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 69
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 69
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: METOLACHLOR 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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METOLACHLOR 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..................................................14 

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

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

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

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

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

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

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

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

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

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

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

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Metolachlor..............................................................8 

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

 Table 3. Androgenic/Anti-Androgenic Pathway for Metolachlor ..............................15 

 Table 4. Thyroid Pathway for Metolachlor .........................................................19 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................31 

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

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

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METOLACHLOR 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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METOLACHLOR 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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METOLACHLOR 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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METOLACHLOR FINAL 

Page 1 of 61 

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 metolachlor 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 July 30, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) 

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

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

metolachlor 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). 

Metolachlor (CGA-24705) is a racemic (1:1) mixture of enantiomers (four stereoisomers). This 

document included data from metolachlor and S-metolachlor (an isomer-enriched formula). 

For the estrogen pathway, there was no convincing evidence of potential interaction with the 

estrogen pathway. Metalochlor was negative in the in vitro estrogen receptor (ER) binding and 

the ER transactivation assay (ERTA), gave an equivocal response in the aromatase assay 

(reduction in activity observed (down to 42% at 10−3

 M), but potential issue with protein 

(receptor) denaturation and/or compound solubility) and induced a marginal increase (1.2- to 1.3-

fold) in estradiol production in the steroidogenesis assay. No treatment-related estrogenic effects 

were seen in the in vivo uterotrophic or female pubertal assays. In the fish short-term 

reproduction assay (FSTRA), the only effects seen, which were at the high concentration, 

occurred in the presence of overt toxicity. There were no treatment-related effects on female 

reproductive parameters in the existing metolachlor Part 158 mammalian studies. While there 

was a 6% increase in cracked eggs (as eggs not cracked/eggs laid) in one Part 158 avian study, 

there were no complementary effects in this study, the other avian reproduction study, or the 

other in vivo studies. 

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METOLACHLOR FINAL 

Page 2 of 61 

Metolachlor was positive for androgen receptor (AR) binding but did not alter testosterone 

production in the in vitro steroidogenesis assay. In the Hershberger assay (dosed 

subcutaneously), there was evidence for an anti-androgenic at the high dose (500 mg/kg/day). 

However, in the Tier 1 male pubertal assay and Part 158 mammalian studies in which animals 

were dosed orally, no androgen-related effects were seen. In the FSTRA, the only effect in the 

absence of overt toxicity was a reported significant decrease in fat pad score in the mid-treatment 

level (0.48 mg a.i./L). Overall, there is no convincing evidence for a potential interaction with 

the androgen pathway. 

There is some evidence for potential interaction of metolachlor with the thyroid pathway in 

mammals based on effects on thyroid histopathology in both sexes in the pubertal assays. These 

effects are further supported by alterations in T4 levels in females and thyroid stimulating 

hormone (TSH) levels in males. In the amphibian metamorphosis assay (AMA), however, the 

results do not suggest a potential for thyroid interaction as there were no effects on thyroid 

histopathology or developmental stage. 

In the EDSP Tier 1 pubertal assays there was evidence for potential interaction with the thyroid 

pathway in studies conducted with adult animals, but no such data exists for potential thyroid 

toxicity in the young animals. 

For mammals, for metolachlor, the current points of departure (POD) for human health risk 

assessment is 9.7 mg/kg/day is based on decreased body weight gain in female dog at 

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

Reference Dose (RfD) for dietary exposure. A second POD of 50 mg/kg/day is based on 

increased incidence of clinical signs, decreased body weight/body weight gain, food 

consumption and food efficiency seen in maternal animals at 500 mg/kg/day (LOAEL) in a 

developmental toxicity study in rats. This POD is used for deriving the Margins of Exposure 

(MOEs) for non-dietary risk assessments. The PODs used for overall risk assessments are 

approximately 5-30-fold lower than the dose (300 mg/kg/day) that induced histopathological 

lesions of the thyroid gland in the female pubertal assay. 

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

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

thyroid toxicity in the young. However in the case of metolachlor, although no data exists for 

potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended 

since: 1) the PODs are lower than the dose that caused thyroid lesions; 2) there was no evidence 

of thyroid toxicity in the data base; 3) in subchronic and chronic toxicity studies in dogs, rats and 

mice, decreased body weight and body weight gain were the most commonly observed effects; 

5) the CTA would be tested at higher doses and would not be expected to yield a POD lower 

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METOLACHLOR FINAL 

Page 3 of 61 

than the level currently used for risk assessment; and 6) the chronic RfD and MOEs are 

protective of the thyroid effects seen in the female pubertal assay. Therefore, additional studies 

to investigate thyroid toxicity in the young are not required. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is not 

recommended for metolachlor since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

For wildlife, as there was no convincing evidence for potential interaction with estrogen, 

androgen pathway in fish or thyroid pathway in amphibians, no additional testing is 

recommended. 

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

duck are available for metolachlor. In mallard ducks no reproductive effects were observed, 

whereas in bobwhite quail an effect on a reproductive parameter was 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 metolachlor exposure. Relative to the 

EDSP, additional testing is not recommended. 

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METOLACHLOR FINAL 

Page 4 of 61 

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 metolachlor 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 July 30, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

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

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

potential interaction of metolachlor 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). 

 Metolachlor (CGA-24705) is a racemic (1:1) mixture of enantiomers (four stereoisomers); the 

(S)- enantiomer (CGA 77102, or α-metolachlor) is the active pesticidal component and the (R)-

enantiomer (CGA 77101) is inactive. In 1997, an isomer-enriched formula called S-metolachlor 

(88% S-isomer and 12% R-isomer was registered. 

Metolachlor has a vapor pressure of 3.14 x 10-5mm Hg at 25°C. The reported water solubility is 

530 mg/L at 20°C with an octanol/water partition coefficient (log Kow) of 3.13 to 3.4. The vapor 

phase is susceptible to photodegradation, and the particulate phase is susceptible to wet and dry 

deposition. Metolachlor is also susceptible to direct photolysis and aquatic biodegradation, but 

minimal biodegradation in soil. Metolachlor has an estimated whole body bioconcentration 

factor in fish ranging from <1 to 69. 

The available information considered in determining the potential interaction of metolachlor with 

the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically 

relevant information (OSRI) such as general toxicity studies and other published articles. These 

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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. 

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 requested to be 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). However, the Test Order recipient elected to 

conduct all eleven Tier 1 screening assays, so no OSRI were submitted in lieu of the Tier 1 

assays. The Agency has also conducted a more 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 hormonal 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 to characterize the main lines of evidence as well any conclusions. Finally, the last step is to 

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

above. 

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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 concordance 

or consistency (complementarity) of the responses in the individual study is evaluated. For the 

Tier 1 in vivo assays, often multiple endpoints are measured in each assay. 

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

relative degree of diagnostic utility of a 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, or T consisting of different 

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

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit 

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

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

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

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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., changes in organ weights or blood chemistry). The circumstances for 

which a dose/concentration was considered overtly toxic for a particular study are described in 

Section IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/chemical properties and its 

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

the E, A or T pathway. Adequately addressing the three main components described above is 

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

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

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

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

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

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

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

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

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

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

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

A. EDSP Tier 1 Screening Assays 

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

below in Table 1. 

Table 1. Tier 1 Screening Assays for Metolachlor 

Tier 1 Assays Test Guideline Test Order Status 

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

(MRID 48617807) 

ERα Transcriptional Activation Assay (Human 

cell line HeLa 9903) 

OSCPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID 48617808) 

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

(MRID 48617806) 

Steroidogenesis Assay (Human cell line 

H295R) 

OSCPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID 48617809) 

Aromatase Assay (human recombinant 

microsomes) OSCPP 890.1200 Requirement Satisfied (MRID 48617805) 

Uterotrophic Assay (Rat) OSCPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID 48617801) 

Hershberger Assay (Rat) OSCPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID 48617802) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied 

(MRID 48617804) 

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied 

(MRID 48617803) 

Fish Short-term Reproduction Assay OSCPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID 48617811)

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID 48617810) 

Page 16 of 69
METOLACHLOR FINAL 

Page 9 of 61 

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

1. Effects on Estrogen Pathway 

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

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

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

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

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

considered further in the WOE assessment. 

Metolachlor was negative (or not interactive) in the Tier 1 in vitro estrogen receptor (ER) 

binding assay and the ERα transcriptional activation assay. Metolachlor was also negative in an 

ERTA considered as OSRI that was conducted in Chinese hamster ovary (CHO) cells (Kojima et 

al., 2004). In the Tier 1 aromatase assay, while enzyme activity was reduced to 42% at the 

highest tested concentration (10−3

 M), the data (Hill slope) indicate a potential issue with protein 

(receptor) denaturation and/or compound solubility, and as such, the results were classified as 

equivocal. In the Tier 1 steroidogenesis assay increases (1.2- to 1.3-fold) in estradiol production 

were observed at the two highest concentrations, 10 and 100 μM. 

Metolachlor was negative in the in vivo uterotrophic assay. In the female pubertal rat assay, there 

were no differences in body weight at VO, or final body weights, in the treated groups relative to 

control. Adjusted age at VO was advanced (approximately two days) at the low dose, but not at 

the high dose. Although a statistically significant decrease in percentage of animals cycling was 

noted at the high dose (93.3%) when compared to controls (100%), this findings is not 

considered treatment-related since this represents only 1/15 animals. There was a decrease in the 

number of regularly cycling females, but there was no dose-response. In the FSTRA, there was a 

98% decrease in fecundity in the high-concentration group (4.7 mg a.i./L), but no effect on 

fertility. Female plasma vitellogenin (VTG) was decreased by 77% in the high-concentration 

along with increased incidences of oocyte atresia, granulosa cell phagocytosis, and decreased 

early and late vitellogenic oocytes. However, these effects at the high concentration occurred in 

conjunction with overt toxicity, based on decreased survival (71%) and clinical signs of toxicity 

(i.e., erratic swimming, loss of equilibrium and lack of feeding). 

In the mammalian Part 158 toxicity studies, a non-statistically significant (NS) and non-dose 

responsive increase in pituitary weights with no corroborating pituitary histopathological 

findings in all dose groups in the chronic dog study was noted. No other estrogen-related effects 

were observed in the Part 158 mammalian studies. 

In the Part 158 avian reproduction studies no treatment-related reproductive effects were 

observed in the study with mallard duck up to 800 mg/kg-diet. In a study with bobwhite quail, 

there was a treatment-related 6% increase in eggs cracked/eggs laid at the high dose (1000 

Page 17 of 69
METOLACHLOR FINAL 

Page 10 of 61 

mg/kg-diet); no other treatment-related reproductive effects or signs of parental overt or systemic 

toxicity were observed. 

In a Part 158 fathead minnow early-life cycle study, body length was decreased (significance not 

reported) ≥ 0.220 mg/L, and decreases in wet weight were observed at ≥0.110 mg/L and in dry 

weight at ≥0.056 mg/L. In another fish early-life cycle study with sheepshead minnow, there 

was a decreasing trend in length and weight at concentrations ≥2.2 mg/L, and increased mortality 

at ≥4.1 mg/L. 

Page 18 of 69
METOLACHLOR FINAL 

Page 11 of 61 

Table 2. Estrogenic/Anti-Estrogenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metolachlor1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity2\

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

EDSP Tier 1 Assay 

ER Binding (MRID 48617807) N ERTA (MRID 48617808) N Aromatase (MRID 48617805) E Steroidogenesis (MRID 48617809) P Uterotrophic (MRID 48617801) N Female Pubertal Rat (MRID 48617804) N N N N N N FSTRA (MRID 48617811) NE N N N N N 

OSRI 

ERTA (Kojima et al., 2004) N 3-Month Subchronic toxicity (Rat; MRID 43928923)

NE N N NE 

3-Month Subchronic toxicity (Rat; MRID 44775401, 44775402)

NE N N NE 
METOLACHLOR FINAL 

Page 12 of 61 

Table 2. Estrogenic/Anti-Estrogenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metolachlor1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity2\

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

3-Month Subchronic toxicity (Dog; (MRID 43928922)

NE N N NE 

6-Month Subchronic toxicity (Dog; MRID 00032174)

NE N N N 

21-day Dermal (Rabbit; MRID 41833101) 

N N NE N 

Developmental toxicity (Rat; MRID 43928925) 

 N N 

Developmental toxicity (Rat; MRID 00151941) 

 NR N 

Developmental toxicity (Rabbit; MRID 43928924) 

 NR N 

Developmental toxicity (Rabbit; MRID 00041283) 

 NR N 

Two-generation reproduction (Rat; MRID 00080897) 

NE NE N NE N 

Chronic toxicity/ carcinogenicity (Rat; MRID 00129377) 

NE N N NE 
METOLACHLOR FINAL 

Page 13 of 61 

Table 2. Estrogenic/Anti-Estrogenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metolachlor1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology

Pituitary Weight 

Estrous Cyclicity2\

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Vitellogenin (VTG) 

Carcinogenicity (Mouse; MRID, 00117597) 

N N N NR 

Chronic toxicity (Dog; MRIDs 40980701, 41164501, 42218601, 42218602) 

NE N N N 

Avian Reproduction (Duck; MRID 44015602) 

N 

Avian Reproduction (Quail; MRID 46508901) 

P5 

(H) 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High  Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated or is n

Changes in weight are absolute unless otherwise indicated. LW= Liver weight; KW= Kidney weight; GGT= gamma-glutamyl tran

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 

4. Erratic swimming, loss of equilibrium, or lethargic swimming, and increased mortality (survival ↓29%) at the high concentration (4

5. Increases in cracked eggs at the high test concentration. P Positive findings N Negative findings (in vitro)/No effect (in vivo) NE Not examined NR Not reported E Equivocal 
METOLACHLOR FINAL 

Page 14 of 61 

2. Effects on Androgen Pathway 

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

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

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

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

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

table and not considered further in the WOE assessment. 

Metolachlor was a positive binder in the Tier 1 in vitro androgen receptor (AR) binding assay. 

Metolachlor reduced competitive binding to the AR to approximately 50% at 10−3 M. However, 

metolachlor was negative for AR agonism and antagonism in an OSRI AR transcriptional 

activation (ARTA) assay conducted in CHO cells (Kojima et al., 2004). Metolachlor did not alter 

testosterone production in the Tier 1 in vitro steroidogenesis assay. 

In the Tier 1 Hershberger assay, the androgenic portion of the assay was negative. In the antiandrogenic portion of the assay, the weights for ventral prostate and LABC were decreased by 25 

and 22%, respectively, at the high dose, and LABC weights also were decreased (↓17%) in the 

mid-dose group (300 mg/kg/day). Terminal body weight decreases in the high-dose metolachlor 

group (500 mg/kg/day) were 10% and 8% in the androgenic and anti-androgenic portions of the 

assay, respectively, with concomitant decreases in body weight gains (45 and 33%, respectively), 

demonstrating that the 500 mg/kg/day dose approached a toxic dose. 

In the male pubertal rat assay, no treatment-related changes in body weights, body weight gains, 

or age and weight at PPS occurred in either treatment group (400 and 800 mg/kg/day). Liver 

weights were increased (p<0.05) by 15 and 20%, and kidney weights were increased (p<0.05) by 

11% each, in both dose groups, respectively. Mean testosterone concentration was decreased in 

a dose-related manner (↓45 and 69%), but the observed difference may be an artifact due to 

unusually high testosterone concentrations (>8.00 ng/mL) in three animals in the control group. 

No histopathological lesions were observed in the testes or epididymides. 

