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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

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

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

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

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

THR O UG H: Je ss Rowland

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

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

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

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals

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

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

CHEMICAL: METALAXYL

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYMETALAXYL FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary..........................................................................................1

I. Introduction.......................................................................................4

II. Sources of Scientific Data and Technical Information......................................5

A. EDSP Tier 1 Screening Assays............................................................5

B. Other Scientifically Rele vant Information (OSRI)......................................5

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

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

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................14

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

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

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

1. Tier 1 in vivo Assays.............................................................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-Estr ogenic Pathway for Metalaxyl ...................................11
Table 3. Androgenic/Anti-Androge nic 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 Metalaxy l WoE Analysis.............................51METALAXYL FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance
AOP Adverse Outcome Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r CFR Code of Federal Regulations

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g enerationMETALAXYL FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Am p hibian Growth and Develo p ment Assa y LC50 Lethal Concentration in 50% of test organisms

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

M Male
MDL Minimum Detection Level MEOGRT Medaka Extended One Generation Re p roduction Test
MH Medium hi g h
ML Medium low MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationMETALAXYL FINAL

v Abbreviation Terminology

MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)METALAXYL FINAL

vi Abbreviation Terminology

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

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidenceMETALAXYL FINAL

Page 1 of 51 Executive Summary

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

In determining whether metalaxyl interacts with E, A or T hormone path ways, the number and type of effects induced, the magn itude and pattern of responses obs erved across studies, taxa, and
sexes were considered. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-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 th e Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WOE) an alysis of the potenti al 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 show ed 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 ER α agonism in the Tier 1 ER transactivation assay (ERTA) assay and negative for both ER α 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 th at assay. Overall metalaxyl does not appear to have a pot ential to interact with the estrogen pathway in mammals. Conversely, metalaxyl appears to po tentially 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: e ffects on gonadal histology (decreased o varian yolk synthesis), decrease d plasma vitellogenin (VTG) concentration, decreased gonadal somatic index (GSI) and finall y, cessation of fecundity (no eggs produced).METALAXYL FINAL

Page 2 of 51 Therefore, while there were largel y negative responses in the Tier 1 in vitro assays and in vivo mammalian dataset, for the FSTRA there were co mplementary responses along several levels of biological organization for the females including a lterations in enzyme activity (plasma VTG) up to the apical endpoint of fecundit y. As such, significant effects of the reproductive capacity of
fish were observed in the FSTRA which suggests that metalaxyl ma y potentially inte ract 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 steriodogenesi s assays were negative) or in vivo mammalian Tier 1 ( i.e ., Hershberger and male pubertal assays were negative) an d Part 158 mammalian or avian studies (no androgen-related e ffects observed). In addition, metalaxyl was negative in two literature studies evaluating AR binding (Bauer et al ., 2002) and AR transc riptional 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 alte red 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 al ong with some indications of potential stress (lethargy and decreased body weight) in the earl y stages of the study, the effect s 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 thyroi d 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 th at there may be a potential for interaction with the estrogen and androgen pathway in fis h. Currently, the chronic fish NOAEC value is 9.1 mg a.i./L based on no effects observed in a fath ead 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 fe males, including a cessa tion in fecundity, at 104 mg a.i./L.METALAXYL FINAL

Page 3 of 51 For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for metalaxyl. In both spec ies, no effects on reproductive parameters were observed. The type of data obtained from Pa rt 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for evaluating potential reproductive effects to birds from metalaxyl exposure. Relative to the EDSP, ad ditional testing is not recommended.

As there was no convincing evid ence of a potential thyroid interaction in amphibians, no additional testing is recommended.METALAXYL FINAL

Page 4 of 51 I. Introduction

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

In determining whether metalaxyl interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pa ttern of responses observed across studies, taxa, and sexes were consider ed. Additionally, th e conditions under which effects occur were considered, in particular , whether or not endoc rine-related responses occurred at dose(s) that also resulted in ge neral systemic toxicity or overt toxicity.

On August 13, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and th e Office of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) analys is of the potential interaction of metalaxyl with the E, A or T hormone pathways. The T1 WoERC 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 ha s a log Kow of 1.65, and bioconcentration is not anticipat ed 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 th e particulate phase is susceptible to wet and dry deposition. Metalaxyl is also susceptible to direct photolysis, and aquatic biodegradation, and rapi d biodegradation in soil.

The available information considered in determin ing 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 toxi city studies and other pu blished articles. These data are summarized in Sections III.A through I II.C. An analysis of the data submitted to theMETALAXYL 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 recomme ndations are presented in Section V.

II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix

1. B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were 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 re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam 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 c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria, and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is tocharacterize the main lines of evidence as well any conclusions . Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.METALAXYL FINAL

Page 6 of 51 As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A, or T pathway. Additionally, the concordance or consistency (complementarity) of the responses in the individual study is evaluated. For the
Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

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

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

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of bi ological 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 systemicMETALAXYL FINAL

Page 7 of 51 toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was gene rally defined in accordance with EPA’s current approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g., organ weights/histopathology), biochemical ( e.g., blood chemistry), and other clinical signs ( e.g., lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e ., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addres sing the three component s described above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availa ble toxicity data into account.METALAXYL FINAL

Page 8 of 51 The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether a substan ce 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)

ER α 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)METALAXYL FINAL

Page 9 of 51 B. Effects on Hypothalamic-P ituitary-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 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This
table also includes HPG-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

Metalaxyl was classified as equivocal in the in vitro estrogen receptor (ER) binding (rat uterine cytosol) assay based on the overall results from th e three assay runs (int eractive in one run and not interactive in the remaining two runs). Metalaxyl was negative in the Tier 1 ER α transcriptional activation (ERTA) and aromatase assays and did not alter estradiol production in the steroidogenesis assay. In a non-guideli ne ERTA considered as OSRI (Kojima et al ., 2004), metalaxyl did not show agonist or antagonist activity for ER α or ER β

.
Metalaxyl did not induce uterine weight increases in the uterotrophic assay and was classified as negative for estrogenic activity. Metalaxyl also sh owed 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, respec tively, but atretic follicle count s in the treated groups were
similar to controls.

In the FSTRA, there was a 100% decrease in fecu ndity in the high-concen tration 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. Wh ile not statistically significant (p=0.092), at the high concentration, there was a 96% decrease in female plasma vitellogenin (VTG) concentrati ons that upon visual observation of the data was considered biologically significant. It is no ted 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 inci dent rate), it was conclu ded that these lesions were probably not tr eatment-related.

