UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

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

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 57
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 57
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: METRIBUZIN 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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METRIBUZIN 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 Relevant Information (OSRI)......................................5 

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

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

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

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

2. Effects on Androgen Pathway..................................................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-Estrogenic Pathway for Metribuzin ..................................10 

 Table 3. Androgenic/Anti-Androgenic 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...........................49 

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METRIBUZIN 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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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METRIBUZIN FINAL 

v 

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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METRIBUZIN FINAL 

vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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METRIBUZIN FINAL 

Page 1 of 49 

Executive Summary 

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

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

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

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

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

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

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

in this weight of evidence (WoE) assessment. 

In determining whether a pesticide chemical interacts with E, A or T hormone pathways, the 

number and type of effects induced, the magnitude and pattern of responses observed across 

studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, whether or not endocrine-related responses occurred at dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On August 27, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the 

Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) 

conducted a weight-of-evidence analysis (WoE) of the potential interaction 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, metribuzin was negative in the in vitro estrogen receptor (ER) 

binding and ER transactivation assay (ERTA) and was equivocal in the aromatase assay. It was 

positive in the steroidogenesis 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 evidence for potential estrogen-related effects in the uterotrophic 

and female pubertal assays. Additionally, there were no treatment-related estrogen-related effects 

observed in females in the fish short-term reproduction assay (FSTRA). Furthermore, there were 

no estrogen-related effects noted in any of the mammalian or wildlife Part 158 studies. 

For the androgen pathway, metribuzin 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 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 the mammalian or wildlife 

Part 158 studies. 

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METRIBUZIN FINAL 

Page 2 of 49 

There was convincing evidence of potential interaction 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, colloid 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 mammalian Part 158 studies. In a rat chronic 

toxicity/carcinogenicity study, dose-dependent increases in absolute thyroid weights and 

significant increases in serum T4 and decreases in serum T3 were observed, as well as thyroid 

follicular cell hyperplasia in males. Finally, in the rabbit subchronic dermal toxicity study, 

serum T3 levels were decreased in males and T4 levels were increased in both sexes without 

concomitant changes in thyroid 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 males. 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 histopathological lesions (males). 

In the Part 158 mammalian chronic toxicity/carcinogenicity study, metribuzin 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 and thyroid follicular cell hyperplasia. 

For metribuzin, the point of departure (POD) of 1.3 mg/kg/ is based on increased thyroid weights 

and thyroid follicular cell hyperplasia seen at 13.8 mg/kg/day (LOAEL) in a chronic 

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 and 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 toxicity in the young. 

Consequently, a special thyroid assay in pregnant animals, fetuses, 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 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 may 

Page 10 of 57
METRIBUZIN 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 (surrogates for aquatic-phase amphibians) is not 

available, as the available data indicated adverse effects at all concentrations 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 requested under registration review for metribuzin. However, as potential 

effects on the thyroid pathway were indicated, to add to the understanding of potential thyroid 

effects, the EDSP Tier 2 Larval Amphibian growth and Development Assay (LAGDA) is 

recommended since there is no chronic aquatic amphibian toxicity data available. 

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

duck are available for metribuzin. In both species, no effects on reproductive parameters were 

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

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

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

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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 potential of chemicals to interact with the estrogen 

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

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

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

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

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

this weight of evidence (WoE) assessment. 

In determining whether a pesticide chemical interacts with E, A or T hormone pathways, the 

number and type of effects induced, the magnitude and pattern of responses observed across 

studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, whether or not endocrine-related responses occurred at dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On August 27, 2014, 2014, the Tier 1 Assay Weight of Evidence Review Committee 

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

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

of 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). 

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 Kow). Based on available 

data, the primary routes of degradation of metribuzin 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 (T1/2's of 106 and 112 days, respectively). 

The available information considered in determining 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 toxicity studies and other published articles. These 

data are summarized in Sections III.A through III.C. An analysis of the data submitted to the 

agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in 

Section IV. The EDSP Tier 2 study recommendations are presented in Section V. 

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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 believed to be relevant to one or more of the Tier 1 

assays were submitted as OSRI by the Test Order recipients and/or the public. This included 

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

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

Report of the Endocrine Disruptor Review Team on 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 considered but not utilized for the WoE analysis are listed in 

Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

above. 

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

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

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 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 include published literature studies as well as 

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

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

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

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

endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit 

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

conditions 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 systemic 

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Page 7 of 49 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

forms of toxicity (e.g., 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/chemical properties and its 

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

the E, A or T pathway. Adequately addressing these three main questions is fundamental to the 

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

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

conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) 

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

needed and why. Assessing the need for additional data is based on a case-by-case analysis 

which took all available toxicity data into account. 

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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 biologically relevant with regard to treatment-related effect which 

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

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

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

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays submitted in response to the agency’s test order for 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) 

ERα 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) 

Page 16 of 57
METRIBUZIN FINAL 

Page 9 of 49 

B. Effects on Hypothalamic-Pituitary-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 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 reported in the table 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 ERα transcriptional activation assay (ERTA). 

However, metribuzin was equivocal in the aromatase assay, and positive in the steroidogenesis 

assay. Aromatase activity was reduced to 63% of control at 10−3

 M. Since the average lowest 

portion of the inhibition curve was between 50-75% of control activity, metribuzin was classified 

as equivocal in this assay. In the steroidogenesis assay, dose-dependent increases in estradiol 

production of 1.56- and 2.20-fold were observed at metribuzin concentrations of 10−5 and 10−4 M, 

respectively. 

