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

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### MEMORANDUM

Date: 6/29/2015

SUBJECT: Metalaxyl: Data Evaluation Records (DERs) for EDSP Tier 1 Assays

PC Code: 113501 

### Decision No.: 461740, 468748 

### Petition No.: NA Risk Assessment Type: NA TXR 

### No.: 0054219 

### DP Barcode: D399227, D404681 

### Registration No.: NA Regulatory Action: NA CaseNo.: NA

MRID 

### No.: See Table 

### CAS No.: 57837-19-1 

### 40CFR: NA

FROM: Greg Akerman, Ph.D. 

# A _Ab---- Immediate Office ;

# ~ Health Effects Division (7509P)



### THROUGH: Jess Rowland

### ~ Deputy Director Health Effects Division



### TO: Jolene Trujillo Biologist/Chemical Review Manager Risk Management and Implementation Branch V Pesticide Re-evaluation Division (7505P)



### I. ACTION REQUESTED Ver.Apr. 2010



### The Pesticide Re-evaluatipn Division (PRD) of OPP has requested that the Health Effects Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays submitted 

### in response to the agency's Test Order for metalaxyl: Test Order# CON-113501-18.

Page 1 of2Page 2 of 2 

### II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for metalaxyl.

III. MRID Table

Chemical: Metalaxyl PC Code: 113501 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48685801 890.1150 Androgen Receptor Binding (Rat Prostate) 48617601 890.1200 Aromatase Assay (Human Recombinant) 48617602 890.1250 Estrogen Receptor Binding 48617607 890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) 48617603

890.1350 Fish Short-Term Reproduction 48685802 890.1400 Hershberger (Rat) 48617604 890.1450 Female Pubertal (Rat) 48701301 890.1500 Male Pubertal (Rat) 48701301 890.1550 Steroidogenesis (Human Cell Line – H295R) 48617605 890.1600 Uterotrophic (Rat) 48617606Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 1 of 67

DER Template Version: 22 September 2011 Data Requirement: EPA DP Barcode 401220

OECD Data Point 231

EPA MRID 48685801

EPA Guideline 890.1100 Amphibian Metamorphosis Assay (Frog)

Test Material: Metalaxyl Purity (%): 98.2%
Common Name Metalaxyl
Chemical Name IUPAC methyl N -(methoxyacetyl)- N -2,6-xylyl)-DL-alaninate; methyl 2-{[2,6-dimethylphenyl)methoxyacetyl]amino}propionate
CAS Name N-(2,6-dimethylphenyl)-N- (methyoxyacetyl)- alanine methyl ester

CAS No. 57837-19-1 Synonyms Metalaxyl Technical, N-(2,6-dimethylphenyl)-N-(methyoxyacetyl)- alanine methyl ester [CAS# 57837-19-1]
methyl N-((methoxyacetyl)-N-2,6-xylyl)-DL-alaninate methyl 2-([2,6-(dimethylphenyl)methoxyacetyl]amino)propionate
2-[(2,6-dimethylphenyl)-(2-methoxy-1-oxoethyl) imino]propanoic acid

methyl ester N-(2,6-dimethylphenyl)-N-(mehtoxyacetyl)-DL-alanine methyl ester

EPA PC Code 113501

Primary Reviewer: John Marton Signature:
Staff Scientist, Cambridge Environmental, Inc. Date: 10/16/2012

Secondary Reviewer: Teri S. Myers Signature:
Senior Scientist, Cambridge Environmental, Inc. Date: 12/20/2012

Primary and Final Additional Reviewer: Robin Sternberg Signature:
Wildlife Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 05/17/2013, 05/27/2015 Digitally signed by ROBIN STERNBERG DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN STERNBERG, dnQualifier=0000039126 Date: 2015.06.01 16:01:22 -04'00'Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 2 of 67

DER Template Version: 22 September 2011 Additional Reviewer: Anita Ullagaddi Signature: No longer with EPA
Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 01/11/2013

Additional Reviewer: Amy Blankinship Signature:
Biologist, USEPA/OCSPP/OPP/EFED/ERB6 Date:

Date Evaluation Completed: 05/27/2015

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

laevis ). Unpublished study performed by Smithers Viscient, Wareham, Massachusetts. Laboratory report number

13694.6146. Study sponsored by Metalaxyl EDSP Consortium, Burr Ridge, Illinois. Study completed February

29, 2012.

The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven
screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the

strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within
the assay, where available, and redundancy across the battery. Thus, the results of each individual assay
should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has
the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay
results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

Disclaimer: The guideline recommendations in this DER template are offered as a general reference to
aid in preparation of the DER. The purpose of these recommendations is not to serve as substitute for
the Test Guidelines, nor to provide any guidance on how the study should be conducted. 

## AMY BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.05 08:57:02 -04'00'Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 3 of 67

DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay 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 NieuwkoopFaber Stage (NF) 51 (80/control and treatment group; 20/replicate) were exposed to nominal concentrations
of 0 (negative control), 1.0, 10, and 100 mg a.i./L; mean-measured concentrations were <0.069 (<LOQ;
negative control), 0.98, 10, and 95 mg a.i./L. The test system was maintained at 22 to 24°C and a pH of

7.8 to 8.2.

By test termination, mortality averaged 8.75% in the negative control and 7.5, 0, and 3.75% in the low, mid,
and high treatment groups, respectively. Spinal curvature was observed in between 15 and 50% of surviving
tadpoles in the negative control and all treatment groups and was not considered to be treatment related. The
clinical sign of lethargy in all tadpoles of the high treatment group was observed on the first two days of

exposure.

Tadpoles at NF stage > 60 were not included in analyses of growth and hind-limb length (HLL). Day 7 body
weight was marginally significantly decreased (p=0.06) by 19% at the high treatment level. Day 21 body weight
was significantly increased (p<0.05) by 15% in the high treatment group and was marginally significantly
decreased (p=0.057) by 9% in the low treatment group.

Metalaxyl significantly decreased (P<0.05) Day 7 and Day 21 normalized (for snout-vent length) HLL at the
high treatment level. There was no significant (p>0.05) acceleration or delay of Day 7 or Day 21 median NF

developmental stage at any treatment level. Although the number of late-stage tadpoles did not warrant a separate analysis (<20% observed), the reviewer noted a concentration-dependent reduction in the number
of late-stage individuals (>NF 60); there were 16 individuals staged NF 61 and 62 in the negative control, 13

in the low treatment group, 11 in the mid treatment group, and only 3 NF 61 tadpoles in the high treatment
group. No asynchronous development was observed. Thyroid gland histopathology observations included mild
follicular cell hyperplasia and hypertrophy in the negative control and all treatment groups and were not

considered to be treatment-related.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 4 of 67

DER Template Version: 22 September 2011 The following performance criteria were not met: (1) negative control Replicate D and 0.98 mg a.i./L

Replicate B both had a total of 3 mortalities by Day 7 which is greater than the guideline criterion of no more than two mortalities per replicate; and (2) temperature of the test solutions (22-24°C) exceeded the acceptable
limits of 22±1°C. On Day 7, the maximum temperature was 23.6°C. Inter-replicate and inter-treatment
differentials did not exceed 0.5 °C for any given day. However, these deviations did not impact the interpretation
of the study.

The assay satisfies the EDSP Tier 1 Test Order requirements for an Amphibian Metamorphosis Assay (OCSPP
Guideline 890.1100).

Results Synopsis:

Test organism NF stage at test initiation: 51 Test organism total length at test initiation (optional): Not reported

Test type: Flow-throughData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 5 of 67

DER Template Version: 22 September 2011 Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects 1,2 in the Amphibian
Metamorphosis Assay (AMA) with Metalaxyl.

Treatment

(mg a.i./L)

[mean- measured] NF Developmental
Stage Hind Limb
Length 3 Asynchronous
Development Thyroid Gross

and
Histopathology

Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21

0.98 No No No No No No No

10 No No No No No No No

95 No No Yes Yes No No No

1 A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless

otherwise specified.

2 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of
the DER. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a boardcertified pathologist.

3 Hind-limb length is normalized to snout-vent length (SVL).Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 6 of 67

DER Template Version: 22 September 2011 I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following the guidelines outlined in the U.S.
Environmental Protection Agency’s Endocrine Disruptor Screening Program
Test Guidelines 890.1100, Amphibian Metamorphosis (Frog); and OECD

Guideline for Testing of Chemicals No. 231, Amphibian Metamorphosis
Assay. The following deviations were noted:

1. Details on parental feeding were not provided.

2. Negative Control Replicate D and 0.98 mg a.i./L Replicate B both had a total of 3 mortalities by
Day 7 which is greater than the criteria of no more than two mortalities per replicate. 3. Temperature of the test solutions (22-24°C) exceeded the recommended values (22±1°C). On
Day 7, the maximum temperature was 23.6°C. Inter-replicate and inter-treatment differentials did
not exceed 0.5 oC for any given day
4. Results from the periodic screening analysis of the dilution water were not provided. However, the
study author reported that no pesticides, PCBs, or toxic metals were detected at concentrations deemed harmful in accordance with ASTM (2005) standard practices.

These deviations do not impact the interpretation of the study.

Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance

statements were provided. This study was conducted in compliance with all
pertinent OECD and U.S. EPA Good Laboratory Practice regulations with the
following exception: routine food and water contaminant screening analyses
were conducted using standard U.S. EPA procedures by GeoLabs, Inc.,
Braintree, Massachusetts and are considered facility records under Smithers
Viscient’s Standard Operating Procedures.

A. Test Material MetalaxylData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

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DER Template Version: 22 September 2011 Description: Solid

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow or
Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : 20100540 (Batch No.)

Purity: 98.2%
For the first 14 days of exposure, nominal concentrations of exposure and stock solution were calculated based on the initial purity (100%). From Day

15 through test termination, nominal concentrations and stock solutions were calculated based on the updated purity (98.2%), recertified on exposure day
14 (20 July 2011).

Impurities: None reported

Stability of Compound: Stable. Mean-measured concentrations yielded recoveries of 95 to 100% of
nominal with coefficients of variation (CV) of 2.6 to 4.3%.

Storage Conditions of

Test Chemicals: Stored at room temperature in the dark.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page8of67 DER Template Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:African clawed frog EPA recommends African clawed frog (Xenopus laevis). Western [Africa] clawed frog Silurana (Xenopus) tropicalis may be used as an alternate species1 ; however, a
list of all of th e necessary protocol deviations to accommodate this species is recommended for inclusion in the study report. The guideline recommends that the performance criteria used to support the reliability of the test be identified.Species scientific name: Xenopus laevisSpecies strain (if stated):Not reported1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the EndocrineDisruptor Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention(OCSPP), Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page9of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained as inhouse stock?YesBrood stock was originally obtained fromNasco, Fort Atkinson, Wisconsin. EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimationconditions same as definitivetest?YesAcclimation period for parentalfrogs (if applicable):>14 daysDetails on parental feeding:Not specifiedDetails on parental health:The brood stock didnot show any sign ofsickness, disease,injuries, orabnormalities from theday of receipt to theday of pre-exposureinitiation.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page10of67 DER Template Version: 22 September 2011Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest single spawn?Yes EPA and OECD recommend that the best 2 – 3 individual spawns, with a minimum of 1500 larvae/spawn, be evaluated to identify the best single spawn, and that the larvae selected for testing originate from the best single spawn (i.e.,the spawns are not comixed)Number of spawns evaluated (ifapplicable):3Number of eggs sampled perspawn:At least 900Embryos were assessed approximately5 days post-fertilization. Criteria wereembryo survival and the quality of theembryos.NF stage at test initiation51 EPA recommends that the definitive study be initiated with larvae at Nieuwkoop – Faber (NF) developmental stage 51 ( 17 days post-fertilization).Age at test initiation:13 days postfertilization (dpf)Mean total length at testinitiation (if reported):Not reportedRange of total length at testinitiation (if reported):Not reportedData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page11of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas the optional size selectionmethod used?NoDetails on larval selection:Larvae were selectedbased on survival andoverall quality withrespect to the threespawns (~1500eggs/spawn).Loading rate (rearing density):10 larvae/L EPA recommends that rearing density (loading rate) not exceed approximately 10 larvae/L culturing system for flow - through systems or 4 tadpoles/L in static - renewal exposure systems.Type of food:Xenopus ExpressTadpole FoodXenopus Express is a commerciallyavailable diet that is suitable for normalgrowth and development of X. laevistadpoles. EPA recommends Sera Micron ® throughout

pre - exposure (after NF stage 45/46) and during the entire 21 - d definitive study. If another diet is used, the study report shouldData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page12of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSource of food:Xenopus Express,Brooksville, Florida provide analysis of iodide content and potential contaminants, and the diet should demonstrate equal performance to Sera