In the Part 158 mammalian toxicity studies, the only organ weight effect s noted were decreased absolute pituitary weights ( ↓ 14-21%; not dose dependent) in F 2B females in all dose groups, decreased (p<0.05) adjusted pitu itary weights in the high-dose F 3B males ( ↓ 40%) and increased (p<0.05) absolute pituitary wei ghts in the mid- and high-dose F 3B females ( ↑ 50% and 25%,METALAXYL FINAL

Page 10 of 51 respectively), in the three-ge neration reproduction study in ra ts. No treatment-related histopathological findings were noted in any of th ese studies.

There were no treatment-related reproductive effect s 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 concentra tions up to 9.1 mg/L.METALAXYL FINALPage 11 of 51Table 2. Estrogenic/Anti-Estrogenic Pathway for MetalaxylLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metalaxyl1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Obsered 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48617607)EERTA(MRID 48617603)NAromatase(MRID 48617602)NSteroidogenesis(MRID 48617605)NUterotrophic(MRID 48617606)NNELW↑24%(H)N4Female Pubertal Rat(MRID 48701301)NNP5N N N↑LW6(L, M,H)NFSTRA(MRID 48685802)NE↓23%(H)P(H)7NFrt: NA orNFcd:↓100% (H)↓96%(H)NOSRIERTA(Kojimaet al., 2004)NThree-generationreproduction (Rat;MRID 00071600)NNNEF2B:↓21, 21,14% (L,M, H)NX(F2B,H)NMETALAXYL FINALPage 12 of 51Table 2. Estrogenic/Anti-Estrogenic Pathway for MetalaxylLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metalaxyl1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Obsered 2 Overt Toxicity Observed 3Developmentaltoxicity (Rat; MRID00144423)NX(M, H)X(M,H)Developmentaltoxicity (Rabbit;MRID 00144372)NX(H)NChronic toxicity/carcinogenicity (Rat;MRID 00098481)NRNRNNRX(H)NCarcinogenicity(Mouse; MRID00103354)NENENNENNChronic toxicity (Dog;MRID 00071598)N N NNX(M, H)N90-Day subchronictoxicity (Rat; MRID48104702)NENNNEX(MH, H)X(MH,H)28-Day subchronictoxicity (Mouse;MRID 48104701)NENN NEX(M, H)N91-Day subchronictoxicity (Dog; MRID00084111)NNNNN NAvian reproduction(Quail; MRID44755001)NN NMETALAXYL FINALPage 13 of 51Table 2. Estrogenic/Anti-Estrogenic Pathway for MetalaxylLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Metalaxyl1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histo p atholo gy Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Obsered 2 Overt Toxicity Observed 3Avian reproduction(Duck; MRID44761701)NN N1.Key to responses: L=Low treatment, ML=Medium-low treatment,M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows (↓or↑) indicate the direction of the response. A shaded cell indicates that this parameter is not routinely evaluated or is not applicable in this assay. Changes inweight are absolute unless otherwise indicated. LW= Liver weight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4.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 days 2 and 35. Total primary follicle counts were increased by 43 and 57%in the 250 and 350 mg/kg/day dose groups, respectively, but no effect was noted in the numberof atretic follicles.6. Unadjusted liver weights were increased by 10, 19, and 23% inthe low-, medium-, and high-dose females, respectively) with similar increases in relativeliver/body weights.7. Decreased yolk synthesis in high-dose females.E EquivocalP Positive findingsN Negative finding (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedMETALAXYL FINAL

Page 14 of 51 2. Effects on Androgen Pathway

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

Metalaxyl was classified as a non-binder in the in vitro androgen receptor (AR) binding (rat prostate cytosol) assay and ne gative in the steroidogenesis assa y. 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 fo r 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 ga ins, 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 th e high-concentration grou p in the FSTRA (score of 6 vs. 32 in control). In addition, there was an increase in the percen tage of spermatogonia in the medium- and high-concentration males a nd asynchronous gonad development at the high
concentration.

There was no evidence of effects on male reproduc tive parameters in the existing metalaxyl Part 158 mammalian toxicity studies. Additionally, th ere were no treatment-related effects observed in the Part 158 avian reproduction studies.METALAXYL FINALPage 15 of 51Table 3. Androgenic/Anti-Androgenic Pathway for MetalaxylLines of Evidence Indicating Potential Interaction withthe 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Obsereved 3EDSP Tier 1 AssayAR Binding (MRID48617601)NSteroidogenesis(MRID 48617605)NHershberger (MRID48617604)NENLW:↑13-17%(H);NMale Pubertal Rat(MRID 48701301)NNNNNNNNNNFSTRA(MRID 48685802)NENP (M,H)4tubercles:↓(H)NNNOSRIAR Binding(Baueret al., 2002)NARTAKojimaet al.(2004)NThree-generationreproduction (Rat;MRID 00071600)NNENNE NN NChronic toxicity/carcinogenicity (Rat;MRID 00098481)N N N NNR NRX(M, H)NMETALAXYL FINALPage 16 of 51Table 3. Androgenic/Anti-Androgenic Pathway for MetalaxylLines of Evidence Indicating Potential Interaction withthe 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 Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Obsereved 3Carcinogenicity(Mouse; MRID00103354)NENNENNENEN NChronic toxicity(Dog; MRID00071598)NNNENNNEX(M, H)N90-Day subchronictoxicity (Rat; MRID48104702)N N NN NE NEX(MH, H)N28-Day subchronictoxicity (Mouse;MRID 48104701)N N N N NE NEX(H)N91-Day subchronictoxicity (Dog; MRID00084111)NN NR NRNNN NAvian reproduction(Quail; MRID44755001)NN NAvian reproduction(Duck; MRID44761701)NN N1.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment..Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that this parameter is not routinely evaluated or is not applicable inthis assay. Changes in weight are absolute unless otherwiseindicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventralprostate (VP), Dorsal prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG),glans penis(GP). LW= Liver weightMETALAXYL FINALPage 17 of 512.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 byan X in this column. For details see Section IV. A3.The overt toxicity in the Tier 1 assays are presented inthis column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column.For details see Section IV. A4. Increased incidence of percentage of spermatogonia in the medium- and high-concentration males, and asynchronous gonad development at the highconcentration.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedMETALAXYL FINAL

Page 18 of 51 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal female and male rat assays, and the AMA (frog).

