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 59
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 59
 

EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: METALAXYL 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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METALAXYL 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.............................................................22 

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

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

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

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

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

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

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

LIST OF TABLES: 

 Table 1. Tier 1 Assays for Metalaxyl.................................................................8 

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

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

 Table 4. Thyroid Pathway for Metalaxyl ...........................................................19 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................29 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................40 

APPENDIX 3. References Not Utilized in the Metalaxyl WoE Analysis.............................51 

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METALAXYL 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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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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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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Page 1 of 51 

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 metalaxyl interacts with E, A or T hormone pathways, the number and 

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

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

in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in 

general systemic toxicity or overt toxicity. 

On August 13, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of 

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

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

metalaxyl with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature). 

For the estrogen pathway, metalaxyl showed an equivocal response in the Tier 1 in vitro estrogen 

receptor (ER) binding assay (interactive in one run and not interactive in the remaining two 

runs); however, it was negative for ERα agonism in the Tier 1 ER transactivation assay (ERTA) 

assay and negative for both ERα agonism and antagonism in a published ERTA study (Kojima et 

al., 2004). Metalaxyl was also negative in the Tier 1 in vitro steroidogenesis and aromatase 

assays. There was no convincing evidence of an interaction with the estrogen pathway in the in 

vivo!mammalian Tier 1 assays!(uterotrophic, female pubertal assays) or in the Part 158 

mammalian or avian studies. Primary follicle counts were increased in the female pubertal 

assay, but no effect was noted in the number of atretic follicles and there were no complementary 

responses in the other estrogen pathway endpoints evaluated in that assay. Overall metalaxyl 

does not appear to have a potential to interact with the estrogen pathway in mammals. 

Conversely, metalaxyl appears to potentially interact with the estrogen pathway in fish. Several 

complementary effects on females were observed in the fish short-term reproduction assay 

(FSTRA) at the high test concentration of 104 mg a.i./L including: effects on gonadal histology 

(decreased ovarian yolk synthesis), decreased plasma vitellogenin (VTG) concentration, 

decreased gonadal somatic index (GSI) and finally, cessation of fecundity (no eggs produced). 

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Page 2 of 51 

Therefore, while there were largely negative responses in the Tier 1 in vitro assays and in vivo

mammalian dataset, for the FSTRA there were complementary responses along several levels of 

biological organization for the females including alterations in enzyme activity (plasma VTG) up 

to the apical endpoint of fecundity. As such, significant effects of the reproductive capacity of 

fish were observed in the FSTRA which suggests that metalaxyl may potentially interact with the 

HPG axis.

There was no convincing evidence of interaction of metalaxyl with the androgen pathway in the 

in vitro (i.e, AR binding and steriodogenesis assays were negative) or in vivo mammalian Tier 1 

(i.e., Hershberger and male pubertal assays were negative) and Part 158 mammalian or avian 

studies (no androgen-related effects observed). In addition, metalaxyl was negative in two 

literature studies evaluating AR binding (Bauer et al., 2002) and AR transcriptional activation 

(Kojima et al., 2004). However, decreased tubercle scores and gonadal histopathological 

changes were seen in males in the FSTRA. As with the findings in the estrogen pathway, while 

the in vitro and mammalian in vivo assays do not indicate potential interaction, the results in the 

male fish, with complementary effects in the female fish, suggest a potential to interact with the 

androgen pathway in fish. 

There was no convincing evidence of interaction of metalaxyl with the thyroid pathway. Thyroid 

parameters were not altered in Tier 1 mammalian in vivo studies, except for a slight reduction in 

thyroid colloidal area in the male pubertal. However, there were no other thyroid-related effects 

observed in this assay or in other mammalian studies. In the AMA, while there was some 

evidence of delayed development in the tadpoles at the high test concentration, given the lack of 

effects on thyroid histopathology along with some indications of potential stress (lethargy and 

decreased body weight) in the early stages of the study, the effects observed in this assay do not 

appear to be due to a potential interaction with the thyroid pathway. 

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

recommended for metalaxyl since there was no evidence of potential interaction with the 

estrogen, androgen or thyroid pathways in mammals. 

Based on the complementary responses observed in the FSTRA, including effects on the apical 

endpoint of fecundity, EDSP Tier 2 Medaka Extended One-Generation Reproduction Test is 

recommended. The results in the FSTRA indicate that there may be a potential for interaction 

with the estrogen and androgen pathway in fish. Currently, the chronic fish NOAEC value is 9.1 

mg a.i./L based on no effects observed in a fathead minnow early life-stage. Effects on male 

gonadal histopathology were observed at 10 mg a.i./L in the FSTRA, with an increase in the 

number of effects observed in the males and females, including a cessation in fecundity, at 104 

mg a.i./L. 

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

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

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

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

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

As there was no convincing evidence of a potential thyroid interaction in amphibians, no 

additional testing is recommended. 

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Page 4 of 51 

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 metalaxyl interacts with E, A or T hormone pathways, the 

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

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

effects occur were considered, in particular, whether or not endocrine-related responses 

occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity. 

On August 13, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of 

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

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

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). 

Metalaxyl is a systemic phenylamide fungicide that is a racemic mixture of R- and Senantiomers: (R)-and (S)-2-[(2,6-dimethylphenyl)-methoxyacetylamino]-propionic acid methyl 

ester. Metalaxyl has a vapor pressure of 5.62×10−6 mm Hg at 25°C and volatilization from soil 

or water is not expected to be a major route of metalaxyl dissipation. The solubility of metalaxyl 

in water at 20°C is 7.1 g/L and is 8.4 g/L at 22°C. Metalaxyl has a log Kow of 1.65, and 

bioconcentration is not anticipated to be significant due to a bioconcentration factor of 

approximately 4. Metalaxyl will exist in both vapor and particulate phases on release to the 

atmosphere. The vapor phase is susceptible to photodegradation, and the particulate phase is 

susceptible to wet and dry deposition. Metalaxyl is also susceptible to direct photolysis, and 

aquatic biodegradation, and rapid biodegradation in soil. 

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

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

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

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

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METALAXYL FINAL 

Page 5 of 51 

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

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

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

Report of the Endocrine Disruptor Review Team on metalaxyl (USEPA, 2010). Since then, the 

Agency has also conducted a more recent search of available scientific literature for any 

additional relevant information. Summaries of the available OSRI are presented in Appendix 2. 

Additionally, literature/studies considered but not utilized for the WoE analysis are listed in 

Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

above. 

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

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

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 

consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

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

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

relevance for informing interactions with the E, A, or T pathway. Additionally, the 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 and T consisting of different 

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

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

the whole organism level. The extent of expression of responses at to 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 

considerations 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 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 metalaxyl are shown below 

in Table 1. 

Table 1. Tier 1 Assays for Metalaxyl 

Tier 1 Assays Test Guideline Test Order Status 

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

(MRID 48617607) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID 48617603) 

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

(MRID 48617601) 

Steroidogenesis Assay (Human cell line 

H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID 48617605) 

Aromatase Assay (human recombinant 

microsomes) OCSPP 890.1200 Requirement Satisfied (MRID 48617602) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID 48617606) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID 48617604) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied 

(MRID 48701301) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied 

(MRID 48701301) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID 48685802) 

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID 48685801) 

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METALAXYL FINAL 

Page 9 of 51 

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

1. Effects on Estrogen Pathway 

The potential for metalaxyl 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. 

Metalaxyl was classified as equivocal in the in vitro estrogen receptor (ER) binding (rat uterine 

cytosol) assay based on the overall results from the three assay runs (interactive in one run and 

not interactive in the remaining two runs). Metalaxyl was negative in the Tier 1 ERα

transcriptional activation (ERTA) and aromatase assays and did not alter estradiol production in 

the steroidogenesis assay. In a non-guideline ERTA considered as OSRI (Kojima et al., 2004), 

metalaxyl did not show agonist or antagonist activity for ERα or ERβ. 

Metalaxyl did not induce uterine weight increases in the uterotrophic assay and was classified as 

negative for estrogenic activity. Metalaxyl also showed no consistent evidence of an estrogenic 

response in the female pubertal rat assay. There were no differences in VO- or estrous-related 

endpoints. In the ovaries, total primary follicle counts were increased by 43 and 57% in the 

medium- and high-dose groups, respectively, but atretic follicle counts in the treated groups were 

similar to controls. 

In the FSTRA, there was a 100% decrease in fecundity in the high-concentration group (104 mg 

a.i./L); as such fertility was not assessed in this treatment. Additionally, treatment-related effects 

included a 23% decrease in gonado-somatic index (GSI), and alterations in ovarian histology 

including decreased yolk synthesis at the high test concentration. While not statistically 

significant (p=0.092), at the high concentration, there was a 96% decrease in female plasma 

vitellogenin (VTG) concentrations that upon visual observation of the data was considered 

biologically significant. It is noted that there was an increase in the incidence of atretic oocytes 

and granulomatous inflammation in females in the high treatment group. However, given that 

these findings were occasionally coincident with the presence of protozoal spores and were also 

observed in control female fish (at a lower incident rate), it was concluded that these lesions 

were probably not treatment-related. 

In the Part 158 mammalian toxicity studies, the only organ weight effects noted were decreased 

absolute pituitary weights (↓14-21%; not dose dependent) in F2B females in all dose groups, 

decreased (p<0.05) adjusted pituitary weights in the high-dose F3B males (↓40%) and increased 

(p<0.05) absolute pituitary weights in the mid- and high-dose F3B females (↑50% and 25%, 

Page 17 of 59
METALAXYL FINAL 

Page 10 of 51 

respectively), in the three-generation reproduction study in rats. No treatment-related 

histopathological findings were noted in any of these studies. 

There were no treatment-related reproductive effects in either the Part 158 bobwhite quail or 

mallard duck one-generation reproduction studies at concentrations up to 900 mg/kg-diet (ppm). 

Additionally, there were no treatment-related effects observed in a fathead minnow fish early-life 

cycle study at concentrations up to 9.1 mg/L.

Page 18 of 59
METALAXYL FINAL 

Page 11 of 51 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Metalaxyl

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metalaxyl1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

EDSP Tier 1 Assay 

ER Binding (MRID 48617607) 

E

ERTA (MRID 48617603) N Aromatase (MRID 48617602) N Steroidogenesis (MRID 48617605) N Uterotrophic (MRID 48617606) N NE Female Pubertal Rat (MRID 48701301) N N 

P5 N N N 

FSTRA (MRID 48685802) NE ↓23% (H) P (H)7 N 

Frt: NA or 

N 

Fcd: 

↓100% (H) 

↓

OSRI 

ERTA (Kojima et al., 2004) N Three-generation reproduction (Rat; MRID 00071600) 

N N NE 

F2B: 

↓21, 21, 

14% (L, 

M, H) 

 N 
METALAXYL FINAL 

Page 12 of 51 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Metalaxyl

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metalaxyl1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Developmental toxicity (Rat; MRID 00144423) 

 N 

Developmental toxicity (Rabbit; MRID 00144372) 

 N 

Chronic toxicity/ carcinogenicity (Rat; MRID 00098481) 

NR NR N NR 

Carcinogenicity (Mouse; MRID 00103354) 

NE NE N NE 

Chronic toxicity (Dog; MRID 00071598) N N N N 90-Day subchronic toxicity (Rat; MRID 48104702) 

 NE N N NE 

28-Day subchronic toxicity (Mouse; MRID 48104701) 

 NE N N NE 

91-Day subchronic toxicity (Dog; MRID 00084111) 

N N N N 

Avian reproduction (Quail; MRID 44755001) 

N 
METALAXYL FINAL 

Page 13 of 51 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Metalaxyl

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metalaxyl1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormone 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility(Frt) 

/Fecundity (Fcd) 

Avian reproduction (Duck; MRID 44761701) 

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. LW= Liver 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. Due to adverse clinical signs seen at the high dose (300 mg/kg/day) on Day 1 the dose was reduced to 200 mg/kg/day for study day

5. Total primary follicle counts were increased by 43 and 57% in the 250 and 350 mg/kg/day dose groups, respectively, but no effect 

of atretic follicles. 