In the FSTRA, there was a significant 63% decrease in male tubercle score in the highconcentration (4.7 mg a.i./L), and reported significant decreases in fat pad scores in the mid-

(0.48 mg a.i./L) and high-concentration groups. There was a marginally significant (p=0.057) 

decrease of 29% in male GSI at the high concentration along with changes in gonadal 

histopathology including an increase in diploid spermatogonia in germinal epithelium. Overall 

survival was decreased 29% in the high-concentration group, and the surviving fish exhibited 

signs of clinical toxicity (erratic swimming, loss of equilibrium, lethargic swimming, and curved 

spines). 

There were no androgen-related effects observed in the Part 158 mammalian or avian studies. 

Page 22 of 69
METOLACHLOR FINAL 

Page 15 of 61 

Table 3. Androgenic/Anti-Androgenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Metolachlor1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

EDSP Tier 1 Assay 

AR Binding (MRID 48617806) P Steroidogenesis (MRID 48617809) N Hershberger (MRID 48617802) NE 

VP 

↓25% 

LABC: 

22% (H) 

Male Pubertal Rat (MRID 48617803) N N N N N N N N FSTRA (MRID 48617811) NE N N Fat pad: ↓20% (M) N 
METOLACHLOR FINAL 

Page 16 of 61 

Table 3. Androgenic/Anti-Androgenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Metolachlor1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

OSRI 

ARTA (Kojima et al., 2004) N 3-Month Subchronic toxicity (Rat; MRID 43928923)

 N N NE N NE NE 

3-Month Subchronic toxicity (Rat; MRID 44775401, 44775402)

 N N N N NE NE 

3-Month Subchronic toxicity (Dog; MRID 43928922)

 N N NE N NE NE 

6-Month Subchronic toxicity (Dog; MRID 00032174)

 N N NE NE N NE 

21-day Dermal (Rabbit; MRID 41833101) 

 N NE N NE N N 

Two-generation reproduction (Rat; MRID 00080897) 

 N N N N NE NE N 

Chronic toxicity/ carcinogenicity (Rat; MRID 00129377) 

N N NE NE NE NE 

Carcinogenicity (Mouse; MRID 00117597) 

N NR NR NR NR N 
METOLACHLOR FINAL 

Page 17 of 61 

Table 3. Androgenic/Anti-Androgenic Pathway for MetolachlorLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Metolachlor1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Age and Weight at 

PPS 

Chronic toxicity (Dog; MRIDs 40980701, 41164501, 42218601, and 42218602) 

N N N N N NE 

Avian Reproduction (Duck; MRID 44015602) 

N 

Avian Reproduction (Quail; MRID 46508901) 

 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 this parameter is not routinely evaluated or is not applicable iweight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prost

(DP), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). BW= Body weight; BWGLW= Liver weight; KW= Kidney weight 

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 

4. Body weight decreased 8% in the antagonist portion of the assay at the high dose (500 mg/kg/day; s.c. injection) with a decrease in 

33%. 

5. Erratic swimming, loss of equilibrium, or lethargic swimming, and increased mortality (survival ↓29%) at the high concentration (4

P Positive findings N Negative findings (in vitro)/No effect (in vivo) NE Not examined NR Not reported 
METOLACHLOR FINAL 

Page 18 of 61 

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

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

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

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

pubertal female and male (rat) assays, and the AMA (frog). 

The potential for metolachlor 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 

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

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

further in the WOE assessment. 

In the female pubertal rat assay with metolachlor, evidence of changes in thyroid function 

included increased (↑25%) total thyroxine (T4) in the high-dose (600 mg/kg/day) females with no 

effect on thyroid stimulating hormone (TSH) and slight changes in the thyroid histopathology. 

In the male pubertal rats serum TSH levels were increased (↑54 and 134%) in both dose groups 

(400 and 800 mg/kg/day) and slight changes in the thyroid histopathology. There no changes in 

serum T4 levels noted at these doses. There were no changes in thyroid weights in either sex. 

In the AMA, metolachlor treatment decreased Day 7 wet weight by 19%, Day 7 snout-vent 

length (SVL) by 7% and Day 21 normalized hind-limb length (HLL) by 23% at the high 

concentration (8.4 mg a.i./L), relative to the negative control. However, no treatment-related 

effects on thyroid histopathology or developmental stage were noted. 

The only effect observed in the mammalian Part 158 toxicity studies that was related to the 

thyroid pathway (HPT axis) was a non-statistically significant (NS) and non-dose responsive 

increase in pituitary weights in all dose groups in the chronic dog study. 

In a published, non-Guideline study, Jin et al., 2011 reported effects on thyroid-related gene 

transcription in juvenile and adult Japanese medaka fish. The findings showed treatment-related 

effects on transcription were gender-, tissue-, and developmental-stage-specific. Effects on 

males occurred in the presence of estrogen only. The study did not evaluate other endocrinerelated endpoints and there is uncertainty in what impact these alterations in gene transcription 

would have on higher-levels of biological organization. A study using spotted marsh frogs 

(Limnodynastes tasmaniensis) reported no effects on developmental stage, body length or sex 

ratio in tadpoles exposed up to 10 μg/L metolachlor; however, as there were no measurements on 

thyroid or thyroid hormones, the study is limited in its ability to inform about potential thyroidrelated interactions (Spolyarich et al. 2010). 

Page 26 of 69
METOLACHLOR FINAL 

Page 19 of 61 

Table 4. Thyroid Pathway for Metolachlor

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Metolachlor1

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, BL, 

SVL) Systemic Toxicity Observed 2

Overt Toxicity Observed3

EDSP Tier 1 Assay 

Female Pubertal Rat 

(MRID 48617804) N P

(H) 

↑25% 

(H) N N 

LW: ↑22% 

KW: ↑10% 

↑GGT 

(H) 

N 

Male Pubertal Rat 

(MRID 48617803) N P (L, H) N 

↑54, 

134% 

(L, H) 

N 

LW: 

↑15, 20% 

KW: 

↑11, 11% 

(L, H) 

N 

AMA (Frog; MRID 

48617810) N 

Day 21 

HLL: 

↓23% 

(H) 

Day 7 

BW: ↓19% 

SVL: ↓7% 

(H) 

 N4 

OSRI 

3-Month Subchronic 

toxicity (Rat; MRID 

43928923)

NE N NE X 

 (MH, H) 

X 

(MH, H) 

3-Month Subchronic 

toxicity (Rat; MRID 

44775401, 

44775402)

NE N NE N X 

(H) 

3-Month Subchronic 

toxicity (Dog; MRID 

43928922)

N N NE X 

(MH, H) N 

6-Month Subchronic 

toxicity (Dog; MRID 

00032174)

N N N X 

(H) 

X 

(M, H) 

21-day Dermal 

toxicity (Rabbit; 

MRID 41833101) 

NE NE N X 

 (H) N 

Two-generation 

reproduction (Rat; 

MRID 00080897) 

N N NE X 

(H) N 

Chronic toxicity/ 

Carcinogenicity 

(Rat; MRID 

00129377) 

NE N NE X 

( H) N 

Carcinogenicity 

(Mouse; MRID 

00117597) 

NR NR NR X 

(H) 

X 

(H) 

Page 27 of 69
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Page 20 of 61 

Table 4. Thyroid Pathway for Metolachlor

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Metolachlor1

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, BL, 

SVL) Systemic Toxicity Observed 2

Overt Toxicity Observed3

Chronic toxicity 

(Dog; MRIDs 

40980701, 

41164501, 

42218601, and 

42218602) 

N N N X 

(L, M, H) N 

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

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

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

unless otherwise indicated. LW= liver weight; KW= kidney weight; GGT= gamma-glutamyl transferase 

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

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

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

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

4. While clinical signs of toxicity (lethargy or surfacing) were reported until day 8, at the high concentration, these 

clinical signs of toxicity did not persist throughout the exposure period. There were also 4 mortalities (out of 

80) observed at this treatment level during the 21 day exposure period. 

P Positive findings 

N Negative findings 

NE Not examined 

NR Not reported 

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METOLACHLOR FINAL 

Page 21 of 61 

IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight of evidence (WoE) determination on the 

potential of metolachlor to interact with endocrine related processes (i.e., E, A or T hormonal 

pathways) as well as recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

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

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

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

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

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

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

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

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

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

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

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

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

whether or not the responses are related through an endocrine pathway, therefore non-endocrine 

toxic responses (including but not limited to mortality or body weight changes) are also 

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

with weight of evidence evaluation of the results of the Tier 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 assays and 

OSRI studies are described below. Generally, one parameter (i.e., death or >10% decrease in 

mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. 

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

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

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

1. Tier 1 in vivo Assays 

All six in vivo assays in the EDSP Tier 1 battery were available for metolachlor. In the 

Hershberger and uterotrophic assays, all animals survived until scheduled termination, and no 

Page 29 of 69
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Page 22 of 61 

test substance-related clinical findings were noted in any of the animals. In the female pubertal 

rat assay, one control animal was sacrificed in extremis; all other rats survived until scheduled 

sacrifice. No treatment-related clinical findings were noted during the daily examinations at any 

dose. In the male pubertal rat assay, one high-dose male was found dead (not treatment related) 

and one control animal was sacrificed in extremis; all other rats survived until scheduled 

sacrifice. Clinical findings in males included salivation prior to dosing, and clear material

around the mouth at approximately 1 hour post-dose in both treatment groups. Clear material 

also was observed around the ventral neck in high-dose animals. Moderate increases in liver and 

kidney weights (approximately ↑10-20%) were also noted in high-dose females and low- and 

high-dose males. 

In the FSTRA, overall survival was decreased (↓29%) in the high-concentration group (4.7 mg 

a.i./L), and the surviving fish showed signs of erratic swimming, loss of equilibrium, lethargic 

swimming, and curved spines. In addition, excess feed was observed in the tanks. In the AMA, 

while clinical signs of toxicity (lethargy or surfacing) were reported until day 8 at the high 

concentration (8.4 mg a.i./L), these clinical signs of toxicity did not persist throughout the 

exposure period. There were also 4 mortalities (out of 80) observed at this treatment level during 

the 21 day exposure period. The decrease in body weight and SVL at day 7 at the high 

concentration (but not at day 21) supports initial signs of stress that did not continue throughout 

the remaining exposure period. Additionally in the AMA, crooked tails were observed in 27 to 

57 tadpoles (out of 80 exposed) in the control and all treatment groups, but did not appear to be 

treatment-related. 

2. OSRI 

There were no mortalities or clinical signs in a subchronic oral toxicity study in rats with 

S-metolachlor. Treatment-related systemic toxicity was observed in the medium-high and highdose groups as decreases in body weight, body weight gain, and food consumption and 

efficiency. Body weights were decreased in medium-high (↓10% at Week 10) and high-dose 

males (↓11-13% from Week 1), respectively, and were decreased in medium-high (↓10% from 

Week 11) and high-dose females (↓12-17% from Week 8), respectively. There were also no 

mortalities or clinical signs in a subchronic oral toxicity study in rats with metolachlor. There 

was no evidence of any treatment-related effect in male rats, but decreased body weights were 

observed in the high-dose females (↓10-11%) from Week 9. 

There were no mortalities in a subchronic oral toxicity study in dogs with S-metolachlor. 

Clinical signs were limited to incidences of few or no stools in the medium-high and high-dose 

dogs. There were also no mortalities or clinical signs in a subchronic oral toxicity study in dogs 

with metolachlor. Mean body weight gain was decreased (NS) during Weeks 0-13 and 0-26 in 

the high-dose males (↓55-63%) and females (↓44-50%), and mean Week 26 body weights were 

decreased in the medium- and high-dose males by 11 and 14%, respectively. 

Page 30 of 69
METOLACHLOR FINAL 

Page 23 of 61 

Some systemic maternal toxicity was observed in a developmental toxicity study in rats with Smetolachlor. All females in the mid-high and high-dose groups and 9/24 females in the mediumlow dose group exhibited discomfort after daily dose administration which consisted of pushing 

their heads through the bedding for approximately one hour. In addition, body weight gains 

were decreased in the mid-high and high dose group during dosing and over the entire study. In 

an earlier developmental study in rats with metolachlor, four dams in the high-dose group died 

on GDs 7, 8, and 10 (n=2). Clinical signs of toxicity were observed in these animals one or more 

days prior to death. Clinical signs, observed primarily in the high-dose dams, included excess 

salivation (25/25), urine-stained abdominal fur (19/25), excess lacrimation (8/25), and 

clonic/tonic (6/25) or clonic (5/25) convulsions. One death in the high-dose group due to 

anorexia and body weight loss was considered treatment-related in a developmental toxicity 

study in rabbits with S-metolachlor. Maternal toxicity, consisting of little, none, or soft stools 

was observed in the medium- and high-dose animals. Mortalities due to anorexia were observed 

in an earlier developmental toxicity study in rabbits with metolachlor. One doe in each the lowand high-dose groups died on study due to persistent anorexia. Two rabbits aborted; one 

abortion occurred in a high-dose doe with persistent anorexia. There was a treatment-related 

increase in the incidence of persistent anorexia in the does in the high-dose group. There was a 

treatment-related decrease in body weight gain in the high-dose group during the dosing period 

(↓500%) and the combined dosing and post-dosing periods (↓133%). 

High-dose females had an increased overall mortality rate due to early deaths during the first few 

weeks of treatment in a carcinogenicity study in mice with metolachlor. Although the deaths 

were attributed to a viral infection, the potential contribution of the test material cannot be 

dismissed. 

No treatment-related mortalities or clinical signs of toxicity were observed in the 21-day dermal 

toxicity study in rabbits, the two-generation reproduction study in rats, the chronic oral toxicity 

studies in rats or dogs or the avian reproduction studies in mallard ducks and bobwhite quail. 

In a fathead minnow early-life cycle study, length was decreased (significance not reported) ≥

0.220 mg/L, and decreases in wet weight were observed at ≥0.110 mg/L and in dry weight at 

≥0.056 mg/L. In another fish early-life cycle study with sheepshead minnow, there was a 

decreasing trend in length and weight at concentrations ≥2.2 mg/L, and increased mortality at 

≥4.1 mg/L. 

B. Estrogen Pathway 

There was no convincing evidence of potential interaction with the estrogen pathway. 

Metalochlor was negative in the in vitro estrogen receptor (ER) binding and the ER 

transactivation assay (ERTA), gave an equivocal response in the aromatase assay (reduction in 

activity observed (down to 42% at 10−3

 M), but potential issue with protein (receptor) 

Page 31 of 69
METOLACHLOR FINAL 

Page 24 of 61 

denaturation and/or compound solubility), and induced a marginal increase (1.2- to 1.3-fold) in 

estradiol production in the steroidogenesis assay. No treatment-related estrogenic effects were 

seen in the in vivo uterotrophic or female pubertal assays. In the fish short-term reproduction 

assay (FSTRA), the only effects seen, which were at the high concentration, occurred in the 

presence of overt toxicity. There were no treatment-related effects on female reproductive 

parameters in the existing metolachlor Part 158 mammalian studies. While there was a 6% 

increase in cracked eggs (as eggs not cracked/eggs laid) in one Part 158 avian study, there were 

no complementary effects in this study, the other avian reproduction study, or the other in vivo

studies. 

C. Androgen Pathway 

Metolachlor was positive for AR binding but did not alter testosterone production in the in vitro

steroidogenesis assay. In the Hershberger assay (dosed subcutaneously), there was evidence for 

an anti-androgenic at the high dose (500 mg/kg/day). However, in the Tier 1 male pubertal assay 

and Part 158 mammalian studies in which animals were dosed orally, no androgen-related effects 

were seen. In the FSTRA, the only effect in the absence of overt toxicity was a reported 

significant decrease in fat pad score in the mid-treatment level (0.48 mg a.i./L). Overall, there is 

no convincing evidence for a potential interaction with the androgen pathway. 