The potential for metalaxyl to interact with thyroid regulation is summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment.

No effects on the thyroid pathway (thyroid we ight, 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 me dium- and high-dose males. In the AMA there was no significant effect on medi an Nieuwkoop-Faber (NF) devel opmental 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 thyr oid gland histopathology were observed at any
treatment concentrations. Normalized hind-li mb length (HLL) was significantly decreased on both Day 7 ( ↓ 8.4%) and Day 21 ( ↓ 13%) at the high test concentrati on (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 concentr ation 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 th e study. While there was some ev idence 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 decr eased body weight) in the early stages of the study, the effects observed in this assay do not a ppear to be due to a potential interaction with the thyroid pathway.

In the mammalian Part 158 toxic ity studies, histopathological ex amination 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 thre e-generation rat reproductive study. In this study, changes in absolute or adjusted pituit ary weights were noted in both adults (F 2B females all dose groups) and offspring (F 3B high-dose males and mid- and high- dose females), but changes were
not consistent across l itters nor dose-dependent.METALAXYL FINAL

Page 19 of 51 Table 4. Thyroid Pathway for Metalaxyl

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

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

EDSP Tier 1 Assay

Female Pubertal Rat (MRID 48701301) N N N N N ↑ LW (L, M, H) 4 N

Male Pubertal Rat (MRID 48701301) N P 5 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) N 7

OSRI

Three-generation reproduction (Rat; MRID 00071600) N NE F 2B females: ↓

21, 21,

14% (L, M, H);

male: N X (F 2B, 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) NMETALAXYL FINAL

Page 20 of 51 Table 4. Thyroid Pathway for Metalaxyl

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

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

91-Day subchronic toxicity (Dog; MRID 00084111) NR N N N N

1. Key to responses: L=Low treatment, ML=Medium-lo w 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 obse rved 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 poten tial 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 reportedMETALAXYL FINAL

Page 21 of 51 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of metalaxyl to interact with endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

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

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses are mediated through an endocrine pathway, therefore nonendocrine toxic responses (incl uding but not limited to mortalit y or body weight changes) are
also considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence evaluation of the results of the 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 presen ce of overt toxicity. All effect s in endocrine se nsitive tissues should be evaluated in terms of primary intera ctions with the endocri ne system vs. secondary effects related to toxicity in non-endocrine or gans or overall disruptions in homeostasis” (USEPA, 2013).

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

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 and OSRI
studies are described below. Generally, one parameter ( i.e. , death or >10% decrease in mammalian body weight) was sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity ( e.g. , changes in organ weights or blood chemistry).METALAXYL FINAL

Page 22 of 51 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 studie s 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 (hi gh dose) on Day 1, adverse clinical signs were observed (decreased motor activity, ptosis, atax ia, lacrimation, hunched posture, intermittent body jerks, chromodacryorrhea and slight excess salivation), as well as reductions in food consumption. Therefore, the 300 mg/kg/day do se was reduced to 200 mg/kg/day on Study Days 2 and 3. After the dose reduction, food consum ption in this group increased and no other adverse clinical signs were observed.

In the FSTRA, while there were significant decr eases in male body weight at all treatments ( ↓ 7, 16 and 16% at 1.1, 10 and 104 mg/L, this effect wa s not dose-responsive and no other treatmentrelated mortalities or clinical signs were observed. Therefor e, this decrease in male body weights were not considered treatm ent-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 margin ally significantly (p =0.06) decreased 19% by Day 7 in this treatment, however, was 15% greate r than controls by day 21. Therefore, while there were indications of potentia l stress in the high treatment gr oup in the early stages of the AMA, these effects appeared to be transient a nd did not persist throughou t 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 s ubchronic (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). A lthough 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 ha d decreases in overall food consum ption 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 sign s including post-dose ataxia, loss of activity, body tremors and reduced righting reflex. A dditionally, twelve high-dose anim als (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 obser ved 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 hea d, face, ventral thorax, abdomen,METALAXYL FINAL

Page 23 of 51 back, anogenital area, and/or forelimbs. Add itional clinical signs obs erved 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 th e 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 pren atal development study, except for one high-dose doe (necropsy unremarkable). Maternal toxicity in the hi gh-dose animals (300 mg/ kg/day) consisted of little/none/soft stools. Body weight gain of does in th e high-dose group was negative (

− 75 g) and was decreased ( ↓ 192%) during the exposure period.

There were no treatment-related mortalities or clini cal signs of toxicity re ported 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 in crease in absolute and/or relative mean liver weight, often accompanied (if examined) by changes in liver enzymes ( e.g. , alkaline phosphatase, GGT, SGPT, SGOT, so rbitol dehydrogenase) or othe r liver-related clinical chemistries ( e.g. , bilirubin) and histopathological changes ( e.g. , hypertrophy). Incidences of fatty liver, or periacinar, v acuolation 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 equivo cal response in the Tier 1 in vitro ER binding assay (interactive in
one run and not interactive in the remaini ng two runs); however, it was negative for ER α agonism in the Tier 1 ERTA assay and negative for both ER α 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 assa ys. There was no convincing evid ence 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 appear s to potentially inter act with the estrogen pathway in fish. Several complementary effect s on females were observed in the FSTRA at the high test concentration of 104 mg a.i./L in cluding: effects on gonadal histology (decreased ovarian yolk synthesis), decrea sed plasma VTG concentration, decreased GSI, and finally cessation of fecundity (no eggs produced). Th erefore, while there were largely negative responses in the Tier 1 in vitro assays and in v ivo mammalian dataset, for the FSTRA there were complementary responses along several levels of biological organization for the females including alterations in enzyme activity (plasma VT G) up to the apical endpoint of fecundity. AsMETALAXYL FINAL

Page 24 of 51 such, significant effects of the reproductive capac ity of fish were observed in the FSTRA which suggests that metalaxyl may potentia lly 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 steriodogenesi s assays were negative) or in vivo mammalian Tier 1 ( i.e ., Hershberger and male pubertal assays were negative) an d Part 158 mammalian and avian studies (no androgen-related e ffects observed). In addition, metalaxyl was negative in two literature studies evaluating AR binding (Bauer et al ., 2002) and AR transc riptional 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 alte red 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 al ong with some indications of potential stress (lethargy and decreased body weight) in the earl y stages of the study, the effect s 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 me talaxyl demonstrates no convincing evidence of potential interaction with the estrogen or a ndrogen 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 thyroi d pathways in mammals.METALAXYL FINAL

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 th at there may be a potential for interaction with the estrogen and androgen pathway in fis h. Currently, the chronic fish NOAEC value is 9.1 mg a.i./L based on no effects observed in a fath ead 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 fe males, including a cessa tion in fecundity, at 104 mg a.i./L.