6. Unadjusted liver weights were increased by 10, 19, and 23% in the low-, medium-, and high-dose females, respectively) with simil

liver/body weights. 

7. Decreased yolk synthesis in high-dose females. E Equivocal P Positive findings N Negative finding (in vitro)/No effect (in vivo) NE Not examined NR Not reported 
METALAXYL FINAL 

Page 14 of 51 

2. Effects on Androgen Pathway 

The potential for metalaxyl 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. 

Metalaxyl was classified as a non-binder in the in vitro androgen receptor (AR) binding (rat 

prostate cytosol) assay and negative in the steroidogenesis assay. Negative results in a nonguideline in vitro AR binding (Bauer et al., 2002) and an androgen receptor transcriptional 

activation (ARTA) assay (Kojima et al., 2004) further support a lack of interaction of metalaxyl 

with the AR. 

Metalaxyl was also classified as negative for androgenic and anti-androgenic activity in the in 

vivo Hershberger. Metalaxyl had minimal effects in the male pubertal rat assay. No treatmentrelated changes in body weights, body weight gains, or age at preputial separation (PPS) 

occurred in either treatment group. Slight non-dose-dependent decreases in body weight at PPS 

were noted (↓6-8%) in all treatment groups. There was no effect on AST weights or serum 

testosterone concentration, and no histopathological lesions were observed in the testes or 

epididymides. 

Median male tubercle score was decreased in the high-concentration group in the FSTRA (score 

of 6 vs. 32 in control). In addition, there was an increase in the percentage of spermatogonia in 

the medium- and high-concentration males and asynchronous gonad development at the high 

concentration. 

There was no evidence of effects on male reproductive parameters in the existing metalaxyl Part 

158 mammalian toxicity studies. Additionally, there were no treatment-related effects observed 

in the Part 158 avian reproduction studies.

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METALAXYL FINAL 

Page 15 of 51 

Table 3. Androgenic/Anti-Androgenic Pathway for Metalaxyl

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Metalaxyl1Study 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 48617601) N Steroidogenesis (MRID 48617605) N Hershberger (MRID 48617604) NE N Male Pubertal Rat (MRID 48701301) N N N N N N N N FSTRA (MRID 48685802) NE N P (M, H)4 tubercles: 

↓ (H) N 

OSRI 

AR Binding (Bauer et al., 2002) N ARTA Kojima et al. (2004) N Three-generation reproduction (Rat; MRID 00071600) 

N NE N NE N 

Chronic toxicity/ carcinogenicity (Rat; MRID 00098481) 

 N N N N NR NR 
METALAXYL FINAL 

Page 16 of 51 

Table 3. Androgenic/Anti-Androgenic Pathway for Metalaxyl

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Metalaxyl1Study 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 

Carcinogenicity (Mouse; MRID 00103354) 

NE N NE N NE NE 

Chronic toxicity (Dog; MRID 00071598) 

N N NE N N NE 

90-Day subchronic toxicity (Rat; MRID 48104702) 

 N N N N NE NE 

28-Day subchronic toxicity (Mouse; MRID 48104701) 

 N N N N NE NE 

91-Day subchronic toxicity (Dog; MRID 00084111) 

N N NR NR N N 

Avian reproduction (Quail; MRID 44755001) 

N 

Avian reproduction (Duck; MRID 44761701) 

N 

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

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated 

this assay. Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Sem

prostate (VP), Dorsal prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gla(GP). LW= Liver weight 
METALAXYL FINAL 

Page 17 of 51 

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

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 

For details see Section IV. A 

4. Increased incidence of percentage of spermatogonia in the medium- and high-concentration males, and asynchronous gona

concentration. 

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

 NE Not examined  NR Not reported 
METALAXYL FINAL 

Page 18 of 51 

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 metalaxyl 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. 

No effects on the thyroid pathway (thyroid weight, histopathology or hormone concentrations) 

were observed in the in vivo male and female pubertal rat assays, except for slight dosedependent reductions in colloid area in the medium- and high-dose males. In the AMA there 

was no significant effect on median Nieuwkoop-Faber (NF) developmental stage at Day 7 or 21 

relative to the negative control. However, at Day 21, there was a dose-dependent decrease in the 

number of late-stage frogs (NF stage >60), i.e., 16, 13, 11, 3 in the control, low, mid, and high 

treatment groups, respectively. No effects on thyroid gland histopathology were observed at any 

treatment concentrations. Normalized hind-limb length (HLL) was significantly decreased on 

both Day 7 (↓8.4%) and Day 21 (↓13%) at the high test concentration (95 mg/L). There also was 

a significant increase in Day 21 snout-to-vent length (SVL; ↑6%) at the high test concentration. 

Day 7 body weight was marginally significantly decreased (p=0.06; ↓19%) at the high 

concentration, with a decrease of 9% (p=0.057) at the low concentration and an increase of 15% 

(p<0.05) at the high concentration on Day 21. It is noted that clinical signs of toxicity (i.e., 

lethargy) were noted on Day 0 and 1 of the study at the high test concentration but were not 

observed over the duration of the study. While there was some evidence of delayed development 

in the tadpoles at the high test concentration, given the lack of effects on thyroid histopathology 

along with some indications of potential stress (lethargy and decreased body weight) in the early 

stages of the study, the effects observed in this assay do not appear to be due to a potential 

interaction with the thyroid pathway. 

In the mammalian Part 158 toxicity studies, histopathological examination of the pituitary did 

not reveal any treatment-related changes in any in vivo studies with metalaxyl. Changes in 

pituitary weights were only noted in the three-generation rat reproductive study. In this study, 

changes in absolute or adjusted pituitary weights were noted in both adults (F2B females all dose 

groups) and offspring (F3B high-dose males and mid- and high-dose females), but changes were 

not consistent across litters nor dose-dependent. 

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METALAXYL FINAL 

Page 19 of 51 

Table 4. Thyroid Pathway for Metalaxyl

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Metalaxyl1

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 Observed2

Overt Toxicity Observed3 

EDSP Tier 1 Assay 

Female Pubertal Rat 

(MRID 48701301) N N N N N ↑LW 

(L, M, H)4 N 

Male Pubertal Rat 

(MRID 48701301) N P5 N N N N N 

AMA (Frog; MRID 

48685801) N 

HLL: 

day 7-

↓8%; 

day 21- 

↓13% 

(H)6

Day 21 

SVL: ↑6% 

(H); 

BW: 

Day 21 

↓9%, ↑15% 

(L, H) 

 N7 

OSRI 

Three-generation 

reproduction (Rat; 

MRID 00071600) 

N NE 

F2B 

females: ↓

21, 21, 

14% 

(L, M, H); 

male: N 

X 

(F2B, H) N 

Developmental toxicity 

(Rat; MRID 00144423) NE NE N X 

 (M, H) 

Developmental toxicity 

(Rabbit; MRID 

00144372) 

NE NE N N 

Chronic toxicity/ 

carcinogenicity (Rat; 

MRID 00098481) 

NR N NR X 

(M, H) N 

Carcinogenicity 

(Mouse; MRID 

00103354) 

NE N NE N N 

Chronic toxicity (Dog; 

MRID 00071598) N N N X 

(M, H) N 

90-Day subchronic 

toxicity (Rat; MRID 

48104702) 

NE N NE X 

(MH, H) 

X 

(MH, 

H) 

28-Day subchronic 

toxicity (Mouse; MRID 

48104701) 

NE N NE X 

(M, H) N 

Page 27 of 59
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Page 20 of 51 

Table 4. Thyroid Pathway for Metalaxyl

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Metalaxyl1

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 Observed2

Overt Toxicity Observed3 

91-Day subchronic 

toxicity (Dog; MRID 

00084111) 

NR N N N 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.BW= Body weight; LW= Liver weight; SVL=Snout ventral length; HLL=hind-limb 

length 

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. Unadjusted liver weights were increased by 10, 19, and 23% in the low-, medium-, and high-dose females, 

respectively) with similar increases in relative liver/body weights. 

5. Slight dose-dependent reductions in colloid area in the medium- and high-dose males 

6. While there was no effect on median NF stage in the treatment groups compared to the control, there was a 

dose-dependent decrease in the number of late stage frogs i.e., 16, 13, 11, 3 in the control, low, mid, and high 

treatment groups, respectively. 

7. Lethargy in all high concentration tadpoles was observed after initial dose administration (first 24 hours). 

Additionally, a marginal decrease (p=0.06) in body weight was observed on Days 7 (↓19%) in the high 

concentration. Therefore, while there were signs of potential stress at the early stages of the study, they did not 

persist over the duration of the test. 

P Positive findings 

N Negative findings 

NE Not examined 

NR Not reported 

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METALAXYL FINAL 

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IV. Committee’s Assessment of Weight of Evidence 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

associated with weight of evidence evaluation of the results of the Tier 1 assays (July, 2013) 

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

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

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

effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis”

(USEPA, 2013). 

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 and OSRI 

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

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

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

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

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

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1. Tier 1 in vivo Assays 

Systemic or overt toxicity in the Tier 1 in vivo studies was generally minimal. No mortality or 

clinical signs of toxicity were observed in the female and male pubertal rat studies at doses up to 

350 mg/kg/day or in the Hershberger assay up to 375 mg/kg/day. In the Uterotrophic assay, after 

administration of metalaxyl at 300 mg/kg/day (high dose) on Day 1, adverse clinical signs were 

observed (decreased motor activity, ptosis, ataxia, lacrimation, hunched posture, intermittent 

body jerks, chromodacryorrhea and slight excess salivation), as well as reductions in food 

consumption. Therefore, the 300 mg/kg/day dose was reduced to 200 mg/kg/day on Study Days 

2 and 3. After the dose reduction, food consumption in this group increased and no other 

adverse clinical signs were observed. 

In the FSTRA, while there were significant decreases in male body weight at all treatments (↓7, 

16 and 16% at 1.1, 10 and 104 mg/L, this effect was not dose-responsive and no other treatmentrelated mortalities or clinical signs were observed. Therefore, this decrease in male body 

weights were not considered treatment-related or a sign of potential stress. In the AMA, lethargy 

was observed on Days 0 and 1 in all high-concentration tadpoles (95 mg a.i./L) but was not noted 

after Day 1. Additionally, body weight was marginally significantly (p=0.06) decreased 19% by 

Day 7 in this treatment, however, was 15% greater than controls by day 21. Therefore, while 

there were indications of potential stress in the high treatment group in the early stages of the 

AMA, these effects appeared to be transient and did not persist throughout the duration of the 

study. Evaluation of potential systemic toxicity was not conducted in either the FSTRA or 

AMA. 

2. OSRI 

Systemic or overt toxicity in the OSRI in vivo studies was also minimal. No effects of treatment 

were observed on mortality or clinical signs in a subchronic (90-day) oral toxicity study in rats. 

Body weight was decreased by 13-18%, resulting in a decrease in total body weight gain (↓43%) 

in high-dose females (1672 mg/kg/day). Although body weight in the medium-high dose 

females was decreased <10%, there was a decrease in total body weight gain of 20%. Female 

rats in these dose groups also had decreases in overall food consumption that were concentration 

dependent. 