D. Thyroid Pathway 

There is some evidence for potential interaction of metolachlor with the thyroid pathway in 

mammals based on effects on thyroid histopathology in both sexes in the pubertal assays. These 

effects are further supported by alterations in T4 levels in females and TSH levels in males. In 

the AMA, while a decrease in HLL was observed at Day 21 at the high test concentration, 

clinical signs of toxicity (related to swimming) and decreased body weight and SVL were 

observed in this concentration during the beginning phases of the study. Overall, the results to 

not suggest a potential for thyroid interaction as there were no effects on thyroid histopathology 

or developmental stage. 

E. Conclusions 

Based on the available data, there is no convincing evidence for potential interaction with the 

estrogen or androgen pathways in mammals or wildlife. For the thyroid pathway, there is 

evidence for potential interaction in mammals, but no convincing evidence for potential 

interaction in wildlife. 

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V. EDSP Tier 2 Testing Recommendations 

In the EDSP Tier 1 pubertal assays, there was evidence for potential interaction with the thyroid 

pathway in studies conducted with adult animals, but no such data exists for potential thyroid 

toxicity in the young animals. 

For metolachlor, the current points of departure (POD) of 9.7 mg/kg/day used for human health 

risk assessment is based on decreased body weight gain in female dog at 32.7 mg/kg/day 

(LOAEL) in a chronic toxicity study. This POD is used for deriving the chronic Reference Dose 

(RfD) for dietary exposure. A second POD of 50 mg/kg/day is based on increased incidence of 

clinical signs, decreased body weight/body weight gain, food consumption and food efficiency 

seen in maternal animals at 500 mg/kg/day (LOAEL) in a developmental toxicity study in rats. 

This POD is used for deriving the Margins of Exposure (MOEs) for non-dietary risk 

assessments. The PODs used for overall risk assessments are approximately 5-30-fold lower than 

the dose (300 mg/kg/day) that induced histopathological lesions of the thyroid gland in the 

female pubertal assay. 

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

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

thyroid toxicity in the young. However in the case of metolachlor, although no data exists for 

potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended 

since: 1) the PODs are lower than the dose that caused thyroid lesions; 2) there was no evidence 

of thyroid toxicity in the data base; 3) in subchronic and chronic toxicity studies in dogs, rats and 

mice, decreased body weight and body weight gain were the most commonly observed effects; 

5) the CTA would be tested at higher doses and would not be expected to yield a POD lower 

than the level currently used for risk assessment; and 6) the chronic RfD and MOEs are 

protective of the thyroid effects seen in the female pubertal assay. Therefore, additional studies 

to investigate thyroid toxicity in the young are not required. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is not 

recommended for metolachlor since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

For wildlife, as there was no convincing evidence for potential interaction with estrogen, 

androgen pathway in fish or thyroid pathway in amphibians, no additional testing is 

recommended. 

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

duck are available for metolachlor. In mallard ducks no reproductive effects were observed, 

whereas in bobwhite quail an effect on a reproductive parameter was observed. The type of data 

obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient 

Page 33 of 69
METOLACHLOR FINAL 

Page 26 of 61 

for evaluating potential reproductive effects to birds from metolachlor exposure. Relative to the 

EDSP, additional testing is not recommended. 

 

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METOLACHLOR FINAL 

Page 27 of 61 

VI. References 

Chang, J.C.F. (1995) CGA-77102 Technical 13-week oral toxicity in rats. Unpublished study 

conducted by Ciba-Geigy Corp, Crop Protection Division, Environmental Health Center for Ciba 

Crop Protection, Ciba-Geigy Corp. Laboratory Study No. F-00191. February 21, 1995. (MRID 

43928923) 

Chang, J.C.F. (1995) CGA-77102 Technical Final report 90-day oral toxicity in dogs. 

Unpublished study conducted by Ciba-Geigy Corp, Crop Protection Division, Environmental 

Health Center for Ciba Crop Protection, Ciba-Geigy Corp. Laboratory Study No. F-00193. June 

14, 1995. (MRID 43928922) 

Estes, F.L. (1980) 6-Month Chronic Oral Toxicity Study in Beagle Dogs. Unpublished study 

conducted by International Research and Development Corporation (IRDC), Mattawan, MI., 

Study Number 382-054, May 21, 1980. (MRID 00032174) 

Fankhauser, H. (1999) CGA-24705 Final Report. 3-month oral toxicity study in rats 

(administration in food). Unpublished study conducted by Novartis Crop Protection AG, 

Toxicology, 4332 Stein, Switzerland. Study ID # 971143, Novartis #1162-98. January 25, 1999. 

(MRID 44775401) 

Fankhauser, H. (1999) CGA-771 02 Final Report. 3-month oral toxicity study in rats 

(administration in food). Unpublished study conducted by Novartis Crop Protection AG, 

Toxicology, 4332 Stein, Switzerland. Study ID # 971144, Novartis #1161-98. January 25, 1999. 

(MRID 44775402) 

Gilles, P.A. and Giknis, M.L.A. (1995) A teratology study of CGA-77102 Technical in New 

Zealand White rabbits. Unpublished study conducted by Ciba-Geigy Corp, Crop Protection 

Division, Environmental Health Center. Laboratory Study No. F00192. April 27, 1995. (MRID 

43928924) 

Hazellette, J.R. and Arthur, A. T. (1989) Metolachlor Technical, 13/52-Week Oral Toxicity 

Study in Dogs (MIN 862253). Unpublished study conducted by the Division of 

Toxicology/Pathology, CIBA-GEIGY, Surmnit, NJ and Methpath Laboratories, Rockville, MD, 

Study Number 862253, January 23, 1989. (MRIDs 40980701, 41164501, 42218601, and 

42218602) 

Jin, Y., Chen, R., Wang, L., Liu, J., Yang, Y., Zhou, C., Liu, W., and Fu, Z. (2011) Effects of 

metolachlor on transcription of thyroid system-related genes in juvenile and adult Japanese 

medaka (Oryzias latipes). Gen Comp Endocrinol. 170(3):487-493. 

Page 35 of 69
METOLACHLOR FINAL 

Page 28 of 61 

Khalil, S. (1995) CGA-77102 Rat oral teratology. Ciba-Geigy Ltd., Basle, Switzerland for Ciba 

Crop Protection, Unpublished study conducted by Ciba-Geigy Corp. Laboratory Study No. 

941058. August 21, 1995. (MRID 43928925) 

Kojima, H., Katsura, E., Takeuchi, S., Niiyami, K., and Kobayashi, K. (2004) Screening for 

estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using 

Chinese hamster ovary cells. Environ. Health Perspect. 112 (5): 524-531. 

Lightkep, G.E. (1980) Teratogenic Potential of CGA-24705 in New Zealand White Rabbits 

Segment II Evaluation. Unpublished study conducted by Argus Research Laboratories, Inc., 

Horsham, P A. Argus Project 203-001, July 16, 1980. (MRID 00041283) 

Lochry, E.A. (1985) Embryo/Fetal Toxicity and Teratogenic Potential Study of CGA-24705 (FL841697) Administered Orally via Gavage to Crl:COBS®CD®(SD) BR Presumed Pregnant Rats. 

Unpublished study conducted by Argus Research Laboratories, Inc., Horsham, P A. Argus 

Project 203- 004, August 6, 1985. (MRID 00151941) 

Mastrocco, F., Huber, K., Schiavo, D.M., Hazelette, J.R. and Green, J.D. (1987) 21-Day Dermal 

Toxicity Study in Rabbits. Unpublished study conducted by the Research Department, 

Pharmaceuticals Division, Ciba-Geigy Corporation. Study Number 862012, November 16, 1987. 

(MRID 41833101) 

Page, J.G. (1981) Two-Generation Reproduction Study in Albino Rats with Metolachlor. 

Unpublished study conducted by Technical. Toxigenics, Decatur, IL. Study Number 450-0272, 

August 31, 1981. (MRID 00080897) 

Palmer, S.J., Schneider, S.Z., Kendall, T.Z., and Krueger, H.O. (2012) Metolachlor: Amphibian 

Metamorphosis Assay for the Detection of Thyroid Active Substances. Unpublished study 

performed by Wildlife International, Ltd., Easton, MD. Laboratory report No. 568A-103. Study 

sponsored by Metolachlor Task Force, Stilwell, KS. Study completed January 24, 2012. (MRID 

48617810) 

Palmer, S.J., Martin, K.H., Kendall, T.Z., and Krueger, H.O. (2012) Metolachlor: Fish Shortterm Reproduction Assay with the Fathead Minnow, (Pimephales promelas). Unpublished study 

performed by Wildlife International, Ltd., Easton, Maryland. Lab Study No.: 568A-102. Study 

sponsored by Metolachlor Task Force, Stilwell, Kansas. Study completed January 23, 2012. 

(MRID 48617811) 

Sloter, E. D. (2012) A Hershberger Assay of Metolachlor Administered Subcutaneously in 

Peripubertal Orchidoepididymectomized Rats. Unpublished study conducted by WIL Research 

Laboratories, LLC, Ashland, OH. Laboratory project ID: WIL-786010, January 25, 2012. 

(MRID 48617802) 

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Page 29 of 61 

Sloter, E.D. (2012) Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal 

Female Rats Following Oral Administration of Metolachlor. Unpublished study conducted by 

WIL Research Laboratories, LLC, Ashland, OH, Laboratory Project ID: WIL-786003, January 

25, 2012. (MRID 48617804) 

Sloter, E.D. (2012) Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal 

Male Rats Following Oral Administration of Metolachlor. Unpublished study conducted by WIL 

Research Laboratories, LLC, Ashland Ohio. WIL 786004, January 25, 2012. (MRID 48617803) 

Sloter, E.D. (2012) A Uterotrophic Assay of Metolachlor Administered Subcutaneously in 

Ovariectomized Rats. Unpublished study conducted by WIL Research Laboratories, LLC, 

Ashland, OH. Laboratory Study No.: WIL-786001, January 3, 2012. (MRID 48617801) 

Sousa, J.V. (2012) S-Metolachlor (CGA-77102): Early Life-Stage Toxicity Test with Fathead 

Minnow (Pimephales promelas). Unpublished study performed by Springborn Laboratories, Inc., 

Wareham, MA. Lab Study No. 178 1.6576. Study sponsored by Novartis Crop Protection, Inc., 

Greensboro, NC. Study completed November 30, 1999. (MRID 44995903) 

Taliaferro, L.C., Taliaferro, M.C., and Miller, V.C. (1996) Metolachlor: The reproductive 

toxicity test of CGA-24705 in Mallard ducks (Anas platyrhynchos). Unpublished study 

conducted by EBA, Inc., Snow Camp, NC. Sponsored by Ciba Geigy Corporation, Greensboro, 

NC. Report ID: 029409. (MRID 44015602) 

Temple, D.L., et al. (2005) Metolachlor technical: A reproduction study with the Northern 

Bobwhite. Unpublished study performed by Wildlife International Ltd., Easton, MD. Laboratory 

Project No. 568-102. Study sponsored by the Metolachlor Task Force, Chemical Consultants 

Intemational, Stilwell, KS. (MRID 46508901) 

Thomas, J.A. (2012) In Vitro Rat Prostate Androgen Receptor Competitive Binding Assay. 

Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory 

Study No.: WIL-786007, January 4, 2012. (MRID 48617806) 

Thomas, J.A. (2012) In vitro inhibition assay with human recombinant aromatase. Unpublished 

study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report No.: 

WIL-786005, January 4, 2012. (MRID 48617805) 

Thomas, J.A. (2012) In Vitro Rat Uterine Estrogen Receptor Competitive Binding Assay. 

Unpublished study conducted by WIL Research Laboratories, LLC, Ashland, OH. Laboratory 

Study No.: WIL-786006, January 4, 2012. (MRID 48617807) 

Tisdel, M. (1982) Carcinogenicity Study with Metolachlor in Albino Mice. Unpublished study 

conducted by Hazleton-Raltech, Inc., Madison, WI. Study Number 79020, August 13, 1982. 

(MRID 00117597) 

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Page 30 of 61 

Tisdel, M. (1983) Two-year Chronic Oral Toxicity and Oncogenicity Study with Metolach1or in 

Albino Rats. Unpublished study conducted by Hazleton-Raltech, Inc., Madison, WI. Study 

Number 80030, May 2, 1983. (MRID 00129377) 

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. 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. 

Wagner, H. (2012) Metolachlor: H295R steroidogenesis assay. Unpublished study conducted by 

CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9179V-100537STER, January 26, 2012. 

(MRID 48617809) 

Ward, S. (1980). Effects of Metolachlor (Dual) on Survival, Growth, and Development of 

Sheepshead Minnows (Cyprinodon variegatus). Unpublished study conducted by EG&G, 

Bionomics Marine Research Laboratory, Pensacola, Florida. Study No. BP-80-5-80. Submitted 

by Ciba-Geigy Corporation, Greensboro, NC. (MRID No. 43044602) 

Willoughby, J.A. (2012) Metolachlor: estrogen receptor transcriptional activation (human cell 

line (HeLa-9903)). Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory 

Report No.: 9179V-100537ERTA, January 25, 2012. (MRID 48617808). 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA); (OCSPP 890.1100) 

The 21-day assay (MRID 48617810) of metolachlor (97.3% purity) on amphibian 

metamorphosis of Xenopus laevis (African clawed frog) was conducted under flow-through 

conditions. Amphibian larvae at developmental Nieuwkoop-Faber (NF) Stage 51 (80/control and 

treatment group; 20/replicate) were exposed to nominal concentrations of 0 (negative control), 

0.09, 0.9, and 9.0 mg a.i./L. Mean-measured concentrations were <0.0500 (<LOQ; negative 

control), 0.075, 0.79, and 8.4 mg a.i./L. The test system was maintained at 22 ± 1 °C and a pH of 

8.1 to 8.3. 

On Day 21, mortality was 3.3 and 6.6% in the mid and high treatment groups, respectively. 

Crooked tails were observed in 27 to 57 tadpoles in the negative control and all treatment groups 

Metolachlor significantly reduced (p<0.05) Day 7 wet weight by 19% and Day 7 snout-vent 

length by 7% at the high treatment group relative to the negative control. Day 21 normalized 

(for snout-vent length) hind leg length (HLL) was also significantly reduced (p<0.05) by 23% at 

the high treatment level relative to the negative control. Metolachlor exposure had no effect on 

the NF developmental stage in all treatment groups relative to the negative control on Days 7 and 

21; no asynchronous development was observed. Day 21 normalized HLL at the high treatment 

level was significantly reduced (p<0.05) by 23% relative to the negative control group. 

Thyroid gland histopathology observations included thyroid gland hypertrophy, thyroid gland 

atrophy, and/or follicular cell hypertrophy in the negative control and all treatment groups. 

Analysis of Day 21 wet weight, SVL, HLL, and normalized HLL excluded late stage tadpoles 

(i.e., NF stage =61-63); there was no pattern in the development of late stage individuals across 

treatment groups, with the proportion of late stage tadpoles ranging from 2 to 6% of all tadpoles 

tested in the negative control and all treatment groups. 