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

As there was no convincing evid ence of a potential thyroid interaction in amphibians, no additional testing is recommended.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 oncogen icity 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 Bi oresearch Laboratories, Inc. Project no. 1545. January 20, 1981. (MRID 00071598)

Carlson, M.B. (2012) A Pubertal Development a nd Thyroid Function Study of Metalaxyl by Oral Gavage in Peripubertal Male and Female Ra ts. Unpublished study conducted by Charles River
Laboratories Preclinical Serv ices, Pennsylvania; Horsham, PA. Laboratory Study No.: 20012408, July 17, 2012. (MRID 48701301)

Carlson, M.B. (2011) Oral (Gavage) Hershberg er Bioassay of Metalaxyl in Male Rats. Unpublished study conducted by Charles River Laborat ories, Preclinical Se rvices, 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 Ra t. Unpublished study conducted by Huntingdon Research, Huntingdon, Cambridgeshire, Engl and for Ciba-Geigy Corporation, Basle, Switzerland, May 23, 1980 Study). (MRID 00071600)

Finn, J.P. (1977) CGA-48988. 91-day dietary toxici ty study in beagle dogs. Unpublished study conducted by Hazleton Laborat ories Europe Ltd. Report No.: 653-380-4. (MRID 00084111)

Hoberman, A.M. (2011) Oral (Gavage) Uterotro phic Bioassay of Metalaxyl in Female Rats. Unpublished study conducted by Charles River Laborat ories, Preclinical Se rvices, 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 de rivative on androgen and estrogen receptor activities. Environ Health Perspect. 111:497–502.METALAXYL FINAL

Page 27 of 51 Kojima, H., Katsura, E., Takeuchi, S., Niiyama, K., and Kobayashi, K. (2004) Screening for estrogen and androgen receptor activi ties in 200 pesticides by in vitr o reporter gene assays using Chinese hamster ovary cells. Environ Health Perspect . 112(5):524-31.

Laughlin, K. (1984) Teratology study in ra bbits: metalaxyl: 382- 097. Unpublished study conducted by International Research and Develo pment 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, submitte d by Ciba-Geigy Corp., Greensboro, N.C.; CDL:244183-K; 244782). (MRID 00071308)

Lee, M.R. (2012) Metalaxyl- Amphibian Meta morphosis 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: Oncoge nicity in dietary administration to mice for two years. Unpublis hed study conducted by Life Science Research. Report no. 8C/01A008/442. June 3, 1981. (MRID 00103354)

Merkel, D. (2003) Metalaxyl: Subchronic Toxi city 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 Toxi city 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 In teractions for Metalaxyl. Metalaxyl EDSP Consortium. Project Identific ation. MEDP-13.(MRID No.49051402).

Pedersen, C.A. (1999) Avian repr oductive toxicity study with meta laxyl technical in bobwhite quail. Unpublished study conducted by Bio-Life Associates, Ltd. Pr oject No.: 164-003-07. (MRID 44755001)

Pedersen, C.A. (1999) Avian repr oductive toxicity study with me talaxyl technical in mallard ducks. Unpublished study conducted by Bio-Life Associates, Ltd. Pr oject No.: 164-004-08. (MRID 44761701)METALAXYL FINAL

Page 28 of 51 USEPA (2010) Metalaxyl - Report of the Endoc rine Disruptor Review Team; Test Order#: EDSP-113501-124- EDSP-113501-126. December 14, 2010. Office of Chemical Safety and
Pollution Prevention. U.S. Environmenta l Protection Agency, Washington, D.C.

USEPA. (2011) Endocrine Disruptor Screeni ng Program – Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

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

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

Wagner, H. (2011) Metalaxyl: H295R Steroi dogenesis Assay. Unpub lished study conducted by CeeTox, Kalamazoo, MI. Study ID: 9143V- 100326STER, December 7, 2011. (MRID 48617605)

Wilga, P.C. (2011) Metalaxyl: Human Reco mbinant 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 Recepto r 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 Re ceptor 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 Recepto r Transcriptional Ac tivation (Human Cell Line (HeLa-9903)) Final Report. Unpublished study conducted by CeeTox, Inc, Kalamazoo, MI. Laboratory Project ID: 9143V-100326E RTA, 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. Ma rch 2, 2012. (MRID 48685802)METALAXYL FINAL

Page 29 of 51 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-Fa ber Stage (NF) 51 (80/control and treatment group; 20/replicate) were exposed to nominal co ncentrations of 0 (neg ative 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 ma intained at 22 to 24°C and a pH of 7.8 to 8.2.

By test termination, mortality averaged 8.75% in the negative contro l and 7.5, 0, and 3.75% in the low, mid, and high treatment groups, respective ly. Spinal curvature was observed in between 15 and 50% of surviving tadpoles in the negative control and all treatm ent 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 significan tly decreased (p=0.06) by 19% at the high treatment level. Day 21 body weight was signifi cantly increased (p<0.05) by 15% in the high treatment group and was marginally significan tly 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 si gnificant (p>0.05) accelerat ion or delay of Day 7 or Day 21 median NF developmental stage at a ny treatment level. Alth ough the number of latestage tadpoles did not warrant a separate anal ysis (<20% observed), the reviewer noted a concentration-dependent reduction in the number of late-stage individual s (>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. Thyroi d gland histopathology observations included mild follicular cell hyperplasia and hypertrophy in the negative control a nd all treatment groups
and were not considered to be treatment-related.