In a rat prenatal development study, one dam at the mid dose (250 mg/kg/day) was sacrificed in 

extremis on GD 13 with clinical signs including post-dose ataxia, loss of activity, body tremors 

and reduced righting reflex. Additionally, twelve high-dose animals (400 mg/kg/day) died or 

were sacrificed in extremis between GDs 6 and 8; most of these animals exhibited convulsions 

within minutes after dosing. Clinical signs observed in the mid- and high-dose animals included: 

post-dose convulsions; dry or wet matter on head, eyes, nose, abdomen, anogenital area, and/or 

vaginal opening; and stained and/or matted hair coat on the head, face, ventral thorax, abdomen, 

Page 30 of 59
METALAXYL FINAL 

Page 23 of 51 

back, anogenital area, and/or forelimbs. Additional clinical signs observed only in high-dose 

animals included: loss of activity, and reduced or loss of righting reflex. Although there were no 

treatment-related changes in body weight noted in any dose groups, body weight gains were 

decreased primarily during the early treatment period. In the mid- and high-dose groups, body 

weight gain was decreased during GDs 6-9 by 160 and 100%, respectively. All animals survived 

until sacrifice in a rabbit prenatal development study, except for one high-dose doe (necropsy 

unremarkable). Maternal toxicity in the high-dose animals (300 mg/kg/day) consisted of 

little/none/soft stools. Body weight gain of does in the high-dose group was negative (−75 g) 

and was decreased (↓192%) during the exposure period. 

There were no treatment-related mortalities or clinical signs of toxicity reported in any other Part 

158 mammalian toxicity studies. A common finding in some of the in vivo Tier 1 studies and 

most of the mammalian toxicity studies was an increase in absolute and/or relative mean liver 

weight, often accompanied (if examined) by changes in liver enzymes (e.g., alkaline 

phosphatase, GGT, SGPT, SGOT, sorbitol dehydrogenase) or other liver-related clinical 

chemistries (e.g., bilirubin) and histopathological changes (e.g., hypertrophy). Incidences of 

fatty liver, or periacinar, vacuolation were noted in the chronic rat and mouse studies. 

There were no treatment-related signs of toxicity in either Part 158 avian reproduction study up 

to 900 ppm, or in the fish early life-cycle toxicity study with fathead minnow up to 9.1 ppm. 

B. Estrogen Pathway 

Metalaxyl showed an equivocal response in the Tier 1 in vitro ER binding assay (interactive in 

one run and not interactive in the remaining two runs); however, it was negative for ERα

agonism in the Tier 1 ERTA assay and negative for both ERα agonism and antagonism in a 

published ERTA study (Kojima et al., 2004). Metalaxyl was also negative in the Tier 1 in vitro

steroidogenesis and aromatase assays. There was no convincing evidence of an interaction with 

the estrogen pathway in the in vivo!mammalian Tier 1 assays!(uterotrophic, female pubertal 

assays) or in the Part 158 mammalian or avian studies. Primary follicle counts were increased in 

the female pubertal assay, but no effect was noted in the number of atretic follicles and there 

were no complementary responses in the other estrogen pathway endpoints evaluated in that 

assay. Overall metalaxyl does not appear to have a potential to interact with the estrogen 

pathway in mammals. Conversely, metalaxyl appears to potentially interact with the estrogen 

pathway in fish. Several complementary effects on females were observed in the FSTRA at the 

high test concentration of 104 mg a.i./L including: effects on gonadal histology (decreased 

ovarian yolk synthesis), decreased plasma VTG concentration, decreased GSI, and finally 

cessation of fecundity (no eggs produced). Therefore, while there were largely negative 

responses in the Tier 1 in vitro assays and in vivo mammalian dataset, for the FSTRA there were 

complementary responses along several levels of biological organization for the females 

including alterations in enzyme activity (plasma VTG) up to the apical endpoint of fecundity. As 

Page 31 of 59
METALAXYL FINAL 

Page 24 of 51 

such, significant effects of the reproductive capacity of fish were observed in the FSTRA which 

suggests that metalaxyl may potentially interact with the HPG axis.

C. Androgen Pathway 

There was no convincing evidence of interaction of metalaxyl with the androgen pathway in the 

in vitro (i.e, AR binding and steriodogenesis assays were negative) or in vivo mammalian Tier 1 

(i.e., Hershberger and male pubertal assays were negative) and Part 158 mammalian and avian 

studies (no androgen-related effects observed). In addition, metalaxyl was negative in two 

literature studies evaluating AR binding (Bauer et al., 2002) and AR transcriptional activation 

(Kojima et al., 2004). However, decreased tubercle scores and gonadal histopathological 

changes were seen in males in the FSTRA. As with the findings in the estrogen pathway, while 

the in vitro and mammalian in vivo assays do not indicate potential interaction, the results in the 

male fish with complementary effects in the female fish, suggest a potential to interact with the 

androgen pathway in fish. 

D. Thyroid Pathway 

There was no convincing evidence of interaction of metalaxyl with the thyroid pathway. Thyroid 

parameters were not altered in Tier 1 mammalian in vivo studies, except for a slight reduction in 

thyroid colloidal area in the male pubertal. However, there were no other thyroid-related effects 

observed in this assay or in other mammalian studies. In the AMA, while there was some 

evidence of delayed development in the tadpoles at the high test concentration, given the lack of 

effects on thyroid histopathology along with some indications of potential stress (lethargy and 

decreased body weight) in the early stages of the study, the effects observed in this assay do not 

appear to be due to a potential interaction with the thyroid pathway. 

E. Conclusions 

The conclusion of the WoE evaluation is that metalaxyl demonstrates no convincing evidence of 

potential interaction with the estrogen or androgen pathways in mammals. However, the 

complementary responses observed in both sexes in the FSTRA suggest a potential for metalaxyl 

to interact with the E and A pathways in fish. There is no convincing evidence for the potential 

for metalaxyl to interact with the T pathway in mammals or wildlife. 

V. EDSP Tier 2 Testing Recommendations 

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

recommended for metalaxyl since there was no evidence of potential interaction with the 

estrogen, androgen or thyroid pathways in mammals. 

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Page 25 of 51 

Based on the complementary responses observed in the FSTRA, including effects on the apical 

endpoint of fecundity, EDSP Tier 2 Medaka Extended One-Generation Reproduction Test is 

recommended. The results in the FSTRA indicate that there may be a potential for interaction 

with the estrogen and androgen pathway in fish. Currently, the chronic fish NOAEC value is 9.1 

mg a.i./L based on no effects observed in a fathead minnow early life-stage. Effects on male 

gonadal histopathology were observed at 10 mg a.i./L in the FSTRA, with an increase in the 

number of effects observed in the males and females, including a cessation in fecundity, at 104 

mg a.i./L. 

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

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

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

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

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

As there was no convincing evidence of a potential thyroid interaction in amphibians, no 

additional testing is recommended. 

 

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METALAXYL FINAL 

Page 26 of 51 

VI. References 

Aldridge, D., Leng, J.M. (1985) Teratology Study in Rats. Unpublished study conducted by 

International Research and Development Corporation, Mattawan, Michigan for Ciba-Geigy 

Corporation, P.O. Box 18300, Greensboro, NC, Laboratory Report # 382-100, January 3, 1985. 

(MRID 00144423) 

Ashby, R. (1980) CGA 48 988: Toxicity and oncogenicity in dietary administration to rats for 

two years. Unpublished study conducted by Life Science Research, Stock, Essex, England. 

Report No. 80/CIA009/315, September 23, 1980. (MRID 00098481) 

Bauer, E.R.S., Bitsch, N., Brunn, H., Sauerwein, H., Meyer, H.H.D. (2002) Development of an 

immunoimmobilized androgen receptor assay (IRA) and its application for the characterization 

of the receptor binding affinity of different pesticides. Chemosphere 46(7):1107-1115. 

Beck, L.S., DeWard, J., Kitchen, D.M., et al. (1981) Six-month chronic oral toxicity study with 

CGA-48988. Unpublished study conducted by Elars Bioresearch Laboratories, Inc. Project no. 

1545. January 20, 1981. (MRID 00071598) 

Carlson, M.B. (2012) A Pubertal Development and Thyroid Function Study of Metalaxyl by Oral 

Gavage in Peripubertal Male and Female Rats. Unpublished study conducted by Charles River 

Laboratories Preclinical Services, Pennsylvania; Horsham, PA. Laboratory Study No.: 

20012408, July 17, 2012. (MRID 48701301) 

Carlson, M.B. (2011) Oral (Gavage) Hershberger Bioassay of Metalaxyl in Male Rats. 

Unpublished study conducted by Charles River Laboratories, Preclinical Services, Horsham, PA. 

Laboratory Project ID number: 20012407, December 9, 2011. (MRID 48617604) 

Cozens, D.D., Allen, P.A., Clark, R., et al. (1980) Effect of CGA-48988 on Reproductive 

Function of Multiple Generations in the Rat. Unpublished study conducted by Huntingdon 

Research, Huntingdon, Cambridgeshire, England for Ciba-Geigy Corporation, Basle, 

Switzerland, May 23, 1980 Study). (MRID 00071600) 

Finn, J.P. (1977) CGA-48988. 91-day dietary toxicity study in beagle dogs. Unpublished study 

conducted by Hazleton Laboratories Europe Ltd. Report No.: 653-380-4. (MRID 00084111) 

Hoberman, A.M. (2011) Oral (Gavage) Uterotrophic Bioassay of Metalaxyl in Female Rats. 

Unpublished study conducted by Charles River Laboratories, Preclinical Services, Horsham, PA. 

Laboratory Project ID: 20012406, December 9, 2011. (MRID 48617606) 

Kojima, H., Iida, M., Katsura, E., Kanetoshi, A., Hori, Y., and Kobayashi, K. (2003) Effects of a 

diphenyl ether-type herbicide, chlornitrofen, and its amino derivative on androgen and estrogen 

receptor activities. Environ Health Perspect. 111:497–502. 

Page 34 of 59
METALAXYL FINAL 

Page 27 of 51 

Kojima, H., Katsura, E., Takeuchi, S., Niiyama, 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):524-31. 

Laughlin, K. (1984) Teratology study in rabbits: metalaxyl: 382-097. Unpublished study 

conducted by International Research and Development Corporation, Matawan, Michigan. Report 

No. 382-097, December 4, 1984. (MRID 00144372) 

LeBlanc, G.A., Mastone, J., Wilson, B.F., et al. (1980) The Toxicity of CGA-48988 to Fathead 

Minnow (Pimephales promelas) Eggs and Fry: Report # BW-80-4-642. Unpublished study 

conducted by EG & G, Bionomics, submitted by Ciba-Geigy Corp., Greensboro, N.C.; 

CDL:244183-K; 244782). (MRID 00071308) 

Lee, M.R. (2012) Metalaxyl- Amphibian Metamorphosis Assay with African Clawed Frog 

(Xenopus laevis). Unpublished study conducted by Smithers Viscient, Wareham, Massachusetts. 

Laboratory report number 13694.6146. Study sponsored by Metalaxyl EDSP Consortium, Burr 

Ridge, Illinois. February 29, 2012. (MRID 48685801) 

McShehy, T., Macrae, S., and Whitney, J. (1981) CGA-48988: Oncogenicity in dietary 

administration to mice for two years. Unpublished study conducted by Life Science Research. 

Report no. 8C/01A008/442. June 3, 1981. (MRID 00103354) 

Merkel, D. (2003) Metalaxyl: Subchronic Toxicity Study (28-Day Dietary Study in Mice). 

Unpublished study conducted by Product Safety Laboratories, Dayton, NJ. Laboratory Study No. 