In the in vivo Tier 1 assays, metribuzin was negative 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 metribuzin. An increase in prevalence and severity for 

incidence of egg debris in the ovaries was observed with increasing concentration as well as an 

increase in the incident of oocyte atresia in the highest treatment, but the pathologist’s report did 

not consider any finding to be treatment-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 were 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 toxicity 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 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. 

Page 17 of 57
METRIBUZIN FINAL 

Page 10 of 49 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Metribuzin 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metribuzin1Study Type/ Literature Citation 

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 

SiTii

EDSP Tier 1 Assay 

ER Binding (MRID 48617902) N ERTA (MRID 48617903) N Aromatase (MRID 48617901) E Steroidogenesis (MRID 48617906) P Uterotrophic (MRID 48617907) N 

(L, 

Female Pubertal Rat (MRID 48672203) N N N N N N 

(

FSTRA (MRID 48672202) NE N N N N N 

OSRI 

Developmental Toxicity (Rat; MRIDs 00163802, 41551801) 

 N6 

(M

Developmental Toxicity (Rabbit; MRIDs 41249201, 41835501) 

 N 

Two-Generation Reproduction Toxicity (Rat; MRID 40838401) 

 NE NE N NE N N 

(M
METRIBUZIN FINAL 

Page 11 of 49 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Metribuzin 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metribuzin1Study Type/ Literature Citation 

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 

SiTii

Chronic Toxicity/ Carcinogenicity (Rat; MRID 42672501) 

 NE N N NE 

(M

Carcinogenicity (Mouse; MRID 00087795) 

 NE N N NE 

Chronic Toxicity (Dog; MRID 00061260) 

 NE N N NE 

Subchronic Toxicity (Dog; MRID 45621001) 

 N N N N 

Subchronic Dermal Toxicity (Rabbit; MRID 43970701) 

 NE N N NE 

Avian Reproduction Toxicity (Duck; MRID 43860501) 

N 

Avian Reproduction Toxicity (Quail; MRID 43926601) 

N 

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

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in weight are absolute unless otherwise indicated. 

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

this column. For details see Section IV. A 

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

details see Section IV. A 
METRIBUZIN FINAL 

Page 12 of 49 

4. 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 m

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) o

throughout the study. Absolute, adjusted, and relative liver weights were increased (p≤0.01) by 21-22% at the high dose; relative 

increased (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 dosincreased 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 in the study, all three doses showed overt toxicity. P Positive findings N Negative findings (in vitro)/No effect (in vivo) E Equivocal response NE Not examined 
METRIBUZIN FINAL 

Page 13 of 49 

2. Effects on Androgen Pathway 

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

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

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

This table also includes HPG-relevant findings from data evaluated as OSRI. This table also 

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

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

considered further in the WOE assessment. 

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 assay. 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 effects observed in the FSTRA. 

There were no androgenic or anti-androgenic effects noted in any of the Part 158 mammalian or 

wildlife studies. 

 

 

Page 21 of 57
METRIBUZIN FINAL 

Page 14 of 49 

Table 3: Androgenic/Anti-Androgenic Pathway for Metribuzin 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Metribuzin1 Study Type/ Literature Citation 

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 

EDSP Tier 1 Assay 

AR Binding (MRID 48617905) N Steroidogenesis (MRID 48617906) N Hershberger (MRID 48617904) NE N Male Pubertal Rat (MRID 48672203) N N N N N N N N FSTRA (MRID 48672202) NE N P5 N N 

OSRI 

Two-Generation Reproduction Toxicity (Rat; MRID 40838401) 

 NE N NE N NE NE N 

Chronic Toxicity/ Carcinogenicity (Rat; MRID 42672501) 

 N N NE N NE 

Carcinogenicity (Mouse; MRID 00087795) 

 N N NE NE NE 

Chronic Toxicity (Dog; MRID 00061260) 

 N N NE NE NE 

Subchronic Toxicity (Dog; MRID 45621001) 

 N N N N N 
METRIBUZIN FINAL 

Page 15 of 49 

Table 3: Androgenic/Anti-Androgenic Pathway for Metribuzin 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Metribuzin1 Study Type/ Literature Citation 

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 

Subchronic Dermal Toxicity (Rabbit; MRID 43970701) 

 N N NE N NE 

Avian Reproduction Toxicity (Duck; MRID 43860501) 

 N 

Avian Reproduction Toxicity (Quail; MRID 43926601) 

 N 

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

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in 

weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral proprostate (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 O

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 

details see Section IV. A 

4. At the high dose (120 mg/kg/day), increased salivation was observed in all rats, and one animal in this group had reduced motor a

respiration on Day 13 and another rat had ocular discharge on Days 20-25. Absolute, adjusted (for body weight on PND 21), and were increased (p≤0.01) by 21-23%. Serum total protein was increased (p≤0.01) by 5%. At necropsy, enlarged liver was observe

were increased numbers of mitoses in 7/11 examined livers, as well as minimal centrilobular hepatocellular hypertrophy (4/11 rat

increased salivation was observed on several occasions throughout the study in 5/15 rats. Relative liver weights were increased (p

enlarged liver was observed in 1/15 rats. 

5. Higher incidence of advanced maturation in the testes at the highest treatment group 

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

NE Not examined 
METRIBUZIN FINAL 

Page 16 of 49 

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

The current EDSP Tier 1 battery does not have a specific in vitro assay to detect chemicals with 

the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it 

does include three in vivo assays that provide data to detect changes in the HPT axis, i.e., the 

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

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

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

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

further in the WOE assessment. 

In the Tier 1 male and female pubertal assays, 120 mg/kg/day metribuzin 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 histopathology showed minimal to slight 

follicular cell hypertrophy, colloid alteration, and an increased number of mitoses (2/15 males). 