Micron ® .Iodide concentration in diet (ifknown):Not reportedFrequency of feeding:2 times/day EPA recommends that feeding occur at least twice per day.Details on feeding regime:Feeding regimeconformed to guidelinerecommendations. It is recommended that food rations during

the pre - exposure period be increased along with larval growth to approximately 30 mg/larva/day by test initiation. EPA and OECD recommend that food rations increase from 30 mg/larva/day at test initiation (Study Day 0 -4) to 80 mg/larva/day in the last week of the test (Study Day 15-21).Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page13of67 DER Template Version: 22 September 2011C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-throughEPA recommends the use of a flowthrough system.Type of flow-through dilutionsystem (if applicable):intermittent flowproportional diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):~27.1 mL/minThe flow rate was calculated based on avolume of 6.5 L and 6 volumereplacements per day. This rate allowedcomplete volume replacement every 4hours. Recommended flow-through rate is 25 mL/min (complete volume replacement ca. every 2.7 hrs).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page14of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems (ifapplicable):Pumps delivered thestock solution into thediluter’s chemical mixingchamber. Theappropriateconcentrations weredelivered to the testvessels using flowsplitting cells.Flow splitting accuracy of the diluter cellswas within ±5% of the nominal value. Theexposure system was operating properlyfor 11 days prior to test initiation. All testchambers were brushed and siphoned dailyto remove detritus and uneaten food duringthe test; cleaning equipment werededicated to treatment and controls toavoid cross contamination.Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2)

Aeration i s not recommended for

mixing;3) A demonstration that the test solution is completely mixed before introduced into the test sys tem is recommended; 4) The recommended flow splitting accuracy is within 10%.Renewal period for staticrenewal (if applicable):NA If static renewal is used, EPA recommends 24 -hr renewal; renewal period is recommended not to exceed 72

hours.Aeration?No EPA recommends maintaining dissolved oxygen concentrations >40% air saturation ( >3.5 mg/L) . Aeration may be maintained through bubblers. It isData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page15of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations recommended to set bubblers at levels that do not cause stress on the tadpoles.Source of dilution water:Dilution water wasFETAX solution,prepared by fortifyinglaboratory well waterbased on the formulationpresented in “StandardGuide for Conducting theFrog EmbryoTeratogenesis AssayXenopus (FETAX).Laboratory well water originated from anon-site 100 m bedrock well. Water waspumped to a concrete reservoir where itwas supplemented on demand withuntreated, de-chlorinated, Town ofWareham well water. The water wasaerated prior to use to ensure properequilibration of dissolved gases. EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants, including known substrates of the iodine transporter of the thyroid gland (e.g., fluoride, chlorate, perchlorate). OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?Yes. See Reviewer’sCommentsData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page16of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsIodide supplementation inwater?No If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 ս g/L, iodide supplementation is recommended. This supplementation is in addition to the recommended dietary source of iodide (e.g.. in Sera Micron).Test vessel type/materials:Each aquarium wasconstructed of glass,silicone sealant, stainlesssteel, and Teflon®.EPA and OECD recommend that watercontact portions of the system not compromise the study (e.g., all g lass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:30 x 14.5 x 20 cmFill volume:6.5 LVolume was maintained by a 14.5 cm highside drain.Additional details on exposuresystem:NAData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page17of67 DER Template Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsHardness (mg/L as CaCO3)150 160 NA weekly EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH7.48.37.79Daily in alternatingreplicate testvesselsEPA recommends pH 7.5 ± 1, interreplicate and inter - treatment differentials should not exceed 0.5.Dissolved oxygen (mg/L)3.8 7.7 5.84Daily in alternatingreplicate testvessels EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration

>3.5 mg/L (>40% air saturation).Temperature222422.2Daily in alternatingreplicate testvessels andcontinuously in onecontrol replicate EPA recommends temperature 22±1 o C;

inter -replicate and inter-treatment differentials should not exceed 0.5 o C.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page18of67 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsIodide7bi-annual samplesof well water EPA recommends aquatic iodide range

0.5 – 10 ս g/L (supplemental iodide should not exceed 2 ս g/L).AmmoniaNot Reported General recommendations for frequency of measurements: EPA recommends that water quality parameters be measured in a control and at one test item concentration at least weekly. In static renewal systems, water quality parameters, including ammonia, should be measured just prior to renewa l. In addition, EPA recommends that DO be measured at each concentration at least weekly and that temperature be measured continuously. OECD recommends that DO and temperature be measured at least weekly and that pHFluoride<0.50bi-annual samplesof well waterPerchlorateNot ReportedChlorateNot ReportedAlkalinity (mg/L as CaCO3)Conductivity (սmhos/cm)741700801900Not ReportedNot ReportedMeasured inreplicate Aof thecontrol, low, andhigh testconcentrations onDay 0 and inalternating replicatesweekly thereafter.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page19of67 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline Recommendations and hardness be measured at least at the beginning and end of the test.D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?YesIf yes, what was the method fordetermining the highest testconcentration in the rangefinder?LC50value of >100mg/L for rainbow trout,carp, and bluegillsunfish EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity

(e.g., <10% mortality), whichever concentration is lowest.Species: Xenopus laevisData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page20of67 DER Template Version: 22 September 2011Life stage:NF 51Test duration:4 daysAdditional details:The test wasconducted under staticrenewal conditions withnominal concentrationsof 6.3, 13, 25, 50, and100 mg/L.No concentration results in more50%mortality and the LC50was thereforeestimated to be >100 mg/L.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in thedefinitive test:Estimated LC50fromthe range-finding study(>100 mg/L). EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicityData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page21of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations (e.g., <10% mortality), whichever concentration is lowest.Reference study citation (ifapplicable):NASeparation of testconcentrations:0.10 EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean-measuredData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page22of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsLimit of quantification (LOQ):0.061-0.069 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not ReportedFrequency of measurement:Samples were collectedfor analysis on Days 0,7, 13, and 20. It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every week thereafter.Number of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA; a solvent controlwas not used EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per testitem treatment level:4 EPA recommends 4 replicates.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page23of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of larvae per treatmentat test initiation:80Was a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA EPA recommends that the solvent not exceed 0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):NA3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review.Aquatic Toxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Serieson Testing and Assessment. No. 23. Paris, France.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page24of67 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12
(light:dark).Light intensity at water’ssurface:0.720 to 0.920 Klux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:NADuring the solubility trial, all stocks wereobserved to be clear and colorless withno visible undissolved test substancefollowing overnight mixing.The sameprocedures were used during testing.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page25of67 DER Template Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Metalaxyl.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0.00<0.061-0.069NANA EPA and OECD recommend that test item concentrations be maintained at a coefficient of variation (CV) 20%.Treatment 11.00.984.3Treatment 210102.6Treatment 3100953.9Abbreviations:CVCoefficient of variation.E. ObservationsBiological Endpoints:Day 7- NF stage, wet weight, snout-vent length (SVL), hind-limb length (HLL), normalized HLLDay 21- NF stage, wet weight, snout-vent length (SVL), hind-limb length (HLL), normalized HLL, thyroid histopathologyDaily- mortality, clinical observations/sub-lethal effectsWere raw (individual) data provided?YesData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page26of67 DER Template Version: 22 September 2011 EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (Days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (D ays 7 and 21); hind limb length (Days 7 and 21); snout-vent length (Days 7 and 21); body weight (test initiation, for optional size-base d larval selection); and thyroid gland gross pathology and histopathology (Day 21). Note the histopathology section of the test guideline also incl udes thyroid gross

pathology observations.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 27 of 67

DER Template Version: 22 September 2011 II. RESULTS AND DISCUSSION

A. Results

By test termination, % mortality averaged 8.75% in the negative control and 7.50, 0, and 3.75% in the mean-measured 0.98, 10, and 95 mg a.i./L treatment groups, respectively (Table 9). Replicate

D in the negative control and Replicate B in the 0.98 mg a.i./L group had a total of three mortalities
which is greater than the performance criteria of no more than 2 mortalities per replicate.

Table 9: Larval Mortality in Xenopus laevis

.

Treatment (mg a.i./L)

[mean-measured] Larval Mortality

Day 7 1 Day 21

n Mortality # Mortality % n Mortality # Mortality %

Negative Control 80 7 2 8.75 60 0 0

0.98 80 4 3 5 60 2 4 3.3

10 80 0 0 60 0 0

95 80 1 5 1 60 2 6 3.3

1 Sample size and cumulative mortality values at Day 7 prior to interim sacrifice.

2 Replicate A and B, one mortality; Replicate C, two mortalities; Replicate D, three mortalities

3 Replicate A, one mortality; Replicate B, three mortalities

4 Replicate C, two mortalities

5 Replicate D, one mortality

6 Replicate A, one mortality; Replicate C, one mortality

Median developmental stage on Day 7 was 54 in the control and all treatment groups (Table 10).

On Day 21, median developmental stage was 60 in the control and all treatment groups. The 10 thData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 and 90 th percentiles did not differ by more than 4 NF stages. No asynchronous development was
observed throughout the test.

Table 10: Larval Development in Xenopus laevis – Developmental Stage and Asynchronous Development.

Treatment (mg a.i./L)

[mean-measured] Developmental Stage

Day 7 Day 21

n Median
Stage # Asynchronous n Median
Stage # Asynchronous

Negative Control 4 54 0 4 60 0

0.98 4 54 0 4 60 0

10 4 54 0 4 60 0

95 4 54 0 4 60 0Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Day 7 HLL ranged from 2.77 mm at 95 mg a.i./L to 0.31 mm at 0.98 mg a.i./L. Day 7 normalized HLL
ranged from 0.14 at 95 mg a.i./L to 0.16 in the negative control and 0.98 mg a.i./L treatment group. Day
21 HLL ranged from 16.95 mm at 0.98 mg a.i./L to 18.39 mm in the negative control. Day 21 normalized
HLL ranged from 0.62 at 95 mg a.i./L to 0.71 in the negative control.

Table 11: Larval Development in Xenopus laevis – Hind Limb Length.

Treatment

(mg a.i./L)

[measured] Hind Limb Length (HLL)

Day 7 Day 21 1

n Mean
(mm) ±SD HLL:

SVL 2 n Mean
(mm) ±SD HLL:

SVL 2

Negative Control 4 3.17 0.22 0.16 4 18.39 0.76 0.71

0.98 4 3.31 0.22 0.16 4 16.95 1.70 0.68

10 4 3.14 0.27 0.15 4 18.07 0.78 0.69

95 4 2.77 0.11 0.14 4 17.03 0.81 0.62

Abbreviations: SD Standard deviation.

1 NF Stage > 60 excluded

2 Summary results for snout-vent length (SVL) are presented in the next table ( Table 12 ).Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Day 7 SVL ranged from 19.35 mm at 95 mg a.i./L to 21.09 mm at 10 mg a.i./L. Day 7 body weight ranged
from 0.44 g at 95 mg a.i./L to 0.58 g at 10 mg a.i./L. Day 21 SVL ranged from 25.38 at 0.98 mg a.i./L
to 27.82 mm at 95 mg a.i./L. Day 21 body weight ranged from 1.13 g at 0.98 mg a.i./L to 1.43 g at 95
mg a.i./L.

Table 12: Larval Growth in Xenopus laevis

.

Treatment
(mg a.i./ L)
[measured] Snout-Vent Length (SVL) Body Weight 2

Day 7 Day 21 1 Day 7 Day 21 1

n Mean
(mm) ±SD n Mean
(mm) ±SD n Mean
(g) ±SD n Mean
(g) ±SD

Negative

Control 4 20.30 0.72 4 26.19 0.18 4 0.54 0.05 4 1.24 0.02

0.98 4 20.45 0.61 4 25.38 0.48 4 0.53 0.04 4 1.13 0.10

10 4 21.09 1.13 4 26.28 0.28 4 0.58 0.09 4 1.22 0.04

95 4 19.35 0.53 4 27.82 0.45 4 0.44 0.03 4 1.43 0.04

Abbreviations: SD Standard deviation.

1 NF Stage > 60 excluded

2 Also referred to as “wet weight” in the test guideline.

No thyroid gland hypertrophy or hyperplasia were observed in the control or treatment group tadpoles (Table 13). Mild follicular cell hypertrophy and hyperplasia were observed in the control and all

treatment groups, though no treatment-related relationship was detected.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus laevis

.

Treatment

(mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Thyroid Gland
Hypertrophy Thyroid Gland
Atrophy Follicular Cell
Hypertrophy Follicular Cell
Hyperplasia

n Incidence n Incidence n Incidence n Incidence

Negative

Control 0 20 20 20 20 20 18 20 17

1 20 0 20 0 20 2 20 3

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.98 0 18 2 18 18 18 18 16 18 17

1 18 2 0 18 0 18 2 18 1

2 18 2 0 18 0 18 0 18 0

3 18 2 0 18 0 18 0 18 0

10 0 19 2 19 19 19 19 16 19 17

1 19 2 0 19 0 19 3 19 2

2 19 2 0 19 0 19 0 19 0

3 19 2 0 19 0 19 0 19 0

95 0 20 20 20 20 20 19 20 17

1 20 0 20 0 20 1 20 3

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

1 Thyroid gland gross pathology and histopathology are gr aded 0 – 3 based on severity: 0=Not remarkable, 1=Mild,

2=Moderate, 3=Severe. See OECD No. 82 for reference.