13634, December 22, 2003. (MRID 48104701) 

Merkel, D. (2003) Metalaxyl: Subchronic Toxicity Study (90-Day Dietary Study in Rats). 

Unpublished study conducted by Product Safety Laboratories, Dayton, NJ. Laboratory Study No. 

13635, December 22, 2003. (MRID 4810470) 

Neal, B., and Williams, A.(2013). EDSP Tier 1 Screening Results: Weight of the Evidence 

Discussion of Potential Endocrine Pathway Interactions for Metalaxyl. Metalaxyl EDSP 

Consortium. Project Identification. MEDP-13.(MRID No.49051402). 

Pedersen, C.A. (1999) Avian reproductive toxicity study with metalaxyl technical in bobwhite 

quail. Unpublished study conducted by Bio-Life Associates, Ltd. Project No.: 164-003-07. 

(MRID 44755001) 

Pedersen, C.A. (1999) Avian reproductive toxicity study with metalaxyl technical in mallard 

ducks. Unpublished study conducted by Bio-Life Associates, Ltd. Project No.: 164-004-08. 

(MRID 44761701) 

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Page 28 of 51 

USEPA (2010) Metalaxyl - Report of the Endocrine Disruptor Review Team; Test Order#: 

EDSP-113501-124- EDSP-113501-126. December 14, 2010. Office of Chemical Safety and 

Pollution Prevention. U.S. Environmental Protection Agency, Washington, D.C. 

USEPA. (2011) Endocrine Disruptor Screening Program – Weight of Evidence: Evaluating 

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

Safety and Pollution Prevention. 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. (2011) Metalaxyl: H295R Steroidogenesis Assay. Unpublished study conducted by 

CeeTox, Kalamazoo, MI. Study ID: 9143V-100326STER, December 7, 2011. (MRID 48617605) 

Wilga, P.C. (2011) Metalaxyl: Human Recombinant Aromatase Assay. Unpublished study 

conducted by CeeTox, Inc., Kalamazoo, MI. Study ID: 9143V-100326AROM, December 7, 

2011. (MRID 48617602) 

Willoughby, J.A. (2011) Metalaxyl: Androgen Receptor Binding Assay (Rat Prostate Cytosol). 

Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No: 9143V100326ARB, December 7, 2011. (MRID 48617601) 

Willoughby, J.A. (2011) Metalaxyl: Estrogen Receptor Binding (Rat Uterine Cytosol). 

Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No: 9143V100326ERB, December 8, 2011. (MRID 48617607) 

Willoughby, J.A. (2011) Metalaxyl: Estrogen Receptor Transcriptional Activation (Human Cell 

Line (HeLa-9903)) Final Report. Unpublished study conducted by CeeTox, Inc, Kalamazoo, MI. 

Laboratory Project ID: 9143V-100326ERTA, December 7, 2011. (MRID 48617603) 

Willoughby, J.A. (2012) Supplemental Information - Laboratory Proficiency Data for ERTA 

assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. 

Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. July 19, 2011. (MRID 48843501) 

York, D.O. (2012) Metalaxyl - Short-term Reproduction Assay with the Fathead Minnow 

(Pimephales promelas). Unpublished study conducted by Smithers Viscient, Wareham, 

Massachusetts, Laboratory Project No. 13694.6143. Submitted by Metalaxyl EDSP Consortium, 

Burr Ridge, Illinois. March 2, 2012. (MRID 48685802) 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100) 

The 21-day assay (MRID 48685801) of metalaxyl (98.2% purity) on amphibian metamorphosis 

of the African clawed frog (Xenopus laevis) was conducted under flow-through conditions. 

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

group; 20/replicate) were exposed to nominal concentrations of 0 (negative control), 1.0, 10, and 

100 mg a.i./L; mean-measured concentrations were <0.069 (<LOQ; negative control), 0.98, 10, 

and 95 mg a.i./L. The test system was maintained at 22 to 24°C and a pH of 7.8 to 8.2. 

By test termination, mortality averaged 8.75% in the negative control and 7.5, 0, and 3.75% in 

the low, mid, and high treatment groups, respectively. Spinal curvature was observed in between 

15 and 50% of surviving tadpoles in the negative control and all treatment groups and was not 

considered to be treatment related. The clinical sign of lethargy in all tadpoles of the high 

treatment group was observed on the first two days of exposure. 

Tadpoles at NF stage >60 were not included in analyses of growth and hind-limb length (HLL). 

Day 7 body weight was marginally significantly decreased (p=0.06) by 19% at the high 

treatment level. Day 21 body weight was significantly increased (p<0.05) by 15% in the high 

treatment group and was marginally significantly decreased (p=0.057) by 9% in the low 

treatment group. 

Metalaxyl significantly decreased (P<0.05) Day 7 and Day 21 normalized (for snout-vent length) 

HLL at the high treatment level. There was no significant (p>0.05) acceleration or delay of Day 

7 or Day 21 median NF developmental stage at any treatment level. Although the number of latestage tadpoles did not warrant a separate analysis (<20% observed), the reviewer noted a 

concentration-dependent reduction in the number of late-stage individuals (>NF 60); there were 

16 individuals staged NF 61 and 62 in the negative control, 13 in the low treatment group, 11 in 

the mid treatment group, and only 3 NF 61 tadpoles in the high treatment group. No 

asynchronous development was observed. Thyroid gland histopathology observations included 

mild follicular cell hyperplasia and hypertrophy in the negative control and all treatment groups 

and were not considered to be treatment-related. 

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

In an androgen receptor (AR) binding assay (MRID 48617601), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, 

which measured the binding of a single concentration of [3

H]-R1881 (1 nM) in the presence of 

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increasing concentrations (10−10 to 10−3

 M) of metalaxyl (98.2% purity, Batch #20100540). 

DMSO was used as the solvent vehicle at a final assay concentration of ca. 3.2%. A total of 

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

R1881 as the ligand reference standard. 

A saturation binding experiment was conducted to demonstrate that the AR in the rat prostate 

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

radiolabeled reference androgen (R1881) prior to routinely conducting AR competitive binding 

experiments. Saturation binding data were not originally provided in the study report; however, 

summarized saturation binding data (MRID 48843501) from the performing laboratory were 

submitted following a request by the Agency. The mean dissociation constant (Kd) for [3

H]-

R1881 was 1.080±0.690 nM, and the estimated Bmax was 1.071±0.167 fmol/100 μg protein for 

the single batch of prostate cytosol that was prepared. Only one of the three Kd values was 

within the range reported in the EPA validation program (0.685 to 1.57 nM), and all Bmax values 

were below the recommended range (7 to 16 fmol/100 μg protein). Confidence in these numbers 

is low based on the large variation in Kd estimates among runs. 

In the competitive binding experiment, the estimated log IC50s for R1881 (−9.0 M, Runs 2 and 3) 

and the weak positive control, dexamethasone (average of −4.4 M), were within the expected 

ranges; however, the log IC50 for R1881 was low in Run 1 (−9.9 M). Due to a laboratory error, 

the R1881 concentrations used in the Run 1 assay differed from the ranges reported in the EPA 

Test Guidelines for AR binding. The relative binding affinity (RBA) for the weak positive 

control averaged 0.0022% across all three runs. All performance criteria were generally met, 

with the exception that bottom % binding could not be determined for dexamethasone for Run 1. 

The deviation was considered minor. No precipitation of the test compound was observed. 

Metalaxyl had no significant effect on specific binding of the [3H]-ligand (>75% bound) over the 

concentration range of 10−10 to 10−3

 M in all runs; therefore, an IC50 and RBA could not be 

calculated for metalaxyl. 

Based on the results of the three assays, metalaxyl is classified as a Non-Binder in the Androgen 

Receptor Binding Assay. 

Aromatase Assay; (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48617602), metalaxyl (100.4% a.i., Batch# 

20100540) in 1% dimethyl sulfoxide (DMSO) was incubated with human recombinant 

aromatase and tritiated androstenedione (([1β3

H(N)]-androst-4-ene-3,17-dione; [3H]-ASDN) for 

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15 minutes at 37 °C to assess the potential of metalaxyl to inhibit aromatase activity. Metalaxyl 

was tested at logarithmic concentrations from 10–10 to 10−3

 M in three independent runs. 

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). 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. 

Aromatase activity in the full activity controls was 0.240 ± 0.070 nmol·mg-protein−1

·min−1. The 

response of each full activity control within a run was between 90 to 110% of the average full 

activity, with two exceptions likely due to an unevenly split sample. Activity in the background 

controls ranged 0.43 to 0.87% and averaged 0.59% of the full activity controls. 

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

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

replicates of each concentration within runs, except that the %CV for the 10−8 M (19%) and 10−9

M (34%) replicates for Run 2 exceeded the recommended limit of 15%. The estimated log IC50

averaged −7.18 M and the Hill slope averaged −1.03 for 4-OH ASDN. 

For metalaxyl, aromatase activity averaged 0.250 ± 0.069 nmol·mg-protein−1

·min−1 at the lowest 

tested concentration 10−10 M and 0.232 ± 0.073 nmol·mg-protein−1·min−1 at the highest tested 

concentration 10−3 M. The average response curve indicated that the aromatase activity of 

metalaxyl at concentrations from 10−10 to 10−3

 M was essentially equivalent (i.e. >75% activity) 

to the activity observed in the full activity control. 

Based on the data from the average response curve, metalaxyl is classified as a Non-inhibitor of 

aromatase activity in this assay. 

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

In an estrogen receptor (ER) binding assay (MRID 48617607) for metalaxyl (98.2% purity, 

Batch #20100540), uterine cytosol from Sprague Dawley rats was used as the source of ER for a 

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

H]-

17β-estradiol (1 nM) in the presence of increasing concentrations (10−10 to 10−3

 M) of metalaxyl. 

DMSO was used as the solvent vehicle at a final concentration of ca. 4%. A total of three 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. 

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

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

radio-labeled reference estrogen prior to conducting ER competitive binding experiments. 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The protein concentrations used in the saturation binding 

runs varied between each run, and were approximately 3- to 6-fold greater than recommended 

(160 to 320 μg versus 50±10 μg). The Kd for [3

H]-17β-estradiol was 0.331±0.061 nM and the 

estimated Bmax was 74.55±3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The Kd

for each run was within the expected Guideline range of 0.03 to 1.5 nM. 

In the competitive binding experiment, the estimated mean log IC50 was −9.0 M for 17β-estradiol 

and −5.6 M for the weak positive control. The mean RBA was 0.0344% for the weak positive 

control. All performance criteria were generally met; however, octyltriethoxysilane displaced 

more than 25% of [3H]-17β-estradiol from the ER at the highest concentration tested (10−3 M) in 

Run 3, and bottom % binding and Hill slope were slightly outside of the tolerance limits for 17βestradiol in Run 2. 

Metalaxyl reduced the mean specific binding of the radiolabeled ligand in a concentrationdependent manner in one of the three runs (declining to <46.8% in Run 2); however, metalaxyl 

had <25% inhibition of binding at concentrations up to 10−3

 M in the other two runs. The 

estimated log IC50 was −3.8 M for metalaxyl in Run 2, with a relative binding affinity (RBA) of 

0.0006%. 

Based on the results from the three runs (interactive in one run and not interactive in the other 

two runs), metalaxyl is classified as Equivocal in the Estrogen Receptor Binding Assay. 