Serum T4 levels were decreased by 43-72% in the 60 and 120 mg/kg/day males and by 44% in 

the 120 mg/kg/day females, while serum TSH levels were significantly increased by 109 and 

236% in the 60 and 120 mg/kg/day in males, respectively. 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 thyroid 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 consumption). 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 weight increases were associated with decreases in serum T4 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 T3 and T4 levels were measured at 3, 6, 12, 

18 and 24 months. Significant increases in serum T4 (10 -57%) and decreases in serum T3 (10 – 

30%) were observed at all dose levels in males and females treated either one or two years 

beginning at 91 days, and continuing until study terminations at 2 years. However, the changes 

in T4 and T3 were not consistently dose related at the various time intervals. 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 weights 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 females 

Page 24 of 57
METRIBUZIN FINAL 

Page 17 of 49 

(↑18% and ↑13%, respectively). Thyroid follicular cell hyperplasia (minimal, bilateral, diffuse) 

was seen in males at the mid (40%) and high (55%) 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 cholesterol levels were noted in the mid- and highdose males and females throughout most of the intervals examined. 

In a dog chronic toxicity study, metribuzin treatment at the high dose (ca. 55 mg/kg/day) caused 

thyroid weight increases of 38% in a surviving male and 20% in a surviving female. There were 

no corresponding changes in thyroid histology or serum T4 and T3 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 T3 levels were decreased (p<0.05) 

by 23% in the high-dose males (1000 mg/kg/day), and T4 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. 

Page 25 of 57
METRIBUZIN FINAL 

Page 18 of 49 

Table 4. Thyroid Pathway for Metribuzin.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Metribuzin1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, SVL) Systemic Toxicity Obsserved2

Overt Toxicity Observed3

EDSP Tier 1 Assay 

Male Pubertal Rat 

(MRID 48672203) 

↑45-46% 

(H) 

P4 

(H) 

↓43%, 

↓72% 

(L, H) 

↑109%, 

↑236% 

(L, H) 

N P5

 

(L, H) N 

Female Pubertal Rat 

(MRID 48672203) 

↑15% 

(H) N ↓44% (H) N N 

P6

 

(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) 

N7 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 

P8

(M, H) 

♂

P9

 

(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=Medium-low treatment, M=Medium treatment, MH=Medium high 

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

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

otherwise indicated. 

Page 26 of 57
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 was 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 several 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. Absolute, 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 T3 and T4 were measured at 3, 6, 12, 18 and 24 months. Beginning at 91 days, statistically significant 

decreases in serum levels of T4 and significant decreases in T3 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 available 

Page 27 of 57
METRIBUZIN FINAL 

Page 20 of 49 

IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight 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 recommendations regarding Tier 2 testing.. The results of the Tier 1 assays 

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

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

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

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

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

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

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

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

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

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

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

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

whether or not the responses are mediated through an endocrine pathway, therefore nonendocrine toxic responses (including but not limited to mortality or body weight changes) are 

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

associated with weight of evidence evaluation of the results of the Tire 1 assays stated that “In 

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

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

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

related to toxicity in 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/concentration to be considered overtly toxic. 

However, in other instances, more than one parameter was needed to determine overt toxicity. 

Effects which were considered to be signs of systemic toxicity were generally less severe forms 

of toxicity (e.g., changes in organ weights or blood chemistry).

Page 28 of 57
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1. Tier 1 in vivo Assays 

In the female pubertal rat assay, systemic toxicity was evident at both the low (60 mg/kg/day) 

and high (120 mg/kg/day) doses, based on clinical signs of increased salivation in 6/15 low-dose 

rats and 7/15 high-dose rats on several occasions 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 based on clinical signs of increased salivation in 5/15 low-dose 

rats on several occasions throughout the study in all high-dose rats. Additionally, one high dose 

rat had reduced motor activity and labored respiration. At the low dose, relative liver weights 

were increased (p≤0.01) by 12%, and enlarged liver was observed 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 observed in 11/15 rats. In addition, 

there were increased numbers of mitoses in 7/11 examined livers, as well as minimal 

centrilobular hepatocellular hypertrophy (4/11 rats). 

In the uterotrophic assay, 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. 

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

2. OSRI 

In a prenatal developmental toxicity 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 treated 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 high dose (750 ppm) in P and F1 males during treatment, and by 22-

35% in the P and F1 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, 

Page 29 of 57
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 

F1 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 from PND 0-21 were noted in the high dose F1

pups. The F2 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/carcinogenicity 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 consumption at the 300 and 900 ppm dose levels, 

supporting a conclusion of test article related toxicity. At Week 52, terminal body weights were 

decreased (p<0.05) by 9% in the 900 ppm females; 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 weights were also increased in the 300 (↑8%) and 900 

(↑12%) males at Week 52, but were similar to controls 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 liver weights were increased (p<0.05) by 47% in the 

high dose (3200 ppm) females and absolute kidney 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 refused. 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 hematological, 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 sacrificed 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. Both dogs were thin and pale with pale yellow 

gingiva, with substantial evidence of anemia, and the female was considered to have an unthrifty 

appearance. There were increases (at one or more intervals) in ALP, AST, ALT, GGT, TBILI, 

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Page 23 of 49 

and bile acids, and very low UDP-GT levels. Microscopic lesions included: bone marrow 

hypoplasia and hyperplasia; myocardial degeneration and hemorrhage; inflammation of liver, 

kidney and small intestine; unusual extramedullary hematopoiesis in liver and spleen; unusual 

pigmentation in the liver; pronounced involution of the thymus; and renal hemorrhage. 