Androgen Receptor (AR) Bindi ng 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 ofMETALAXYL FINAL

Page 30 of 51 increasing concentrations (10

− 10 to 10

− 3 M) of metalaxyl (98. 2% purity, Batch #20100540).
DMSO was used as the solvent vehicl e 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 wa s functioning with appropriate affinity for the radiolabeled reference androgen (R1881) prior to routinely conducting AR competitive binding experiments. Saturation binding data were not or iginally provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agenc y. The mean dissociation constant (K d ) for [ 3 H]- R1881 was 1.080±0.690 nM, and the estimated B max was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. Only one of the three K d values was within the range reported in the EPA valid ation program (0.685 to 1.57 nM), and all B max values were below the recommended range (7 to 16 fmo l/100 μg protein). Confidence in these numbers is low based on the large variation in K d estimates among runs.

In the competitive binding experiment, the estimated log IC 50 s 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 IC 50 for R1881 was low in Run 1 (

− 9.9 M). Due to a laboratory error,
the R1881 concentrations used in the Run 1 assa y 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 no t be determined for dexamethasone for Run 1. The deviation was considered minor. No preci pitation of the test compound was observed.

Metalaxyl had no significant effect on specific binding of the [ 3 H]-ligand (>75% bound) over the concentration range of 10

− 10 to 10

− 3 M in all runs; therefore, an IC 50 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 tritiate d androstenedione (([1 β

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

Page 31 of 51 15 minutes at 37 °C to assess the potential of metalaxyl to inhib it 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. Tritia ted water was quantified using liquid scintillation counting (LSC). Each run included a fu ll 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 c ontrol within a run was between 90 to 110% of the average full activity, with two exceptions likely due to an une venly 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 IC 50 , 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 IC 50 averaged

− 7.18 M and the Hill slope averaged

− 1.03 for 4-OH ASDN.

For metalaxyl, aromatase activity av eraged 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 indi cated 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 th e 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) Bindi ng Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding a ssay (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]-

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

− 10 to 10

− 3 M) of metalaxyl.
DMSO was used as the solvent vehi cle 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.METALAXYL FINAL

Page 32 of 51 A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and wa s functioning with appropriate affinity for the radio-labeled reference estrogen prior to conduc ting ER competitive binding experiments. Saturation binding data were not originally pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e 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 approxima tely 3- to 6-fold greater than recommended (160 to 320 μg versus 50±10 μg). The K d for [ 3 H]-17 β -estradiol was 0.331±0.061 nM and the
estimated B max was 74.55±3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The K d 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 IC 50 was

− 9.0 M for 17 β -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 gene rally met; however, octylt riethoxysilane displaced more than 25% of [ 3 H]-17 β -estradiol from the ER at the highest concentration tested (10

− 3 M) in Run 3, and bottom % binding and Hill slope were s lightly outside of the tolerance limits for 17 β

estradiol in Run 2.

Metalaxyl reduced the mean spec ific binding of the radiolabel ed ligand in a concentrationdependent manner in one of the three runs (decl ining 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 IC 50 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 (interacti ve in one run and not in teractive in the other two runs), metalaxyl is classified as Equi vocal in the Estrogen Receptor Binding Assay.

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

In an estrogen receptor transcript ional activation assay (MRID 48617603), hER α -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. Lu ciferase 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 17 α

methyltestosterone in the first r un prevented observation of its log PC 50 . The mean RPC Max forMETALAXYL FINAL

Page 33 of 51 metalaxyl was 0.5% for the first run and 1.1% for the second, and the associated PC Max was 10

− 5

M for the first run and 10

− 7 M for the second run.

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

methyltestosterone was lower than the expected values, indicating a decrea sed sensitivity of the assay to very weak agonists. Although the conditi ons of this assay were not optimal to detect very weak activity, metalaxyl responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 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 concentrati on possible based on cytotoxicity. Because the

RPC Max < PC 10 in both assay runs, metalaxyl was cons idered 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 (MRI D 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 w eeks old) were exposed to metalaxyl (100.4% purity) at nominal concentrati ons of 0 (negative control), 1.0, 10.0, and 100 mg a.i./L; meanmeasured concentrations were <LOQ (<0.066, ne gative control), 1.1, 10.0, and 104 mg a.i./L. The test system was maintained at 25 to 26 o 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 c oncentration-dependent reduc tion of 7 to 16% across all treatment levels compared to the negative c ontrol. 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/f emale/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 st atistically significan t (p=0.092). However upon visual observation of the data, this reduction in VTG was consideredto be biologically significant. Female gonadosomatic index (GSI) was significantly redu ced (p<0.05) by 23% at the high treatment levelMETALAXYL FINAL

Page 34 of 51 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 spermat ogonia 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. Ther e was an increase in th e incidence of atretic oocytes and granulomatous inflammation in female s 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 testos terone and 17ß-estradiol were not measured in this study.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger Assay (MRID 48617604) screeni ng for androgenic activity, metalaxyl (98.2% a.i., Batch/lot # 20100540) in 2.0% aqueous carb oxymethylcellulose (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-a ndrogenic activity, metalaxyl (98.2% a.i.) in 2.0% aqueous CMC was administered daily via oral gavage to gr oups of eight 54- or 60- day old, castrated male Sprague Dawley rats at dose le vels 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 ne gative control.

For both components of the assa y, body weights and food consump tion were determined daily. The animals were dosed for 10 consecutive days a nd terminated approximately 24 hours after the final dose administration. At necropsy, the five androgen-dependent tissu es, liver, and kidneys were collected and weighed.

All animals survived until scheduled termination. No adverse clinical si gns of toxicity were observed in the androgen agonist assay. In th e anti-androgen assay, trea tment related clinical signs of toxicity (urine-stain ed abdominal fur) were observe d in three rats from the 375 mg/kg/day group.METALAXYL FINAL

Page 35 of 51 In the androgen agonist assay, mean body wei ghts and body weight gains in the metalaxyl treatment groups were comparable to the vehicl e 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. Over all average food consumption for both metalaxyl treatment groups were comparable to the vehicl e controls; however, overall food consumption in the TP positive control group was increased (p ≤ 0.01) by 10%. No treatme nt-related effects were observed in any of the accessory sex organs or kidneys of metala xyl-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 preputia l 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 an i-bulbocavernosus (LABC); 645% in Cowper’s glands; and 71% in glans penis. The performa nce criteria indicated that this assay was performing as expected.

In the anti-androgen assay, mean body weights a nd overall body weight gains of the metalaxyl treatment groups were comparable to the vehi cle controls. Animals dosed with TP + FT (positive control) had mean body weights and over all mean body weight gains comparable to the
corn oil vehicle controls. Overall av erage 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 obse rved 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 we re less than the maximum guideline-specified %CV values, except for the % CV for glans peni s of the vehicle control (CV = 18%; maximum permissible CV = 17%).