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

In an estrogen receptor transcriptional activation assay (MRID 48617603), hERα-HeLa-9903 

cells cultured in vitro were exposed to metalaxyl (≥98.2% a.i., Lot #20100540) at logarithmically 

increasing concentrations from 10−4

 to 10−11 M in 0.1% DMSO in two independent runs. The 

experiments were performed using 96-well plates and each metalaxyl concentration was tested in 

6 wells/plate in each run. Cells were exposed to the test agent for 24±2 hours to induce reporter 

(luciferase) gene products. Luciferase expression in response to activation of the estrogen 

receptor by metalaxyl was measured using a proprietary luciferase activity assay. 

Metalaxyl was tested up to the limit of cytotoxicity 10−4

 M. Cytotoxicity of 17αmethyltestosterone in the first run prevented observation of its log PC50. The mean RPCMax for 

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metalaxyl was 0.5% for the first run and 1.1% for the second, and the associated PCMax was 10−5

M for the first run and 10−7 M for the second run. 

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, metalaxyl 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 16.5-52.8% PC. Metalaxyl was only able to reach a maximum of 0.5-1.1% 

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

RPCMax < PC10 in both assay runs, metalaxyl 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 48685802) of metalaxyl with fathead minnows 

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

groups; 2 males and 4 females in each group; 21 weeks old) were exposed to metalaxyl (100.4% 

purity) at nominal concentrations of 0 (negative control), 1.0, 10.0, and 100 mg a.i./L; meanmeasured concentrations were <LOQ (<0.066, negative control), 1.1, 10.0, and 104 mg a.i./L. 

The test system was maintained at 25 to 26o

C and a pH of 6.9 to 7.6. 

Adult male and female survival was unaffected by treatment with metalaxyl; survival in males 

was 100, 88, 100, and 88% and survival in females was 100, 100, 100, and 94% in the negative 

control, low, mid, and high treatment groups, respectively. Male body weight exhibited a 

significant (p<0.05), generally concentration-dependent reduction of 7 to 16% across all 

treatment levels compared to the negative control. There were no notable differences in 

secondary sex characteristics or clinical signs for any treatment group compared to the negative 

control. 

In the negative control, fecundity was 19 eggs/female/day/replicate, and fertilization success 

averaged 96%. Fecundity and fertility were not significantly affected at the low and mid 

treatment levels. However, there were no eggs available for assessment at the high treatment 

level. 

At the high treatment level, there was a 96% decrease in female plasma vitellogenin (VTG) 

concentrations that was not statistically significant (p=0.092). However upon visual observation 

of the data, this reduction in VTG was considered to be biologically significant. Female gonadosomatic index (GSI) was significantly reduced (p<0.05) by 23% at the high treatment level 

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compared to the negative control. Male tubercle score was significantly reduced (p<0.05) at the 

high treatment level compared to the negative control. 

There were no notable differences in gonadal stage. In male fish, there was an increased 

incidence of increased percentage of spermatogonia at the mid and high treatment levels and 

asynchronous gonad development at the high treatment level. In female fish, there was decreased 

yolk synthesis at the high treatment level. There was an increase in the incidence of atretic 

oocytes and granulomatous inflammation in females in the high treatment group. However, given 

that these findings were occasionally coincident with the presence of protozoal spores and were 

also observed in negative control females (at a lower incident rate), these lesions were not 

considered to be treatment related. Plasma testosterone and 17ß-estradiol were not measured in 

this study. 

Hershberger Assay; (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48617604) screening for androgenic activity, metalaxyl (98.2% 

a.i., Batch/lot # 20100540) in 2.0% aqueous carboxymethylcellulose (CMC) was administered 

daily via oral gavage to groups of eight 57-day old, castrated male Sprague Dawley rats at dose 

levels of 0 (vehicle), 125, or 375 mg/kg/day. An androgenic positive control group consisted of 

eight castrated rats exposed to 0.4 mg/kg/day of testosterone propionate (TP) by subcutaneous 

(s.c.) injection. 

To screen for potential anti-androgenic activity, metalaxyl (98.2% a.i.) in 2.0% aqueous CMC 

was administered daily via oral gavage to groups of eight 54- or 60-day old, castrated male 

Sprague Dawley rats at dose levels of 0 (vehicle), 40, 125, or 375 mg/kg/day in conjunction with 

a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic 

positive control group consisted of eight castrated rats exposed to 0.4 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 and food consumption 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, liver, and kidneys 

were collected and weighed. 

All animals survived until scheduled termination. No adverse clinical signs of toxicity were 

observed in the androgen agonist assay. In the anti-androgen assay, treatment related clinical 

signs of toxicity (urine-stained abdominal fur) were observed in three rats from the 375 

mg/kg/day group. 

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In the androgen agonist assay, mean body weights and body weight gains in the metalaxyl 

treatment groups were comparable to the vehicle controls throughout the assay. Animals in the 

positive control (TP) group had increased (p≤0.01) overall (Days 1-11) body weight gains 

(↑31%) compared to the vehicle controls. Overall average food consumption for both metalaxyl 

treatment groups were comparable to the vehicle controls; however, overall food consumption in 

the TP positive control group was increased (p≤0.01) by 10%. No treatment-related effects were 

observed in any of the accessory sex organs or kidneys of metalaxyl-dosed animals, metalaxyl. 

However, mean liver weights were increased (p<0.01) by 17% in the 375 mg/kg/day group 

compared to the vehicle controls. No gross lesions were observed at necropsy, but one animal in 

the 375 mg/kg/day group did not achieve preputial separation by necropsy. Animals in the TP 

group had increased (p≤0.01) accessory sex organ weights as follows: 500% in seminal vesicles; 

691% in ventral prostate; 177% in levator ani-bulbocavernosus (LABC); 645% in Cowper’s 

glands; and 71% in glans penis. The performance criteria indicated that this assay was 

performing as expected. 

In the anti-androgen assay, mean body weights and overall body weight gains of the metalaxyl 

treatment groups were comparable to the vehicle controls. Animals dosed with TP + FT 

(positive control) had mean body weights and overall mean body weight gains comparable to the 

corn oil vehicle controls. Overall average food consumption was decreased (p≤0.01) in animals 

treated with metalaxyl at 125 mg/kg/day (↓6%) compared to the vehicle controls. Overall 

average food consumption in the TP + FT group was decreased (p≤0.01) by 7% compared to the 

corn oil vehicle control. No treatment-related effects were observed in any of the accessory sex 

organs or kidneys of metalaxyl-dosed animals. However, mean liver weights were increased 

(p≤0.01) by 13% in the 375 mg/kg/day group compared to the vehicle controls. No gross lesions 

were observed at necropsy, but the left Cowper’s gland was not present in one animal in the 40 

mg/kg/day group. Animals in the TP + FT group had decreased (p≤0.01) accessory sex organ 

weights as expected: 78% in seminal vesicles; 70% in ventral prostate; 44% in LABC; 53% in 

Cowper’s gland; and 19% in glans penis. The %CV values for each accessory sex organ of the 

vehicle control and metalaxyl high dose group were less than the maximum guideline-specified 

%CV values, except for the % CV for glans penis of the vehicle control (CV = 18%; maximum 

permissible CV = 17%). 

The high dose of metalaxyl selected for this study (375 mg/kg/day) was appropriate based on 

data from earlier studies showing clinical signs of systemic toxicity at doses ≥500 mg/kg/day and 

the increased liver weights observed at 375 mg/kg/day in the current assay. 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. Metalaxyl was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

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Female Pubertal; (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48701301), 15 female Sprague-Dawley (Crl:CD(SD)) 

rats/dose group were treated daily via oral gavage with metalaxyl (>98% a.i., Lot # 20100540) in 

2.0% aqueous carboxymethylcellulose at doses of 0, 125, 250, or 350 mg/kg/day from postnatal 

day (PND) 22 to 42 or 43. Animals were examined for vaginal opening (VO) daily beginning on 

PND 22, and age and weight at day of attainment were recorded. Following sacrifice on PND 42 

or 43, blood was collected for clinical chemistry analyses, and total thyroxine (T4) and thyroid 

stimulating hormone (TSH) levels were determined using radioimmunoassays. Adrenal, liver, 

pituitary, thyroid, and urogenital organs were weighed and examined macroscopically. The 

uterus, ovary, thyroid, and kidney were examined microscopically. 

No treatment-related related effects were observed on survival, clinical signs, body weights, 

body weight gains, or gross pathology. No treatment-related histological lesions were noted in 

the thyroid or uterus, and the colloid area and follicular cell height in the thyroids of the treated 

groups were generally similar to control. Age of attainment of VO, body weight at VO, and 

proportion/incidence of unopened vagina in the treated groups were similar to control animals. 

No treatment-related effect was observed on the mean age of first vaginal estrus, mean cycle 

length, % cycling, % regularly cycling, and the cycle status at necropsy. Serum T4 and TSH 

levels in the treated groups were similar to the control values. 

The absolute and relative liver weights were increased (p≤0.05 or 0.01) dose-dependently in all 

treated groups by 8-23% compared to control. Gamma-glutamyl transpeptidase levels were 

increased (p≤0.01) by 175 and 305% at 250 and 350 mg/kg/day, respectively; however, the 

serum levels were reported to be within the historical control range. In the ovaries, total primary 

follicle counts were increased (p≤0.05 or ≤0.01) by 43 and 57% at 250 and 350 mg/kg/day, 

respectively, but atretic follicle counts in the treated groups were similar to control values. 

The high dose (350 mg/kg/day) was determined to be adequate based on generalized summaries 

from several ranging-finding studies and findings in the previously reviewed uterotrophic study 

(MRID 48617606) which showed decreased motor activity, ptosis, ataxia, lacrimation, and 

hunched posture in female rats in the 300 mg/kg/day dose group. 

Male Pubertal; (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48701301), 15 male Sprague-Dawley (Crl:CD(SD)) rats/dose 

group were treated daily via oral gavage with metalaxyl (>98% a.i., Lot # 20100540) in 2.0% 

aqueous carboxymethylcellulose at doses of 0, 125, 250, or 350 mg/kg/day from postnatal day 

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(PND) 23 to 53 or 54. Animals were examined for preputial separation (PPS) daily beginning on 

PND 30, and age and weight at day of attainment were recorded. Following sacrifice on PND 53 

or 54, blood was collected for clinical chemistry analyses, and testosterone, total thyroxine (T4), 

and thyroid stimulating hormone (TSH) levels were determined using radioimmunoassays. 

Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed and examined 

macroscopically. The testes, epididymides, right kidney, and thyroid were examined 

microscopically. 

No adverse, treatment-related effects were observed on survival, clinical signs, body weights, 

body weight gains, organ weights, clinical chemistry, or gross pathology. No adverse 

histological lesion was observed in the testes, epididymides, or kidneys. Mean serum 

testosterone, T4, and TSH levels in the treated groups were similar to control, and the thyroid 

follicular cell heights in the treated groups were generally similar to control. Although colloid 

area in the thyroid was slightly reduced in a dose-related manner, there were no concomitant 

effects noted in thyroid weights or hormone concentrations. 

Body weights, body weight gains, age of attainment of PPS, body weight at PPS, and 

proportion/incidence of separated prepuce in the treated groups were similar to control with the 

exception of minor decreases (p≤0.05) in the adjusted body weight at PPS at 125 mg/kg/day 

(↓6%) and unadjusted and adjusted body weights at PPS at 250 and 350 mg/kg/day (↓6-8%). 

These minor decreases were not dose dependent in magnitude. No effect was noted on daily 

body weight measurements. 

All organ weight values in the treated groups were similar to control, except for a minor percent 

increase in relative liver weight at 350 mg/kg/day (5.4%) compared to control (4.9%). This 

effect was minor, without a significant effect on absolute or adjusted weight, and without 

corroborating gross pathology or clinical chemistry findings of hepatotoxicity. Consequently, 

this effect was considered incidental. 