Additionally, in the males, findings indicative of slight anemia were evident at the high dose at 

termination. Discoloration of the liver was noticed 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 controls). In the females, pigmentation was observed in the liver in 

the high dose (1/4 treated 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 absolute 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 reproduction studies at dietary 

concentrations up to 420 ppm. In an early life-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 steroidogenesis 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 evidence for potential estrogen-related effects in 

the uterotrophic and female pubertal assays. Additionally, there were no treatment-related 

estrogen-related effects observed in females in the FSTRA. Furthermore, there were no 

estrogen-related effects noted in any of the mammalian or wildlife Part 158 studies. 

C. Androgen Pathway 

Metribuzin 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 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 the mammalian or wildlife Part 158 studies. 

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D. Thyroid Pathway 

There was convincing evidence of potential interaction 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 exhibited enlarged thyroids and minimal to slight follicular cell 

hypertrophy, colloid alteration, and an increased 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 mammalian Part 158 studies. In a rat chronic 

toxicity/carcinogenicity study, dose-dependent increases in absolute thyroid weights and 

significant increases in serum T4 and decreases in serum T3 were observed, as well as thyroid 

follicular cell hyperplasia in males. Finally, in the rabbit subchronic dermal toxicity study, 

serum T3 levels were decreased in males and T4 levels were increased in both sexes without 

concomitant changes in thyroid weight or histopathology. 

E. Conclusions 

There was no convincing evidence for potential interaction with the estrogen or androgen 

pathways. The available data indicate that metribuzin potentially 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 chronic 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 and 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 toxicity 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 of 

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METRIBUZIN FINAL 

Page 25 of 49 

this special study is to generate data to establish a POD (i.e., NOAELs and LOAELs or 

benchmark doses) that may be used for human health risk assessment. The POD from this special 

study would address the concern for the potential ability of metribuzin to disrupt thyroid function 

in pregnant females and in the fetus and newborn. 

Currently, a definitive chronic endpoint for fish (surrogates for aquatic-phase amphibians) is not 

available, as the available data indicated adverse effects at all concentrations 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 requested under registration review for metribuzin. However, as potential 

effects on the thyroid pathway were indicated, to add to the understanding of potential thyroid 

effects, the EDSP Tier 2 Larval Amphibian growth and Development Assay (LAGDA) is 

recommended since there is no chronic aquatic amphibian toxicity data available. 

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

duck are available for metribuzin. In both species, no effects on reproductive parameters were 

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

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

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

 

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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. Laboratory 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) Teratology study in the rabbit with Sencor technical 

(metribuzin). Miles, Inc., Toxicology Department, 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 with 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 (common 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 toxicity of SENCOR technical to the waterflea 

(Daphnia magna) under flow-through conditions. Miles Incorporated, Stilwell, KS. Laboratory 

Report No. 103248, completion date not provided. MRID 42447802. Unpublished. 

Gagliano, G, and Bowers, L (1993) Chronic toxicity of SENCOR technical to the waterflea 

(Daphnia magna) under flow-through conditions. Miles Incorporated, Stilwell, KS. Laboratory 

Report No. 105023, completion date not provided. MRID 42731201. Unpublished. 

Page 34 of 57
METRIBUZIN FINAL 

Page 27 of 49 

Hancock, G (1995a) Effect of technical SENCOR on mallard reproduction. Bayer Corporation, 

Agriculture Division, Stillwell, KS. Laboratory Report No. 106983, October 6, 1995. MRID 

43860501. Unpublished. 

Hancock, G (1995b) Effect of technical SENCOR on northern bobwhite reproduction. Bayer 

Corporation, Agriculture Division, Stillwell, KS. Laboratory Report No. 106984, February 1, 

1996. MRID 43926601. Unpublished. 

Hayes, R (1981) Metribuzin® (Sencor): oncogenicity study in mice. Mobay Chemical Corp., 

Kansas City, Mo. Laboratory Study No. CDL:246397-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 Corporation, 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 Laboratories Inc., Toxicology Department, 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 (metribuzin) 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: chronic toxicity studies on dogs (two-year feeding 

experiment). Bayer AG, Department of Toxicology, Wuppertal, West Germany. Laboratory 

Report No. 4887 & 41814, September 24, 1974. MRID 00061260. Unpublished. 

Porter, M. Jasty, and Hartnagel, R (1988) A two-generation reproduction study in rats with 

Sencor technical (metribuzin). Miles, Inc., Toxicology Department, Elkhart, 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. Laboratory 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. Laboratory Study No.: SA 11213, December 2, 2011. 

MRID 48617902. Unpublished. 

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Sheet, L and Hall, T. (2012). Weight of evidence to evaluate the potential for metribuzin to affect 

estrogen, androgen or thyroid systems. Bayer CropScience, Research 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 the Hershberger bioassay. Bayer S.A.S., Sophia 

Antipolis Cedex, France. Laboratory Study 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 Endocrine 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 Disruptor Screening Program – Weight of Evidence: Evaluating 

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

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

Protection Agency, Washington, DC. 

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

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

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

Agency, Washington, DC. 

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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) Binding 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 saturation binding experiment was conducted to 

demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was 

functioning with appropriate affinity for the radio-labeled reference androgen (R1881) prior to 

routinely conducting AR competitive binding experiments. 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 weak positive control and R1881 as the ligand 

reference standard. 

In the saturation binding experiment, the mean maximum binding capacity (Bmax) was 14 

fmol/100 μg protein and the mean dissociation constant (Kd) was 1.01 nM. The Kd obtained 

from Run #2 (0.59 nM) was below recommended values for saturation binding, but the Kds from 

the other two runs were within the recommended range (0.685 to 1.57 nM), and the Scatchard 

plots for the three Saturation assays were generally linear. The specific binding reached a 

plateau, and non-specific binding (NSB) was <20% of total binding (4.6-12.6%). 