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

In an androgen receptor (AR) binding assay (MRID 48617806), ventral prostate cytosol from 

previously castrated (23-24 hours prior to collection) Sprague Dawley rats, was used as the 

source of AR to conduct saturation and competitive binding experiments. The saturation binding 

experiment was conducted to demonstrate that the AR in the rat prostate was present in 

reasonable numbers and was functioning with appropriate affinity for the radio-labeled reference 

androgen ([3

H]-R1881) prior to conducting the competitive binding experiments. The 

competitive binding assay measured 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 metolachlor (97.3% purity, Lot# 

20081115). Ethanol was used as the solvent vehicle at a final assay concentration of 2.6%. A 

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total of three runs were performed, and each run included dexamethasone as a weak positive 

control, and R1881 as the ligand reference standard. 

In the saturation binding experiment, the maximum binding capacity (Bmax) was 14.1 fmol/100 

μg protein and the dissociation constant (Kd) was 0.83 nM. The Kd and Bmax values were within 

the range reported in the EPA validation program. Confidence in these numbers is high 

according to the goodness of fit (R2

 = 0.985); however, only one run was performed. The 

specific binding reached a plateau, and non-specific binding (NSB) was ≤8.8% of total binding 

The Scatchard plot for the saturation assay was not linear for the entire [3

H]-R1881 concentration 

range tested; however, it was linear over the range from 0.5 to 10 nM, which encompasses the 

ligand concentration used in the competition binding experiment.

In the competitive binding experiment, the estimated mean log IC50s for R1881 (−8.8 M) and the 

weak positive control, dexamethasone (−4.2 M), were within the expected ranges. The mean 

relative binding affinity (RBA) for the weak positive control was 0.0026%. The solvent control 

responses indicated no drift in the study assay. All performance criteria were generally met. 

In each run, there were no apparent reduction in average [3H]-R1881 binding at metolachlor 

concentrations from 10−10 to 10−4 M (87.7-106.8%), but average binding of the radiolabeled 

ligand declined to 48.96-50.99% at 10−3

 M. The average binding was approximately 50% in 

Runs 2 and 3 (50.94 and 50.99%), the average binding curve for metolachlor across all three runs 

indicates that ~50% displacement (competitive binding to the AR) occurred at 10−3 M. The 

average log IC50 and RBA for metolachlor were −2.994 M and 0.00016%, respectively. 

Based on the results from the three runs, metolachlor is classified as a Binder in the Androgen 

Receptor Binding Assay. 

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP19) assay (MRID 48617805), metolachlor (97.3% a.i., Lot # 

20081115) was incubated with human recombinant aromatase and tritiated androstenedione (1-β

[

3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in 1% ethanol at log concentrations from 10−10 to 

10−3

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

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

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

using liquid scintillation counting (LSC). 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 chemical series 

(10−10 to 10−3

 M) with three repetitions per concentration. 

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Aromatase activity in the full activity controls ranged from 0.2747 to 0.4910 nmol·mgprotein−1

·min−1 for the 3 test runs, with a mean and standard deviation of 0.3924±0.0224 

nmol·mg-protein−1·min−1. Activity in the background controls ranged from 0.23 to 1.17% and 

averaged 0.49% of the full activity controls. The responses of the full activity controls and 

background controls were acceptable for each run. 

For the positive control substance (4-OH ASDN) aromatase activity was within the 

recommended ranges for the top and bottom of the curve, Hill slope, log IC50 and %CV for 

replicates of each concentration within runs. The estimated log IC50 averaged −7.3 M and the 

Hill slope averaged −0.93. 

For metolachlor, aromatase activity averaged 87.86±6.2% of the full activity control at the 

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

tested concentration of 10−3

 M. Aromatase activity at the second highest tested concentration 

(10−4 M) averaged 84.53±2.9% of the full activity control. For Runs 1 and 3, the report indicated 

that the log IC50 values were "ambiguous" but values of −3.79 and −3.81 M, respectively, as well 

as Hill slope values of –8.05 and –8.31, respectively, were reported. A log IC50 value and Hill 

slope were not determined for Run 2 as the concentration response curve did not fit the 4-

parameter model. 

Although >50% inhibition was observed at the highest test concentration (10−3

 M) in all three 

runs, the Hill slope (-8.4) for the average response curve was steeper than -2. The unusually 

steep slope may indicate an issue with protein denaturation or solubility at this test concentration. 

Therefore, metolachlor is classified as Equivocal for aromatase inhibition in this assay. 

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

In an estrogen receptor (ER) binding assay (MRID 48617807) for metolachlor (97.3% purity, 

Lot # 20081115), uterine cytosol from previously ovariectomized Sprague Dawley rats was used 

as the source of ER to conduct saturation binding and competitive binding experiments. The 

saturation binding experiment was conducted to demonstrate that the ER in the rat uterine 

cytosol was present in reasonable numbers and was functioning with appropriate affinity for the 

radiolabeled reference estrogen prior to conducting the competitive binding experiments. The 

competitive binding experiment measured the binding of a single concentration of [3

H]-17βestradiol (1 nM) in the presence of increasing concentrations (logarithmic increase from 10−10 to 

10−3

 M) of metolachlor. Ethanol was used as the solvent vehicle at a final concentration of 2%. 

A total of 5 runs were performed, and each run included 19-norethindrone as a weak positive 

control, octyltriethoxysilane as a negative control, and 17-β-estradiol as the natural ligand 

reference material. 

In the saturation binding experiments, the mean maximum binding capacity (Bmax) was 31.7 

fmol/100 μg protein and the mean dissociation constant (Kd) was 0.075 nM. The Bmax value was 

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within the recommended range and demonstrated that the estrogen receptor was present in a 

reasonable concentration. The Scatchard plots for the three Saturation assays were linear. The 

specific binding appeared to reach a plateau, and NSB was generally less than 20% of total 

binding (1.97 to 21.51%). 

For the competitive binding experiment, the estimated mean log IC50 was −8.675 M for 17βestradiol and −5.254 M for the weak positive control. The mean RBA was 0.045% for the weak 

positive control. Confidence in these numbers is high due to the small variation. All 

performance criteria were generally met; however, parameters for 19-norethindrone were outside 

of the tolerance limits for a weak positive control in Run 5, and a technical error occurred with 

the negative control, octyltriethoxysilane, in Run 2. 

Metolachlor had no significant effect on specific binding of the [3H]-ligand to the ER in runs 2-5; 

therefore, a log IC50 and RBA could not be calculated for metolachlor (specific binding of [3

H]-

17β-estradiol was ≥72.28% in Run 1). 

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

Receptor Binding Assay. 

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

In an estrogen receptor transcriptional activation assay (MRID 48617808), hERα-HeLa-9903 

cells cultured in vitro were exposed to metolachlor (97.3% a.i., Lot #20081115) at 

logarithmically increasing concentrations from 10−11 to 10−4 M in DMSO (0.1%) for 

approximately 24 hours in two independent runs. The experiments were performed using 96-

well plates and each metolachlor concentration was tested in 6 wells/plate in each run. Cells 

were exposed to the test agent for 24±2 hours at 37±2°C to induce reporter (luciferase) gene 

products. Luciferase expression in response to activation of the estrogen receptor was measured 

upon using a proprietary luciferase assay 

Metolachlor was tested up to a cytotoxic level, which was observed at concentrations ≥10−4

 M. 

The mean RPCMax was 3.3% for the first run and 0.6% for the second, and the associated PCMax

was 10−7

 M for both. 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions of this assay were not optimal to detect 

very weak activity, metolachlor responses were similar to those of the negative control 

corticosterone and not comparable to the responses of 17α-methyltestosterone, which was able to 

reach a maximum of 36.5-46.3% PC. Metolachlor was only able to reach a maximum of 0.6-

3.3% PC when tested up to the highest concentration possible based on cytotoxicity. Because 

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the RPCMax < PC10 in both assay runs, metolachlor was considered negative for estrogen receptor 

transcriptional activation in this test system. 

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

The 21-day short-term reproduction assay (MRID 48617811) of metolachlor with fathead 

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

spawning groups, 2 males and 4 females in each group; 5.5 months old) were exposed to 

metolachlor (97.3% purity) at nominal concentrations of 0 (negative control), 0.050, 0.50, and 

5.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.0300, negative control), 0.046, 

0.48, and 4.7 mg a.i./L. The test system was maintained at 24.1 to 25.2o

C and a pH of 7.0 to 8.2. 

Overall survival was significantly reduced (p<0.05) by 66.7% for males, 73.3% for females, and 

70.8% for the combined sexes at the high treatment level compared to the negative control. 

Additionally, male and female fish in the high treatment level showed signs of erratic swimming, 

loss of equilibrium, lethargic swimming, and curved spine; excess feed was observed in high 

treatment tanks. There were no significant differences (p>0.05) for male or female growth 

parameters (weight and length); however, there was a non-statistically significant decrease 

(p>0.05) of 23% in male body weight at the high treatment compared to the negative control. 

Mean spawning frequency and fecundity in the negative control were at least every 4 days and 

28 eggs/female/day (range 20.2-33.9), respectively; fertilization success in the negative control 

was 97.9%. There was a significant reduction (p<0.05) in fecundity of 98% at the high treatment 

level relative to the negative control; only one replicate in the high treatment level produced 

eggs, and there was 100% fertilization success in that replicate. 

Female vitellogenin (VTG) levels were significantly reduced (p<0.05) by 77% at the high 

treatment level relative to the negative control. Male VTG levels were not significantly different 

(p>0.05) from the negative control. There were no significant differences in female gonadosomatic index (GSI) compared to the negative control, but there was a marginally significant 

increase (p=0.057; Mann-Whitney) of 29% in male GSI at the high treatment level compared to 

the negative control. There was a significant reduction (p<0.05, Mann-Whitney) in male tubercle 

score at the high treatment level (median tubercle score: 5) compared to the negative control 

(median tubercle score: 22). No tubercles were observed in female fish. The study authors also 

reported a statistically-significant decrease (p<0.05; Jonckheere-Terpstra trend test) in mean 

male fatpad scores in the mid and high treatment groups compared to the negative control. Sex 

steroids were not measured. 

Gonadal histopathologic changes in females included increased oocyte atresia related to 

increases in granulosa cell hypertrophy and hyperplasia and increased granulosa cell 

phagocytosis as well as decreased early and late vitellogenic oocytes and ovary size at the high 

treatment level. In males of the high treatment group, germinal epithelium showed an increased 

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presence of diploid spermatogonia with a decreased presence of spermatocysts containing 

spermatocytes and spermatids. One in six males in the high treatment group showed moderate 

alterations in these ratios and could not be staged under normal criteria. These treatment-related 

changes appeared to result from an inability of spermatogonia in the testes of affected fish to 

enter into spermatogenesis. 

Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48617802) screening for androgenic activity, metolachlor 

(97.3% a.i., Lot #20081115) in corn oil was administered daily via subcutaneous (s.c.) injection 

to groups of six 54- to 56-day old, castrated male Sprague Dawley rats at dose levels of 0 

(vehicle), 100, 300, or 500 mg/kg/day. An androgenic positive control group consisted of six 

castrated rats exposed to 0.2 mg/kg/day of testosterone propionate (TP) by s.c. injection. 

To screen for potential anti-androgenic activity, metolachlor (97.3% a.i.) in corn oil was 

administered daily via s.c. injection to groups of six 54- to 56-day old, castrated male Sprague 

Dawley rats at dose levels of 0 (vehicle), 100, 300, or 500 mg/kg/day in conjunction with a daily 

dose of reference androgen TP at 0.2 mg/kg/day by s.c. injection. The anti-androgenic positive 

control group consisted of six castrated rats exposed to 0.2 mg/kg/day TP (s.c. injection) and 

3 mg/kg/day flutamide (FT, via oral gavage). 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, adrenal glands, kidney, and 

liver were collected and weighed. One animal in the 100 mg/kg/day group in the androgen 

agonist assay was inadvertently given FT on the last day of dose administration; therefore, this 

animal was removed from the study. 

One animal in the 300 mg/kg/day group in the anti-androgen assay was found dead on Day 10, 

within one hour of dosing; no macroscopic findings were noted at necropsy. All other rats 

survived until scheduled sacrifice. No animals exhibited any dose-related clinical signs of 

toxicity and there were no treatment-related gross pathological findings. 

In the androgen agonist assay, overall body weight gains were decreased compared to controls 

by 18% (not significant [NS]) and 45% (p<0.01) in the 300 and 500 mg/kg/day groups, 

respectively. Body weights were also decreased on Days 6-11 in the 500 mg/kg/day group; 

however, the decreases were not statistically significant. Body weights in the 100 and 300 

mg/kg/day dose groups and the positive control group were comparable to controls throughout 

the duration of the assay. There were no treatment related effects in accessory sex organs, 

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adrenal glands, kidneys, or liver for any metolachlor treatment group. Animals in the positive 

control group had increased (p<0.01) accessory sex organ weights as follows: 327% in seminal 

vesicles; 381% in ventral prostate; 132% in levator ani-bulbocavernosus (LABC); 322% in 

Cowper’s gland; and 53% in glans penis. Adrenal glands, kidney, and liver weights in the 

positive control group were comparable to the vehicle control. 

Percent CVs for the vehicle control and the 500 mg/kg/day treatment groups were compared to 

the performance criteria in the Guidelines. All %CV values were less than the maximum 

permissible values, with the exception of the %CV for ventral prostate for the 500 mg/kg/day 

group (CV = 48.7%; maximum permissible CV = 45%). 

In the anti-androgen assay, overall body weight gains were decreased by 16% (NS) and 33% 

(p<0.01) in the 300 and 500 mg/kg/day groups, compared to controls. Body weights were also 

decreased on Days 6-11 in the 500 mg/kg/day group; however, the decreases were not 

statistically significant. Body weights in the 100 and 300 mg/kg/day dose groups and the 

positive control group were comparable to controls throughout the duration of the assay. At 500 

mg/kg/day, the weights for ventral prostate and LABC were decreased (p<0.05) by 25% and 

22%, respectively. LABC weights were also decreased (p<0.05) in the 300 mg/kg/day group 

(↓17%). There were no treatment related effects in seminal vesicles, Cowper’s glands, glans 

penis, adrenal glands, kidneys, or liver for any metolachlor group. Animals dosed with TP + FT 

(positive control) had decreased (p<0.01) accessory sex organ weights as follows: 74% in 

seminal vesicles; 74% in ventral prostate; 58% in LABC; 75% in Cowper’s gland; and 33% in 

glans penis. Adrenal glands, kidney, and liver weights in the positive control group were 

comparable to the vehicle control. The performance criteria indicated that this assay was 

performing as expected. 

The high dose of metolachlor selected for this study (500 mg/kg/day) is appropriate because it 

induced anti-androgenic effects but did not cause significant toxicity. In addition, reductions in 

body weight grains of 16-18% (NS) at 300 mg/kg/day and 33-45% (p<0.01) at 500 mg/kg/day 

indicate that the doses selected were adequate. 

Metolachlor was negative for androgenic activity in the Hershberger assay as no statistically 

significant increases in organ weights were seen in two or more of the five androgen sensitive 

tissues in the androgen agonist assay. Metolachlor was positive for potential anti-androgenic 

activity in this Hershberger assay as statistically significant decreases in organ weights were seen 

in two or more of the androgen sensitive tissues in the androgen antagonist assay. 

Female Pubertal Assay; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48617804), 15 Crl:CD(SD) Sprague-Dawley rats/dose group 

were treated daily via oral gavage (5 mL/kg) with metolachlor technical (97.3% a.i., Lot 

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#20081115) in corn oil at doses of 0, 300 or 600 mg/kg/day from post-natal day (PND) 22 to 42. 