One 350 mg/kg/day male (#349) was found dead on PND 40. Although the study authors 

considered this death to be treatment related, none of the data presented supported that 

conclusion. The cause of death was not determined, but was not due to gavage error (perforation 

of lungs, trachea, or esophagus). All tissues appeared normal at necropsy, and no adverse 

clinical signs were noted in this animal on any day during the study. 

Although there was limited evidence of toxicity at the high dose (350 mg/kg/day) in this study, 

the selection of the high dose was appropriate based on systemic toxicity in the dose-range 

finding studies and findings in the uterotrophic assay (MRID 48617606) which showed 

decreased motor activity, ptosis, ataxia, lacrimation, and hunched posture in female rats in the 

300 mg/kg/day dose group. In addition, mean liver weights were significantly increased in male 

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rats in the Hershberger assay (MRID 48617604) at 375 mg/kg/day supporting the selection of 

350 mg/kg/day as an appropriate high dose in the male pubertal assay. 

Steroidogenesis; (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48617605), H295R cells cultured in vitro in 24-well plates 

were incubated with metalaxyl (98.2% a.i., Batch# 20100540) at log concentrations from 0.0001 

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

assay concentration of 0.05%. 

Testosterone and estradiol levels were measured using HPLC/MS/MS using internal standards. 

Four independent experiments were performed; however, only Runs 2 through 4 were deemed 

successful. 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 levels; positive controls included a known inhibitor (prochloraz) and known 

inducer (forskolin) of estradiol and testosterone production. 

All guideline acceptability recommendations and requirements were met, including lack of 

cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low 

coefficients of variation, %CV), and appropriate induction and inhibition with positive controls. 

There were no significant changes in testosterone production in Run 2 at any concentration tested 

or in Run 4 at ≤10 μM of metalaxyl, although there was a slight decrease in testosterone 

production (0.88-fold, p<0.05) at 100 μM. In Run 3, there was a consistent, slight increase in 

testosterone production (1.15- to 1.17-fold; p<0.05) at metalaxyl concentrations of 0.0001 to 10 

μM, but the increase at 100 μM was not significant (1.09-fold, NS). For estradiol, there were no 

significant changes in production in Runs 2 or 4 at any tested concentration, and there was only a 

slight increase (1.18- to 1.19-fold; p<0.05) in estradiol production in Run 3 at the three lowest 

tested concentrations (0.0001 – 0.01 μM). Changes in testosterone and estradiol were not 

reproducible or concentration dependent. 

Based on the hormone responses in each of the three independent runs, EPTC treatment did not 

result in statistically significant and reproducible alterations in testosterone or estradiol 

production. 

Uterotrophic; (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48617606) conducted to screen for potential estrogenic activity, 

metalaxyl (98.2% a.i., Batch/lot # 20100540) in 2.0% aqueous carboxymethylcellulose (CMC) 

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was administered daily via oral gavage to groups of six ovariectomized female Sprague Dawley 

rats at dose levels of 0 (vehicle), 100 or 300/200 mg/kg/day on post-natal days (PND) 57-59 or 

63-65. A positive control group was treated with a daily dose of 17α-ethynyl estradiol (EE) at 5 

μg/kg/day by subcutaneous (s.c.) injection. Body weights and food consumption were 

determined daily. All animals were terminated and necropsied on PND 60 or PND 66 

approximately 24 hours after the final dose administration to determine wet and blotted uterine, 

and liver weights. 

All animals survived until scheduled termination. Following administration of metalaxyl at 300 

mg/kg/day on Day 1, adverse clinical signs were observed (decreased motor activity, ptosis, 

ataxia, lacrimation, hunched posture, intermittent body jerks, chromodacryorrhea, and slight 

excess salivation), as well as reductions in food consumption. Therefore, the 300 mg/kg/day 

dose level was reduced to 200 mg/kg/day on Study Days 2 and 3. Following the dose reduction, 

food consumption in this group increased and no other adverse clinical signs were observed. 

Body weights throughout the dosing period and overall (Days 1-4) body weight gain (BWG) in 

the metalaxyl treated groups were comparable to controls. Overall (Days 1-4) food consumption 

for both metalaxyl treatment groups was decreased (p≤0.05) by 6-8% compared to the vehicle 

controls. Uterine weights in the metalaxyl treated groups were comparable to the vehicle 

controls. Absolute and relative liver weights in the 300/200 mg/kg/day group were increased 

(p≤0.05) compared to vehicle controls (↑24-25%); relative liver weights were increased (p≤0.05) 

in the 100 mg/kg/day group (↑13%). 

Food consumption in the EE positive control group was decreased (p≤0.01) throughout the 

study, for a decrease (p≤0.01) of 19% in overall average food consumption and a decrease 

(p≤0.01) of 54% in the overall BWG compared to the vehicle controls. Absolute wet and blotted 

uterus weights for the positive control (EE) group were increased (p≤0.01) by 547% and 207%, 

respectively, as expected. 

The high dose used in this study was adequate based on adverse clinical signs of toxicity 

observed following a single dose of metalaxyl at 300 mg/kg and increased liver weights in the 

300/200 mg/kg/day group. 

No statistically significant changes were seen in uterine weight in this assay. Metalaxyl is 

negative in the uterotrophic assay. 

 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of metalaxyl with the endocrine system are summarized below. Given the 

breadth of toxicity data available on metalaxyl, the only Part 158 studies discussed in this 

appendix are those that fulfill current guideline requirements and are directly relevant to 

assessing potential endocrine effects (i.e., multiple-generation rodent reproductive studies, 

development studies, chronic toxicity studies, etc.). 

Androgen Receptor Binding Assay 

In a published, non-Guideline study (Bauer et al., 2002), metalaxyl (99.3% purity) was tested 

(as part of a larger group of pesticides) in an AR binding study with an immuno-immobilized 

recombinant human AR (rhAR) system. Metalaxyl was dissolved in ethanol at a concentration 

dependent on its maximal solubility (the concentration of the stock solution and the 

concentration range tested for metalaxyl were not reported). Specific binding was calculated as 

the difference of total binding and non-specific binding in the presence of a 200-fold surplus of 

unlabelled DHT. The relative binding affinity of metalaxyl was compared to DHT by using the 

inhibition constants (Ki) of each compound to calculate the relative binding affinity 

(RBA=Ki DHT×100 / Ki metalaxyl). Statistical analyses were not conducted. 

No binding was observed for metalaxyl at its maximum soluble concentration (not reported); 

therefore, the RBA was reported to be <0.001%. 

Receptor Transactivation Assays

In a published, non-Guideline study (Kojima et al., 2004), the potential of metalaxyl (>95% 

purity) 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. The human ER (hERα) and AR (hAR) expression vectors (pcDNAERα and 

pZeoSV2AR, respectively) were constructed as described previously (Kojima et al., 2003). The 

hERβ expression vector was constructed by cloning the hERβ cDNA with reverse transcriptasepolymerase chain reaction from human placental RNA. The sequence was verified and inserted 

into the mammalian expression vector pcDNA3.1Zeo(-) creating pcDNAERβ. The construction 

of the estrogen or androgen responsive elements containing reporter plasmid (pGL3-tkERE and 

pIND-ARE, respectively) was described previously (Kojima et al., 2003). pRL-SV40 containing 

the Renilla luciferase gene was used as an internal control for transfection efficiency. 

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. For measurement of antagonistic activity to hERα, 

hERβ and hAR, either 10−11 or 10−10 M 17β-estradiol (E2, >97% purity) or 10−10 M 

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5α-dihydrotestosterone (DHT, 95% purity) 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 against ERα, ERβ, and AR, respectively. 

Metalaxyl was found to be negative for agonist or antagonist activity against ERα and ERβ, and 

negative for agonist or antagonist activity against AR. 

28–Day Oral Toxicity in Rodents (Mouse; OCSPP 870.3050) 

In a 28-day oral toxicity study (MRID 48104701, 2003), metalaxyl (98.4% purity) was 

administered in the diet to four groups of CRL:CD-1® (ICR) BR mice (6/sex/dose group) at 

nominal concentrations of 0, 250, 1250, or 7000 ppm (equivalent to 0/0, 52/65, 242/290, and 

1364/1960 mg/kg/day for males/females, respectively) for 28 days. 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 

vesicles, uterus, vagina, and parathryoid/thyroid (control and high-dose groups only examined 

for all organs; liver examined for all groups). 

No adverse, treatment-related effects were observed on mortality, clinical signs, neurological 

evaluations, body weight, food consumption, hematology, clinical chemistry, organ weights, or 

gross or histological pathology. 

Although not calculated or statistically analyzed by the study authors, overall body weight gains 

(0-28 days) for male and female mice at the high dose were decreased by 26 and 14%, 

respectively. Bilirubin concentrations were decreased in males and females in the medium- (30 

and 50%, respectively) and high-dose (40 and 69%, respectively) groups. Hepatic enzyme 

concentrations were increased in females at the medium- and high-doses for alanine 

aminotransferase (69 and 67%, respectively) and sorbitol dehydrogenase (47 and 42%, 

respectively). In the high-dose group, increases occurred in absolute liver weight (37%), and 

liver weight relative to body weight (40%) and brain weight (38%) of male mice. Increases in 

absolute liver weight occurred in females at the medium- and high-doses (28 and 36%, 

respectively), as well as increased liver weight relative to body weight (22 and 33%, 

respectively) and liver weight relative to brain weight (42 and 56%, respectively). Minimal 

diffuse hepatocyte hypertrophy was observed in the high-dose males (6/6) and females (4/6) 

compared to control (0-1/6). In the absence of other clinical or pathological findings, the 

increases in liver weights in conjunction with the observed decreases in bilirubin concentrations 

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and hepatocyte hypertrophy were considered to be adaptive due to xenobiotic exposure and not 

adverse. 

A LOAEL is not established. The NOAEL is 7000 ppm (the limit dose; equivalent to 

1364/1960 mg/kg/day in males/females, respectively). 

90–Day Oral Toxicity in Rodents (Rat; OCSPP 870.3100) 

In a subchronic oral toxicity study (MRID 48104702, 2003), metalaxyl (98.4% purity) was 

administered to CRL:CD® (SD) IGS BR rats (10/sex/dose) in the diet at concentrations of 0, 

250, 1250, 7500, or 20,000 ppm (equivalent to 0, 16/19, 83/101, 496/595, or 1304/1672 

mg/kg/day for males/females, respectively) for 13 weeks. 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 

vesicles, uterus, mammary gland, pituitary, and parathyroid/thyroid (control and high-dose 

groups only examined for all organs, liver examined for all groups, and kidneys examined in all 

males). 

No effects of treatment were observed on mortality, clinical signs, or gross pathology. In the 

high-dose females, body weight was decreased (p<0.01) by 13-18%, resulting in a decrease (NS) 

in total body weight gain (↓43%). Body weight in the medium-high dose females generally was 

decreased (p<0.05) by 7-9% with a decrease (NS) in total body weight gain of 20%. Female rats 

in these dose groups also had decreases (NS) in overall food consumption that were 

concentration dependent. Body weight in high-dose males was decreased during Weeks 1 and 2 

of the study (p<0.01, ↓8 and p<0.05, ↓6%, respectively) with an overall body weight gain 

decrease (NS) of 14%. Food consumption was also decreased (p<0.01 or p<0.05) in males 

during Weeks 1, 8, and 11 of the study, resulting in an overall decrease (p<0.05, ↓6%) in total 

food consumption. 