2 Insufficient tissue was available. Therefore, the sample size was appropriately adjusted.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page32of67 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.Treatment(mg a.i./L)[measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular CellHeight (Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control020202020NANANANANANA1200200NANANANANANA2200200NANANANANANA3200200NANANANANANA0.98018181818NANANANANANA1180180NANANANANANA2180180NANANANANANA3180180NANANANANANA10019191919NANANANANANA1190190NANANANANANA2190190NANANANANANA3190190NANANANANANA95020202020NANANANANANAData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page33of67 DER Template Version: 22 September 2011Treatment(mg a.i./L)[measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular CellHeight (Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence1200200NANANANANANA2200200NANANANANANA3200200NANANANANANAAbbreviations:NANot applicable.1Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 forreference.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Table 14a: Additional Thyroid Gland Histopathology Observations in Xenopus laevis

.

Treatment

(mg a.i./L)

[measured] Additional Qualitative Observations 1

Severity Colloid Depletion Changes in Colloid
Quality

n Incidence n Incidence

Negative Control 0 20 20 20 20

1 20 0 20 0

2 20 0 20 0

3 20 0 20 0

0.98 0 18 18 18 18

1 18 0 18 0

2 18 0 18 0

3 18 0 18 0

10 0 19 19 19 19

1 19 0 19 0

2 19 0 19 0

3 19 0 19 0

95 0 20 20 20 20

1 20 0 20 0

2 20 0 20 0

3 20 0 20 0

1 Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See

OECD No. 82 for reference.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Beginning on Day 7 and continuing throughout the remainder of the exposure period, spinal curvature
was observed among tadpoles in the control and treatment groups. At test termination, spinal curvature
was observed in 37% of control tadpoles and 40, 50, and 15% of tadpoles in the 0.98, 10, and 95 mg a.i./L treatment groups, respectively (Table 15). There was no clear treatment-related response,

and the study author reported that for previous studies conducted at Smithers Viscient, there was no
correlation between the presence of spinal deformities and the increase in CV% for potentially affected endpoints ( e.g. , SVL, HLL, and wet weight).

In an amended study report, the study author added that: “Shortly following exposure initiation, all tadpoles within the 95 mg/L treatment level began to exhibit a sublethal response (e.g., lethargy).
These observations continued the following day (day 1). After day 1, these observations were no

longer observed and the tadpoles appeared to behave normally. All other tadpoles at the remaining
treatment levels and control appeared to behave normally during the entire exposure period.”

Table 15: Clinical Signs in Xenopus laevis

.

Treatment

(mg a.i./L)

[mean- measured] Clinical Signs 1

Type n Incidence 2

Negative Control Spinal curvature 60 37%

0.98 Spinal curvature 58 40%

10 Spinal curvature 60 50%

95 Spinal curvature
Lethargy 58

80 15%

100% 3

1 Note that asynchronous development (unable to stage) is reported previously in Table 10.

2 Represents observations on Day 21.

3 Observed on Day 0 and 1.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 B. Study Author’s Analysis and Conclusions

Larval mortality was analyzed using the step-down Cochran-Armitage test when a monotonic trend
was present. When a there was not a monotonic trend, mortality data were analyzed using Fisher’s
Exact test with the Bonferroni-Holm adjustment. Developmental stage, SVL, normalized HLL, and wet weight were analyzed using the Jonckheere-Terpstra test (step-down) when there was a monotonic
trend. Otherwise, these variables were tested for normality using the Shapiro-Wilks ( Į = 0.01) test
and for homogeneity of variance using the Bartlett’s test ( Į = 0.01). If the data met these assumptions,
analyses were conducted using Dunnett’s Multiple Comparison test. When the data did not meet the
assumptions of normality and homogeneity of variance, analyses were performed using TamhaneDunnett test or the Mann-Whitney test with Bonferroni-Holm adjustment. All analyses were conducted
using CETIS-Comprehensive Environmental Toxicity Information System TM Verion 1.8.1.1. Unless

otherwise noted, all tests were conducted at Į = 0.05.

On Day 7, HLL, normalized HLL, and wet weight were all significantly reduced at the mean-measured 95 mg a.i./L treatment group relative to the negative control. By test termination (Day 21), SVL and

wet weight were significantly promoted at the mean-measured 95 mg a.i./L treatment group, whereas
HLL, normalized HLL, and developmental stage were significantly reduced at this level relative to the
negative control. Based on the significant reductions in normalized HLL at the highest treatment group,
Metalaxyl can be considered “likely thyroid active” to Xenopus laevis larvae under conditions used in

the current test.

In an amended study report, the study author added: “The impacts observed at the high dose (95
mg/L) appear to be indicative of a general toxic response. The increases observed at the 21-day wet

weight and 21-day snout-vent lengths indicate that the tadpoles are delayed in reaching
metamorphosis. When approaching metamorphosis, tadpoles begin to reduce in overall size and weight
by resorbing tissue and reducing absolute water content. The decreases in 7-day hind limb length, 7- day hind limb length (normalized by SVL), 7-day wet weight, 21-day developmental stage, 21-day
hind limb length and 21-day hind limb length (normalized by SVL) also indicate a delay in development.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 These developmental delays are likely attributed to the sublethal responses observed during the first
two days. The animals exposed at this dose were unable to adapt to the exposure concentrations
within a reasonable timeframe, resulting in a two-day developmental delay. These results coupled with
the lack of findings with the thyroid gland histology support this reasoning.”

C. Reviewer’s Analysis and Conclusions

Statistical Methods: All growth and developmental variables were tested for normality using the Shapiro-Wilks test ( Į = 0.01) and for homogeneity of variance using Levene’s test ( Į = 0.01). If the

data exhibited a monotonic trend, analyses were conducted using the Jonckheere-Terpstra test. If the
data were not monotonic but met the assumptions of parametric statistics, Dunnett’s test was used to
test for significant differences between control and treatment means. If the data were not monotonic
and did not meet the assumptions of normality and homogeneity of variance, means were tested using
the Mann-Whitney test. Day 7 wet weight, SVL, and HLL were analyzed using Dunnett’s test, whereas

Day 7 normalized HLL was analyzed using the Jonckheere-Terpstra test. Day 7 developmental stage
was not analyzed because all replicate medians were the same. Day 21 means and medians were
calculated twice, once with including all tadpoles and once excluding tadpoles at NF stage  61. Day21 developmental stage and Day 21 wet weight (excluding NF > 60) were analyzed using the MannWhitney test. Day 21 wet weight (all tadpoles), Day 21 SVL, Day 21 HLL, and Day 21 normalized
HLL (excluding NF >60) were analyzed using Dunnett’s test. Day 21 normalized HLL including all

tadpoles was analyzed using the Jonckheere-Terpstra test. No asynchronous development was
observed. Unless otherwise indicated, effects were considered statistically significant at p < 0.05.

Conclusions:

On Day 7, normalized HLL was the only endpoint significantly reduced (8% reduction, JonckheereTerpstra, p = 0.008); all other endpoints were comparable between the treatment groups and control.
Day 21 developmental stage was comparable between the control and treatment groups (MannWhitney, p > 0.05). The reviewer, however, noted a dose-dependent reduction in the appearance of
late stage individuals (>NF 60); the abundance of late stage tadpoles did not warrant a separateData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 analysis, but there were 16 individuals staged NF 61 and 62 (50:50; 61:62) in the control, 13 (54:46)
in the 0.98 mg a.i./L level, 11 (73:27) in the 10 mg a.i./L level, and only 3 NF 61 tadpoles in the
95 mg a.i./L level. When NF >60 tadpoles were excluded from Day 21 endpoints (other than NF
stage), no differences were found in Day 21 wet weight (Mann-Whitney, p > 0.05).

Day 21 SVL was significantly increased at the mean-measured 95 mg a.i./L treatment group (6% ,
Dunnett’s, p < 0.001). Further, when NF > 60 tadpoles were excluded, Day 21 SVL was significantly
reduced (3.1% reduction) at the mean-measured 0.98 mg a.i./L treatment group (Dunnett’s, p = 0.024). Day 21 HLL was unaffected by treatment (Dunnett’s, p > 0.05). Body weight at Day 21 was
significantly increased by 15% in the high treatment (95 mg a.i./L; Mann-Whitney p<0.05) and was
marginally significantly (p=0.057) decreased by 9% in the lowest treatment group (0.98 mg a.i./L).
Day 21 normalized HLL was significantly reduced (13%; p=0.028) at the mean-measured 95 mg

a.i./L treatment group. The clinical sign of lethargy on days 0 and 1 in all of the tadpoles of the
mean-measured 95 mg a.i./L treatment group may be indicative of overt toxicity.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis AssayEPA MRID Number 48685801Page39of67 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Metalaxyl.Treatment(mg a.i./L)[mean-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 214Day 7Day 21Day 21Median pMedian p% Diff. p%Diff. p%Diff. p%Diff. pTreatment-RelatedEffects? (Yes/No)Negative Control54--60------------------No0.9854--601.03.70.88-4.080.66--------No1054--601.0-4.30.15-1.740.96--------No9554--600.48-8.40.008-12.80.028--------NoStatistical TestNAMann-WhitneyJonckheereDunnett’s------Abbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavilyweighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.3Hind-limb length is normalized to snout-vent length (SVL).4NF > 60 excludedData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Table 17: Growth Endpoints 1,2 in the AMA with Metalaxyl.

Treatment

(mg a.i./L)

[measured] Snout-Vent Length Body Weight

Day 7 Day 21 3 Day 7 Day 21 3

% Diff. p % Diff. p % Diff. p %

Diff. p

Negative Control -- -- -- -- -- -- -- --

0.98 0.73 0.99 -3.08 0.024 -2.26 0.98 -8.92 0.057

10 3.89 0.38 0.33 0.97 7.48 0.63 -1.65 0.63

95 -4.66 0.26 6.22 <0.001 -19.21 0.06 15.07 0.029

Statistical Test Dunnett’s Dunnett’s Dunnett’s Mann-Whitney

Abbreviations: Diff. Difference.

1 Unless otherwise indicated, effects are reported based on comparison to the negative (clean water) control.

2 Unless otherwise specified, effects are considered statistically significant at p < 0.05.

3 NF > 60 excluded

E. Study Deficiencies

There were deviations from the guideline as noted in Section I. Materials and Methods of the DER.
The following performance criteria were not met:



### 1. Negative Control Replicate D and 0.98 mg a.i./L Replicate B both had a total of 3 mortalities by
Day 7 which is greater than the criteria of no more than two mortalities per replicate. 2. Temperature of the test solutions (22-24°C) exceeded the recommended values (22±1°C). On
Day 7, the maximum temperature was 23.6°C. Inter-replicate and inter-treatment differentials did
not exceed 0.5 oC for any given day.
Although the mortality in two replicates exceeded the criteria of no more than 2 mortalities, the reported
measurements for these replicates were similar to measurements in the other replicates. Therefore,
these replicates were retained in the analysis and did not impact the interpretation of the results.

These deviations do not impact the interpretation of the study.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 F. Reviewer’s Comments

Prior to the range-finding and definitive exposure assays, a solubility trial was conducted using nominal
concentrations of 100, 200, 500, and 1000 mg a.i./L in FETAX solution. All solutions were observed
to be clear and colorless with no visible undissolved test substance following overnight mixing. Solubility
samples were handled according to the same procedures using during definitive testing and analytical

recoveries were 100% in all four treatment levels.

Representative samples of the well water source were analyzed bi-annually for the presence of
pesticides, PCBs, and toxic metals by GeoLabs, Inc., Braintree, Massachusetts using U.S. EPA
standard methods. None of these compounds were detected at concentrations considered toxic in any of the water samples analyzed, in agreement with ASTM (2005) standard practices.

III. REFERENCES

Ives, M. 2009. CETIS, Comprehensive Environmental Toxicity Information System TM . User’s Guide.
Tidepool Scientific Software, McKinleyville, California.

Niuewkoop, P.D. and J. Faber. 1994. Normal Table of Xenopus laevis. Garland Publishing, New York.

Mount, D.I. and W.A. Brungs. 1967. A simplified dosing apparatus for fish toxicological studies. Water

Research 1: 21-29.

Sprague, J.B. 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity. Water

Research 3: 793-821.Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

test for amphib metamorph screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR01 ( 7-d wet weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.975 0.909 2.523 0.107 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.54 0.05 0.02 8.71 0.47, 0.62 Dose1 4 0.53 0.04 0.02 6.67 0.47, 0.59 Dose2 4 0.58 0.09 0.05 15.59 0.44, 0.73 Dose3 4 0.44 0.03 0.02 7.46 0.39, 0.49

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.54 0.50 0.60 . . Dose1 0.54 0.48 0.56 97.74 2.26 Dose2 0.59 0.47 0.67 107.48 -7.48 Dose3 0.43 0.41 0.48 80.79 19.21

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 4.66 0.022

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.54 . 0.55 . . . . . . Dose1 0.53 0.980 0.55 0.679 . . . . . Dose2 0.58 0.629 0.55 0.714 0.568 . . . . Dose3 0.44 0.058 0.44 0.014 0.154 0.016 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 7.92 0.048

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.54 . . Dose1 0.54 0.678 0.282 Dose2 0.59 0.678 0.721 Dose3 0.43 0.067 0.046Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 4.66 0.022

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -0.54 . -0.52 . . . . . . Dose1 -0.53 0.980 -0.52 0.764 . . . . . Dose2 -0.58 0.629 -0.52 0.797 0.568 . . . . Dose3 -0.44 0.058 -0.52 0.814 0.154 0.016 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 7.92 0.048

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -0.54 . . Dose1 -0.54 0.678 0.718 Dose2 -0.59 0.678 0.279 Dose3 -0.43 0.067 0.954

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR02 ( 7-d stage (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value
. . . . NO DATA FOR TEST

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 54.00 0.00 0.00 0.00 . , . Dose1 4 54.00 0.00 0.00 0.00 . , . Dose2 4 54.00 0.00 0.00 0.00 . , . Dose3 4 54.00 0.00 0.00 0.00 . , .Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 54.00 54.00 54.00 . . Dose1 54.00 54.00 54.00 100.00 0.00 Dose2 54.00 54.00 54.00 100.00 0.00 Dose3 54.00 54.00 54.00 100.00 0.00

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value . . . 1 . . . . . . .