Animals were examined for vaginal opening daily beginning on PND 22, and weight at day of 

attainment was recorded. Following sacrifice on PND 42, total thyroxine (T4) and thyroid 

stimulating hormone (TSH) levels were analyzed using an electrochemiluminescent 

immunoassay (T4) and a magnetic [125I]rTSH gamma counter immunoassay (TSH). Kidneys, 

liver, adrenal glands, thyroid, pituitary, and ovary weights were recorded, and microscopic 

examination of the thyroid, kidneys, ovary and uterus were performed. 

One animal in the control group was sacrificed in extremis on PND 25 due to impairment of the 

left forelimb. All other rats survived until scheduled sacrifice. No treatment-related clinical 

findings were noted during the daily examinations at any dose level. There were no significant 

differences in body weight at vaginal opening or final body weights in the treated groups relative 

to controls. Adjusted age at VO was significantly earlier at 300 mg/kg/day (p<0.05) but not at 

600 mg/kg/day. One female in the control group failed to attain vaginal opening. There were no 

statistically significant differences in mean age at first vaginal estrous or mean cycle length. The 

cycle status at necropsy was similar among groups. Serum TSH was not affected by treatment. 

Unadjusted, adjusted and relative liver weights were increased at 600 mg/kg/day (p<0.05, ↑20-

22%); only relative liver weight was significantly increased at 300 mg/kg/day (p<0.05, ↑9%). 

Unadjusted, adjusted and relative kidney weights were increased at 600 mg/kg/day (p<0.05, ↑8-

10%). No other effects on organ weights were reported. The percent of females cycling (93.3%) 

and regularly cycling (77.8%) were lower at 600 mg/kg/day (p<0.05) than the controls (100% for 

both). Percent regularly cycling was also reduced at 300 mg/kg/day (p<0.05, 53.8%). There 

were dose-related increases in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day 

groups, respectively; the increase was significant (p<0.05) at 600 mg/kg/day only. Mean 

globulin levels were significantly higher in the 300 mg/kg/day (p<0.05, ↑7%) and 600 mg/kg/day 

(p<0.05, ↑7%) groups; there were corresponding reductions in A/G ratios of 8% and 12%, 

respectively, with only the high dose being significant (p<0.05). GGT was significantly elevated 

at 600 mg/kg/day (p<0.05, ↑700%). 

There was an increased incidence of Grade 2 follicular cell height at 600 (11/14) mg/kg/day 

relative to control (4/14); also, one animal at the high dose was observed with Grade 3. There 

was an increased incidence of Grade 4 colloid area at 600 mg/kg/day (2/14, 4/15 and 11/14 in the 

control, 300 and 600 mg/kg/day respectively). The differences in follicular cell height and 

colloid area were significant at 600 mg/kg/day (p=0.005). The ovaries appeared normal 

histopathologically. There were no microscopic findings in the uterus related to treatment with 

the test material. Renal tubular cysts were increased in the 300 and 600 mg/kg/day groups (1/14, 

2/15 and 4/15 in the 300 and 600 mg/kg/day groups, respectively). 

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No effects were observed on body weights or body weight gain in the study. Increased liver 

weights accompanied by elevated GGT levels and increased kidney weights demonstrate organ 

toxicity as a result of treatment. However, histopathology was not conducted on the liver and 

microscopic findings in the kidney were minimal, so the degree of toxicity in these organs is 

unclear. Age at vaginal opening was not affected by treatment. There was a dose-related 

increase in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, respectively; 

the increase was significant (p<0.05) at 600 mg/kg/day only. Serum TSH was unaffected by 

treatment. The dosing levels tested in this assay are adequate based on systemic toxicity 

(increased liver and kidney weights) at the high dose. 

Male Pubertal Assay; (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48617803), 15 Crl:CD(SD) Sprague-Dawley rats/dose group 

were treated daily via oral gavage with metolachlor technical (97.3% a.i., Lot #20081115) in 

corn oil at doses of 0, 400 or 800 mg/kg/day from post-natal day (PND) 23 to 53. Animals were 

examined for preputial separation (PPS) daily beginning on PND 30 and weight at day of 

attainment was recorded. Following sacrifice on PND 53, blood was taken for total thyroxine 

(T4), testosterone, thyroid stimulating hormone (TSH) and clinical chemistry analysis. Although 

the hormones were reportedly analyzed by radioimmunoassay (RIA) or chemiluminescence, the 

specific details of the methods were not provided. Weights were recorded for kidneys, liver, 

adrenal glands, thyroid, pituitary, epididymides, ventral prostate, dorsal prostate, seminal vesicle, 

levator ani plus bulbocavernosus (LABC) muscles and testes, and microscopic examinations 

were performed on the thyroid, kidneys, testis and epididymides.

One male in the 800 mg/kg/day group (Animal No. 79812-07) was found dead on PND 40; no 

significant clinical or macroscopic findings were observed in this animal. One male in the 

control group (Animal No. 79823-03) was sacrificed in extremis on PND 47 following clinical 

findings of a swollen right forelimb resulting in impaired use; gross examination of this animal at 

necropsy revealed a punctured esophagus probably due to a dosing error. All other rats survived 

until scheduled sacrifice. Clinical findings included salivation prior to dosing and clear material 

around the mouth approximately 1 hour post-dosing in the 400 and 800 mg/kg/day dose groups. 

At 800 mg/kg/day, clear material was also observed around the ventral neck. 

No treatment-related changes in body weights, body weight gains, or age and weight at PPS 

occurred in either treatment group. Liver weights were increased (p<0.05) by 15-20% and 

kidney weights were increased (p<0.05) by 11% each at 400 and 800 mg/kg/day, respectively. A 

significant trend for increased thyroid weights was observed, although pairwise comparisons 

were not significant; the increasing trend in thyroid weights did not correlate with higher TSH 

levels or microscopic findings. Although a significant trend for decreasing right epididymis 

weights was observed, it was not significant by pairwise comparison and was not observed in the 

left epididymis. 

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TSH levels were increased (p<0.05) by 54 and 134% at 400 and 800 mg/kg/day, respectively. 

There was no corresponding effect on T4 levels, and the increased TSH levels did not 

consistently correlate with thyroid gland weight or histopathological findings on an individual 

animal basis. 

Mean testosterone levels were decreased in a dose-related manner (↓45%, NS and ↓69%, p<0.05) 

in the 400 and 800 mg/kg/day groups, respectively. The control group had three animals with 

unusually high testosterone levels (>8.00 ng/mL), and the control mean was above the 

performance criteria maximum (3.96 ng/mL) when these animals are included in the analysis. 

When these animals were excluded from the analysis, the mean value for the control group was 

2.27 ng/mL, which is similar to the values for the treated groups (2.29 and 1.35 ng/mL) although 

a statistical analysis was not conducted using the adjusted mean. 

At the scheduled euthanasia, no treatment-related findings were noted. A slight treatment-related 

effect on thyroid morphology is considered possible based the larger number of animals with 

follicular height Grade 1 and colloid area Grade 5 in the control group relative to the 400 and 

800 mg/kg/day animals; this is consistent with a higher number of treated animals with follicular 

height and colloid area scores of 3. These findings are consistent with higher mean thyroid 

weights and elevated TSH levels in the treated animals; however, these endpoints were not 

correlated when evaluated on an individual animal basis. Additionally, increased follicular 

height was not necessarily correlated with decreased colloid area in individual animals. There 

were no treatment-related findings in the testes, epididymides or kidneys. 

The high dose tested was considered to be adequate based on the results of a study in which a 

metolachlor dose of 1000 mg/kg/day (route of administration not reported) resulted in overt 

toxicity (all of the males found dead, euthanized in extremis or terminated early). In addition, 

organ toxicity was observed in the current study with both liver and kidney weights significantly 

increased at 400 and 800 mg/kg/day. 

Steroidogenesis Assay; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48617809), H295R cells cultured in vitro in 24-well plates 

were incubated with metolachlor, (97.3% a.i., Batch # 20081115) at log concentrations from 

0.0001 to 100 μM in triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle, 

at a final concentration was 0.05%. 

Testosterone and estradiol concentrations were measured by HPLC/MS-MS with positive ion 

multiple reaction monitoring. Three independent experiments were performed. A Quality 

Control (QC) plate was run concurrently with each independent run of a test chemical plate to 

demonstrate that the assay responded properly to positive control agents at two concentration 

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levels; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of 

estradiol and testosterone production. The highest suitable concentration of metolachlor in all 

three runs was 100 μM. 

Guideline recommendations were met, including adequate production of testosterone and 

estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with 

positive controls. Cytotoxicity greater than 20% was not observed. 

Statistically significant changes in testosterone production after incubation with metolachlor 

were not observed, although the mean fold-difference was decreased 17% for 100 μM 

metolachlor. Statistically significant increases in estradiol production were observed in two of 

the three runs at 10 μM, and in all three runs at the 100 μM concentration. The mean folddifferences were approximately 1.2- and 1.3-fold, respectively, and showed a concentrationdependent response. 

Based on the hormone responses in each of the three independent runs, metolachlor treatment 

resulted in statistically significant and reproducible increases in estradiol production, but did not 

result in a statistically significant and reproducible alterations in testosterone production. 

Uterotrophic Assay; (OCSPP 890.1600) 

In an uterotrophic assay (MRID 48617801) conducted to screen for potential estrogenic activity, 

metolachlor (97.3% a.i., lot # 20081115) in corn oil was administered daily via subcutaneous 

(s.c.) injection to groups of six ovariectomized Sprague Dawley rats at dose levels of 0 

(vehicle), 100, 300, or 1000 mg/kg/day on post-natal days (PND) 57-59. A positive control 

group was treated with a daily dose of 17α-ethynyl estradiol (EE) at 0.5 μg/kg/day by s.c. 

injection. Body weights were determined daily. All animals were terminated and necropsied on 

PND 60 approximately 24 hours after the final dose administration to determine wet and blotted 

uterine weights. 

All animals survived until scheduled termination. No test substance-related clinical findings 

were noted in any of the animals during the study. Body weights were comparable to the 

controls throughout the duration of the assay, but overall (Days 1-4) body weight gains were 

decreased (p<0.01) in the 300 mg/kg/day (↓42%) and 1000 mg/kg/day (↓53%) metolachlor 

groups compared to the controls. Body weight gains in the positive control (EE) group were also 

decreased (p<0.01) by 57% compared to the controls. 

Uterine weights in the metolachlor treated groups were comparable to the controls. Absolute wet 

and blotted uterus weights for the positive control (EE) group were increased (p<0.05) as 

expected by 288% and 175%, respectively. 

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Based on the observed decreases (42% and 53%) in body weight gain at 300 and 1000 

mg/kg/day, adequate dose levels were achieved in this study. 

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

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

ER/AR Transcriptional Activation 

In a published, non-Guideline study (Kojima et al., 2004), the potential of 200 chemicals 

(including metolachlor; purity 95-100%) to interact with two human ER subtypes (hERα and 

hERβ) and one human androgen receptor (hAR) in transactivation assays with Chinese hamster 

ovary cells (CHO-K1 cells) was investigated. For detection of hERα or hERβ activity, cells were 

transfected with pcDNAERα or pcDNAERβ, pGL3-tkERE, and pRL-SV40. For detection of 

hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flutolanil 

was tested over a concentration range from 10−8 to 10−5 M, and the responses were compared to 

the activity of 17β-estradiol (E2; purity >97%) or 5α-dihydrotestosterone (DHT; purity 95%) for 

the ER or AR assays, respectively. For measurement of antagonistic activity to hERα, hERβ and 

hAR, either 10−11 or 10−10 M E2 or 10−10 M DHT was added to the cell cultures along with the 

test compound, respectively. Agonistic activities were evaluated by the relative activity 

expressed as 20% relative effective concentration (REC20), the concentration of the compound 

demonstrating 20% of the activity of 10−10 M E2, 10−9 M E2, or 10−9 M DHT for ERα, ERβ, and 

AR, respectively. Antagonistic activities were evaluated similarly by the relative activity 

expressed as 20% relative inhibitory concentration (RIC20), the concentration of the compound 

showing 20% inhibition of the activity of E2 or DHT against ERα, ERβ, and AR, respectively. 

Metolachlor did not show agonist or antagonist activity for ERα, ERβ, or AR in this study. 

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

In a subchronic oral toxicity study in rats (MRID 43928923), S-metolachlor (89.6% purity; 

93.7% S-isomer) was administered to Crl: COBS (SD) BR rats (15/sex/group, except for females 

in the low-, medium-low, and medium-high dose groups; n=10/group) in the diet at nominal 

concentrations of 0 (acetone-treated diet), 30, 300, 3000, or 10,000 ppm for 13 weeks. Estimated 

compound intakes (by standard conversion) were 0, 1.5, 15, 150, and 500 mg/kg/day. Five 

rats/sex in the control and high-dose groups were permitted a 28-day recovery period after the 

13-week treatment period. Organs that were weighed included the liver, kidneys, testes, ovaries, 

and adrenals. Gross and microscopic pathological examinations were conducted on these tissues 

as well. 

No deaths occurred during the study, and no treatment-related effects on appearance, behavior, 

or physical examinations were noted in any treatment group. Treatment-related systemic toxicity 

was observed in the medium-high and high-dose groups as decreases in body weight, body 

weight gain, and food consumption and efficiency. Body weights were decreased (p<0.05 and 

0.01) in medium-high (↓8-10% from Week 2) and high-dose males (↓11-13% from Week 1), 

respectively, and were decreased (p<0.05 and 0.01) in medium-high (↓8-10% from Week 8) and 

high-dose females (↓5-17% from Week 1), respectively. Body weight gains were decreased by 

15.7 and 24.3% in males and 18.8 and 40.8% in females in the medium-high and high-dose 

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groups, respectively. No apparent treatment-related effects were noted in ophthalmological 

examinations, or in hematology, clinical chemistry, or urinalysis parameters. Decreased (p<0.05 

and 0.01) absolute adrenal weights were noted in high-dose animals ((↓17 and 22%), and kidney 

weights were increased (p<0.01) in the medium-high and high-dose males (↑18%), with 

increases in relative kidney weights in males and females. No gross pathology findings were 

noted, and the only histopathological finding was an increased incidence of eosinophilic 

intracytoplasmic inclusion bodies in high-dose males (6/10 vs. 0/10 control). 

The systemic toxicity NOAEL is 300 ppm (15 mg/kg/day) and the LOAEL is 3000 ppm (150 

mg/kg/day) based on decreased body weight, body weight gain, and food consumption and 

efficiency in both sexes and increased kidney weights in males. 

In another subchronic oral toxicity study in rats (MRIDs 44775401 and 44775402), metolachlor 

(97.7% purity) was administered to Crl:CD (SD) BR rats (20/sex for control, 10/sex/treated 

group) in the diet at nominal concentrations of 0, 30, 300, or 3000 ppm (mean-measured 

concentrations of 0, 2.00, 20.2, and 210 mg/kg/day for males and 0, 2.32, 23.4, and 259 

mg/kg/day for females). Organs that were weighed included the liver, kidneys, testes, 

epididymides, ovaries, and adrenals. Gross and microscopic pathological examinations were 

conducted on these tissues, as well as the prostate, seminal vesicle, uterus, vagina, pituitary, and 

thyroids. 

No treatment-related deaths or clinical signs occurred during the study (two deaths occurred 

unrelated to treatment). In addition, there were no treatment-related effects on ophthalmologic 

parameters, water consumption, urinalysis, food efficiency, or gross/microscopic pathology in 

either males or females. There was no evidence of any treatment-related effect in male rats. 