In the competitive binding experiment, specific [3

H]-R1881 binding was >75% at all 

concentrations of metribuzin tested in all three runs. An IC50 and relative binding affinity (RBA) 

could not be calculated for metribuzin. 

The estimated average log IC50s 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 Kd from run 2, all performance criteria were met.

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

Androgen Receptor Binding Assay. 

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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 recombinant aromatase and tritiated androstenedione 

(1-β [3H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in dimethyl sulfoxide (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 control, a background activity control, a positive control series (10−10 to 

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

metribuzin series (10−10 to 10−3 M) with three repetitions per concentration. 

For the positive control (4-OH ASDN), aromatase 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 IC50 and %CV. For 

4-OH ASDN, the estimated log IC50 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 full activity control. 

For metribuzin, aromatase activity averaged 0.318±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, aromatase 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, indicating 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) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (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 was conducted to demonstrate that the ER in the 

rat uterine cytosol was present in reasonable numbers and was functioning with appropriate 

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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]-17β-estradiol (1 nM) in the presence 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 17β-estradiol as the natural ligand reference material. 

For the saturation binding experiment, the appropriate protein concentration for the saturation 

binding assay was determined to be 0.5 mg/mL as it bound 25-35% of the [3

H]-17β-estradiol. 

The mean Kd for [3

H]-17β-estradiol was 0.070 nM, and the estimated Bmax was 0.034 nM for the 

prepared rat uterine cytosol. 

For the competitive binding experiment, the data for metribuzin did not fit the non-linear 

regression model and percent binding was >75% at all concentrations tested (≤10−3 M) in all 

three runs. Therefore, a mean log IC50 and relative binding affinity (RBA) could not be 

calculated for metribuzin. 

The estimated mean log IC50 for 17β-estradiol and 19-norethindrone was −8.8 M and −5.0 M, 

respectively, and the mean RBA for 19-norethindrone was 0.017% compared to the natural 

ligand. Several performance criteria were not met for 17β-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 concentration range that fully defined the top of the curve and the 

maximum percent binding was within approximately 25 percentage points of the lowest 

concentration of the estradiol standard. Each of the three runs was classified as not interactive. 

The lowest average percent radiolabeled estradiol 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. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48617903), hERα-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, 

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in two independent runs conducted on separate days. The experiments were performed using 96-

well plates and each metribuzin concentration was tested in triplicate (3 wells/plate). Cells were 

exposed to the test agent for 23±1 h to induce reporter (luciferase) gene products. Luciferase 

expression in response to activation of the estrogen 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 compounds demonstrated that the 

assay was functioning properly. The only deviation was a weak positive response in one run to 

corticosterone (negative control compound), otherwise the performance criteria were met for 

17β-estradiol and corticosterone. For 17α-estradiol (weak agonist), the Log PC10 was lower than 

the expected range in the first assay, as was the Hill Slope. For 17α-methyltestosterone (very 

weak agonist), the Log PC50 was lower than the expected range in run 2, and the Log PC10 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 RPCMax for metribuzin 

was 0.7% for the first run and 14.1% for the second run, and the associated PCMax was 10−4

 M 

for the first run and 10−6 M for the second run. Although the RPCMax ≥PC10 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 considered negative for estrogen receptor 

transcriptional activation in this test system. 

Fish Short Term Reproduction Assay (FSTRA; OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 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 group; ca. 6.5 months old) were exposed to 

metribuzin (94.4% purity) at nominal concentrations 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 maintained 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 and 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 behavior or changes in appearance (specifically 

color/banding, ovipositor appearance, or size of dorsal nape pad) in the negative control or 

treatment groups. There were no significant differences (p>0.05) in male or female body weight 

or length compared to the negative control. 

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In the negative control replicates, spawning occurred at least every 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 was not significantly reduced (p>0.05) in any 

treatment group compared to the negative control. There was no significant difference (p>0.05) 

in fertilization success between the negative 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 negative control. Plasma vitellogenin (VTG) was not significantly 

different (p>0.05) in males or females. For males, it is noted that at the 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 advanced 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 exposure could not be excluded, the high treatment level did have 

increased female mortality, and therefore, the maturation 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 histopathological results that could be correlated with exposure to 

the test substance were noted. 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48617904) screening for androgenic activity, metribuzin (94.4% 

a.i., batch/lot K285067) in an aqueous solution 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 testosterone propionate (TP) by 

subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, metribuzin in an aqueous solution of 0.5% 

methylcellulose 400 + 0.4% Tween 80 was also administered daily via oral gavage to groups of 

58-day old, castrated male Sprague Dawley rats (6/group) at dose levels 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 positive 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. 

Page 41 of 57
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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 daily. The animals were dosed 

for 10 consecutive days and terminated approximately 24 hours after the final dose 

administration. At necropsy, the five androgen-dependent tissues were collected and weighed.

In the androgen agonist assay, 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 treatment-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 individual CVs in the control 

and high dose treatment groups exceeded the guideline performance criteria values. 

In the anti-androgen assay, body weights 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 follows: 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 adequate 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 weight changes were not seen in two or more of the five androgen 

sensitive issue weights. Metribuzin was negative 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 were 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 (T4) and thyroid stimulating hormone (TSH) levels were 

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determined using enzyme immunoassay kits. Adrenals, liver, pituitary, thyroid, and urogenital 

organ weights were recorded and microscopic examination 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 cycling; on the weights of kidneys, pituitary, adrenals, ovaries, or 

uterus (wet or blotted); or on levels of creatinine, blood urea nitrogen, total protein, total 

bilirubin, aspartate aminotransferase, or alkaline phosphatase. There were no treatment-related 

histopathological findings in the thyroid gland, ovaries, uterus, or kidneys. 