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 54.00 . . . . . . . . Dose1 54.00 . . . . . . . . Dose2 54.00 . . . . . . . . Dose3 54.00 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 0.00 1.000

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 54.00 . . Dose1 54.00 1.000 . Dose2 54.00 1.000 . Dose3 54.00 1.000 .

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose1

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value . . . 1 . . . . . . .

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -54.00 . . . . . . . . Dose1 -54.00 . . . . . . . . Dose2 -54.00 . . . . . . . .Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 45 of 67

DER Template Version: 22 September 2011 Dose3 -54.00 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 0.00 1.000

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -54.00 . . Dose1 -54.00 1.000 . Dose2 -54.00 1.000 . Dose3 -54.00 1.000 .

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose1 Jonckheere Dose1

test for amphib metamorph screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR03 ( 7-d sn-vent length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.950 0.485 0.690 0.576 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 20.30 0.72 0.36 3.55 19.15, 21.44 Dose1 4 20.45 0.61 0.30 2.98 19.48, 21.41 Dose2 4 21.09 1.13 0.57 5.37 19.29, 22.89 Dose3 4 19.35 0.53 0.27 2.75 18.50, 20.20

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 20.47 19.31 20.95 . . Dose1 20.43 19.73 21.19 100.73 -0.73 Dose2 21.39 19.50 22.08 103.89 -3.89 Dose3 19.29 18.78 20.06 95.34 4.66

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 3.34 0.056

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 20.30 . 20.61 . . . . . . Dose1 20.45 0.987 20.61 0.793 . . . . .Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 46 of 67

DER Template Version: 22 September 2011 Dose2 21.09 0.383 20.61 0.824 0.662 . . . . Dose3 19.35 0.256 19.35 0.073 0.251 0.038 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 7.17 0.067

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 20.47 . . Dose1 20.43 0.889 0.614 Dose2 21.39 0.235 0.906 Dose3 19.29 0.156 0.152

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 3.34 0.056

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -20.30 . -20.30 . . . . . . Dose1 -20.45 0.987 -20.30 0.585 . . . . . Dose2 -21.09 0.383 -20.30 0.619 0.662 . . . . Dose3 -19.35 0.256 -20.30 0.638 0.251 0.038 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 7.17 0.067

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -20.47 . . Dose1 -20.43 0.889 0.386 Dose2 -21.39 0.235 0.094 Dose3 -19.29 0.156 0.848

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR04 ( 7-d hind-limb length (mm) )Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 47 of 67

DER Template Version: 22 September 2011 TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.974 0.905 1.176 0.360 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3.17 0.22 0.11 7.05 2.81, 3.52 Dose1 4 3.31 0.22 0.11 6.55 2.97, 3.65 Dose2 4 3.14 0.27 0.13 8.48 2.72, 3.56 Dose3 4 2.77 0.11 0.05 3.95 2.59, 2.94

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 3.24 2.84 3.34 . . Dose1 3.33 3.05 3.53 104.58 -4.58 Dose2 3.09 2.90 3.49 99.21 0.79 Dose3 2.77 2.63 2.89 87.36 12.64

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 4.81 0.020

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 3.17 . 3.24 . . . . . . Dose1 3.31 0.659 3.24 0.767 . . . . . Dose2 3.14 0.997 3.14 0.545 0.676 . . . . Dose3 2.77 0.051 2.77 0.012 0.016 0.110 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 8.69 0.034

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 3.24 . . Dose1 3.33 0.494 0.807 Dose2 3.09 0.889 0.385 Dose3 2.77 0.103 0.010

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

********************************************************************************Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 48 of 67

DER Template Version: 22 September 2011 PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 4.81 0.020

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -3.17 . -3.10 . . . . . . Dose1 -3.31 0.659 -3.10 0.762 . . . . . Dose2 -3.14 0.997 -3.10 0.795 0.676 . . . . Dose3 -2.77 0.051 -3.10 0.812 0.016 0.110 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 8.69 0.034

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -3.24 . . Dose1 -3.33 0.494 0.193 Dose2 -3.09 0.889 0.615 Dose3 -2.77 0.103 0.990

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR05 ( 7-d norm hind-limb )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.949 0.475 0.403 0.754 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.16 0.01 0.00 4.12 0.15, 0.17 Dose1 4 0.16 0.01 0.00 4.06 0.15, 0.17 Dose2 4 0.15 0.01 0.00 5.95 0.13, 0.16 Dose3 4 0.14 0.00 0.00 3.51 0.13, 0.15

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.16 0.15 0.16 . . Dose1 0.16 0.15 0.17 103.70 -3.70 Dose2 0.15 0.14 0.16 95.66 4.34 Dose3 0.14 0.14 0.15 91.64 8.36Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 49 of 67

DER Template Version: 22 September 2011 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 5.66 0.012

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.16 . 0.16 . . . . . . Dose1 0.16 0.518 0.16 0.802 . . . . . Dose2 0.15 0.398 0.15 0.119 0.096 . . . . Dose3 0.14 0.050 0.14 0.012 0.010 0.585 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 8.98 0.030

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.16 . . Dose1 0.16 0.343 0.877 Dose2 0.15 0.335 0.151 Dose3 0.14 0.101 0.008

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 5.66 0.012

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -0.16 . -0.15 . . . . . . Dose1 -0.16 0.518 -0.15 0.837 . . . . . Dose2 -0.15 0.398 -0.15 0.865 0.096 . . . . Dose3 -0.14 0.050 -0.15 0.880 0.010 0.585 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 8.98 0.030Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 50 of 67

DER Template Version: 22 September 2011 MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -0.16 . . Dose1 -0.16 0.343 0.123 Dose2 -0.15 0.335 0.849 Dose3 -0.14 0.101 0.992

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR06 ( 21-d stage (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.565 <.001 9.000 0.002 USE NON-PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 60.00 0.00 0.00 0.00 . , . Dose1 4 60.00 0.00 0.00 0.00 . , . Dose2 4 60.00 0.00 0.00 0.00 . , . Dose3 4 59.75 0.50 0.25 0.84 58.95, 60.55

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 60.00 60.00 60.00 . . Dose1 60.00 60.00 60.00 100.00 0.00 Dose2 60.00 60.00 60.00 100.00 0.00 Dose3 60.00 59.00 60.00 99.58 0.42

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 1.00 0.426

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 60.00 . 60.00 . . . . . . Dose1 60.00 1.000 60.00 0.583 . . . . . Dose2 60.00 1.000 60.00 0.618 1.000 . . . . Dose3 59.75 0.389 59.75 0.119 0.515 0.515 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 3.00 0.392Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 51 of 67

DER Template Version: 22 September 2011 MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 60.00 . . Dose1 60.00 1.000 . Dose2 60.00 1.000 . Dose3 60.00 0.478 0.090

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 1.00 0.426

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -60.00 . -59.94 . . . . . . Dose1 -60.00 1.000 -59.94 0.723 . . . . . Dose2 -60.00 1.000 -59.94 0.757 1.000 . . . . Dose3 -59.75 0.389 -59.94 0.775 0.515 0.515 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 3.00 0.392

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -60.00 . . Dose1 -60.00 1.000 . Dose2 -60.00 1.000 . Dose3 -60.00 0.478 0.910

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR07 ( 21-d wet weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.969 0.821 4.866 0.019 USE NON-PARAMETRIC TESTSData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 52 of 67

DER Template Version: 22 September 2011 ********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.24 0.02 0.01 1.81 1.21, 1.28 Dose1 4 1.13 0.10 0.05 8.45 0.98, 1.29 Dose2 4 1.22 0.04 0.02 2.95 1.17, 1.28 Dose3 4 1.43 0.04 0.02 2.94 1.36, 1.50

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.25 1.22 1.27 . . Dose1 1.15 1.02 1.22 91.08 8.92 Dose2 1.24 1.17 1.24 98.35 1.65 Dose3 1.42 1.39 1.49 115.07 -15.07

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 19.69 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.24 . 1.26 . . . . . . Dose1 1.13 0.042 1.26 0.722 . . . . . Dose2 1.22 0.918 1.26 0.756 0.161 . . . . Dose3 1.43 0.001 1.26 0.774 <.001 0.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 11.42 0.010

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 1.25 . . Dose1 1.15 0.103 0.022 Dose2 1.24 0.676 0.330 Dose3 1.42 0.067 0.975

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 19.69 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOECData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 53 of 67

DER Template Version: 22 September 2011 Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.24 . -1.19 . . . . . . Dose1 -1.13 0.042 -1.19 0.950 . . . . . Dose2 -1.22 0.918 -1.22 0.809 0.161 . . . . Dose3 -1.43 0.001 -1.43 <.001 <.001 0.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 11.42 0.010

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -1.25 . . Dose1 -1.15 0.103 0.978 Dose2 -1.24 0.676 0.670 Dose3 -1.42 0.067 0.025

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose3 Jonckheere Dose3

test for amphib metamorph screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR08 ( 21-d sn-vent length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.959 0.643 2.663 0.095 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 26.19 0.18 0.09 0.69 25.90, 26.48 Dose1 4 25.38 0.48 0.24 1.90 24.61, 26.15 Dose2 4 26.28 0.28 0.14 1.05 25.84, 26.72 Dose3 4 27.82 0.45 0.22 1.61 27.11, 28.53

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 26.21 25.95 26.39 . . Dose1 25.40 24.82 25.92 96.92 3.08 Dose2 26.23 26.02 26.63 100.33 -0.33 Dose3 27.90 27.26 28.22 106.22 -6.22

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 30.58 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trendData Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 54 of 67

DER Template Version: 22 September 2011 Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 26.19 . 26.42 . . . . . . Dose1 25.38 0.024 26.42 0.872 . . . . . Dose2 26.28 0.974 26.42 0.897 0.022 . . . . Dose3 27.82 <.001 26.42 0.910 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 12.75 0.005

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 26.21 . . Dose1 25.40 0.066 0.010 Dose2 26.23 0.889 0.558 Dose3 27.90 0.066 0.990

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 30.58 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -26.19 . -25.79 . . . . . . Dose1 -25.38 0.024 -25.79 0.964 . . . . . Dose2 -26.28 0.974 -26.28 0.469 0.022 . . . . Dose3 -27.82 <.001 -27.82 <.001 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 12.75 0.005

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -26.21 . . Dose1 -25.40 0.066 0.990 Dose2 -26.23 0.889 0.442 Dose3 -27.90 0.066 0.010Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 55 of 67

DER Template Version: 22 September 2011 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose3 Jonckheere Dose3

test for amphib metamorph screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR09 ( 21-d hind-limb length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.935 0.290 1.260 0.332 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 18.39 0.76 0.38 4.15 17.17, 19.60 Dose1 4 16.95 1.70 0.85 10.03 14.25, 19.66 Dose2 4 18.07 0.78 0.39 4.34 16.82, 19.32 Dose3 4 17.03 0.81 0.41 4.76 15.74, 18.32

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 18.40 17.61 19.14 . . Dose1 16.43 15.55 19.39 92.20 7.80 Dose2 18.09 17.10 19.01 98.29 1.71 Dose3 17.16 16.08 17.74 92.65 7.35

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 1.78 0.205

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 18.39 . 18.39 . . . . . . Dose1 16.95 0.201 17.51 0.166 . . . . . Dose2 18.07 0.955 17.51 0.177 0.493 . . . . Dose3 17.03 0.237 17.03 0.067 1.000 0.553 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 4.46 0.216

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 18.40 . . Dose1 16.43 0.346 0.124 Dose2 18.09 0.889 0.442Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 56 of 67

DER Template Version: 22 September 2011 Dose3 17.16 0.156 0.131

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 1.78 0.205

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -18.39 . -17.61 . . . . . . Dose1 -16.95 0.201 -17.61 0.899 . . . . . Dose2 -18.07 0.955 -17.61 0.920 0.493 . . . . Dose3 -17.03 0.237 -17.61 0.931 1.000 0.553 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 4.46 0.216

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -18.40 . . Dose1 -16.43 0.346 0.876 Dose2 -18.09 0.889 0.558 Dose3 -17.16 0.156 0.869

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR10 ( 21-d norm hind-limb )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.983 0.984 0.912 0.464 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.71 0.03 0.01 4.16 0.66, 0.75 Dose1 4 0.68 0.06 0.03 9.28 0.58, 0.78 Dose2 4 0.69 0.03 0.02 4.35 0.64, 0.74 Dose3 4 0.62 0.04 0.02 6.26 0.55, 0.68Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 57 of 67

