after 2 years. All deaths were attributed to pneumonia, except for the male in the 1500 ppm group after 2 years
which died of severe malnutrition.

The 1500 ppm test group showed slightly redu ced food intake and ga ined significantly less weight than the controls, but after approximately 65 weeks only 1 male and 1 female survived in the 1500 ppm test group and the female showed normal weight gain. Furthermore, significant differences in the hematological, and liver a nd kidney function tests were observed at the 1500 ppm dose level.METRIBUZIN FINAL

Page 45 of 49 No treatment-related changes were seen in absolute or relative weights of testes, ovaries, and adrenal glands. An apparent in crease in relative thyroid weight was noted in both of the surviving 1500 ppm animals ( ↑ 38% male; ↑ 20% female), and also a very slight increase in the relative heart weight of the male. The 1500 ppm male also had increased relative liver and pancreas weight. An increase in relative spleen weight occurred in all males of the 3 test groups compared to controls, and the females showed an increase in relative pancreas weight while there was a very slight dose related d ecrease in relative kidney weight.

The animals that died early in the study with a diagnosed cause of death of severe pneumonia exhibited the expected histological signs (small foci of lympho-histocytic infiltrates, perivascular and focal lympho-histocytic inf iltrates). Relating histologic al findings to hematological observations, the changes observed in hematologi cal tests may be related to changes in blood forming organs as evidenced by the increase in megakaryocytes observed in the bone marrow and spleen of the high dose group. No treatment-related histopathological lesions were seen in the testes, prostate, ovaries, uterus, mammary gl ands, thyroid, adrenal or pituitary glands.

The results of the clinic al tests along with th e increase in liver weights of the 1500 ppm test group may indicate liver damage. Further evidence is the observation of parenchymal necrosis,
interstitial infiltration and other changes not observed in the control or the 25 and 100 ppm test groups. The other histological findings were either si ngular in nature or occurred in equal incidence in all treatment groups and consiste d mostly of lymphocytic infiltration due to inflammation ("non-specific'') involving the hear t, lungs, liver, lymph nodes, kidneys, testes, prostate, adrenals and thyroid gland.

The NOAEL for this study is 100 ppm (3.44/3.56 mg /kg/day, M/F), and the LOAEL is 1500 ppm (55.65/55.3 mg/kg/day, M/F), based on mortality, decreased body wei ght, increased liver weight and changes in clinical chemistry parameters.

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

In a subchronic oral toxicity study ( MRID 45621001 ), 4 beagle dogs/sex/group were administered metribuzin (93.9- 94.8% purity) daily via the diet at doses of 0, 75, 300, or 1200 ppm (equivalent to 0/0, 2.0/1.9, 8.4/8.6, and 26.2/30.2 mg/kg/day in males/females) for 13 weeks (90 days).

One 1200 ppm male and one 1200 ppm female were sacrificed on Days 59 and 58, respectively, due to marked body weight decrease. The male lost 2.3 kg from Day 42 to 56, and severely reduced food consumption was noted beginning on Day 43. The female lost 1.4 kg from Day 42 to 56, and severely reduced food consump tion was observed beginning on Day 52. These animals were thin and pale with pale yellow ging iva, and the female was considered to have an unthrifty appearance. In the male, there were incr eases (at one or more intervals) in ALP, AST, ALT, GGT, TBILI, and bile acids, and very lo w UDP-GT levels. In the female, there were onlyMETRIBUZIN FINAL

Page 46 of 49 increases in ALT and very low UDP-GT. Ther e was substantial eviden ce of anemia in both animals. Microscopic lesions specific to th ese two animals included: bone marrow hypoplasia and hyperplasia; myocardial de generation and hemorrhage; infl ammation of liver, kidney, and small intestine; unusual extramedullary hematopoi esis in liver and spleen; unusual pigmentation in the liver; pronounced involution of the thymus; and renal hemorrhage.