Clinical chemistry results included increased (p<0.05 and NS) cholesterol in the medium- and 

high-dose males (↑39 and ↑51%, respectively) and increased (p<0.05) cholesterol and 

triglycerides in the high-dose females (↑94 and ↑46%, respectively). There were no pathological 

changes correlated with the serum lipid changes, but the increases in serum lipids in females 

were considered to be potentially adverse due to the magnitude of the change. Decreased 

bilirubin concentrations in the medium- and high-dose males and females (p<0.05, ↓14-36%) and 

decreased alkaline phosphatase activity in the medium- and high-dose females (p<0.05, ↓24-

39%) were correlated with increased (p<0.01 and p<0.001) liver weights (both sexes; ↑24-75%) 

and hepatocyte hypertrophy (females; all dose groups). The decreased bilirubin and hepatocyte 

hypertrophy were considered to be secondary to treatment (enzyme induction) and non-adverse. 

No other treatment-related effects were noted on absolute organ weights. Terminal body weights 

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were decreased (NS) in medium-dose females (↓8%) and high-dose males and females (↓9 and 

22%, respectively). 

For males, the LOAEL is 1250 ppm (83 mg/kg/day) based on increased incidence of 

nephropathy; the NOAEL is 250 ppm (16 mg/kg/day). For females, the LOAEL is 7500 ppm 

(595 mg/kg/day) based on decreased body weight, body weight gain, and food consumption, and 

the NOAEL is 1250 ppm (101 mg/kg/day). 

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

In a subchronic toxicity study in dogs (MRID 00084111, 1977), metalaxyl (technical; purity not 

available) was administered to Beagle dogs, 3/sex/group, at concentrations of 50 or 250 ppm in 

the diet (estimated compound intakes by standard conversion were 1.25 or 6.25 mg/kg/day) or to 

dogs at 4/sex/group at concentrations of 0 (negative control) or 1250 ppm in the diet (estimated 

compound intakes by standard conversion were 0 or 31.3 mg/kg/day) for 91 days. Three dogs 

from each treatment group were sacrificed at the end of the treatment period. The remaining 

single dogs (per sex) from the control and 1250 ppm groups were further observed for one month 

without treatment. 

No mortality was observed during the study, and no changes in animal behavior were reported. 

Body weight/body weight gain data were not reported. Serum alkaline phosphatase 

concentrations were slightly increased in one male and one female in the high-dose group at 

Weeks 4, 8, and 12 (identities of affected animals were not reported). Serum alkaline 

phosphatase concentrations were normal in the recovery animals at Week 16. Macroscopic 

findings were not treatment related. There were no dose-related trends in absolute weights, or 

organ weight/body weight and organ weight/brain weight ratios for any examined organs 

including ovaries, uterus, testes, prostate, adrenal, or pituitary glands. In addition, there were no 

dose-related necropsy or microscopic findings in any of these tissues or the thyroid and 

mammary glands. Clinical chemistry, hematology, and urine analyses indicated no treatmentrelated findings, and ophthalmoscopic examinations were also negative for treatment effects. 

The NOAEL is 250 ppm (approximately 6.25 mg/kg/day), and the LOAEL is 1250 ppm 

(approximately 31.25 mg/kg/day) based on the increases in serum alkaline phosphatase 

concentrations. 

Prenatal Development in Rodents (Rat; OSCPP 870.3700) 

In a developmental toxicity study in rats (MRID 00144423; 1985), metalaxyl technical (purity 

not reported) in 1% aqueous methylcellulose was administered by gavage to COBS® CD® rats 

(27 or 38/dose) at concentrations of 0, 50, 250, or 400 mg/kg/day on GDs 6 through 15. All 

dams were sacrificed on GD 20. An initial high dose of 575 mg/kg/day yielded excessive 

toxicity. The dose was reduced to 500 mg/kg/day; surviving animals were sacrificed and 

additional females were mated. The 500 mg/kg/day dose also yielded excessive toxicity. The 

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dose was reduced further to 400 mg/kg/day; surviving animals were sacrificed and additional 

females were mated and the number of females was increased to 38. 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 with a modified Dawson’s method. 

All control and low-dose animals survived to scheduled sacrifice. One dam at the medium dose 

was sacrificed in extremis on GD 13 after displaying clinical signs including post-dose ataxia, 

loss of activity, body tremors, and reduced righting reflex. Twelve high-dose animals died or 

were sacrificed in extremis between GDs 6 and 8; most of these animals exhibited convulsions 

within minutes after dosing. The following clinical signs were observed: post-dose convulsions; 

dry or wet matter on head, eyes, nose, abdomen, anogenital area, and/or vaginal opening; and 

stained and/or matted hair coat on the head, face, ventral thorax, abdomen, back, anogenital area, 

and/or forelimbs. Additional clinical signs observed only in high-dose animals included: loss of 

activity, and reduced or loss of righting reflex. No treatment-related changes in body weight 

were noted at any dose tested. Body weight gains in the medium-dose group were reduced (NS) 

during GDs 6-9 (↓160%) and the overall treatment interval (↓11%, GDs 6-15). Body weight 

gains in the high-dose group were reduced (NS) during GDs 6-9 (↓100%), the overall treatment 

interval (↓16%, GDs 6-15), and the overall study interval (↓10%, GDs 0-20). 

When compared to concurrent control, no treatment-related changes in body weight, food 

consumption, gross pathology, or reproductive parameters (pregnancy rate, number of corpora 

lutea, mean number of implantations/litter, pre- or postnatal implantations, early or late 

resorptions, number of live fetuses/litter, or fetal sex ratio) were noted at any dose tested. The 

maternal toxicity LOAEL is 250 mg/kg/day, based on clinical signs of toxicity including postdose convulsions. The maternal toxicity NOAEL is 50 mg/kg/day. 

There were no treatment-related developmental external or visceral variations noted at any dose 

level. Several variations were noted during skeletal examinations of the high-dose fetuses that 

were outside the historical control ranges: 25 presacral vertebrae; fewer than normal (13 full 

pair) ribs; reduced ossification of the skull; and unossified pubic bones. The developmental 

toxicity LOAEL is 400 mg/kg/day based on increased incidence of skeletal variations. The 

developmental toxicity NOAEL is 250 mg/kg/day. 

Prenatal Development in Non-Rodents (Rabbit; OCSPP 870.3700) 

In a developmental toxicity study in rabbits (MRID 00144372, 1984), metalaxyl (96.7% purity) 

was administered to artificially-inseminated Dutch Belted rabbits (18/dose group) by gavage at 

doses of 0, 30, 150, and 300 mg/kg/day from GDs 7 through 19. On GD 28, 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 

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visceral abnormalities, then eviscerated and examined for skeletal abnormalities according to the 

method of Dawson. 

All animals survived until sacrifice, except for one high-dose doe (necropsy unremarkable). 

Maternal toxicity, consisting of little/none/soft stools was observed in the high-dose animals 

(7/18 vs. 2-3/18 in the control and lower dose groups). Body weight gain of dams in the highdose group was negative (−75 g) and was decreased (↓192%) compared to control during the 

exposure period. Food consumption in the high-dose dams was decreased (NS) by 30% 

compared to control during the exposure period; in general, food consumption was considered 

comparable throughout the study for all groups. No treatment-related changes were seen in 

pregnancy rate, number of corpora lutea, mean number of implantations/litter, pre-or post natal 

implantations, early or late resorptions, number of live fetuses/litter, fetal sex ratio, or soft tissue 

abnormalities at any dose. There were no developmental effects observed at any dose. The 

maternal LOAEL is 300 mg/kg/day based on body weight gain, and the maternal NOAEL is 

150 mg/kg/day. The developmental NOAEL is 300 mg/kg/day (HDT); the LOAEL is not 

determined. 

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

In a three-generation reproduction study in rats (MRID 00071600; 1980), metalaxyl 

(93.5% purity) was administered continuously in the diet to Crl: COBS CD (SD) BR rats 

(25 rats/sex/dose for the first and second generations, 12 males and 24 females/dose for the third 

generation) at concentrations of 0, 50, 250 or 1250 ppm (estimated compound intakes by 

standard conversion were 0, 2.5, 12.5, or 62.5 mg/kg/day). The P animals were exposed to 

metalaxyl beginning at approximately 5 weeks of age and exposure was continued for 91 days 

prior to mating to produce the F1A litters. The P animals were mated 3 times. The F1A pups were 

sacrificed at weaning; the F1B generation was used to select pups (25/sex/dose) to produce the 

F2 generations; the F1C generation was sacrificed in utero at gestation day (GD) 20 and examined 

for developmental effects and the dams examined for reproductive effects. The F1B pups were 

exposed to the same dosage as their parents on postnatal day (PND) 21 and exposure was 

continued for at least 90 days prior to mating to produce the F2A litters. The F1B animals were 

mated 3 times. The F2A pups were sacrificed at weaning; the F2B litters were used to provide 

pups for the F3 generation; the F2C litters were sacrificed at weaning. In addition, ten 

F1B litters/dose were sacrificed in utero at GD 20 and examined for developmental effects and 

the dams examined for reproductive effects. At weaning, 12 F2B rats/sex/dose were maintained 

on their respective diets for at least 90 days, then given a detailed necropsy and subjected to 

organ weight analysis. Another 12 males and 24 females/sex/dose from the F2B litters were 

mated at 90 days of age to produce the F3 litters. The F3A litters were sacrificed at PND 21. The 

F3B litters were sacrificed at PND 21 and 10 pups/sex/dose were given a detailed necropsy and 

subjected to organ weight analysis. Exposure of all animals to the test material was continuous 

throughout the study. Organs that were weighed included the liver, kidneys, testes, ovaries, 

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uterus, pituitary, adrenals, and thyroid in F2B and F3B rats. Gross and microscopic pathological 

examinations were conducted on these tissues as well as the prostate, seminal vesicles, and 

mammary gland in F2B and F3B rats. 

There was no evidence of treatment-related changes in clinical signs, mortality, body weight, 

body weight gains, water consumption, food consumption, or food efficiency observed in the P, 

F1, or F2 adults. There were no treatment-related gross pathological findings in adult rats. There 

were no differences of toxicological concern in P or F1 adult organ weights. An increase 

(p<0.05) in adjusted (to body weight) liver weight compared to control occurred in the high-dose 

F2B females (↑11%). This finding was considered treatment-related and of toxicological concern 

due to the observation of hepatomegaly in females at the same dose in the range-finding study. 

Absolute pituitary weights were decreased (p<0.05 or 0.01) in low-, medium-, and high-dose F2B

females (↓21, 21, and 14%, respectively); these changes were not considered to be 

toxicologically relevant because the decreases were not dose dependent (no histopathological 

examinations were conducted). The parental systemic toxicity LOAEL in females is 1250 ppm 

(approximately equivalent to 62.5 mg/kg/day) based on increased relative (to body) liver 

weights, and the NOAEL in females is 250 ppm (approximately equivalent to 12.5 mg/kg/day). 

A parental systemic toxicity LOAEL is not determined in males; the NOAEL in males is 

≥1250 ppm (approximately equivalent to 62.5 mg/kg/day). 

No treatment-related effects were observed in male or female mating or fertility indices, 

gestation index, gestation length, live birth index, lactation index, parturition, or fetal sex ratio of 

the P, F1, or F2 adults. No treatment-related changes were seen in number of corpora lutea, mean 

number of implantations/litter, or fetuses in the litters from the third mating of F1 adults and 

second mating of F2 adults sacrificed on GD 20. Sexual maturation was not measured. A 

reproductive toxicity LOAEL is not determined; the reproductive toxicity NOAEL is ≥1250 ppm 

(approximately equivalent to 62.5 mg/kg/day). 