DER Template Version: 22 September 2011 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.70 0.67 0.74 . . Dose1 0.67 0.61 0.76 95.92 4.08 Dose2 0.70 0.65 0.72 98.26 1.74 Dose3 0.62 0.58 0.66 87.23 12.77

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 3.55 0.048

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.71 . 0.71 . . . . . . Dose1 0.68 0.664 0.68 0.303 . . . . . Dose2 0.69 0.955 0.68 0.324 0.945 . . . . Dose3 0.62 0.028 0.62 0.007 0.226 0.095 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 7.17 0.067

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.70 . . Dose1 0.67 0.494 0.193 Dose2 0.70 0.889 0.385 Dose3 0.62 0.067 0.016

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 3.55 0.048

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -0.71 . -0.67 . . . . . . Dose1 -0.68 0.664 -0.67 0.913 . . . . . Dose2 -0.69 0.955 -0.67 0.933 0.945 . . . . Dose3 -0.62 0.028 -0.67 0.942 0.226 0.095 . . .Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 58 of 67

DER Template Version: 22 September 2011 ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 7.17 0.067

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -0.70 . . Dose1 -0.67 0.494 0.807 Dose2 -0.70 0.889 0.615 Dose3 -0.62 0.067 0.984

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 59 of 67

DER Template Version: 22 September 2011 21 Day Body Weight (Mann-Whitney exact test)



### Obs **conc endpoint** 

**1** 

### 0 1.234

**2** 

### 0 1.218

**3** 

### 0 1.260

**4** 

### 0 1.266

**5** 

### 1 1.197

**6** 

### 1 1.098

**7** 

### 1 1.224

**8** 

### 1 1.015

**9** 

### 2 1.170

**10** 

### 2 1.244

**11** 

### 2 1.244

**12** 

### 2 1.238

**13** 

### 3 1.418

**14** 

### 3 1.430

**15** 

### 3 1.390

**16** 

### 3 1.490Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 60 of 67

DER Template Version: 22 September 2011 

### Wilcoxon Scores (Rank Sums) for Variable endpoint **Classified by Variable conc**

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score** 

**0** 

### 4 35.0 34.0 8.240146 8.750

**1** 

### 4 13.0 34.0 8.240146 3.250

**2** 

### 4 30.0 34.0 8.240146 7.500

**3** 

### 4 58.0 34.0 8.240146 14.500
**Average scores were used for ties.**

**Kruskal-Wallis Test**
**Chi-Square** 

### 11.4212

**DF** 

### 3
**Asymptotic Pr > Chi-Square** 

### 0.0097
**Exact Pr >= Chi-Square** 

### 8.605E-04Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 61 of 67

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 62 of 67

DER Template Version: 22 September 2011 

### Wilcoxon Scores (Rank Sums) for Variable endpoint **Classified by Variable conc**

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score** 

**0** 

### 4 25.0 18.0 3.464102 6.250

**1** 

### 4 11.0 18.0 3.464102 2.750

**Wilcoxon Two-Sample Test**
**Statistic (S)** 

### 25.0000

**Normal Approximation**

**Z** 

### 1.8764

**One-Sided Pr > Z** 

### 0.0303

**Two-Sided Pr > |Z|** 

### 0.0606

**t Approximation**

**One-Sided Pr > Z** 

### 0.0514
**Two-Sided Pr > |Z|** 

### 0.1027

**Exact Test**

**One-Sided Pr >= S** 

### 0.0286

**Two-Sided Pr >= |S - Mean|** 

### 0.0571

**Z includes a continuity correction of 0.5.**

**Kruskal-Wallis Test**
**Chi-Square** 

### 4.0833

**DF** 

### 1
**Pr > Chi-Square** 

### 0.0433Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 63 of 67

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 64 of 67

DER Template Version: 22 September 2011 

### Wilcoxon Scores (Rank Sums) for Variable endpoint **Classified by Variable conc**

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score** 

**0** 

### 4 20.0 18.0 3.443420 5.0

**2** 

### 4 16.0 18.0 3.443420 4.0
**Average scores were used for ties.**

**Wilcoxon Two-Sample Test**
**Statistic (S)** 

### 20.0000

**Normal Approximation**

**Z** 

### 0.4356

**One-Sided Pr > Z** 

### 0.3316

**Two-Sided Pr > |Z|** 

### 0.6631

**t Approximation**

**One-Sided Pr > Z** 

### 0.3381
**Two-Sided Pr > |Z|** 

### 0.6762

**Exact Test**

**One-Sided Pr >= S** 

### 0.3143

**Two-Sided Pr >= |S - Mean|** 

### 0.6286

**Z includes a continuity correction of 0.5.**

**Kruskal-Wallis Test**
**Chi-Square** 

### 0.3373

**DF** 

### 1
**Pr > Chi-Square** 

### 0.5614Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 65 of 67

DER Template Version: 22 September 2011Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 66 of 67

DER Template Version: 22 September 2011 

### Wilcoxon Scores (Rank Sums) for Variable endpoint **Classified by Variable conc**

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score** 

**0** 

### 4 10.0 18.0 3.464102 2.50

**3** 

### 4 26.0 18.0 3.464102 6.50

**Wilcoxon Two-Sample Test**
**Statistic (S)** 

### 10.0000

**Normal Approximation**

**Z** 

### -2.1651

**One-Sided Pr < Z** 

### 0.0152
**Two-Sided Pr > |Z|** 

### 0.0304

**t Approximation**

**One-Sided Pr < Z** 

### 0.0336
**Two-Sided Pr > |Z|** 

### 0.0671

**Exact Test**

**One-Sided Pr <= S** 

### 0.0143
**Two-Sided Pr >= |S - Mean|** 

### 0.0286

**Z includes a continuity correction of 0.5.**

**Kruskal-Wallis Test**
**Chi-Square** 

### 5.3333

**DF** 

### 1
**Pr > Chi-Square** 

### 0.0209Data Evaluation Record on the Toxicity of Metalaxyl to Amphibians, Metamorphosis Assay

EPA MRID Number 48685801

Page 67 of 67

DER Template Version: 22 September 2011

# DATA EVALUATION RECORD

METALAXYL

Study Type: OCSPP 890.1150, Androgen Recep tor Binding (Rat Prostate Cytosol)

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012

(Revisions to MRID 48617601 to include laboratory proficiency data; Main study was originally reviewed under TA 2-46-2012)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Daniel R. Hunt, M.S. Date: 5/22/2012

Secondary Reviewer Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 7/20/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/27/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/27/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page 1 of 17 METALAXYL/ 113501 OCSPP 890.1150/0ECD None


### Primary Reviewer: Vincent Chen. M.S.

# Signature~~ Risk Assessment Branch III, Health Effects Division (7509P) Date: 2 

### '-i "Ji) N 

### zo,J...S 

### Secondary Reviewer: Greg Akerman, Ph.D. 

### Signature: 

# 4. /2--=
Health Effects Division (7509P) Date:

- J,.- 

## f, r Template version 08/2011 

### DATA EVALUATION RECORD

STUDY TYPE: Androgen Receptor Binding (Rat Prostate Cytosol); OCSPP 890.1150



### PC CODE: 113501 

### DP BARCODE: D399227



### TXR#: 0054219 

### CAS No.: 57837-19-1



### TEST MATERIAL (PURITY): Metalaxyl (98.2%)



### SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate



### CITATION: Willoughby, J.A. (2011) Metalaxyl: Androgen Receptor Binding Assay (Rat Prostate Cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study No: 9143V100326ARB, December 7, 2011. MRID 48617601. Unpublished.



### SPONSOR: 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. CeeTox, Inc., Kalamazoo, MI. July 19, 2011. MRID 48843501. Unpublished.

Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL



### TEST ORDER#: CON-113501-18



### EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48617601), ventral prostate cytosol from Sprague Dawley rats was used 

## as the source of AR to conduct a competitive binding experiment, which measured the binding of a single concentration of

[ 3 

### H]-R1881 (1 nM) in the presence of increasing concentrations (10

- 10 

### to 10- 3 

### M) ofmetalaxyl (98.2% purity, Batch #20100540). DMSO was used 

## as the solvent vehicle at a final assay concentration of 

### ca. 3.2%. A total of three runs were performed, and each run included de x amethasone 

## as a weak positive control, and RI 881 

## as the ligand reference standard.

A saturation binding experiment 

## was conducted to demonstrate that the AR in the rat prostate cytosol 

## was present in reasonable numbers and was functioning with appropriate affinity for the radiolabeled reference androgen (RI 881) prior to routinely conducting AR competitive binding
exp e riments. Saturation bindin g data were not originally provided in the study report; however, summari ze d saturation bindin g data (MRID 48843 5 01) from th e performin g laborato ry were submitted following a request by the Agency. The mean dissociation constant 

### (Kd) for [3H]- Rl 881 

## was 1.080±0.690 nM, and the estimated Bmax 

## was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that 

## was prepared. Only one of the**three~** 

### values 

## was within the range reported in the E PA validation pro gr am (0.685 to 1.57 nM)

, and all Bmax valuesAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None


### 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.

The assay satisfies the EDSP Tier 1 Test Order require ments for an Androgen Receptor Binding Assay (OCSPP 890.1150).

COMPLIANCE: Signed and dated Data Confidentiali ty, GLP Compliance, Quality Assurance, and Certification of Authenticity statements were provided. All phases of the study were conducted in compliance with FIFRA GLP regulations except that dose con centrations of test chemical and control chemicals were not verified using analytical methods.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc.
Location: Kalamazoo, MI Study Director: J.A. Willoughby Other Personnel: K. Rutherford, Director of Laboratory Operations D. Blakeman, Senior Scien tist/Endocrine Group Leader C. Haines, Scientist S. McColley, Scientist C. Toole, Director of Project Management Study Period: May 26, 2011 to December 7, 2011



### 2. Test substance: Metalaxyl
Description: Solid Source: Midwest Research Institute Batch #: 20100540 Purity: 98.2 ± 0.3% Solubility: Not reported (NR) Volatility: NR Stability: NR Storage conditions: Ambient CAS #: 57837-19-1 Molecular weight: 279.3 g/mol Structure:

CH 3 N O

CH 3 O CH 3 CH

3 O O CH

3



### 3. Non-labeled ligand: R1881 Supplier: Sigma Aldrich, St. Louis, MO Catalog #: Lot #: R0908 060M4638 Purity: 98% CAS #: 965-93-5



### 4. Radioactive ligand: [ 3 

### H]-R1881 Supplier: Perkin-Elmer, Boston, MA Catalog and Lot #: NET590 (653698) Date of production: February 24, 2011 Date of use: September 10, 2011 to September 17, 2011 Radiochemical purity: >97% Specific activity: 85.1 Ci/mmol Concentration of stock: 10 nMAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### 5. Positive control: Dexamethasone Supplier: Sigma Aldrich, St. Louis, MO Catalog # Lot #: D1756 077K1050 Purity: 99% CAS # : 50-02-2



### 6. Solvent/vehicle control: Dimethyl sulfoxide (DMSO)
Justification for choice of solvent: Issues with pipetting and vol atilization prevented the use of ethanol as the solvent. DMSO is one of the recommended solvents according to the EPA Guideline (OPPTS 890.1150) Final Concentration: ca . 3.2%



### B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: Male Sprague Dawley rats (number not specified) from Charles River Laboratories were castrated at 90 days of age and the ventral prostates were excised <1 day following castr ation, weighed, and placed in ice-cold TEDG (Tris, EDTA, DTT, glycerol) buffer (inclu sion of protease inhi bitor not reported), homogenized, and centrifuge d for 30 min at 30,000 × g at 4oC. The supernatants were pooled, and the protein concentration of the cy tosol was determined to be 6.2 mg/mL using a commercially available protein kit compa tible with DTT in the TEDG buffer (Bradford Method). The cytosol was divided in to aliquots and stored frozen at

− 80°C until use.

2. Saturation Radioligand Binding Experiment: A saturation binding experiment measuring total and non-specific binding of [ 3 

### H]-R1881 was performed to demonstrate that the AR was present in reasonabl e concentrations and had the appropriate affinity for the R1881 ligand (MRID 48843501). The conditions for the saturation binding experiment are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a Source of receptor Rat ventral prostate cytosol Concentration of radioligand (as serial dilutions) 0.25 to 10 nM Concentration of non-labeled ligand (100X [radioligand]) 2 to 1000 nM Optimization of receptor concentration Sufficient to bind 25 to 35% of radioligand at 0.25 nM Temperature 4o C Incubation time 16 to 20 hours Composition of assay buffer (TEDG) Tris 10 mM (pH 7.4)
EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride (PMSF) 1 mM
DTT 1 mM Sodium Molybdate 1 mM a Data were obtained from page 2 of the study report (MRID 48843501).