Decreased (p<0.05 or 0.01) body weights were observed in the high-dose females (↓8-11%); 

decreases (p<0.05) in the medium-dose group were not considered toxicologically relevant (↓7-

9%). Decreased (p<0.01, ↓22%) overall body weight gain was observed in the high-dose 

females, but decreased (NS) body weight gains in the low- and medium-dose groups displayed 

no dose response (↓13 and 12%, respectively). Decreased (p<0.05 or 0.01) food consumption 

was reported in all female dose groups during Week 1 (↓12-14%), and in the low- and mediumdose groups throughout the study (p<0.05, ↓10-13%). Overall mean food consumption was 

decreased (p<0.01) similarly in the low-, medium-, and high-dose females (↓11, 11, and 12%). 

The toxicological effect of treatment on food consumption is questionable as there was no doseresponsive effect and food efficiency was not affected. 

Changes in absolute and relative organ weights were limited to decreased liver weight in the 

low- and medium-dose females (↓11 and 12%, respectively), and increased relative liver (highdose females) and kidney weights (all treated females). These effects were not considered 

toxicologically relevant due to the small magnitude and the lack of accompanying histopathology 

changes. The LOAEL for female rats is 259 mg/kg/day based on decreased body weight and 

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body weight gain; the NOAEL for females is 23.4 mg/kg/day). The LOAEL for male rats is not 

established (≥210 mg/kg/day), and the NOAEL for >210 mg/kg/day. 

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

In a subchronic oral toxicity study in dogs (MRID 43928922), S-metolachlor (95.4% S-isomer) 

was administered to Beagle dogs (4/sex/group) in the diet at nominal concentrations of 0 

(acetone-treated diet), 300, 500, 1000, or 2000 ppm for 13 weeks. Due to palatability issues at 

the highest dose, high-dose dogs were administered the test compound by capsule (700 mg/day) 

starting at Week 3, and the study was extended for three weeks to achieve capsule dosing for 14 

weeks (high dose) and test diet exposure for 16 weeks (lower doses). Mean-measured compound 

intakes were 0, 9.0, 15.1, 31.1, and 62 mg/kg/day for males and 0, 10, 17.2, 31.5, and 74 

mg/kg/day for females. Organs that were weighed included the liver, kidneys, testes, ovaries, 

thyroid, and adrenals. Gross and microscopic pathological examinations were conducted on 

these tissues, as well as the pituitary, mammary gland, epididymides, prostate, vagina, and 

uterus. 

No animals died during the study, and clinical signs were limited to incidences of few or no 

stools in the medium-high and high-dose dogs. No significant treatment-related effects were 

noted on body weight, although body weight gains were decreased (NS; −0.48 and −0.43 kg, 

respectively) in the medium-high and high-dose males during Week 1, and were negative 

(p<0.01; −0.98 kg) in the high-dose females during Week 1. Food consumption was decreased 

in the medium-high dose males and high-dose males and females during Weeks 1 and 2, but 

recovered during Weeks 13-16. No treatment-related effects were noted for ophthalmological 

examinations, in hematology, clinical chemistry, or urinalysis parameters, or for organ weights 

or gross and microscopic pathology. 

The systemic toxicity NOAEL is ≥2000 ppm (62 and 74 mg/kg/day in males and females, 

respectively) and the LOAEL is >2000 ppm based on a lack of biologically-relevant findings at 

doses up to 2000 ppm (700 mg/day) except for the palatability issue. 

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

In a subchronic oral toxicity study in dogs (MRID 00032174), metolachlor (purity not reported?) 

was administered in the diet to Beagle dogs (8/sex/group for control and high dose groups; 

6/sex/group for low- and mid-dose groups) at nominal concentrations of 0, 100, 300, or 1000 

ppm (mean-measured doses of 0, 2.92, 9.71 and 29.61 mg/kg/day, respectively, in males, and 0, 

2.97, 8.77, and 29.42 mg/kg/day, respectively, in females) for six months. Organs that were 

weighed included the liver, kidneys, testes, ovaries, pituitary, thyroid, and adrenals. Gross and 

microscopic pathological examinations were conducted on these tissues, as well as the mammary 

gland, prostate, and uterus. 

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There were no deaths or clinical signs of toxicity. Mean body weight gain was decreased (NS) 

during Weeks 0-13 and 0-26 in the high-dose males (↓55-63%) and females (↓44-50%), and 

mean Week 26 body weights were decreased in the medium- and high-dose males by 11 and 

14%, respectively. Mean overall food consumption was not affected in the high-dose males, but 

was slightly decreased (↓9%) in the high-dose females. Findings of decreased activated partial 

thromboplastin time and increased alkaline phosphatase concentrations were not considered 

toxicologically significant and were not related to the endocrine system. No treatment-related 

effects were noted in the opthalmological examinations and urinalysis investigations. No 

treatment-related effects were noted in organ weights, gross pathology, or histopathological 

examinations. The LOAEL is 1000 ppm (29.61/29.42 mg/kg/day in males and females, 

respectively) based on decreased body weight gain. The NOAEL is 300 ppm (9.7l/8.77 

mg/kg/day in males and females, respectively). 

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

In a 21-day dermal toxicity study (MRID 41833101), metolachlor (96.4% purity) was applied to 

the shaved skin of New Zealand White rabbits (5/sex/dose group) at nominal doses of 0, 10, 100, 

or 1000 mg/kg/day, 6 hours/day, over a 21-day period. Organs that were weighed included the 

liver, kidneys, adrenals, pituitary, ovaries, testes, prostate, epididymides, and uterus. Gross and 

microscopic pathological examinations were conducted on the liver and kidneys. 

All animals survived the treatment. There were no treatment-related effects on clinical signs, 

body weight/body weight gain, food consumption, ophthalmoscopic examinations, hematology, 

or necropsy examinations. Significant increases in total bilirubin were observed in the mediumand high-dose females (↑68 and 72%, respectively), but these increases were not considered 

toxicologically significant. Absolute liver weight was increased (p<0.05, ↑64%) in the high-dose 

males, but there no accompanying laboratory or necropsy findings. There was evidence of skin 

irritation in all treated groups. Very slight erythema and dry skin were observed in all treated 

animals; one female in the low-dose group had fissuring. Fissuring increased in the mediumdose group (n=2/sex) and one male had moderate erythema. Fissuring and wrinkling of the skin 

were noted in all high-dose animals. Histopathological examination showed hyperkeratosis in all 

animals (except low-dose females, n=3), with dose-dependent parakeratosis, congestion of the 

dermis, edema, and subacute lymphocytic infiltration reported in some or all of the treated 

animals. The systemic LOAEL is not established, and the NOAEL is 1000 mg/kg/day (HDT). 

The dermal irritation LOAEL is 10 mg/kg/day (LDT) based on very slight erythema, dry skin, 

and fissuring (one animal). The NOAEL is not established. 

Prenatal Development in Rodents (Rat; OCSPP 870.3700) 

In a developmental toxicity study in rats (MRID 43928925), S-metolachlor (95.6% S-isomer) 

was administered by gavage to mated female Tif: RAI f (SPF) rats (24/group) at doses of 0 

(0.5% carboxymethylcellulose), 5, 50, 500, or 1000 mg/kg/day on GDs 6 to 15, inclusive. The 

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rats were sacrificed at GD 21 of gestation and subjected to gross necropsy. All fetuses were 

weighed, sexed, and examined for gross external alterations. One-half of the fetuses in each 

litter were examined for visceral soft tissue alterations by the Wilson technique. The remaining 

fetuses in each litter were examined for skeletal alterations. 

No maternal toxicity was observed in this study. All females in the medium-high and high-dose 

groups, and 9/24 females in the medium-low dose group exhibited discomfort after daily dose 

administration (e.g., pushing head through bedding for approximately one hour). Decreased 

(p<0.01) body weights were observed in the high-dose dams on GDs 15 (↓7%) and 21 (↓8%), 

and decreases (p<0.01) in body weight gains of 19 and 28%, respectively, were observed in the 

medium-high and high-dose dams during GDs 6-16. Body weight gains in the higher dose 

groups were not decreased during GDs 16-21, and overall body weight gains were affected to a 

lesser degree (p<0.05 and 0.01) with decreases of 12 and 19%, respectively. Treatment-related 

decreases in food consumption were also noted during dose administration (GDs 6-11 and 11-

16); no differences were observed for GDs 16-21. There were no treatment-related findings at 

necropsy. 

Reproductive (cesarean section) parameters were generally not affected by treatment with 

metolachlor; there were no abortions or early deliveries. A decrease in litter size was noted at 

the high dose (12.8 vs. 15.0 control; live fetuses/dam), but was not significant due to increased 

variability. No treatment-related effects were noted during external, visceral, or skeletal 

examinations. The maternal LOAEL is 500 mg/kg/day, based on decreased body weight, body 

weight gain, and food consumption and efficiency, and the maternal NOAEL is 50 mg/kg/day. 

The developmental LOAEL is not determined (>1000 mg/kg/day), and the developmental 

NOAEL is ≥1000 mg/kg/day. 

In an earlier developmental study in rats (MRID 00151941), metolachlor (96.4% purity) in 0.5% 

aqueous carboxymethylcellulose was administered by gavage to Crl: COBS (SD) BR rats 

(25/group) at doses of 0, 30, 100, 300, or 1000 mg/kg/day on GDs 6 to 15, inclusive. The rats 

were sacrificed at GD 20 of gestation and subjected to gross necropsy. All fetuses were 

weighed, sexed, and examined for gross external alterations. One-half of the fetuses in each 

litter were examined for visceral soft tissue alterations by the Wilson technique. The remaining 

fetuses in each litter were examined for skeletal alterations. 

Four dams in the high-dose group died on study on GDs 7, 8, and 10 (n=2); clinical signs of 

toxicity were observed in these animals one or more days prior to death. Treatment-related 

increases (p<0.01) in clinical signs in the high-dose dams included excess salivation (25/25), 

urine-stained abdominal fur (19/25), excess lacrimation (8/25), and clonic/tonic (6/25) or clonic 

(5/25) convulsions. Excess salivation was also observed in the medium-high dose animals 

(16/25). Because the increased salivation may result from gastric irritation, this finding is not 

considered toxicologically relevant. Body weight gain was decreased in the high-dose group for 

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the dosing period (p<0.05, ↓17%) as well as for the entire gestation period (p<0.01, ↓12%) and 

the dosing plus post-dosing periods combined (p<0.05, ↓12%). Treatment-related decreases in 

food consumption were also noted for these same periods (NS, ↓3-5%). Gross pathological 

findings at necropsy were not reported. 

Reproductive (cesarean section) parameters were generally not affected by treatment with 

metolachlor; there were no abortions or early deliveries. Non-significant decreases in the 

number of implantations/dam (14.6 vs. 15.8 control) and litter size (13.8 vs. 15.2 control) were 

noted at the high dose. Mean fetal weight was also decreased (p<0.05, ↓4%) in the high-dose 

group. No treatment-related effects were noted during external, visceral, or skeletal 

examinations. The maternal LOAEL is 1000 mg/kg/day, based on deaths and increased 

incidences of clinical signs, and the maternal NOAEL is 300 mg/kg/day. The developmental 

LOAEL also is 1000 mg/kg/day based on reductions in the number of implantations per dam and 

the resulting decreased litter size, as well as the decrease in mean fetal weight. The 

developmental NOAEL is 300 mg/kg/day. 

Prenatal Development in Non-Rodents (Rabbit; OCSPP 870.3700) 

In a developmental toxicity study in rabbits (MRID 43928924), S-metolachlor (89.6% purity; 

93.7% S-isomer) was administered to artificially-inseminated, female New Zealand White 

rabbits (19/dose group) by gavage at doses of 0 (3% corn starch with 0.5% Tween 80 

suspension), 20, 100, or 500 mg/kg/day from GDs 7-19, inclusive. On GD 29, all surviving does 

were sacrificed, and all fetuses were weighed, sexed, and external examinations were conducted. 

Each fetus was weighed, examined for gross external observations, sacrificed, examined for 

visceral abnormalities, then eviscerated, stained with Alizarin Red S and examined for skeletal 

abnormalities. 

One high-dose rabbit died after anorexia and body weight loss, and was considered treatment 

related. One death in the low-dose group was related to aborting, and a medium-dose doe was 

sacrificed after breaking a hindlimb. Maternal toxicity, consisting of little/none/soft stools was 

observed in the medium- and high-dose animals (14/19 and 19/19, respectively vs. 6/19 control). 

Decreased (p<0.05) body weight was observed in the high-dose animals on GD 19 (↓8%) with a 

slight decrease on GD 29 (NS; ↓3%). Body weight gain was decreased during the dosing period 

(GDs 7-19) at the high dose (↓121%), with a compensatory increase during GDs 19-29 (↑254%); 

the overall decrease in body weight gain was 29% in the high-dose group. Decreases in food 

consumption and efficiency were also noted in the high-dose group, primarily during the 

treatment period. There were no treatment-related findings at necropsy. 

Reproductive (cesarean section) parameters were generally not affected by treatment with Smetolachlor. Two does aborted during the study (one each in the low- and medium-dose 

groups), but a relation to treatment was not established. No treatment-related effects were noted 

during external, visceral, or skeletal examinations. The maternal LOAEL is 100 mg/kg/day, 

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based on clinical signs, and the maternal NOAEL is 20 mg/kg/day. The developmental LOAEL 

is not determined (>500 mg/kg/day), and the developmental NOAEL is ≥500 mg/kg/day. 

In an earlier developmental toxicity study in rabbits (MRID 00041283), metolachlor (95.4% 

purity) in 0.75% aqueous hydroxymethylcellulose was administered by gavage to assumedpregnant New Zealand White rabbits (16/group) from GDs 6-18, inclusive, at doses of 0, 36, 

120, or 360 mg/kg/day. The animals were sacrificed on GD 30 and the fetuses were weighed, 

sexed, and examined for evidence of developmental effects. Each fetus was weighed, examined 

for gross external observations, sacrificed, examined for visceral abnormalities excluding the 

brain, then eviscerated, stained with Alizarin Red S, and examined for skeletal abnormalities. 

One doe in each the low- and high-dose groups died on GDs 24 and 29, respectively; the cause 

of death in both animals was attributed to persistent anorexia. Two rabbits aborted, one in the 

medium-dose group (GD 25) and a second doe with persistent anorexia in the high-dose group 

(GD 17). One rabbit in each group (control and treatment) reportedly delivered prior to GD 30. 

There was a treatment-related increase in the incidence of persistent anorexia in the does in the 

high-dose group; anorexia was defined as consumption of less than one-half of the daily food 

allotment. Food consumption data were not provided to support this finding. There was a 

treatment-related decrease in body weight gain in the high-dose group for the dosing period 

(p<0.0l, ↓500%) and the combined dosing and post-dosing periods (NS, ↓133%). There was no 

treatment-related increase in gross pathological findings in maternal animals at necropsy. 

Reproductive (cesarean section) parameters were not affected by treatment with metolachlor, and 

no treatment-related external, visceral, or skeletal developmental effects were observed. 

The maternal toxicity LOAEL is 360 mg/kg/day based on an increased incidence of clinical 

observations (persistent anorexia) and decreased body weight gain; the NOAEL is 120 

mg/kg/day. The developmental toxicity LOAEL is not established (>360 mg/kg/day), and the 

NOAEL is ≥360 mg/kg/day. 