At 120 mg/kg/day, increased salivation was observed 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 significant; NS) compared to the controls. 

Overall body weight gains were comparable to the control. Absolute, adjusted (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 observed in 1/15 rats and enlarged liver was 

observed in 5/15 rats in this group; there were increased numbers of mitoses in 4/5 examined 

livers. Serum T4 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 salivation was observed in 6/15 rats on several occasions throughout 

the study. Final body weight and body weight gain, 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 comparable to the controls at this dose level. At necropsy, enlarged 

liver was observed in 1/15 rats and increased numbers of mitoses were observed in this liver. 

Mean TSH levels were increased (NS) by 29% at 60 mg/kg/day; however, serum T4 and alanine 

aminotransferase levels in this group were comparable to the controls. 

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

study. 

Male Pubertal Assay (OCSPP 890.1500) 

In a Male 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) 23 to 53. Animals were examined for preputial separation (PPS) 

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daily beginning on PND 30 and age and weight at day of attainment were recorded. Following 

sacrifice on PND 53, total serum testosterone, thyroxine (T4), and thyroid stimulating hormone 

(TSH) levels were analyzed using enzyme immunoassay kits. Adrenals, liver, pituitary, thyroid, 

and urogenital organ weights were recorded and 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 weight gain, or age and 

body weight at PPS; on the weights of kidneys, pituitary, adrenals, seminal vesicles plus 

coagulating gland (with or without fluid), ventral prostate, dorsolateral prostate, levator anibulbocavernosus muscle complex (LABC), epididymides, 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 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 and another rat had ocular discharge on Days 20-25. All rats in 

this group achieved PPS before study termination. Absolute, adjusted (for body weight on PND 

21), and relative liver weights 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 T4 levels were 

decreased (p≤0.01) by 72% in rats dosed at 120 mg/kg/day; 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 and enlarged liver was observed in 11/15 rats. 

Minimal to slight follicular cell hypertrophy and colloid alteration were observed in the thyroid 

glands; an increased number 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 hepatocellular hypertrophy (4/11 rats). 

At 60 mg/kg/day, increased salivation 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 were comparable to the controls 

at this dose level. Serum T4 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 metribuzin selected for this study (120 mg/kg/day) was adequate based on the 

increased liver weights observed at this dose level. 

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Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48617906), H295R cells cultured in vitro in 24-well plates 

were incubated with metribuzin (94.4% purity, Lot # AE F055208-01-13) at serial log 

concentrations of 10−10 to 10−4

 M for 48 hours in triplicate in three 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 metribuzin. A Quality Control (QC) plate was run concurrently with each 

independent run of the test chemical plate to demonstrate that the assay responded properly to 

positive control agents at two concentration levels. The positive controls included a known 

inhibitor (prochloraz) and a known inducer (forskolin) of estradiol and testosterone production. 

Precipitation of the test compound, and cytotoxicity, were not observed at any concentration 

tested (≤10−4

 M). Guideline acceptability recommendations and requirements were generally 

met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptable 

reproducibility (low coefficients of variation, %CV), and appropriate induction and inhibition 

with positive controls. Exceptions included that the 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 testosterone production (0.81- to 0.97-fold change) were 

noted between 10−10 and 10−4

 M. No effects of metribuzin were 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 independent runs, metribuzin treatment resulted in 

statistically significant and reproducible 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) conducted 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 17α-ethynyl estradiol (EE) in corn oil by gavage at a dose level of 

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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 uterine 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 body weight gains for Days 1-3 were decreased 

in all metribuzin treated groups. The body weight gains were decreased (p<0.01) by 33, 40, and 

46% at 10, 40, and 120 mg/kg/day, respectively. Uterine weights in the metribuzin treated 

groups were comparable to the controls. Absolute 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 adequate 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. 

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APPENDIX 2: Other Scientifically Relevant Information (OSRI) 

Developmental Toxicity (Rat; OCSPP 870.3700) 

In a prenatal developmental 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 (T3) and T4, and the thyroids were removed, weighed, and fixed. The 

remaining dams (28/group) were sacrificed on GD 20. Blood was collected for maternal T3 and 

T4 measurements and the thyroids were collected. The abdominal and thoracic organs of all 

dams were examined grossly. For dams sacrificed on GD 20, the abdomen was opened, the 

ovaries removed, and corpora lutea were counted. The gravid uterus was then removed, 

weighed, and the fetuses were collected by caesarean section. Implantation and resorption sites 

were also determined. The fetuses were examined for viability, sexed, and weighed. Individual 

placentas were also weighed. All fetuses were examined for gross external anomalies. One half 

of the fetuses were examined internally for abdominal 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 processed 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 groups 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. Gravid uterine weights were decreased by 4-13% 

with no difference in litter size. This observation 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, T4 levels were decreased by 49% and 84% at the mid and high dose, 

respectively; however, on GD 20, T4 levels were increased by 26% and 48% at the mid and high 

dose, respectively. The levels of T3 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 pregnancy 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 soft 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 decreased (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 skeletal elements for the high-dose group, which was statistically 

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significant for both the fetal and litter incidence. The delay in ossification corresponds to the 

reduction in fetal weight for the high-dose group and should be considered further evidence of 

developmental toxicity. 