### On the day of the assay, the specif ic activity of the stock solution [ 3 

### H]-R1881 (originally 85.1 Ci/mmol as manufactured on February 24, 2011) was adjusted for decay over time (adjusted specific activities were not reported), and seri al dilutions in low-saltAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None


### TEDG+PMSF buffer were prepared to achieve th e final concentrations in cytosol of 0.25,0.50, 0.70, 1.0, 1.5, 2.5, 5.0, and 10 nM to determine total binding. To determine nonspecific binding, solutions of non-labeled R1881 were prepared in a similar manner to achieve concentrations that were 100-fold greater than each re spective radiolabeled concentration, resulting in final concentr ations in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1000 nM. In the absence of cytosol, th e radiation found in 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

### H]-R1881 and 7.5, 15, or 30 μL of 100 nM [ 3 

### H]-R1881 was measured.

For each batch of cytosol, the optimal protei n concentration was determined by calculating specific binding to differing amounts of protei n per tube with 0.25 nM radiolabeled R1881. The optimal protein concentration was determin ed to be 1.86 mg protein/assay tube, which resulted in the binding of approximately 25-35% of the total radioactivity added. Cytosolic protein used in this assay was thawed fresh fo r this experiment at 4°C, and maintained at 4°C during the binding assay. Each saturation binding experiment consisted of three noncurrent runs (conducted on September 9, 11, and 16, 2011, respectively). Each run contained three replicates at each concentration, re sulting in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment Run a ,b Total Binding Non-Specific Binding Radioligand alone Tubes 1-24 c Tubes 25-48 d Tubes 49-72 e [ 3 H]-R1881 Final conc. (nM) [ 3 H]-R1881 Final conc. (nM) R1881 Final conc. (nM) [ 3 H]-R1881 Initial conc. (nM) [ 3 H]-R1881 (μL)
0.25 0.25 25 10 7.5 0.50 0.50 50 10 15 0.70 0.70 70 10 21 1.0 1.0 100 10 30 1.5 1.5 150 10 45 2.5 2.5 250 100 7.5 5.0 5.0 500 100 15 10 10 1000 100 30 a Data were obtained from page 3 of the study report (MRID 48843501). b Each concentration was run in triplicate for a total of 72 samples. c Tubes 1-24 contained 50 μL of tr iamcinolone acetonide and 7.5-45 μL [ 3 H]-R1881. Samples were dried, and 300 μl of prostate cytosol were added. d Tubes 25-48 contained 50 μL of tr iamcinolone acetonide and 7.5-45 μL [ 3 H]-R1881. R1881 was added in a 100-fold mola r excess of [ 3 H]-R1881 in a volume of 7.5-45 μL. Samples were dried, and 300 μL of prostate cytosol were added. e Tubes 49-72 contained only 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 H]-R1881 or 7.5, 15, 21, or 30 μL of 100 nM [ 3 H]-R1881 without cytosol or other component s to determine the total counts added.



### Following addition of triamcinolone acetonide, [ 3 

### H]-R1881, and/or R1881, the tubes were dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor for 20 hours at approximately 4°C. Samples were maintained at temperatures of approximately 4°C except during whole rack vortexing. To separa te bound from free R1881, hydroxyapatite (HAP) slurry was added to each tube and vortexed 5 times with 4-minute intervals over 20 minutes. The samples were then centrifuged, and the s upernatant was aspirate d and discarded. The samples were washed 4 times in 2 mL of ice cold TEDG+PMSF buffer, followed byAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None


### vortexing and centrifugation for 3 minutes at 700 x g. Following the last wash and decanting of the Tris buffer pellets were then extracted by additional of 2 mL of ethanol. The samples were vortexed and centrifuged for 10 minutes at 1000 x g. Samples were maintained on ice at all times between vortexing . After the final centrifugation, the ethanol supernatants were decanted into scintillation vials that each contained 14-mL portions of scintillation cocktail, and the radiation was quantified by liquid scin tillation counting. A total of three runs were performed.

3. Competitive Binding Experiment: A summary of the assay conditions for the competitive binding experiment is included in Table 3.

TABLE 3. Summary of Conditions for Competitive Binding Experiment a Source of receptor Rat ventral prostate cytosol Concentration of radioligand 1 nM Optimization of receptor concentration Sufficient to bind 10-15% of radioligand b Concentration of test substance (as serial dilutions) 10

− 10 to 10

− 3 M Incubation Temperature 4 oC Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% DTT 1 mM Sodium molybdate 1 mM Protease Inhibitor (PI) c 0.5% a Data were obtained from pages 9, and 14-16 of the study report. b The protein concentration was not reported. c The recommended PI in th e Guideline is 1 mM PMSF. The PI was identified in the study protocol as Calbiochem Protease Inhibitor Cocktail, Set III, EDTA fre e. It is a mixture of six PIs that includes 4-(2- aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBS F has similar specificity to PMSF with greater stability at lower pH.



### The competitive binding experiment was performed according to the protocol provided in the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures the binding of a singl e concentration of [ 3 

### H]-R1881 (adjusted specific activity of 82.6 Ci/mmol for Run 1; and 82.5 Ci/mmol for Runs 2 and 3) to the AR in the presence of increasing concentrations of a test substance.

DMSO was used as the solvent vehicle, a nd no precipitation of me talaxyl was visually observed at concentrations ≥ 10
− 3 

### M. The protein concentration used in the competitive binding experiment was not reported; however, it was reported to be sufficient to bind 10- 15% of the [ 3 

### H]-R1881.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### Dilutions of the test subs tance, reference standard (R1881), weak positive control (dexamethasone), and solvent control (DMSO) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of th ree independent runs on three different days, and each run contained three replicates at each concentration plus si x replicates of the [ 3 

### H]-R1881, non-specific binding (NSB), and solven t controls, resulting in a total of 81 samples per run.

TABLE 4. Competitor Final Molar (M) Con centrations in Competitive Binding Assay a, b
Solvent Control Reference standard We ak positive control Test Chemical None DMSO R1881 Dexamethasone Metalaxyl

Tubes 7-12 Tubes 13-33 c Tubes 37-60 Tubes 61-84 Tubes 1-6 Run 1 Runs 2 & 3 d Run 1 Runs 2 & 3 d
1×10

− 7.5 1×10

− 6 1×10

− 4 1×10

− 3 1×10

− 3
1×10

− 8.5 1×10

− 7 1×10

− 5 1×10

− 4 1×10

− 4
1×10

− 9.5 1×10

− 8 1×10

− 6 1×10

− 5 1×10

− 5
1×10

− 10.5 1×10

− 9 1×10

− 7 1×10

− 6 1×10

− 6
1×10

− 11.5 1×10

− 10 1×10

− 8 1×10

− 7 1×10

− 7
1×10

− 12.5 1×10

− 11 1×10

− 9 1×10

− 8 1×10

− 8
-- -- 1×10

− 10 1×10

− 9 1×10

− 9
-- -- 1×10

− 11 1×10

− 10 1×10

− 10 a Data were obtained from pages 12-14, 16, and 20 of the study report. b Each concentration of each chemical was run in tripli cate for a total of 81 tubes per run (tubes were numbered 1-84,but tubes 34-36 were excluded from the sequence). Tubes 1-84 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 7- 84 also contained 10 μL of the solvent control, reference standard (non-radiolabeled R-1881), weak positive control, or test substance, with the exception of Tubes 13-18 that contained 30 μL of non-radiolabeled R1881 (used to evaluate non-specific binding). Tubes 1-6 contained only 30 μL of [ 3 H]-R1881. c Tubes 13-18 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881. d The reason cited for the deviation fr om the EPA Test Guidelin e (890.1150) was the accide ntal prepping of the wrong stock concentration (pp. 65-66).



### To separate bound from free R1881, HAP slurry was added to each tube and vortexed once every 5 minutes for 20 minutes. The samples were then centrifuged, and the supernatant was discarded. The samples were washed 4 times in 50 mM TRIS buffer. Following the last wash and decanting of the Tris buffer, th e pellets were then ex tracted by addition of 2- mL portions of ethanol. The samples we re vortexed at 5 minute intervals for ca . 15-20 minutes. Samples were centrifuged for 10 minutes at 700 × g . Supernatants were radioassayed by scintillation counting.

4. Data Analysis: For the saturation binding experi ment, total binding and non-specific binding data were modeled via non-linear regression by using Graph Pad Prism v. 5 (GraphPad Software, Inc., La Jolla, CA)], incorporating automatic outlier elimination according to the method of Motulsky and Brown (2006) 1 

### implemented by using the ROUT procedure in Prism v. 5 

### with a Q value of 1.0. Receptor binding data plots were corrected for ligand depletion with the method of 

### Swillens (1995) 2 

### . For the competitive binding assay,

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outlie rs when fitting data with nonlinear regression- a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics, Vol 7, pp 123-142. 2 Swillens, S. (1995) Interpretation of binding curves obtain ed with high receptor concentrations: practical aid forAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None


### similar methods of nonlinear regression (X LfitTM Model 204, IDBS, Guilford, UK) were used to fit a curve (for R1881, the positive c ontrol, and the test substance) to the Hill equation formula which incorporated log IC 50 

### as a parameter to be estimated. For parameters reported from the saturation binding experiment ( 

### K d 

### and B max 

### ) and competitive binding experiment (log IC 50 

### and RBA), m ean and standard deviation were calculated for each run and mean and standard error were calculated for the composite three runs using Microsoft Excel 2007 (v. 12.0.6557.5000; Microsoft Corporation, Redmond, WA), and mean and standard error were calculated for the composite three runs with Microsoft Excel 2010.



### 5. Definitions

a. Classification of test material

If the data fit a 4-parameter non linear regression model, the test chemical is classified as:

Binder: The average curve for the test chemical across runs crosses 50% of radioligand bound, and the Hill Slope is between

− 0.6 to

− 1.4.

Equivocal: The average lowest portion of curves across runs is between 50% and 75% radioligand binding ( i.e. radioligand displacement is at le ast 25% but less than 50%), or the Hill slope for the curve falls outside th e range for the weak positive control (

− 0.6 to

− 1.4).

Non-Binder: The average lowest portion of curves acr oss runs is greate r than 75% activity ( i.e. less than 25% displacement of radioliga nd), or the data do not fit the model.

Untestable: If the test compound is not soluble above 1×10

− 6 

### M and the binding curve does not cross 50%, the chemical is judged to be untestable.

b. Descriptors for receptor binding

B max 

### : maximal binding capacity K d 

### : dissociation constants IC 50 

### : Concentration of the test substance at which 50% of radioligand is displaced from the AR by the competitor 

### Relative Binding Affinity (RBA): ( IC 50 

### of R1881 ÷ IC 50 

### of test substance) × 100 Log RBA: Log 10 

### (IC 50 

### of R1881 ÷ IC 50 

### of test substance)

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters are presented in Table 5. The mean 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 th e single batch of prostate cytosol that was prepared. Only one of the three K d 

### values was within th e range reported in the EPA validation program (0.685 to 1.57 nM); the mean value was above the acceptable

computer analysis. Molec. Pharmacol. 47(6):1197-1203.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None


### range. Confidence in these numbers is low based on the large variation in K d 

### estimates
among runs.

TABLE 5. Saturation Binding Experiment of [ 3 H]-R1881 with Androgen R eceptor from Ra t Prostate Cytosol a
Parameter Run 1 Run 2 Run 3 Mean ± SE b
R 2 (unweighted) 0.880 0.986 0.893 0.880-0.986
B max (nM) 0.010 0.008 0.011 0.010±0.002
B max (fmol/100 μ g protein) 1.087 0.896 1.229 1.071±0.167
K d (nM) 0.855 0.531 1.855 1.080±0.690 a Data were obtained from page 4 of the study report (MRID 48843501). b The range of R 2 is reported and the mean ± SE is reported for the other parameters. R 2 Goodness of fit for curve calculated for specific binding



### Figure 1 illustrates the non-specific, sp ecific, and total binding curves for [ 3 

### H]-R1881 to the androgen receptor for the three independent runs . The specific binding reached a plateau in each run, and non-specific binding was less than 20% of total binding. Figure 2 contains the Scatchard plots that illustrate the binding of [ 3 

### H]-R1881 to the androgen receptor. The data fits generally result ed in linear plots.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### FIGURE 1. Binding of [ 3 

### H]-R1881 to the Androgen Receptor during the Saturation Binding Experiment.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### FIGURE 2. Scatchard Pl ots of the Binding of [ 3 

### H]-R1881 to the Androgen Receptor.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### B. COMPETITIVE BINDING EXPERIMENT: Competitive binding experiment parameters are presented in Table 6 and shown graphically in Figures 3-5.

The estimated average log IC 50 

### was

− 9.3 M for R1881 and

− 4.4 M for the weak positive control (dexamethasone). Compared to R1881, the average RBA for the weak positive control was 0.0022%. Confidence in these numbers is high; however it is noted that the variations between Run 1 compared to Runs 2 and 3 are likely due to the concentration differences used for R1881 and dexamethasone. The curve for dexamethasone was not fully defined in the first run and ther efore, the calculation for the IC 50 

### may be affected. The range of concentrations for R1881 in the first run fu lly defined the curve, but the concentration range was off by more than an order of magn itude. In addition, all solvent control tubes were run at the beginning of each experiment , preventing the reviewer from determining drift in the assay from the be ginning to the end of the run.

Metalaxyl had no effect on specifi c binding (>75% binding) of the [ 3 

### H]-ligand at concentrations up to 10

− 3 

### M in all three runs. An IC 50 

### and RBA for metalaxyl could not be determined. Based on the results of the three valid assays, metalaxyl is classified as a “nonbinder” with the androgen receptor under the conditions of this assay.