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

In a two-generation reproduction study in rats (MRID 00080897), metolachlor (95.4% purity) 

was administered in the diet to two consecutive generations of CD albino rats (15 males/30 

females) at nominal concentrations of 0, 30, 300, or 1000 ppm (mean-measured intake over 15-

week premating period: 0, 2.4, 23.5, and 75.8 mg/kg/day in F0 males and 0, 2.5, 26.0, and 85.7 

mg/kg/day in F0 females; mean-measured intake over 17-week premating period: 0, 2.3, 23.7, 

and 76.6 mg/kg/day in F1 males and 0, 2.6, 25.7, and 84.5 mg/kg/day in F1 females). Animals 

were observed daily of mortality, morbidity and overt signs of toxicity. Food consumption was 

monitored weekly in parental animals during the pre-mating periods. Parental animals were 

weighed weekly during pre-mating, and males were weighed monthly thereafter. Pregnant 

females were weighed on GD 0, 6, 15 and 20 and LD 0, 4, 7, 14 and 21. Pups were weighed 

individually on LD 4, 7, 14 and 21. Body weights of all animals were also determined at 

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sacrifice. Parent males were sacrificed at 157-167 days of age and female were sacrificed at 

196-208 days of age. At sacrifice, the testes of all parental males (F1 and F0) were examined 

histopathologically for spermatogenesis Organ weights for adult animals were determined for 

the adrenals, brain, heart, kidneys, liver, testes, and thyroid. Histopathological examinations 

were conducted on these organs and others, including the testes (with epididymis), prostate, 

ovaries, uteri, mammary gland, and pituitary and thyroid glands. 

There were no deaths in the F0 generation. Two females of the F1 generation died during the 

premating period (one each in the medium- and high-dose groups). One female in the mediumdose group was found dead on GD 19 and a control group female was sacrificed in a moribund 

condition on LD 1. Based on necropsy examinations, none of the deaths were considered 

treatment-related. There were no treatment-related clinical signs of toxicity in either generation. 

Body weight, body weight gain, and food consumption were unaffected in the F0 generation. In 

the F1 generation, food consumption was decreased (p<0.05 or 0.01) in females of the high-dose 

group at Weeks 1, 8, and 15; however, there was no effect on body weight/body weight gain. 

There were no treatment-related effects on organ weights in parental animals in either 

generation, including the adrenals, liver, kidneys, brain, testes (with epididymides) and thyroid 

gland. There were also no treatment-related histopathological finding in either generation in the 

ovaries, uterus, mammary glands, testes, prostate, epididymides, thyroid and pituitary glands. 

There also was no evidence of a treatment-related effect on any of the reproductive parameters 

for either generation. 

Offspring body weight was decreased (p<0.05 or 0.01) in the F1 litters on LDs 14 and 21 (↓4-

9%) and in the F2 litters on LDs 4, 7, 14, and 21 (↓5-8%). These offspring weight loss findings 

are considered toxicologically significant. The parental toxicity LOAEL is not established, and 

the NOAEL is 1000 ppm (75.8/85.7 mg/kg/day in F0 males and females, respectively; 76.6/84.5 

mg/kg/day in F1 males and females, respectively). The reproductive toxicity LOAEL is not 

established, and the NOAEL is 1000 ppm (75.8/85.7 mg/kg/day in F0 males and females, 

respectively; 76.6/84.5 mg/kg/day in F1 males and females, respectively). The offspring LOAEL 

is established at 1000 ppm (75.8/85.7 mg/kg/day in F0 males and females, respectively; 76.6/84.5 

mg/kg/day in F1 males and females, respectively) based on decreased offspring body weights in 

the F1 and F2 litters. The NOAEL is 300 ppm (23.5/26.0 mg/kg/day in F0 males and females, 

respectively; 23.7/25.7 mg/kg/day in F1 males and females, respectively). 

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

In a chronic oral toxicity/carcinogenicity study in rats (MRID 0012937748617809), metolachlor 

(95.3% purity) was administered to Crl:CD (SD) BR albino rats (70/sex/group for control and 

high-dose groups; 60/sex/group for low- and mid-dose groups) at nominal concentrations of 0, 

30, 300, or 3000 ppm (0, 1.5, 15, or 150 mg/kg/day based on standard conversion) for two years. 

The additional ten rats/sex/group in the control and high-dose groups were kept on study for 

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twelve months; five rats/sex/group were sacrificed after twelve months of treatment, and the 

remaining five/sex/group were fed the negative control diet for four weeks and sacrificed. 

Organs that were weighed included the liver, kidneys, heart, spleen, testes, and ovaries. Gross 

and microscopic pathological examinations were conducted on these tissues and others, 

including the prostate, uterus, mammary gland, pituitary, adrenals, and thyroid glands. 

Comparable mortality rates were observed in the treated and control animals. There were no 

treatment-related clinical signs of toxicity, although signs of sialodacryoadenitis viral infection 

were noted in approximately half of the study animals (all groups and sexes). Mean body weight 

gains were decreased (NS) in the high-dose females (↓11-17%) from Week 26 to the end of the 

study, and mean food consumption was slightly decreased (NS, ↓4-9%) throughout the study in 

the high-dose females. Absolute and relative liver weights were increased (↑7 and 13%, 

respectively) in the high-dose males, and these increases persisted in the high-dose males after 

the four-week recovery period. However, the toxicological significance of the finding is 

questionable as there were no accompanying clinical pathology or histological changes. No 

treatment-related effects were noted on other organ weights in males, or for organ weights in 

females, including the testes and ovaries. In addition, no histopathological findings were noted 

in the kidneys, adrenal gland, ovaries, uterus, mammary glands, testes, prostate, or thyroid gland. 

The LOAEL is 3000 ppm (estimated 150 mg/kg/day) for females based on decreased body 

weight gain and food consumption. The NOAEL is 300 ppm (estimated 15 mg/kg/day) for 

females. A LOAEL is not established for males, but the NOAEL is 3000 ppm (estimated 150 

mg/kg/day). 

In addition, the HED Cancer Peer Review Committee evaluated the carcinogenic potential of 

metolachlor on several occasions. Administration of metolachlor in the diet up to 3000 ppm 

(150 mg/kg/day) yielded increased incidences of liver adenomas and combined 

adenomas/carcinomas in female rats. Males showed a statistical trend (but no pair-wise 

significance) for liver tumors. Metolachlor is classified as a Group C possible human carcinogen 

due to liver tumors in rats, with risk quantitated with a Margin of Exposure approach. 

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200) 

In a carcinogenicity study in mice (MRID 00117597), metolachlor (reported to be 95% purity) 

was administered in the diet to CD-1 mice (68/sex/group) at nominal concentrations of 0, 300, 

1000, or 3000 ppm (0, 45, 150, or 450 mg/kg/day, based on standard conversion). Based on the 

calculated mean intakes in treatment groups at Weeks 26, 52, 78, and 104, the overall meanmeasured intakes were 50, 170, and 526 mg/kg/day, respectively, in males and 64, 224, and 704 

mg/kg/day, respectively, in females. Eight mice/sex/group were sacrificed at 12 and 18 months. 

Information regarding the organs/tissues selected for terminal weights and histopathological 

examination were not provided. 

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High-dose females had an increased (p<0.05) overall mortality rate due to a number of deaths 

during the first few weeks of treatment (34/52 vs. 24/52 control, at termination). Although the 

early deaths were attributed to a viral infection, the potential contribution of the test material 

cannot be dismissed. Body weight was decreased (p<0.01 or 0.05) from Week 2 in the high-dose 

males (↓5-9%) and decreased (p<0.05) at approximately two-thirds of the time points from Week 

32 in high-dose females (↓5-7%). Body weight gains were consistently decreased in the highdose males (↓12-51%) and females (↓14-41%). Food consumption was comparable between 

treated and control groups until Week 90 of treatment, at which time the consumption in the 

high-dose males was decreased by 10%; occasional statistical significance was mentioned. 

There was no effect on food consumption in treated females. There were no treatment-related 

effects on hematology, clinical chemistry, or urinalysis parameters. Organ weights were not 

affected except for a dose-related decrease (significance not reported) in the absolute and relative 

weights of the seminal vesicles of high-dose males. However, there was no effect on testes 

weight and no accompanying histological changes were noted in the seminal vesicles; therefore, 

the toxicological significance of the finding is questionable. There were no treatment-related 

microscopic changes, and no treatment-related increases in tumor incidence in the study. 

The LOAEL is 3000 ppm (526/704 mg/kg/day in males and females, respectively) based on 

possible treatment-related deaths in females and decreased body weight/body weight gain in 

males and females. The NOAEL is 1000 ppm (170/224 mg/kg/day in males and females, 

respectively). 

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

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

1989), metolachlor (97% purity) was administered to Beagle dogs (6/sex/group in the control and 

high-dose groups and 4/sex/group in the low- and medium-dose groups) in the diet at 

concentrations of 0, 100, 300, or 1000 ppm for up to 52 weeks (mean-measured doses of 0, 3.5, 

9.7, and 32.7 mg/kg/day, respectively, in males and 0, 3.6, 9.7, and 33.0 mg/kg/day, respectively, 

in females). Two dogs/sex in the control and high-dose groups were designated as recovery 

animals and allowed a four-week recovery period after the 52-week treatment period. An 

additional 4 dogs/sex/group were administered the same treatments and sacrificed after Week 13. 

Organs that were weighed included the liver, kidneys, testes, epididymides, ovaries, pituitary, 

thyroid, and adrenals. Gross and microscopic pathological examinations were conducted on 

these and other tissues, including the mammary glands, prostate, vagina, and uterus. 

There were no treatment-related deaths or clinical signs of toxicity. Mean body weight gains 

were decreased (NS) in the high-dose females for animals sacrificed at Week 13 (↓16%) and at 

Week 52 (↓17%). Decreases in food consumption were noted in high-dose males and females, 

but were not generally associated with decreased body weights. Alkaline phosphatase 

concentrations were increased (p<0.05, 0.01, or NS) in the high-dose females throughout the 

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study (↑27-81%); however, the increases were not considered toxicologically significant due to 

the lack of accompanying necropsy findings. There was a treatment-related decreased in kidney 

weights of males in the low- (NS, ↓16%), medium- (p<0.05, ↓19%), and high-dose (p<0.01, 

↓27%) groups, with no treatment effects observed on kidneys in females and no accompanying 

necropsy findings. No other treatment-related changes were noted in organ weights although 

pituitary weights were increased (NS) in both males and females at the low-, mid- and high- dose 

levels (males, ↑31, 56 and 22%; females, ↑13, 22, 40%). No treatment-related gross or 

microscopic pathology changes were noted in any tissues, including the ovaries, uterus, vagina, 

mammary gland, testes, epididymides, prostate, adrenals, thyroid or pituitary glands. 

The LOAEL is 1000 ppm for females (33.0 mg/kg/day) based on decreased body weight gain, 

and the NOAEL is 300 ppm (9.7 mg/kg/day). The LOAEL for males is not established, and the 

NOAEL for males is 1000 ppm (32.7 mg/kg/day). 

Avian Reproduction (OCSPP 850.2300) 

In an avian reproduction study (MRID 44015602), 24-week old Mallard ducks (Anas 

platyrhynchos) were administered metolachlor (97.3% purity) in the feed at nominal 

concentrations of 0 (no solvent required), 50, 200, or 800 ppm (mg/kg) diet for 20 weeks (ten 

weeks prior to the start of egg production and ten weeks of egg production). Mean dietary doses 

were not calculated for the study. Each treatment group was comprised of sixteen mating pairs, 

with one pair per pen. A progressive photo period was employed in this study: 7 hours/day 

through Week 9 and 17 hours/day until the end of the study. Temperature was maintained at 

15.6-27.8°C throughout the study and humidity was not reported. Body weights were measured 

at test initiation, end of Week 3, during Weeks 7 and 10, and at test termination. Overall weight 

gain for males and females was determined from initial and final body weights. Food 

consumption in each replicate was measured bi-weekly throughout the pre-treatment and 

treatment periods of the adult phase. Individual chick body weights were recorded on Day 14 

after hatching; egg shell thickness was measured every week during egg production (alternating 

from even and odd numbered pens during even and odd numbered weeks). Adult birds and 

chicks were observed routinely for mortality and clinical signs. 

No overt signs of toxicity or treatment-related mortalities were reported. There was a significant 

difference in 14-day survivor weights at the low and high concentrations compared to control. 

However, because the mean 14-day survivor weight at the high concentration was increased 

(p<0.05, ↑11%), the decreased (p<0.05, ↓9%) weight at the low concentration was not 

considered to be treatment related. There were no treatment-related effects on any of the 

measured reproductive endpoints. The slight decrease in the percentage of 14-day-survivors of 

normal hatchlings at the medium concentration was not considered to be treatment related (93 vs. 

97% control) because the percentages of viable embryos of eggs set and of normal hatchlings of 

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live 3-week embryos were slightly decreased (NS) compared to the control group. Therefore, the 

NOAEC is established as 800 pprn and the LOAEC is not determined (≥800 ppm). 

In another avian reproduction study (MRID 46508901), 26-week old Bobwhite quail (Colinus 

virginianus) were administered metolachlor (97.2% purity) in the feed at nominal concentrations 

of 0 (acetone, 0.2% v:w and corn oil, 0.3% v:w), 160, 400, or 1000 mg/kg diet for 20 weeks (ten 

weeks prior to the start of egg production and ten weeks of egg production). Calculated mean 

dietary doses were 13.9, 34.6, and 85.9 mg/kg/day, respectively for the low-, medium-, and highdose groups. Each treatment group was comprised of sixteen mating pairs, with one pair per 

pen. A progressive photo period was employed in this study: 8 hours/day through Week 7 and 

17 hours/day until the end of the study. Temperature was maintained at 24.1±0.7°C throughout 

the study and humidity was maintained at 54.9±15.7%. Individual adult body weights were 

recorded at Week 0, 2, 4, 6, 8, and 20, and body weight changes for the study period were 

determined. Food consumption in each replicate was measured weekly throughout the pretreatment and treatment periods of the adult phase. Individual chick body weights were recorded 

after hatching and on Day 14 after hatching; egg shell thickness was measured every week 

during egg production (alternating from even and odd numbered pens during even and odd 

numbered weeks). Adult birds and chicks were observed routinely for mortality and clinical 

signs. 

There was no reported treatment-related effect on adult mortality; there were three incidental 

deaths during the study in the high-dose group. No overt signs of toxicity were observed due to 

treatment, and all birds appeared normal throughout the study, except for incidental clinical 

findings. Adult body weight changes and food consumption during the treatment period were 

generally similar in all groups, and there was no evidence of a treatment-related effect. 

Occasional increases in food consumption were noted in the low-and medium-dose groups, but 

overall mean food consumption was 17-18 g/bird/day throughout the study. No treatmentrelated findings were observed at necropsy. 

No treatment-related effects were observed on egg production or quality, fertility, embryonic 

development, hatchability, or survival of hatchlings at the low or medium treatment 

concentrations. At the low treatment concentration, there was slight increase (p<0.05) in egg 

production (percentage of eggs laid of maximum number laid) that also resulted in increases 

(p<0.05) in hatchlings and 14-day old survivors. Because the increase in egg production at the 

low concentration was slight, not concentration dependent, and represented an improvement in 

reproductive performance, the difference was not considered to be treatment related. There were 

no differences between the control and the medium treatment groups for any of the reproductive 

parameters measured; however, there was a slight reduction in the percentage of viable embryos 

of eggs set (86 vs. 91% control). This decrease was due to data from one pen with only 2/46 

eggs set containing viable embryos. The percentage of viable embryos (91%) was identical to the 

control value if the data from this one pen was not used. 