The high dose of metalaxyl sel ected for this study (375 mg/kg/ day) was appropriate based on
data from earlier studies s howing clinical signs of sy stemic 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.METALAXYL FINAL

Page 36 of 51 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 gava ge 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 exam ined 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 ch emistry analyses, and total thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were determin ed using radioimmunoassa ys. 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 obser ved on survival, clinical signs, body weights, body weight gains, or gross pathology. No treatme nt-related histological lesions were noted in the thyroid or uterus, and the colloid area and folli cular cell height in the thyroids of the treated groups were generally similar to control. Ag e of attainment of VO, body weight at VO, and proportion/incidence of unopened vagi na in the treated groups were similar to control animals.
No treatment-related effect was observed on the m ean age of first vaginal estrus, mean cycle length, % cycling, % regularly cycling, and the cycle stat us at necropsy. Serum T 4 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 histor ical 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 we re similar to control values.

The high dose (350 mg/kg/day) was determined to be adequate based on generalized summaries from several ranging-finding studi es and findings in the previous ly 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 ma le Sprague-Dawley (Crl:CD(SD)) rats/dose group were treated daily via or al 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 dayMETALAXYL FINAL

Page 37 of 51 (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 (T 4 ), and thyroid stimulating hormone (TSH) levels were determin ed using radioimmunoassays. Adrenal, liver, pituitary, thyroid, and ur ogenital organs were weighed and examined macroscopically. The testes, epididymides , right kidney, and thyr oid were examined microscopically.

No adverse, treatment-related effects were obs erved on survival, clinical signs, body weights, body weight gains, organ weights, clinical chemistry, or gross pathology. No adverse histological lesion was observ ed in the testes, epididymides, or kidneys. Mean serum testosterone, T 4, and TSH levels in the treated groups we re 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 weight s or hormone concentrations.

Body weights, body weight gains, age of a ttainment of PPS, body weight at PPS, and proportion/incidence of separated pr epuce in the treated groups were similar to c ontrol 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 we ights 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 tr eated groups were similar to cont rol, except for a minor percent
increase in relative liver wei ght at 350 mg/kg/day (5.4%) compar ed to control (4.9%). This effect was minor, without a sign ificant effect on absolute or adjusted weight, and without corroborating gross pathology or c linical chemistry findings of he patotoxicity. Consequently,
this effect was considered incidental.

One 350 mg/kg/day male (#349) was found d ead on PND 40. Although the study authors
considered this death to be treatment rela ted, none of the data pr esented supported that
conclusion. The cause of death was not determine d, 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 wa s 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, lacrima tion, and hunched posture in female rats in the 300 mg/kg/day dose group. In addi tion, mean liver weights were si gnificantly incr eased in maleMETALAXYL FINAL

Page 38 of 51 rats in the Hershberger assay (MRID 48617604) at 375 mg/kg/day supporti ng 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 (M RID 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 μ M 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 assa y responded properly to positive control agents at two concentration levels; positiv e controls included a known i nhibitor (prochloraz) and known inducer (forskolin) of estradiol and test osterone production.

All guideline acceptability recomm endations and requirements we re met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptabl e reproducibility (low coefficients of variation, %CV) , and appropriate induction and i nhibition with pos itive controls.

There were no significant changes in testosterone production in R un 2 at any concentration tested
or in Run 4 at ≤ 10 μ M of metalaxyl, although there was a slight decrease in testosterone production (0.88-fold, p<0.05) at 100 μ M. 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

μ M, but the increase at 100 μ M 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 μ M). Changes in testoster one and estradiol were not reproducible or concentration dependent.

Based on the hormone responses in each of the th ree independent runs, EPTC treatment did not result in statistically signifi cant 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 carboxym ethylcellulose (CMC)METALAXYL FINAL

Page 39 of 51 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-nata l days (PND) 57-59 or 63-65. A positive control group was tr eated with a daily dose of 17 α -ethynyl estradiol (EE) at 5

μ g/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 administ ration to determine wet and blotted uterine, and liver weights.

All animals survived until sche duled termination. Following admi nistration of metalaxyl at 300 mg/kg/day on Day 1, adverse clin ical signs were observed (dec reased 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 Stud y 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 over all (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 rela tive liver weights in the 300/200 mg/kg/day group were increased (p ≤ 0.05) compared to vehicle controls ( ↑ 24-25%); relative liver we ights 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 th e vehicle controls. Ab solute 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.METALAXYL FINAL

Page 40 of 51 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral 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 bind ing was calculated as the difference of total binding and non-specific bi nding 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 (K i ) of each compound to calculate th e relative binding affinity (RBA=K i DHT×100 / K i metalaxyl). Statistical an alyses were not conducted.

No binding was observed for metalaxyl at its maximum soluble concentr ation (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 (hER α and hER β ) and one human androgen receptor (hAR) in transactivati on assays with Chinese hamster ovary cells (CHO-K1 cells) was investigated. The human ER (hER α ) and AR (hAR) expression vectors (pcDNAER α and pZeoSV2AR, respectively) were construc ted as described previously (Kojima et al. , 2003). The
hER β expression vector was cons tructed by cloning the hER β cDNA with reverse transcriptasepolymerase chain reaction from human placental R NA. The sequence was verified and inserted into the mammalian expression ve ctor pcDNA3.1Zeo(-) creating pcDNAER β . The construction of the estrogen or androgen responsive elements containing re porter 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 inte rnal control for transfection efficiency.

For detection of hER α or hER β activity, cells were tr ansfected with pcDNAER α or pcDNAER β

, pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. For measurement of antagonist ic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M 17 β -estradiol (E 2 , >97% purity) or 10

− 10 MMETALAXYL FINAL

Page 41 of 51 5 α -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 (REC 20 ), the concentration of the compound demonstrating 20% of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β , and AR, respectively. Antagonistic activ ities were evaluated similarly by the relative activity expressed as 20% relative inhibitory concentration (RIC 20 ), the concentration of the compound showing 20% inhibition of the activity of against ER α , ER β , and AR, respectively.