The maternal LOAEL is 25 mg/kg/day, based 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 fetal 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 administered 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 removed intact and weighed, then opened, and fetuses 

and resorptions removed, and each implant noted. Abdominal and thoracic viscera were 

examined in maternal rabbits and any gross anatomical changes recorded. Each fetus was 

examined for viability, then blotted dry, weighed, and subjected to a complete external 

examination. Following external examination, fetuses were individually identified and subjected 

to a complete internal examination of the abdominal and thoracic viscera. Skin was removed 

from the head for purposes of examining the eyes, and a transverse section 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 the result of either dosing trauma or systemic infection. 

Administration of metribuzin to pregnant rabbits resulted in maternal 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 maternal 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 unrelated to treatment were observed in all fetuses with external 

abnormalities, including control fetuses. These included 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 any dose group. Skeletal abnormalities in fetuses 

were determined within the laboratory historical incidence of the skeletal abnormalities and were 

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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 developmental LOAEL is 85 mg/kg/day, based on 

increased incidence of irregular spinous process 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% purity) in the diet at dose levels of 0, 30, 150 or 

750 ppm (equivalent to 0, 1.5, 7.5, or 37.5 mg/kg/day) for 2 generations. P animals were treated 

for 10 weeks prior to mating and continued throughout lactation until lactation day (LD) 21, 

when they were sacrificed. F1 pups selected for mating (for F2 offspring) received the treatment 

diet for ten weeks prior to mating. The F1 males were sacrificed when the F2 pups were born, 

and the F1 females were sacrificed at LD 21. The F2 pups were culled at PND 4 and the 

remaining pups were sacrificed on PND 21. Ten randomly selected P and F1 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, seminal vesicles, prostate, 

pituitary, liver and any tissue with significant pathology in the P and F1 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 livers from low- and mid-dose 

animals and prostate glands from low- and mid-dose males of the P generation were examined. 

At 750 ppm, body weight gains were decreased (p<0.05) by 9-11% in the P and F1 males during 

treatment, and by 22-35% in the P and F1 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, body weight gains were decreased (p<0.05) by 11-14% in the 150 and 750 F1

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, gestation length, lactation index, 

parturition, or fetal sex ratio) of the P or F1 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 F1 pups. The F2 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. 

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No treatment related necropsy findings were noted in the P and F1 parents, or F1 and F2 stillborn 

or culled neonates. No treatment related effects were noted in microscopic examination of the 

reproductive organs, pituitary, or tissues with gross pathology. Since the investigators noted 

elevations of GGT activity in F1 females, livers were examined microscopically and a 

dose-related increase in hypertrophy of hepatocytes of the centrilobular and midzonal regions 

were noted with the most relevant increases in high dose males, and mid and high dose females. 

No other biologically relevant observations were noted. 

The LOAEL for parental toxicity is 150 ppm (equivalent to 7.5 mg/kg/day), based on decreased 

body weight gain and hypertrophy of the hepatocytes. 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 body 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 reproductive toxicity is 750 ppm (equivalent to 37.5 

mg/kg/day). 

Chronic Toxicity/Carcinogenicity (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 metribuzin (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 additional 10 rats/sex/dose level 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, lungs, spleen, testes, and ovaries. 

There were no treatment-related effects on mortality, 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 continuing 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 

consumption at the 300 and 900 ppm dose levels, supporting a conclusion of test article related 

toxicity. At Week 104, an increase in corneal neovascularization was observed in the 300 and 

900 ppm males. 

The most biologically relevant change in serum chemistry involved increases in levels of T4 and 

decreases in levels of T3. In general, an increase in T4 levels and a decrease in T3 levels could be 

observed at 30 and 300 ppm for male and female rats, but this trend did not continue at the 900 

ppm dose. In addition, it was difficult to identify a potential mode of action for metribuzin in 

this respect, as TSH levels were not measured. Although the changes in T4 and T3 occurred at 

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the lowest dose level (30 ppm), these were not considered biologically relevant by the registrant, 

as other systemic effects were not observed at this dose. 

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, or 

adrenal glands. At Week 52, terminal body weights were decreased (p<0.05) by 9% in the 900 

ppm females; 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. 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 treatment. At Week 104, absolute thyroid weights 

were increased by 24% in the 900 ppm males and by 18% and 13% in the 300 and 900 ppm 

females, respectively. The increases were supported by microscopic evidence of follicular cell 

hyperplasia in male rats at 300 and 900 ppm. Absolute liver weights were increased (p<0.05) in 

the 300 (↑8%) and 900 (↑12%) males at Week 52, but were similar to controls at Week 104. 

Absolute kidney weights were increased (p<0.05) by 13% at 900 ppm in both sexes at Week 104. 

Observations at gross necropsy included increased size of the thyroid, increased incidence of 

ovarian cysts, exophthalmos, enlarged adrenals, discolored zone in the liver, and epididymal 

masses. However, none of these observations achieved 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 hyperplasia (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/day, M/F) based on decreased 

body weight gains in the females, increased thyroid weights in males, 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 dose 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 observed on mortality, body weights, or gross pathology. 

It was stated that the hemoglobin and hematocrit values were below the expected range for the 

3200 ppm female mice, and concluded that these results were treatment-related. Absolute liver 

weights were increased (p<0.05) by 47% in the 3200 ppm females; absolute kidney weights were 

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also increased (p<0.05) by 13% in these animals. No treatment-related changes in absolute 

weights were seen in the testes, ovaries or adrenal glands. 