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Effects were noted in four of the parameters evaluated: eggshell thickness, Day 14 survivor 

weight, eggs cracked, and percentage eggs not cracked/eggs laid. Eggshell thinning occurred at 

all treatment concentrations (p<0.01, 0.22 mm vs. 0.23 mm control). Because mean eggshell 

thickness was the same (0.22 mm) for all treatment groups, eggshell thinning was considered to 

be not biologically significant at the low and medium concentrations (in the absence of other 

adverse effects (e.g., cracking, reduced viability). Eggshell thinning occurred at the high 

concentration in conjunction with an increase in eggs cracked/eggs laid (p<0.05, 6 vs. 1%). A 

decrease (p<0.05) in Day 14 survivor weight was noted at the medium concentration (↓5%), but 

this response was not dose dependent. 

The highest dose (85.9 mg/kg/day) is the LOAEC for the Bobwhite quail due to an increased 

incidence in eggs cracked, and 34.6 mg/kg/day is the NOAEC. 

Fish Early Life-Stage Toxicity (OCSPP 850.1400) 

In an early life-stage estuarine/marine fish study (MRID 43044602, 1980), Sheepshead minnow 

(Cyprinodon variegatus) embryos were obtained by stripping eggs from adult fish that were 

collected wild. Egg production was enhanced by addition of human chorionic gonadotrophin, 

and a sperm suspension prepared from macerated testes was added to fertilize the eggs. Fish 

(100 embryos/treatment) were exposed to metolachlor (97.0% purity) at nominal concentrations 

of 0 [negative and solvent (TEG; 130 mg/L) controls], 0.62, 1.2, 2.5, 5.0, or 10 mg/L (meanmeasured concentrations were 0.55, 1.0, 2.2, 4.1, and 8.6 mg/L). Embryo mortality and time 

hatched were observed, and survival of juveniles was monitored for 22 days. The photoperiod 

was 12 hours light/12 hours dark, instead of the recommend 16 hours light/8 hours dark. 

Standard length and weight were measured at study termination. 

The mean-measured concentrations ranged from 82 to 89% of the nominal concentrations. 

Hatching was not affected at concentrations ≤8.6 mg/L. Hatching was >90% complete by Day 7 

in all treatments (and ≥99% complete by Day 8), with no delay in hatch observed. Several 

embryonic development abnormalities (not reported) were observed, but were not attributed to 

metolachlor. The test was terminated at Day 26; it was reportedly because fish were dying as a 

result of the artificial salt introduced in the system, but increased mortality was not evident 

across all of the treatments. Mortality was significant at concentrations ≥4.1 mg/L after Days 8, 

15, and 22 post-hatch, with overall mortalities of 40 and 100% in the 4.1 and 8.6 mg/L treatment 

groups, respectively. Length was decreased (p<0.05) in the 4.1 mg/L treatment group (no 

survivors at the highest concentration). Weight was not significantly affected, although a trend 

in decreasing weight and length was observed at concentrations ≥2.2 mg/L. Therefore, the 

LOAEC is 2.2 mg/L based on decreased weight and length, and the NOAEC is 1.0 mg/L. 

In an early life-stage freshwater fish study (MRID 44995903), Fathead minnow (Pimephales 

promelas) embryos were obtained from an in-house culture. Fish (120 embryos/treatment, 

thinned to 80 embryos/treatment) were exposed to S-metolachlor (98.6% purity) at nominal 

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concentrations of 0 (negative control), 31, 63, 130, 250, 500, or 1000 μg/L (mean-measured 

concentrations were 30, 56, 110, 220, 450, and 870 μg/L). Embryo mortality and time to hatch 

were observed, and survival of juveniles was monitored for 22 days. The photoperiod was the 

recommend 16 hours light/8 hours dark. Standard length, and wet and dry weights, were 

measured at study termination. 

The mean measured concentrations ranged from 85 to 97% of the nominal concentrations. No 

sublethal signs of toxicity were reported. Hatching was not affected at concentrations 

≤1000 μg/L. The mean percent hatch ranged from 85-90%, and no delay in hatch was observed. 

The 30-day post-hatch percent survival ranged from 93-100%. Length was decreased 

(significance not reported) ≥220 μg/L. Treatment-related decreases in wet weight were observed 

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

decreased dry weight, and the NOAEC is 30 μg/L. 

Mechanistic Studies: Thyroid System-Related Genes (Fish) 

In a published, non-Guideline study (Jin et al., 2011), the effect of metolachlor treatment on the 

thyroid system of juvenile and adult Japanese medaka fish was determined. In place of 

plasma/serum hormone determination, transcriptional analyses of genes regulating the 

hypothalamic-pituitary-thyroid (HPT) axis were conducted on RNA isolated from whole juvenile 

fish and from the brains and livers of adult fish. 

The Japanese medaka fish used in the study were propagated at the study laboratory. Newlyhatched fish were maintained in glass tanks (with charcoal-dechlorinated tap water at a constant 

temperature) until they reached juvenile stage (1 month) or adult stage (4 months). For exposure 

testing, male and female juvenile fish were raised in two beakers/sex/dose group (n=4 

fish/beaker) containing metolachlor (98% purity) at nominal concentrations of 0 (control), 1, 10, 

100, and 1000 μg/L in 0.1% ethanol in water (v:v). Adult male and female fish were dosed in 

separate aquariums, two/dose group, containing six fish each; adult fish were dosed at the same 

nominal concentrations as juvenile fish. All fish were exposed to metolachlor for 14 days at 

ambient temperature (261°C); the rearing water and exposure water were changed daily. Fish 

were anesthetized on ice prior to sample collection. Each whole juvenile fish was considered to 

be one sample, yielding eight samples/sex/dose group. The brain and liver were collected from 

each adult fish, and the tissues from three same sex fish from each exposure tank were combined, 

yielding four samples of each tissue/sex/dose group. 

The effect of estrogen exposure, with or without metolachlor exposure, on juvenile male medaka 

fish was also examined. Fish were exposed to estrogen at 25, 50, and 100 ng/L with or without 

metolachlor at a concentration of 100 μg/L. Two vessels were created for each exposure group, 

each containing four fish. This study was conducted concurrently with the metolachlor study so 

that the 100 μg/L and 0 μg/L metolachlor-exposure groups served as the positive and negative 

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controls, respectively. Fish were exposed as described previously, and each whole juvenile fish 

was considered to be one sample. 

Total RNA was isolated from individual juvenile fish and from the brains and livers of adult fish; 

the RNA was denatured and then a reverse transcriptase kit was used to synthesize cDNA. The 

expression of five genes were determined: Rpl-7 (used as a house-keeping gene to standardize 

results), Trh (thyrotropin-releasing hormone), Dio2 (deiodinase enzyme), Thrα (thyroid 

receptor), and Thrβ (thyroid receptor). 

No fish died during exposure. In female juvenile fish, metolachlor exposure increased (p<0.05) 

the expression of Trh at 100 μg/L and the expression of Dio2, Thrα, and Thrβ at all treatment 

concentrations ≥10 μg/L. In male juvenile fish, there were no differences in the expression of 

Trh, Dio2, Thrα, and Thrβ due to metolachlor treatment. In the brains of female adult medaka, 

metolachlor exposure at 100 and 1000 μg/L increased (p<0.05) the expression of all four genes 

related to the thyroid system, with no effects in male adult medaka. Expression of Dio2 in adult 

female livers was increased (p<0.05) at 1000 μg/L, with no effects of metolachlor exposure on 

the expression of Dio2 in adult male livers or the expression of Thrα and Thrβ in adult male or 

female livers. In juvenile male medaka exposed to metolachlor at 100 μg/L in the presence of 

estrogen, expression of Dio2 was increased (p<0.05) at the 50 ng/L concentration, and 

expression of Dio2, Thrα, and Thrβ was increased (p<0.05 or 0.01) at 100 ng/L. No effects of 

metolachlor exposure in the presence of estrogen were observed on expression of Trh. 

The study authors concluded that exposure to metolachlor (10, 100, and 1000 μg/L for juvenile 

female medaka, and 100 and 1000 μg/L for adult female medaka) increased transcription of 

genes related to the thyroid system; transcription induced by metolachlor was gender-, tissue-, 

and developmental stage-specific. Estrogen co-exposure was found to increase transcription of 

thyroid system genes in male juvenile medaka treated with metolachlor. 

Amphibian Development: Spolyarich et al. 2010 (ECOTOX 153638) 

Spotted marsh frog larvae (Limnodynastes tasmaniensis), which is indigenous to Australia, were 

rear in 80-L aquaria filled with dechlorinated tap water with complete water renewal every 14 

days. Larvae were fed every 2nd day with a 1:1 mix of Sera Micron® and Nutrafin® Fry Food 

with increasing food ration as the animals matured. The larval development assay was based on 

Xenopus Metamorphosis Assay (XEMA) with exposure starting at early limb bud emergence 

(Gosner stage 28) and continuing until the majority of larvae were undergoing tail resorption 

(Gosner stage 42). Each replicate contained 10 larvae in 10 L of water within 14.5-L glass 

aquaria fitted with a glass lid to limit evaporation. Each aquarium was aerated and was on a 

12:12 light:dark cycle. Exposure commenced just prior to gonadal differentiation (Gosner stage 

30). Animals were removed at Gosner stage 44 or 45. Spiking took place after every water 

renewal after every 3rd or 4th day of exposure. DO, temperature and pH were measured daily. 

Sex ratios initially based on gross gonadal morphology. Male gonads from Tests 1, 6, 7 (atrazine 

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only) and Test 4 (atrazine, metolachlor and thiobencarb mixture) were examined histologically 

for the presence of oocytes. Atrazine (98% ai), metolachlor (99.5% ai), thiobencarb (99% ai) 

concentrations were analyzed weekly in the 4-day water following the water renewal. Tadpoles 

were exposed to metolachlor at nominal concentrations of 0.1, 1 and 10 μg/L. No atrazine, 

metolachlor or thiobencarb were detected in controls. Recovery of atrazine was on average 

121.5%; average recovery of metolachlor was 90.8%; average recovery of thiobencarb was 

34%. Thiobencarb recovery was 27.2% in a mixture with atrazine and 28.1% in a mixture with 

atrazine and metolachlor. The authors conclude the exposure of spotted march frog lavae to 

atrazine (0.1, 1, 3 and 30 μg/L, metolachlor (0.1, 1 and 10 μg/L) and thiobencarb (90, 180, and 

360 μg/L) from Gosner stage 28 to 42 under controlled lab conditions had no significant effect 

on tadpole growth, development and sex ratio. Binary mixtures of atraine and thiobencarb as 

well as ternary mixtures of all three also had not significant effect on the developing larvae; 

abnormal gonad morphology was observed on two occassions, both from atrazine at 0.1 μg/L 

with one larvae with testicular oovarian follicle but this did not exhibit a concentration associated 

response. In this study, the reviewer noted that the study used wild-caught animals; therefore, 

prior exposure history is unknown. Also, histology was only conducted on male gonads. Sex 

was apparently based on gross gonadal morphology alone and was not verified across all of the 

test animals with histology. 

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APPENDIX 3: References Not Utilized in the Metolachlor 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 Metolachlor 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. 

Aït-Aïssa, S., Laskowski, S., Laville, N., Porcher, J. M., and Brion, F. (2010) Anti-androgenic 

activities of environmental pesticides in the MDA-kb2 reporter cell line. Toxicol In Vitro. 24: 

1979-1985. 

Buccafusco, R.J.; Stiefel, C.; Sullivan, D.M.; et al. (1978) Acute Toxicity of CGA-24705 to 

Rainbow Trout (Salmo gairdneri): Report #BW-78-6-186. (Unpublished study received Jul 13, 

1978 under 100-597; prepared by EG&G, Bionomics, submitted by Ciba- Geigy Corp., 

Greensboro, N.C.; CDL:234396-B) (MRID 00018722) 

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Buccafusco, R.J.; Stiefel, C.; Sullivan, D.M. (1978) Acute Toxicity of CGA-24705 to Bluegill 

(Lepomis macrochirus): Report #BW- 78-6-181. (Unpublished study received Jul 13, 1978 under 

100-597; prepared by EG&G, Bionomics, submitted by Ciba-Geigy Corp., Greensboro, N.C.; 

CDL:234396-C) (MRID 00018723) 

Fink, R. (1974) Final Report: Eight-Day Dietary LC50--Mallard Ducks: Project No. 108-111. 

(Unpublished study received on unknown date under 5G1553; prepared by Truslow Farms, Inc. 

in cooperation with Maryland, Dept. of Agriculture, Div. of Inspection and Regulation, 

submitted by Ciba-Geigy Corp., Greensboro, N.C.; CDL:094220-O) (MRID 00016425) 

Fink, R. (1974) Final Report: Eight-Day Dietary LC50--Bobwhite Quail: Project No. 108-110. 

(Unpublished study received on unknown date under 5G1553; prepared by Truslow Farms, Inc. 

in cooperation with Maryland, Dept. of Agriculture, Div. of Inspection and Regulation, 

submitted by Ciba-Geigy Corp., Greensboro, N.C.; CDL:094220-P) (MRID 00016426) 

Fink, R.; Beavers, J.B.; Brown, R.; et al. (1981) Final Report: Acute Oral LD50--Mallard Duck: 

Project No. 108-200. (Unpublished study received Mar 31, 1981 under 100-587; prepared by 

Wildlife International, Ltd. and Washington College, submitted by Ciba-Geigy Corp., 

Greensboro, N.C.; CDL:244691-B) (MRID 00062465) 

Mai, H., Cachot, J., Brune, J., Geffard, O., Belles, A., Budzinski, H., and Morin, B. (2012). 

Embryotoxic and genotoxic effects of heavy metals and pesticides on early life stages of Pacific 

oyster (Crassostrea gigas). Mar Pollut Bull. 64(12):2663-2670. 

Mathias, F. T., Romano, R. M., Sleiman, H. K., de Oliveira, C. A., and Romano, M. A. (2012). 

Herbicide metolachlor causes changes in reproductive endocrinology of male Wistar rats. ISRN 

Toxicology. 2012:130846. 

Murphy, T. (1987). Dermal Absorption ofMetolachlor in Rats. CIBA-GEIGY 

Corporation, Agricultural Division, Metabolism Department, Greensboro, NC and WIL Research 

Laboratories, Ashland, Ohio. Laboratory Study No. ABR-87051, August 25, 1987. MRID No. 

41833102. 

Pereira, S. P., Fernandes, M. A., Martins, J. D., Santos, M. S., Moreno, A. J., Vicente, J. A., 

Videira, R. A., and Jurado, A. S. (2009). Toxicity assessment of the herbicide metolachlor 

comparative effects on bacterial and mitochondrial model systems. Toxicol In Vitro. 23(8):1585-

1590. 

Reylea, R.A. (2009) A cocktail of contaminants: how mixtures of pesticides at low 

concentrations affect aquatic communities. Oecologia: 159, 363-376. DOI 10.1007/s00442-008-

1213-9. 

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Spolyarich, N., R. Hyne, S. Wilson, C. Palmer and M. Byrne. 2010. Growth, development and 

sex ratios of Spotted Marsh Frog (Limnodynastes tasmaniensis) larvae exposed to atrazine and a 

herbicide mixture. Chemosphere 78: 807 - 813. (ECOTOX Ref. No. 153638) 

Williams, B. K. and Semlitsch, R. D. (2010). Larval responses of three midwestern anurans to 

chronic, low-dose exposures of four herbicides. Arch Environ Contam Toxicol. 58(3):819-827. 

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