TABLE 6. Competitive Binding Assay of Meta laxyl with AR from Rat Prostate Cytosol a
Parameter Run 1 Run 2 Run 3 Mean ± SE b

R 2 (unweighted) R1881 NR NR NR NA Dexamethasone NR NR NR NA Metalaxyl NR NR NR NA Log IC 50 (M) R1881

− 9.9

− 9.0

− 9.0

− 9.3 ± 0.30 Dexamethasone

− 4.3

− 4.4

− 4.6

− 4.4 ± 0.09 Metalaxyl NA NA NA NA IC 50 (M) b R1881 1.26 × 10

− 10 1.00 × 10

− 9 1.00 × 10

− 9 7.09 × 10

− 10 (± 2.91) Dexamethasone 5.01 × 10

− 5 3.98 × 10

− 5 2.51 × 10

− 5 3.83 × 10

− 5 (± 0.73) Metalaxyl NA NA NA NA Log RBA b Dexamethasone

− 5.60

− 4.60

− 4.40

− 4.87 ± 0.37 Metalaxyl NA NA NA NA RBA (%) b Dexamethasone 0.0003 0.0025 0.0040 0.0022 ± 0.0011 Metalaxyl NA NA NA NA a Data were obtained from page 21 of the study report. b All values are reviewer- calculated from the log IC 50 values reported in the study report. For means expressed in scientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean value. SE Standard Error NA Not applicable. NR Not reported R 2 Goodness of fit RBA (%) relative binding affinityAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### FIGURE 3. Percentage [ 3 

### H]-R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethas one, or Metalaxyl (Run 1).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### FIGURE 4. Percentage [ 3 

### H]-R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethas one, or Metalaxyl (Run 2).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 15 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### FIGURE 5. Percentage [ 3 

### H]-R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethas one, or Metalaxyl (Run 3).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 16 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay was functioning properly, each r un was evaluated using the criteria shown in Table 7.

TABLE 7. Criterion a Tolerance Limit(s) b Value Yes No
Ligand depletion is minimal. The recommended ratio of total binding in the absence of competitor to total amount of [ 3 H]-R1881 added per assay tube. ≤ 15% ≤ 12.7 X

Test chemical Top (% binding) c 80 to 115 95.1 to 107.5 X R1881 fitted curve parameters Top (% binding) 82 to 114 96 to 103 X Bottom (% binding)

− 2.0 to 2.0 0 X Hill Slope

− 1.2 to

− 0.8

− 1.0 X Weak positive control (dexamethasone) fitted curve parameters Top (% binding) 87 to 106 101 to 105 X Bottom (% binding)

− 12 to 12 NA; -1 to 7 X d Hill Slope

− 1.4 to

− 0.6

− 1.0 to

− 0.6 X Saturation Binding Experiment K d (nM) 0.685 to 1.57 0.531 to 1.855 e X f

Non-specific binding (%) ≤ 10.0 0.28 to 0.50 g X a Data were obtained from pages 23-28, and 52-57 of the study report. b These values represent ranges from the validation study. c 10

− 9 to 10

− 10 M d A value could not be determined for Run 1 due to the aforementioned laboratory error with the prepared concentration of dexamethasone. Bo ttom % binding was within the accepta ble range in the other two runs. e MRID 48843501 f One value was below the limit, one value was within the limit, and one value was above the limit. g Calculated by the study reviewer. NR Not reported



### All performance criteria were met, with the exception that bottom % binding could not be determined for dexamethasone for Run 1 due to a laboratory error in th e preparation of the concentration used in the assay (resulting in dexamethasone concentrations ranging from 10
− 11 

### to 10
− 4 

### M instead of 10

− 10 

### to 10
− 3 

### M). The resulting curve for dexamethasone fit two of the three criteria (Hil l Slope and top plateau) and the st udy author stated that the log IC 50 

### for dexamethasone performed within CeeTox histor ical data. The devi ation was considered minor.

Additionally, the curve for the reference material showed that increasing concentrations of unlabeled R1881 displaced [ 3 

### H]-R1881 in a manner consiste nt with one-site binding, as indicated by a descent from 84.4-91.9% to 4.9- 8.2% binding over approximately an 81-fold increase in concentration of R1881 (i.e., cove ring approximately 2 log units). Examination across the runs indicated consistency of th e Hill slope, placement along the X-axis, and top and bottom plateaus.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 17 of 17 METALAXYL / 113501 OCSPP 890.1150/ OECD None



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Metalaxyl was classified as a "non-binder" in all three valid independent runs of the assay. T hus, metalaxyl has a fina l classification of "nonbinder" to the AR.

B. AGENCY COMMENTS: In the saturation binding assay, the mean K d 

### for [ 3 

### H]-R1881 was 1.080 nM, and the mean estimated B max 

### was 1.071 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 validation program (0.685 to 1.57 nM); the mean value was above the acceptable range.

In the competitive binding experiment, metalaxyl was classified as a non-binder in each run as there were no test substance-related effects on the [ 3 

### H]-R1881 binding to the AR at concentrations up to 10

− 3 

### M under the conditions of this assay.

In the competitive binding experiment, the estimated average log IC 50 

### was

− 9.3 M for R1881 and

− 4.4 M for the weak positive control (dexamethasone). Compared to R1881, the mean RBA for dexamethasone was 0.0022%. C onfidence in these numbers is generally high due to the small variation between the runs . However, it is not ed that variations between Run 1 compared to Runs 2 and 3 are likely due to the concentration differences used for R1881 and dexamethasone. The bottom of the curve for dexamethasone was not fully defined in the first run. The range of concentrations for R1881 in the first run fully defined the curve, but the concentration range was off by more than an order of magnitude. The concentrations prepared for R1881 and de xamethasone were lower than the targeted (guideline) levels because of an error in the stock preparation. Therefore, non-specific binding was not evaluated with 100X of cold (non-radiolabeled) R1881 but with 30X cold R1881, and the resulting curve for dexamethasone fit only two of the three criteria (Hill slope and Top plateau) in the first run. All performance criteria were met for the other two

runs.

The bottom percent binding for dexamethasone was not achieved in the first run, leaving only two valid runs instead of the required three. However, as the performance was consistent between the other two runs, it is unlik ely that information from an additional run would change the interp retation of this study.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The bottom percent binding fo r dexamethasone was not achie ved in the first run. The concentrations prepared for R1881 and dexa methasone were lower than the targeted (guideline) levels because of an error in the stock preparation.

 Curves were not provided showing the averag e binding of each test substance across all three runs.

# DATA EVALUATION RECORD

METALAXYL

Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-46-2012 (MRID 48617602)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Sandra Hastings Date: 4/27/2012

Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 5/14/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 6/05/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 6/05/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).Aromatase (Human Recombinant) Assay (2011) I Page 1 of 13 METALAXYL / 113501 OCSPP 890.1200 I OECD None


### Primary Reviewer: Vincent Chen, M.S. 

### Signature:

# ~ Risk Assessment Branch III, Health Effects Division (7509P) Date: -Z'i -;J'-J.rJ "Z-O.L-5 

### Secondary Reviewer: Greg Akerman, Ph.D. 

### Signature: 

# .4**~** 

### Health Effects Division (7509P) Date: 

## a. /.J.o- 

# hr= Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200



### PC CODE: 113501 

### DP BARCODE: D399227

TXR#: 0054219 

### CAS No.: 57837-19-1



### TEST MATERIAL (PURITY): Metalaxyl (100.4% a.i.)



### SYNONYMS: Alanine, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-, methyl ester



### CITATION: Wilga, P.C. (2011) Metalaxyl: Human Recombinant Aromatase Assay. CeeTox, Inc., Kalamazoo, MI. Study ID: 9143V-100326AROM, December 7, 2011. MRID 48617602. Unpublished.



### SPONSOR: Metalaxyl EDSP Consortium; 20 Ambriance Drive, Burr Ridge, IL 60527, USA



### TEST ORDER#: CON-113501-18



### EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48617602), metalaxyl (100.4% a.i., Batch# 20100540) in 1 % dimethyl sulfoxide (DMSO) was incubated with human recombinant aromatase and tritiated androstenedione (([1**~- H(N)]-androst-4-ene**  

### 3, 17-dione; [ 3 

### H]-ASDN) for 15 minutes at 37 °C to assess the potential of metalaxyl to inhibit
aromatase activity. Metalaxyl was tested at logarithmic concentrations from 10- 10 

### to 10- 3 

### Min three independent runs.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Each run included a full activity control, a background activity control, a positive control series (10- 10 

### to 10- 5 

### M) using a known inhibitor (4- hydroxyandrostenedione; 4-0H ASDN), and the test chemical series (10- 10 

### to 10- 3 

### M) with three repetitions per concentration.

Aromatase activity in the full activity controls was 0.240 ± 

### 0.070 nmol·mg-protein- 1 

### ·min- 1 • 

### The
response of each full activity control within a run was between 90 to 110% of the average full activity, with two exceptions likely due to an unevenly split sample. Activity in the background controls ranged 0.43 to 0.87% and averaged 0.59% of the full activity controls.

For the positive control substance ( 4-0H ASDN), aromatase activity was within the recommended ranges for the top of the curve, bottom curve, Hill slope, log ICso, and %CV for replicates of each concentration within runs, except that the %CV for the 1 

# o-s M ( 19%) and 10- 9Aromatase (Human Recombinant) A ssay (2011) / Page 2 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None


### 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.

The study satisfies the Test Order requirement for an Aromatase Assay (OCSPP 890.1200).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Aromatase (Human Recombinant) A ssay (2011) / Page 3 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Metalaxyl Description: White, sugar like Source: Nation’s Ag, LLC (Burr Ridge, IL) Lot/Batch #: 20100540 (expiry date: July 26, 2011) Purity: 100.4% a.i. Volatility: Not reported Storage conditions: Room temperature (ambient) Stability: Not reported Solvent: DMSO Solubility (in test solvent): Not reported Highest Concentration Tested: 10

− 3 M in all runs Stock Solution Preparation: Dissolved in DMSO to obtain a stock concentration of 100 mM Molecular weight: 279.3 CAS #: 57837-19-1 Structure:

CH 3 N O

CH 3 O CH 3 CH

3 O O CH

3



### 2. Non-Labeled Substrate: Androstenedione (ASDN)
CAS # : 63-05-8 Source (catalog #): Steraloids, Inc. (catalog # L1712) Lot/Batch # (expiration date): L1712 (expiry date: April 2016) Purity: 99.8%



### 3. Radiolabeled Substrate: 1- β [ 3 

### H(N)]-Androst-4-ene-3,17-dione; ([ 3 

### H]ASDN) Source (catalog #): Perkin Elmer (catalog # NET-926) Lot/Batch # (expiration date): 619344 (expiry date: January 10, 2012) Radiochemical Purity (Supplier): >97% Specific activity: 26.3 Ci/mmol Radiochemical Purity (In-lab determination): Not reported



### 4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN)
CAS # : 566-48-3 Source (catalog #): Sigma-Aldrich (catalog # F2552) Batch # (expiration date): 081K2133 (expiry date: March 2015) Purity: 99.6%



### 5. Solvent (Vehicle Control): Dimethyl sulfoxide (DMSO)
Source: Lot # (expiration date): Sigma-Aldrich (Catalog # D2650) RNBB7617 (expiry date: February 2013) Justification for choice of solvent DMSO was listed as one of the vehicl es acceptable for use in OCSPP 890.1200 guideline; DMSO was not as volatile as ethanol and so evaporation was less of a concern in the assay, and DMSO was more accurate to pipette because of densit y and viscosity Concentration (% of total volume in assays): ≤ 1%Aromatase (Human Recombinant) A ssay (2011) / Page 4 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None



### 6. Test Microsomes: Human recombinant aromatase (CYP19) microsomes Source (catalog #): BD Gentest TM , Woburn, MA (catalog # 456260) Lot/Batch # (expiration date): 03897 (expiry date: February 2014)
Protein concentration: 7.4 mg protein per mL Cytochrome C reductase activity: 290 nmole/mg protein/minute Aromatase activity: 6.0 pmol/pmol P450/min



### B. METHODS

1. Assay Components and Preparations: A mixture of non-labe led and radiolabeled [ 3 

### H]ASDN was prepared such that the final concentration of ASDN in the assay was approximately 0.1 μM, and the amount of tri tium added to each incubation tube was 0.1 μ Ci.

The test chemical(s) were formulated in DM SO such that the volume of DMSO used per assay was no more than 1% v/v of the total as say volume. DMSO was selected because it is listed as one of the solvents of choice detail ed in the EPA guideline; it not as volatile as ethanol and so evaporation was less of a concern in the assay, and is more accurate to pipette because of its density and viscosity.