Mice sacrificed at the remainder 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 lesions included glandular hyperplasia 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 treatment-related histopathological 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 levels 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. Histopathological examinations were made on the following tissues: 

brain, pituitary gland, eyes, thyroid gland, salivary glands, cervical lymph nodes, tonsils, thymus, 

heart, aorta, lung, diaphragm, liver, gall bladder, 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 animals 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 occurred (in the 25 ppm test group) after 1 year. An 

additional 2 deaths (1 male and 1 female) occurred 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 reduced food intake and gained 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 and kidney function tests were observed at the 1500 

ppm dose level. 

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No treatment-related changes were seen in absolute or relative weights of testes, ovaries, and 

adrenal glands. An apparent increase 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 decrease 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 infiltrates). Relating histological findings to hematological 

observations, the changes observed in hematological 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 glands, thyroid, adrenal or pituitary glands. 

The results of the clinical tests along with the 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 singular in nature or occurred in equal 

incidence in all treatment groups and consisted mostly of lymphocytic infiltration due to 

inflammation ("non-specific'') involving the heart, 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 weight, 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 consumption was observed beginning on Day 52. These 

animals were thin and pale with pale yellow gingiva, and the female was considered to have an 

unthrifty appearance. In the male, there were increases (at one or more intervals) in ALP, AST, 

ALT, GGT, TBILI, and bile acids, and very low UDP-GT levels. In the female, there were only 

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increases in ALT and very low UDP-GT. There was substantial evidence of anemia in both 

animals. Microscopic lesions specific to these two animals included: bone marrow hypoplasia 

and hyperplasia; myocardial degeneration and hemorrhage; inflammation of liver, kidney, and 

small intestine; unusual extramedullary hematopoiesis 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 neurological evaluations, 

urinalysis, ophthalmoscopic examinations, and organ weights (including endocrine organs). 

Aside from findings noted in the moribund sacrificed animals, no adverse, treatment-related 

effects were observed on clinical signs, food consumption, and clinical chemistry. No 

pathological evidence of an adverse effect 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; although, the dose-dependency of the effect was 

unclear. Findings indicative of slight anemia were 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 the reported historical control ranges. Equivocal 

findings were observed in the liver 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 pigmentation was observed in both sexes 

(1/4 treated vs 0/4 controls). 

No treatment-related changes were seen in absolute or relative weights of the testes, 

epididymides, ovaries, uterus, thyroid, adrenal or pituitary glands. No treatment-related 

histopathological lesions were seen in the testes, epididymides, 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, decreased 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 dermally to technical metribuzin (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 evaluations on Day 28 and 36. Two or three days after 

the final treatment, or after the post administration observation period, the rabbits were 

euthanized by exsanguination under anesthesia and necropsied. The brain, thyroid, heart, lung, 

liver, kidneys, adrenal glands, spleen, testes and ovaries were weighed. The following organs or 

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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. Organ 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 (macroscopic 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 noted; however in clinical chemistry evaluations, 

serum cholesterol levels were increased in the 1000 mg/kg/day males (↑31%) and females 

(↑32%). T3 was decreased (p<0.05) by 23% in the 1000 mg/kg/day males, and T4 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. There was a slight decrease in 1000 mg/kg/day male absolute and 

relative adrenal weights and a decrease in 1000 mg/kg/day female absolute and relative spleen 

weights, but there was no corroborating pathology 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, no treatment related effects during Days 28 and 36 were noted in 

mortality, clinical observations, body weight and body weight gains, hematological or liver 

enzyme parameters, gross or histopathological observations. The high dose females consumed 

slightly less food than the control animals. No effects on clinical chemistry parameters were 

noted in the 1000 mg/kg/day males, while the females had decreased alkaline phosphatase levels, 

increased urea and cholesterol and decreased T3 along with decreased absolute 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). 

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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 metribuzin (93.5% purity) at nominal 

concentrations of 0, 70, 210, or 420 ppm (analytical 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 treatment-related. No effects on reproduction or 

survival were seen up to and including the highest treatment level, 420 ppm. Additionally, there 

were no significant differences in any of the tested endpoints with metribuzin at the tested 

concentrations compared to controls. A LOEAC was not identified. 

Avian Reproduction Toxicity (Quail; OCSPP 850.2300) 

In another avian reproduction 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 during 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 getting its head caught in the pen. No treatment-related effects 

were found at necropsy. No treatment-related effects 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 controls at all metribuzin concentrations tested; however, there 

was no dose-response relationship for this effect, and the differences between the treatment 

means and the control mean were slight (↓6-8%) and not considered treatment-related. Terminal 

weights of the 70 ppm females were significantly reduced; however, this was considered 

incidental. All other tested endpoints with metribuzin at the tested concentrations were not 

significantly different from controls. 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 embryos 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 hatchability. Length was significantly 

reduced at all test concentrations and weight was significantly reduced at 11.7 and 23.5 mg 

a.i./L. 

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APPENDIX 3: References Not Utilized in the Metribuzin WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant information (OSRI) was provided in the Report of the Endocrine 

Disruptor Review Team (USEPA, 2010). 

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies 

previously submitted to the agency for registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE 

analysis for Metribuzin but were not utilized due to one or more of the following reasons: 1) the 

article was not available in English; 2) the compound of interest was not used in the study; 3) the 

test material was not adequately described; 4) a formulated end-use product or mixture of 

chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 

6) the experimental conditions were not adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) 

the study dealt only with non-EDSP assay development; 12) no specific endocrine-related 

endpoints were assessed in the study; and 13) the study contained only data on invertebrates. 

Modra, H., Haluzova I., Blahova, J., Havelkova, M., Kruzikova, K., Mikula, P., and Svobodova, 

Z. (2008). Effects of subchronic metribuzin exposure on common carp (Cyprinus carpio). 

Neuro Endocrinol Lett. 29(5): 669-74. 

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