No adverse, treatment-related effect was observed during the neur ological evaluations, urinalysis, ophthalmoscopic examinations, and organ weights (including endocrine organs). Aside from findings noted in the moribund sacrif iced animals, no adverse, treatment-related
effects were observed on clini cal signs, food consumption, an d clinical chemistry. No pathological evidence of an adverse e ffect was noted on the endocrine organs.

Aside from the moribund sacrificed animals, decreased body weights and body weight gains were noted in each female treated group; al though, the dose-dependency of the effect was unclear. Findings indicative of slight anemia we re evident in the 1200 ppm males at termination. Erythrocytes, hemoglobin, and hematocrit were decreased (p<0.05) by 22-25% on Day 87. The values for these parameters were also outside th e reported historical control ranges. Equivocal findings were observed in the live r at 1200 ppm. Discoloration of the liver was noticed in two males, one of which was moribund sacrificed (vs 0 controls). Excluding the moribund sacrifices, congestion was noted in 1 male (vs 0 controls), and pigmentati on was observed in both sexes (1/4 treated vs 0/4 controls).

No treatment-related changes were seen in ab solute or relative we ights of the testes, epididymides, ovaries, uterus, thyroid, adrenal or pituitary glands. No treatment-related histopathological lesions were seen in the testes, epidi dymides, prostate, ovaries, uterus, mammary, thyroid, adrenal or pituitary glands.

The LOAEL is 1200 ppm (equivalent to 26.2/30.2 mg/kg/day, M/F), based on findings in the moribund sacrificed animals (clinical signs, de creased body weight, body weight gain, and food consumption, changes in blood chemistry, anemia , and microscopic findings). The NOAEL is 300 ppm (equivalent to 8.4/8.6 mg/kg/day in males/females).

Subchronic Dermal Toxicity (Rabbit; OCSPP 870.3200)

In a subchronic 21-day dermal toxicity study ( MRID 43970701, 1989 ), groups of five New Zealand (HC:NZW) rabbits/sex were exposed derma lly to technical metri buzin (94.0% purity) in 2% (v/v) Cremophor EL at 0, 40, 200 or 1000 mg/kg/day for 6 hours daily over a 15-day period (5 days/week for 3 weeks). An additional five rabbits/sex were exposed as above to 0 or 1000 mg/kg/day and used for post-application evaluati ons on Day 28 and 36. Two or three days after the final treatment, or afte r the post administration observa tion period, the rabbits were euthanized by exsanguination under anesthesia a nd necropsied. The brain, thyroid, heart, lung, liver, kidneys, adrenal glands, spleen, testes and ovaries were weighed. The following organs orMETRIBUZIN FINAL

Page 47 of 49 organ samples were fixed in Bouin's fluid for histological examination: untreated and treated skin, thyroid gland, heart, lung, liver, kidneys, spleen, adrenal glands, testes, epididymides, ovaries, uterus and sternum. Or gan samples of kidneys were also fixed in formol-calcium for histopathological examination.

No mortalities or treatment-related clinical observations were reported. Additionally, no treatment-related dermal effects were noted (macr oscopic examination of the skin and skinfold thickness). No treatment-related effects on body weights or food consumption were observed. No treatment related hematology effects were note d; however in clinical chemistry evaluations,
serum cholesterol levels were incr eased in the 1000 mg/kg/day males ( ↑ 31%) and females ( ↑ 32%). T 3 was decreased (p<0.05) by 23% in the 1000 mg/kg/day males, and T 4 was increased (p<0.05) in the 1000 mg/kg/day males ( ↑ 33%) and females ( ↑ 53%). Additionally liver chemistry analyses demonstrated increased (p<0.05) N -demethylase activity and cytochrome P-450 in the 1000 mg/kg/day males; females showed slight, non- significant increases in similar parameters.

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, thyroid and adrenal glands. Ther e was a slight decrease in 1 000 mg/kg/day male absolute and relative adrenal weights and a d ecrease in 1000 mg/kg/day female absolute and relative spleen weights, but there was no corroborating path ology reported. Absolute and relative kidney weights were reduced in all treated females, but in a non-dose-related manner. No treatmentrelated histopathological lesions were seen in the testes, epididymides, ovaries, uterus, thyroid and adrenal glands.