Metalaxyl was found to be negative for agonist or antagonis t activity against ER α and ER β , and negative for agonist or antagoni st 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 th ese tissues as well as the prostate, seminal vesicles, uterus, vagina, and parathryoid/thyroid (control a nd high-dose groups only examined for all organs; liver examined for all groups).

No adverse, treatment-related effects were obs erved on mortality, clinical signs, neurological evaluations, body weight, food consumption, hemato logy, 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 d ecreased 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) group s. Hepatic enzyme
concentrations were increased in females at the medium- and high-doses for alanine aminotransferase (69 and 67%, respectively) and sorbitol dehydroge nase (47 and 42%, respectively). In the high-dose group, increases occurred in absolute liver weight (37%), and liver weight relative to body wei ght (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 we ight relative to body weight (22 and 33%, respectively) and liver weight relative to brain weight (42 an d 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 th e observed decreases in bi lirubin concentrationsMETALAXYL FINAL

Page 42 of 51 and hepatocyte hypertrophy were considered to be adaptive due to xeno biotic 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/fe males, 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/se x/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, ova ries, and adrenals. Gross and microscopic pathological examinations were conducted on th ese tissues as well as the prostate, seminal vesicles, uterus, mammary gland, pituitary, and parathyroid/thyroid (c ontrol and high-dose groups only examined for all organs, liver examin ed for all groups, and kidneys examined in all males).

No effects of treatment were observed on mortalit y, clinical signs, or gr oss pathology. In the high-dose females, body weight was decreased (p< 0.01) by 13-18%, resulti ng 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 tota l 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 wa s also decreased (p<0.01 or p<0.05) in males during Weeks 1, 8, and 11 of the study, re sulting 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- a nd high-dose females (p<0.05, ↓ 24- 39%) were correlated with increased (p< 0.01 and p<0.001) liver we ights (both sexes; ↑ 24-75%) and hepatocyte hypertrophy (females; all dose groups ). The decreased bilir ubin 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 weightsMETALAXYL FINAL

Page 43 of 51 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/da y). 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 (negat ive 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 th e 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 an imals were not reported). Serum alkaline phosphatase concentrations were normal in the recovery animals at Week 16. Macroscopic findings were not treatment relate d. There were no dose-related tre nds 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, hemato logy, 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 wa s administered by gavage to COBS® CD® rats (27 or 38/dose) at concentra tions 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/da y dose also yielded exce ssive toxicity. TheMETALAXYL FINAL

Page 44 of 51 dose was reduced further to 400 mg/kg/day; surviving animals were sacrificed and additional
females were mated and the number of females wa s 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 tissu e alterations by the Wilson techni que. The remaining fetuses in
each litter were examined for skeletal al terations 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 clin ical signs were observe d: 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 obs erved 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 gain s 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 ove rall study interval ( ↓ 10%, GDs 0-20).

When compared to concurrent control, no tr eatment-related changes in body weight, food consumption, gross pathology, or reproductive pa rameters (pregnancy rate, number of corpora lutea, mean number of implanta tions/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, base d on clinical signs of toxicity including postdose convulsions. The maternal toxicity NOAEL is 50 mg/kg/day.

There were no treatment-related developmental exte rnal 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 c ontrol ranges: 25 presacral verteb rae; fewer than normal (13 full pair) ribs; reduced ossification of the skull; a nd unossified pubic bones. The developmental toxicity LOAEL is 400 mg/kg/day based on increas ed incidence of skelet al variations. The developmental toxicity NOAEL is 250 mg/kg/day.

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

In a developmental toxicity study in rabbits ( MRID 00144372, 1984 ), metalaxyl (96.7% purity) was administered to artificially-inseminated Dutc h Belted rabbits (18/dose group) by gavage at doses of 0, 30, 150, and 300 mg/kg/day from GD s 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 ex ternal observations, sacrificed, examined forMETALAXYL FINAL

Page 45 of 51 visceral abnormalities, then eviscerated and exam ined 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 obs erved in the high-dose animals (7/18 vs. 2-3/18 in the control a nd 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 hi gh-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 nu mber of implantations/l itter, 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 genera tions, 12 males and 24 females/dose for the third generation) at concentrati ons 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 F 1A litters. The P animals were mated 3 times. The F 1A pups were sacrificed at weaning; the F 1B generation was used to select p ups (25/sex/dose) to produce the

F 2 generations; the F 1C generation was sacrificed in utero at gestation day (GD) 20 and examined for developmental effects and the dams ex amined for reproductive effects. The F 1B pups were exposed to the same dosage as their parent s on postnatal day (PND) 21 and exposure was continued for at least 90 days prior to mating to produce the F 2A litters. The F 1B animals were
mated 3 times. The F 2A pups were sacrificed at weaning; the F 2B litters were used to provide pups for the F 3 generation; the F 2C litters were sacrificed at weaning. In addition, ten

F 1B litters/dose were sacrificed in utero at GD 20 and examined for developmental effects and the dams examined for reproductive effects. At weaning, 12 F 2B rats/sex/dose were maintained on their respective diets for at l east 90 days, then given a deta iled necropsy and subjected to organ weight analysis. Another 12 ma les and 24 females/sex/dose from the F 2B litters were mated at 90 days of age to produce the F 3 litters. The F 3A litters were sacrificed at PND 21. The

F 3B 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 liv er, kidneys, testes, ovaries,METALAXYL FINAL

Page 46 of 51 uterus, pituitary, adrenals, and thyroid in F 2B and F 3B rats. Gross and microscopic pathological
examinations were conducted on these tissues as well as the prostate, seminal vesicles, and mammary gland in F 2B and F 3B rats.