No differences of toxicological concern in clinical signs, viability, body weight, body weight 

gains, or litter weights were observed at any time in the F1, F2, or F3 litters. No treatment-related 

changes in absolute or relative weights of the testes, seminal vesicle, ovaries, uterus, or adrenal 

gland were seen in F2B or F3B offspring. Adjusted pituitary weights were decreased (p<0.05) in 

the high-dose F3B males (↓40%) and absolute pituitary weights were increased (p<0.05) in the 

mid- and high-dose F3B females (↑50% and 25%, respectively). These findings were considered 

not of toxicological concern due to the lack of histological findings in both sexes and lack of 

dose-dependence in females. There were no other differences of toxicological concern in organ 

weights in the F3B pups. There was an increase in the degree of hepatic fat deposition in the 

high-dose F3B female pups (high-dose: trace - 1/10, minimal - 6/10, moderate - 3/10; vs control: 

trace - 2/10, minimal - 2/10). There were no other findings of toxicological concern in the F1, F2, 

or F3 pups. The offspring systemic LOAEL is 1250 ppm (approximately equivalent to 

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62.5 mg/kg/day) based on histopathological changes in the livers of female pups. The offspring 

systemic NOAEL is 250 ppm (approximately equivalent to 12.5 mg/kg/day). 

There were no differences of toxicological concern in fetal viability, body weight, or 

development (external, visceral, or skeletal). A developmental toxicity LOAEL is not 

determined. The developmental toxicity NOAEL is ≥1250 ppm (approximately equivalent to 

62.5 mg/kg/day). 

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

In a chronic toxicity/carcinogenicity study in rats (MRID 00098481, 1980), metalaxyl (93% and 

94.6% purity) was administered to SD rats (80/sex/dose) in the diet at nominal concentrations of 

0, 50, 250, and 1250 ppm (male: 0, 1.88, 9.43, and 46.6 mg/kg/day; female: 0, 2.2, 9.95, and 

55.0 mg/kg/day) for two years. Organs that were weighed included the liver, kidneys, testes with 

epididymides, and adrenals. Gross and microscopic pathological examinations were conducted 

on these tissues as well as the prostate, uterus, mammary gland, pituitary, and 

thyroid/parathyroids. 

There were no treatment-related effects on clinical signs, mortality, body weight, food 

consumption, ophthalmologic examination, hematology, or urinalysis. Transient increases 

(p<0.05 and NS, respectively) in SGOT and SGPT were observed in females in the high-dose 

group at Week 78 (↑44% and 30%, respectively). Relative liver weight was increased (p<0.01) 

in high-dose females at Week 55 (↑16%), and relative and absolute liver weights were increased 

(NS and p<0.001, respectively) in high-dose females at Week 105 (↑12 and 22%, respectively). 

Relative liver weight was increased (p<0.001) in medium-dose males at Week 105 (↑19%), and 

absolute and relative liver weights were increased (NS and p<0.01) in high-dose males at Week 

105 (↑11 and 16%, respectively). No treatment-related changes were seen in absolute or relative 

weights of the testes or epididymides. The incidence of periacinar vacuolation of hepatocytes 

was increased in males of all dose groups (18/80, 19/80, and 19/80) in females of the mediumand high-dose groups (43/80 and 44/80) compared to control (7/80 M and 18/80 F). No 

treatment-related histopathological lesions were seen in any other examined tissues. Although 

there is no increased risk of cancer in this study, it was concluded that the MTD was not reached 

in this study. 

The LOAEL is 1250 ppm (male 46.6 mg/kg/day, female 55.0 mg/kg/day) based on increased 

liver weights and histopathology of the liver. The NOAEL is 250 ppm (male 9.43 mg/kg/day; 

female 9.95 mg/kg/day). 

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200) 

In a chronic oncogenicity study in mice (MRID 00103354, 1981), metalaxyl (93.0% and 94.6% 

purity) was administered to Swiss mice (60/sex/dose group) in the diet for 104 weeks at 0, 50, 

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250, and 1250 ppm (0, 5.00, 24.85, 128.89 mg/kg/day for males; 0, 5.68, 29.59, and 

148.16 mg/kg/day for females). Organ weights were not determined in this study. The tissues 

subjected to histological examination included liver, kidneys, testes, epididymides, prostate, 

seminal vesicles, ovary, mammary gland, pituitary, thyroid/parathyroids, and uterus. 

There were no effects on mortality, clinical signs, and food consumption due to metalaxyl 

treatment. There were only transient reductions in body weight gain in the high-dose animals 

(Weeks 11-30 in males; ↓35% and Weeks 31-56 in females; ↓33%) with mean body weight 

decreases of <10%. Dose-dependent increases in fatty infiltration of the liver was observed in 

males and females of the high-dose compared to control. No treatment-related histopathological 

lesions were seen in the any other examined tissues. The relatively high incidence of fatty 

vacuolation of hepatocytes indicates that the mice might not have tolerated a higher dose without 

increasing mortality and or body weight gain. Although there is no increased risk of cancer in 

this study, it was concluded that the MTD was not reached in this study. 

The LOAEL is 1250 ppm (male 128.89 mg/kg/day, female 148.16 mg/kg/day) based on liver 

histology (hepatocytic large fatty vacuolation). The NOAEL is 250 ppm (male 24.85 mg/kg/day, 

female 29.59 mg/kg/day). 

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

In a chronic toxicity study in dogs (MRID 00071598; 1981), metalaxyl (92% purity) was 

administered to Beagle dogs (6/sex/dose) in the diet at nominal concentrations of 0, 50, 250, and 

1000 ppm (0, 1.57, 7.80, and 30.63 mg/kg/day for males; 0, 1.71, 7.41, and 32.36 mg/kg/day for 

females) for 6 months. Two additional dogs/sex were fed the 0 or 1000 ppm diets, and were 

observed for a 30-day recovery period after the termination of treatment. Organs that were 

weighed included the liver, kidneys, testes, ovaries, pituitary, adrenals, and thyroid. Gross and 

microscopic pathological examinations were conducted on these tissues as well as the 

epididymides, prostate, uterus, and mammary gland. 

Administration of metalaxyl for six months did not affect body weight, body weight gain, food 

consumption, hematology, urinalysis, ophthalmologic examinations, and gross pathology. At 

Days 120, 150, and 180 of treatment with metalaxyl, alkaline phosphatase levels were increased 

(p<0.05) in high-dose males (↑52, 81, and 84%, respectively). In contrast, alkaline phosphatase 

levels were decreased (p<0.05) in high-dose females on Days 120 and 150 (↓13 and 21%, 

respectively) and increased (p<0.05) in high-dose females on Day 180 (↑19%). Absolute liver 

weights and relative liver weights were increased (NS) in males and females by 13-14% at the 

mid dose and 13-20% at the high dose. No treatment-related histopathological lesions were seen 

in the thyroid, adrenal or pituitary glands. 

After the 30-day recovery period, body weight in the high-dose male dogs was decreased (NS) 

by 24% (due to 36% increase in recovery body weight in control males vs. terminal body 

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weight). Female dogs in the control and high-dose groups had similar body weights at study 

termination and at the end of the recovery period. 

The LOAEL is 1000 ppm (male 30.63 mg/kg/day, female 32.36 mg/kg/day) based on increased 

liver weights and clinical chemistry changes (alkaline phosphatase). The NOAEL is 250 ppm 

(male 7.80 mg/kg/day, female 7.41 mg/kg/day).

Avian Reproduction (OCSPP 850.2300) 

In an avian reproduction study (MRID 44755001, 1999), Bobwhite quail (Colinus virginianus; 

25-weeks old) were administered metalaxyl (88.7% purity) in the feed at nominal concentrations 

of 0 (1% acetone), 100, 300, or 900 ppm (corrected for 88.7% purity) for 22 weeks (10 weeks 

prior to the start of egg production and during 12 weeks of egg production). 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 8 and 17 hours/day until the end of the 

study. Mean temperature was maintained at 21°C throughout the study and mean humidity was 

maintained at 52%. Individual adult body weights were recorded at study initiation, biweekly 

through Week 8, and at study termination. Food consumption in each replicate was measured 

biweekly throughout the pre-treatment 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 biweekly during egg production. Adult birds and chicks were observed routinely for 

mortality and clinical signs. 

There were no overt signs of toxicity or treatment-related reductions in food consumption or 

body weight at any test concentration compared to control. In addition, there were no effects on 

any reproductive parameter measured at any test concentration compared to control. 

In another avian reproduction study (MRID 44761701, 1999), Mallard ducks (Anas 

platyrhynchos; age not reported) were administered metalaxyl (88.7% purity) in the feed at 

nominal concentrations of 0 (1% acetone), 100, 300, or 900 ppm for 23 weeks (10 weeks prior to 

the egg production and during 13 weeks of egg production). 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 8 and 17 hours/day until the end of the study. 

Mean temperature was maintained at 23°C throughout the study and mean humidity was 

maintained at 63%. Individual adult body weights were recorded at study initiation, biweekly 

through Week 8, and at study termination. Food consumption in each replicate was measured 

biweekly throughout the pre-treatment 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 biweekly during egg production. Adult birds and chicks were observed routinely for 

mortality and clinical signs. 

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There were no overt signs of toxicity or treatment-related mortalities at any test concentration, 

and there were no reductions in adult body weight or feed consumption. There was a decrease in 

the percentage of normal hatchlings of viable embryos in the low-concentration treatment group; 

this decrease was not considered to be treatment related as the value for percentage of normal 

hatchlings was equivalent to the historical control for this parameter. Additionally, the effect 

was not dose dependent, although a similar decrease was also noted in the high-concentration 

group with no effect at the medium concentration. There were no other treatment-related effects 

on any study parameters determined at any test concentrations. 

Fish Early Life-Cycle Toxicity (Fathead minnow, OCSPP 850.1400) 

In a fish early life-cycle toxicity study (MRID 00071308, 1980), groups of fathead minnow 

(Pimephales promelas) embryos (60 embryos/aquarium; 2 aquaria/treatment concentration) were 

exposed to metalaxyl (90.1% purity) at nominal concentrations of 0 (control), 0.62, 1.2, 2.5, 5, 

and 10 mg/L. Mean measured concentrations were <0.04, 0.59, 1.1, 2.1, 4.6, and 9.1 mg/L (84-

95% of nominal). After hatching, forty fry were impartially selected from each aquarium to 

begin the 30-day exposure period. Fry were counted twice weekly beyond Day 6 post-hatch. At 

Day 30 post-hatch, all fry were anaesthetized and percent survival, mean total length, and 

average wet weight were determined; fish were measured individually but weighed as groups 

from each aquarium. There were no effects on the percentage hatch of eggs, survival, mean total 

length, or average wet weight of fry at any of the tested concentrations of metalaxyl. 

 

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APPENDIX 3: References Not Utilized in the Metalaxyl 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 Metalaxyl 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. 

Ciba-Geigy, Ltd. (1984). Expanded report on a segment II reproduction study in the rabbit with 

CGA48988 technical. Ciba-Geigy, Ltd. Study No. 784868. MRID 00161404. Unpublished. 

Ciba-Geigy, Ltd. (1984). Expanded report on the segment II reproduction study with CGA48988 technical in the rat. Ciba-Geigy, Ltd. Study No. 227716. MRID 00161405. Unpublished. 

Hayes, T.B., Case, P., Chui, S., Chung, O., Haeffele, C., Haston, K., Lee, M., Mai, V.P., 

Marjuoa, Y., Parker, J., and Tsui, M. (2006). Pesticide mixtures, endocrine disruption, and 

amphibian declines: Are we underestimating the impact? Environmental Health Perspectives. 

114:Supp 1 (40-5). 

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