In post-administration animals, n o treatment related effects during Days 28 and 36 were noted in mortality, clinical observations, body weight an d body weight gains, hematological or liver enzyme parameters, gross or histopathological ob servations. The high dose females consumed slightly less food than the contro l animals. No effects on clinical chemistry parameters were noted in the 1000 mg/kg/day males, while the fema les had decreased alkaline phosphatase levels,
increased urea and choles terol and decreased T 3 along with decreased abso lute and relative heart weights (no related pathology).

The systemic NOAEL is 200 mg/kg/day, and the LOAEL is 1000 mg/kg/day based on increased plasma cholesterol and liver enzyme activities. The dermal NOAEL is ≥ 1000 mg/kg/day (HDT).METRIBUZIN FINAL

Page 48 of 49 Avian Reproduction Toxicity (Duck; OCSPP 850.2300)

In an avian reproduction toxicity study ( MRID 43860501, 1995 ), groups of 30 Mallard ducks ( Anas platyrhynchos ) were fed diets containing metr ibuzin (93.5% purity) at nominal concentrations of 0, 70, 210, or 420 ppm (analy tical concentrations were 0, 61, 185, and 368 ppm) for 20 weeks (10 weeks pre-laying and 10 weeks during egg laying). The birds were
housed with one male and one female per pen, with 15 pens per dose group.

No adult mortalities were noted. Enlarged livers and/or spleens were found at necropsy in each treatment group, but this was not considered tr eatment-related. No effects on reproduction or survival were seen up to and including the highe st treatment level, 420 pp m. Additionally, there were no significant differences in any of the te sted endpoints with metribuzin at the tested concentrations compared to contro ls. A LOEAC was not identified.

Avian Reproduction Toxicity (Quail; OCSPP 850.2300)

In another avian reprodu ction toxicity study ( MRID 43926601 ), groups of 36 quail ( Colinus virginianus ) were fed diets containing metribuzin (93.5% purity) at nominal concentrations of 0, 70, 210, or 420 ppm (analytical concentrations were 0, 62, 196, and 385 ppm) for 22 weeks (12 weeks pre-laying and 10 weeks durin g egg laying). The birds were housed with one male and one female per pen, with 18 pens per dose group.

One control female died after ge tting its head caught in the pen. No treatment-related effects were found at necropsy. No treatment-related eff ects on reproduction or survival were seen up to and including the highest treatment level, 420 ppm. Day 14 hatchling survivor weight was significantly reduced from the cont rols at all metribuzin concentrations tested; however, there was no dose-response relationship for this effect, and the diffe rences between the treatment means and the control mean were slight ( ↓ 6-8%) and not considered trea tment-related. Terminal weights of the 70 ppm females were significan tly reduced; however, this was considered incidental. All other tested endpoints with metr ibuzin at the tested c oncentrations were not significantly different from contro ls. A LOAEC was not identified.

Fish Early Life-Stage Study (Rainbow trout, OCSPP 850.1400)

Rainbow trout embryos and larvae were exposed to metribuzin in an early life-stage study at mean concentrations of 3.0, 5.7, 11.7, 23.5, and 48 mg a.i./L (MRID 42447801, 1992). The study was initiated with newly fertilized em bryos and exposure continued for 95 days. According to the reviewer’s analysis, survival at test termination was significantly reduced at the highest concentration tested, and there was no effect on hatchabil ity. Length was significantly
reduced at all test concentrations and wei ght was significantly re duced at 11.7 and 23.5 mg
a.i./L.METRIBUZIN FINAL

Page 49 of 49 APPENDIX 3: References Not Utiliz ed in the Metribuzin 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 Metribuzin 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.

Modra, H., Haluzova I., Blahova, J., Havelkova, M., Kruzikova, K., Mikula, P., and Svobodova, Z. (2008). Effects of subchronic me tribuzin exposure on common carp ( Cyprinus carpio ).
Neuro Endocrinol Lett . 29(5): 669-74.