There was no evidence of treatment-related ch anges in clinical signs, mortality, body weight, body weight gains, water consumption, food consum ption, or food efficiency observed in the P,

F 1 , or F 2 adults. There were no treatment-related gro ss pathological findings in adult rats. There
were no differences of toxico logical concern in P or F 1 adult organ weights. An increase (p<0.05) in adjusted (to body weight) liver weight compared to control occurred in the high-dose

F 2B females ( ↑ 11%). This finding was considered treatm ent-related and of toxicological concern due to the observation of hepatomegaly in fema les 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 F 2B females ( ↓ 21, 21, and 14%, respectively); these ch anges were not considered to be toxicologically relevant because the decrease s were not dose dependent (no histopathological examinations were conducted). The parental sy stemic toxicity LOAEL in females is 1250 ppm (approximately equivalent to 62.5 mg/kg/day) based on increased rela tive (to body) liver weights, and the NOAEL in females is 250 ppm (a pproximately 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 equiva lent 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, l actation index, parturition, or fetal sex ratio of the P, F 1 , or F 2 adults. No treatment-related changes were seen in number of corpora lutea, mean number of implantations/litt er, or fetuses in the litter s from the third mating of F 1 adults and second mating of F 2 adults sacrificed on GD 20. Sexual maturation was not measured. A reproductive toxicity LOAEL is not determ ined; the reproductive toxicity NOAEL is ≥ 1250 ppm (approximately equivale nt 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 F 1 , F 2 , or F 3 litters. No treatment-related changes in absolute or relative weights of the te stes, seminal vesicle, ovaries, uterus, or adrenal gland were seen in F 2B or F 3B offspring. Adjusted pituitary we ights were decreased (p<0.05) in the high-dose F 3B males ( ↓ 40%) and absolute pituitary weights were increased (p<0.05) in the mid- and high-dose F 3B females ( ↑ 50% and 25%, respectively). Thes e findings were considered not of toxicological concern due to the lack of histological findi ngs in both sexes and lack of dose-dependence in females. There were no other differences of toxicolo gical concern in organ weights in the F 3B pups. There was an increase in the de gree of hepatic fat deposition in the high-dose F 3B female pups (high-dose: trace - 1/10, minimal - 6/10, moderate - 3/10; vs control: trace - 2/10, minimal - 2/10). There were no othe r findings of toxicological concern in the F 1 , F 2 , or F 3 pups. The offspring systemic LOAEL is 1250 ppm (approximately equivalent toMETALAXYL FINAL

Page 47 of 51 62.5 mg/kg/day) based on histopath ological changes in the livers of female pups. The offspring systemic NOAEL is 250 ppm (approximate ly equivalent to 12.5 mg/kg/day).

There were no differences of toxicological concern in feta l 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/Carcinogenici ty in Rodents (Rat; OCSPP 870.4300)

In a chronic toxicity/carci nogenicity study in rats ( MRID 00098481, 1980 ), metalaxyl (93% and 94.6% purity) was administered to SD rats (80/sex/dos e) in the diet at nomi nal 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 microsco pic pathological examin ations 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, hemato logy, 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 increas ed (NS and p<0.01) in high-dose males at Week 105 ( ↑ 11 and 16%, respectively). No tr eatment-related changes were se en in absolute or relative weights of the testes or epididymides. The in cidence 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) compar ed to control (7/80 M and 18/80 F). No treatment-related histopathological lesions were seen in any ot her examined tissues. Although
there is no increased risk of cancer in this st udy, 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 Swi ss mice (60/sex/dose group) in the diet for 104 weeks at 0, 50,METALAXYL FINAL

Page 48 of 51 250, 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). Orga n weights were not determined in this study. The tissues subjected to histological exam ination included liver, kidneys, te stes, epididymides, prostate, seminal vesicles, ovary, mammary gland, pitu itary, thyroid/parat hyroids, and uterus.

There were no effects on mortal ity, 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 c ontrol. No treatment -related histopathological lesions were seen in the any other examined tissues. The re latively high incidence of fatty vacuolation of hepatocytes indicat es that the mice might not have tolerated a higher dose without increasing mortality and or body we ight 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 fa tty vacuolation). The NOAEL is 250 ppm (male 24.85 mg/kg/day, female 29.59 mg/kg/day).

Chronic Oral Toxicity in No n-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 th e diet at nominal concen trations of 0, 50, 250, and 1000 ppm (0, 1.57, 7.80, and 30.63 mg/kg/day for ma les; 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 te rmination of treatment. Organs that were weighed included the liver, kidneys , testes, ovaries, pituitary, adre nals, 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 di d not affect body weight, body weight gain, food consumption, hematology, urinalysis, ophthalmol ogic 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 treatm ent-related histopathologi cal 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 cont rol males vs. terminal bodyMETALAXYL FINAL

Page 49 of 51 weight). Female dogs in the control and hi gh-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, fe male 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 f eed at nominal concentrations of 0 (1% acetone), 100, 300, or 900 ppm (corre cted for 88.7% purity) for 22 weeks (10 weeks prior to the start of egg produc tion 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 thr ough Week 8 and 17 hours/da y 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 stud y initiation, biweekly through Week 8, and at study termination. Food consumption in each replicate was measured biweekly throughout the pre-treatm ent and treatment periods of th e 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 t oxicity or treatment-related re ductions 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 te st concentration compared to control.

In another avian reproduction study ( MRID 44761701, 1999 ), Mallard ducks ( Anas platyrhynchos ; age not reported) were administered me talaxyl (88.7% purity) in the feed at nominal concentrations of 0 (1 % acetone), 100, 300, or 900 ppm fo r 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 progres sive 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 stud y initiation, biweekly through Week 8, and at study termination. Food consumption in each replicate was measured biweekly throughout the pre-treatm ent and treatment periods of th e 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.METALAXYL FINAL

Page 50 of 51 There were no overt signs of toxi city or treatment-related morta lities at any test concentration,
and there were no reductions in adult body weight or feed consump tion. There was a decrease in the percentage of normal hatchlings of viable embryos in the low-concen tration 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 para meter. 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 concentratio n. 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-cy cle 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 hatchi ng, forty fry were impartially selected from each aquarium to begin the 30-day exposure period. Fry were coun ted twice weekly beyond Day 6 post-hatch. At Day 30 post-hatch, all fry were anaesthetized an d 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.METALAXYL FINAL

Page 51 of 51 APPENDIX 3: References Not Utiliz ed in the Metalaxyl WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt information (OSRI) was provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for Metalaxyl but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only data on invertebrates.

Ciba-Geigy, Ltd. (1984). Expanded report on a se gment II reproduction study in the rabbit with CGA48988 technical. Ciba-Geigy, Ltd. St udy 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, Lt d. Study No. 227716. MRID 00161405. Unpublished.

Hayes, T.B., Case, P., Chui, S., Chung, O., H aeffele, C., Haston, K., Lee, M., Mai, V.P., Marjuoa, Y., Parker, J., and Tsui, M. (2006). Pesticide mixtures, e ndocrine disruption, and amphibian declines: Are we underestimating the impact? Environmental Health Perspectives

. 114:Supp 1 (40-5).