A stock solution of the positive control substance, 4-OH ASDN, was formulated in DMSO. Fresh dilutions of the stock solution were prepar ed in the same solvent on the day of use. Dilutions were prepared such that the target concentrations of the pos itive control substance (10

− 10 

### to 10
− 5 

### M; Table 4) were achieved by the additi on of 20 μL of the dilution for a final assay volume of 2 mL.

Human recombinant microsomes were purchased from BD Gentest TM 

### , and stored at

− 80 ± 10oC. Before use of the microsomes, the ba tch was thawed, diluted with sodium phosphate pH 7.4 buffer to 0.008 mg/mL microsomal protei n (to achieve a final concentration of approximately 0.004 mg/mL microsomal protein per assay tube), and sub-aliquoted into individual vials before being refrozen to mini mize freeze-thaw cycles to no more than one.

Other assay components sodium phosphate buffer, propylene glycol, and NADPH are reported in Table 1.

TABLE 1. Assay Components and Conditions a Assay Factor Values 0.1M sodium phosphate buffer (pH 7.4) Microsomal Protein 0.004 mg/mL NADPH 0.3 mM [ 3 H]ASDN 100 nM Propylene Glycol 5% Temperature 37°C Incubation Time 15 min a Data were obtained from page 16 of the study report.Aromatase (Human Recombinant) A ssay (2011) / Page 5 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None


### 2. Suitability Assessments: The protein concentration of the microsomes (Lot # 03897) was supplied by the vendor as 7.4 mg/mL. Aromat ase activity of the microsomes was also provided by the vendor as 6.0 pmol/min/pmol P450. The minimum aromatase activity in the full activity control samples was de termined to be 0.160 nmol·mg-protein

− 1 

### ·min

− 1 

### , whichwas greater than the minimum recommended aromatase activity of 0.1 nmol·mgprotein

− 1 

### ·min

− 1



### .
3. Aromatase Assay: Each assay run contained 4 tube s for the full enzyme activity and 4 tubes for the background activity controls. Two tubes of each control were run at the beginning of the assay, and two tubes of each cont rol were run at the end of the assay. A full concentration curve in duplicate for the pos itive control, and a full concentration curve in triplicate for the test s ubstance were established. Th e aromatase assay was conducted generally according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10).

The amount of 3 

### H 2 

### O in the aqueous fraction was quantified for each assay tube by LSC, and aromatase activity was reported in units of nmol·mg-protein

− 1 

### ·min

− 1



### .
4. Demonstration of Proficiency: It was stated that profic iency testing of the CYP19 aromatase assay was conducted. Each of the proficiency chemicals (atrazine, econazole, fenarimol and nitrofen) were incubated alongside the positive control inhibitor (4-OH ASDN) on three separate occasions using the same methods employed in the current study. Proficiency records were archiv ed at CeeTox, Inc. and the actu al data from the proficiency testing were not include d in the study report.

a. Positive Control

(1) Initial Demonstration of Laboratory Proficiency: The positive control data for the laboratory met the following criteria:

 Mean aromatase activity in the absence of an inhibito r was at least 0.1 nmol·mgprotein

− 1 

### ·min

− 1



### .

 Mean background control activity was ≤ 15% of the full activity control.

 Performance criteria (Table 2) were met, and served as guidance in identifying runs that provided parameters in the preferred ranges.

The coefficient of variation (CV) for replicates within each sample type and concentration of 4-OH ASDN was generally <15%, except for Run 2 in which the CV was >15% for the 10
− 8 

### M duplicate samples (19%), and for the 10

− 9 

### M duplicate samples ( 34%). The 15% CV maximum is a suggested limit, and the positive control met all other criteria.

(2) Demonstration of Proficiency of Ne w Technician for Conducting Assay (when applicable): The positive control data met the criteria as listed in section (i) of OCSPP 890.1200.Aromatase (Human Recombinant) A ssay (2011) / Page 6 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None

TABLE 2. Performance Criteria for the Positive Control a Parameter Lower Limit Criteria Upper Limit Crit eria Actual Lower Limit Actual Upper Limit Slope

− 1.2

− 0.8

− 1.12

− 0.98 Top (%) 90 110 97.42 101.05 Bottom (%)

− 5 +6 0.46 1.63 Log IC 50 (M)

− 7.3

− 7.0

− 7.29

− 7.10 a Data were obtained from pages 23 and 27 of the study report.



### b. Proficiency Chemicals: It was stated that the prof iciency of the laboratory was demonstrated prior to running any test chemi cals by conducting three full scale test runs on each of the proficiency chemicals as listed in Table 3. The data were not included in the study report, but it was reported that all assays were performed by CeeTox personnel that have demonstrated proficiency performing th e assay as outlined in the test guideline.

TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations
Econazole 24169-02-6 Not reported Not reported Fenarimol 60168-88-9 Not reported Not reported Nitrofen 1836-75-5 Not reported Not reported Atrazine 1912-24-9 Not reported Not reported



### 5. Determination of Aromatase Ac tivity with Test Chemical(s): The response of aromatase activity to the presence of eight concentrations of metalaxyl per run, in triplicate, was tested during three independent runs. Solubility was visually assess ed (presence of cloudiness or a precipitate). No precipitation was observed at any concentration in the first run. The response of each full activity c ontrol within a run was between 91% to 108% of the average full activity, with the exception of the duplicate full activity control responses (129% and 62%) from the end of Run 2 that were outsi de of the guideline recommended range. The laboratory reported that it was likely the samp le was unevenly split between the two tubes, and had no impact on the study.Aromatase (Human Recombinant) A ssay (2011) / Page 7 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None

TABLE 4. Test Chemical Study Design for each Test Run a

Sample Type Repetitions (Tubes) Description Reference or Chemical (M) Full Activity Control 4 All test components b plus solvent vehicle (DMSO) N/A Bkgd Activity Control 4 Same as above without NADPH N/A 4-OH ASDN Conc 1 2 All test components plus 4-OH ASDN 1×10

− 5
4-OH ASDN Conc 2 2 All test components plus 4-OH ASDN 1×10

− 6
4-OH ASDN Conc 3 2 All test components plus 4-OH ASDN 1×10

− 6.5
4-OH ASDN Conc 4 2 All test components plus 4-OH ASDN 1×10

− 7
4-OH ASDN Conc 5 2 All test components plus 4-OH ASDN 1×10

− 7.5
4-OH ASDN Conc 6 2 All test components plus 4-OH ASDN 1×10

− 8
4-OH ASDN Conc 7 2 All test components plus 4-OH ASDN 1×10

− 9
4-OH ASDN Conc 8 2 All test components plus 4-OH ASDN 1×10

− 10

Metalaxyl Conc 1 3 All test components plus Metalaxyl 1×10

− 3 Metalaxyl Conc 2 3 All test components plus Metalaxyl 1×10

− 4 Metalaxyl Conc 3 3 All test components plus Metalaxyl 1×10

− 5 Metalaxyl Conc 4 3 All test components plus Metalaxyl 1×10

− 6 Metalaxyl Conc 5 3 All test components plus Metalaxyl 1×10

− 7 Metalaxyl Conc 6 3 All test components plus Metalaxyl 1×10

− 8 Metalaxyl Conc 7 3 All test components plus Metalaxyl 1×10

− 9 Metalaxyl Conc 8 3 All test components plus Metalaxyl 1×10

− 10 a Data were obtained from page 19 of the study report. b The complete assay contained buffer, pr opylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH.



### C. DATA ANALYSIS

1. Raw Data: Raw data were converted to aromatase activity (nmol·mg-protein

− 1 

### ·min

− 1 

### ) and percent control for the positive control and metalaxyl. The following raw data and calculated endpoints for each run were in cluded in the report (Table 5).

TABLE 5. Raw and Calculated Data
Raw/Calculated Data Included (X) DPM/mL for each portion of extract ed aqueous incubation mixture X Average DPM/mL for each aqueous portion (after extraction) X Total DPM for each aqueous portion (after extraction) X The total DPM present in the assay tube at initiation X The percentage of substrate converted to product X Total DPM after extraction corrected for background X Aromatase activity expressed in nmol·mg protein

− 1 ·min

− 1 X Average aromatase activity in the full activity control tubes X Percentage of control activity remaining in the presence of various inhibitor concentrations X
DPM Disintegrations per minute



### 2. Statistical Methods: Basic statistical analysis was pe rformed on the data, which included means of replicates, standard deviation of the mean, standard error of the mean, and coefficient of variation.

The response curves were fitted by weighted least squares nonlinear regression analysis with weights equal to 1/Y. Model fits were carried out using a 4-parameter regression model (XLfit; IDBS; Version 5.2.0.0; Fit Model 208) and Tukey’s Bi-Weight statistical analysisAromatase (Human Recombinant) A ssay (2011) / Page 8 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None


### for outlier analysis. Concentration response tr end curves were fitted to the percent of control activity values wi thin each of the replicate tubes at each test chemical concentration. Concentration was expressed on the log or ha lf-log scale. For each run the individual percent of control values were plotted versus logarithm of the test chemical concentration. The fitted concentration response curves were superimposed on the plot. Individual plots were prepared for each run. Additional plots were prepared to compare the percent of control activity values across runs. For each run the average percent of control values versus logarithm of test chemical concentra tion were plotted on the same plot. The fitted concentration response curves for each run were superimposed on the plots. On a separate plot the average percent of cont rol values for each run were plo tted versus logarithm of test substance concentration. Th e average concentration response curve across runs was superimposed on the same plot.

3. Interpretation of Results: Interpretation of the assay results was based on the average of three runs, using the categor ies presented in Table 6.

TABLE 6. Interpret ation of Results Criteria Interpretation Data fit 4-parameter nonlinear regression model Average curve across runs crossed 50% a Inhibitor Average lowest portion of curves across runs is between 50% and 75% activity b Equivocal

Average lowest portion of curves across runs is greater than 75% activity b Non-inhibitor

Data do not fit model --- a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near

− 1. Unusually steep curves may indicate protein denaturing or solubility issues. If the slope of the curve is steeper than

− 2.0, the result is classified as equivocal. b If the test compound was not soluble above 10

− 6 M and the inhibition curve does not cr oss 50%, the chemical is typically determined to be untestabl e in the aromatase assay.



### II. RESULTS

A. CONTROL ACTIVITY: Aromatase activity in the full activity controls ranged from 0.160 to 0.331 nmol·mg-protein

− 1 

### ·min

− 1 

### for the three test runs, with a mean and standard deviation of 0.240 ± 0.070 nmol·mg-protein

− 1 

### ·min

− 1 

### . Activity in the background controls ranged 0.43 to 0.87% and averaged 0.59% of the fu ll activity controls. The response of each full activity control with in a run was between 90 to 110% of the average fu ll activity, with the exception of the duplicate fu ll activity control responses (129% and 62%) from the end of Run 2 that were outside of the guidelin e recommended range. The laboratory reported that it was likely the sample was unevenly spl it between the two tubes, and had no impact on the study. The response of the full activ ity controls and bac kground controls were considered acceptable for each run.

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.249 ± 0.065 nmol·mg-protein

− 1 

### ·min

− 1 

### at the lowest tested concentration 10
− 10 

### M and 0.002 ± 0.001 nmol·mg-protein

− 1 

### ·min

− 1 

### at the highest tested concentration 10

− 5 

### M. The mean aromatase activity of the pos itive control (expresse d as % full control activity) for each concentration tested across a ll three runs is presented in Table 7, alongAromatase (Human Recombinant) A ssay (2011) / Page 9 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None


### with the overall standard deviation, standard error of the mean, and % CV. Inhibition response curves for the positive control from each run and the average of all runs are shown in Figure 1. These results were generally with in the recommended ranges for the top of the curve, bottom curve, Hill Slope, log IC 50 

### , and coefficient of variation for replicates of each concentration, with the following exceptions: the %CV for the 10

− 8 

### M (19%) and 10

− 9 

### M (34%) replicates for Run 2 exceed ed the recommended limit of 15%.

TABLE 7. Effect of 4-OH ASDN an d Metalaxyl on Aromatase Activity (as percent of control) from Independent Runs a

Chemical Concen. (Log M) # Runs Overall Mean Overall SD Overall SEM Overall %CV
4-OH ASDN (positive control) − 5 3 0.77 0.08 0.03 10.0

− 6 3 7.10 1.29 0.53 19.0

− 6.5 3 18.76 2.93 1.20 16.3

− 7 3 38.62 4.79 1.96 12.9

− 7.5 3 68.07 7.57 3.09 11.6

− 8 3 88.54 10.65 4.35 12.5

− 9 3 92.69 14.45 5.90 16.2

− 10 3 102.62 9.80 4.00 10.0 Metalaxyl

− 3 3 95.45 14.46 4.82 15.6

− 4 3 102.10 4.13 1.36 4.2

− 5 3 99.79 3.06 1.02 3.1

− 6 3 99.93 3.68 1.23 3.8

− 7 3 101.07 4.96 1.65 5.0

− 8 3 104.54 11.23 3.74 11.0

− 9 3 104.03 10.45 3.98 10.3

− 10 3 103.33 10.71 3.57 10.6 a Values were calculated by the reviewers based on data provided on pages 41-44, 46-49, and 51-54. SD Standard Deviation SEM Standard Error of the Mean CV Coefficient of VarianceAromatase (Human Recombinant) A ssay (2011) / Page 10 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None


### FIGURE 1. Inhibition Respon se Curves for 4-OH ASDN.

C. TEST SUBSTANCE: For metalaxyl, aromatase activity averaged 0.250 ± 0.069 nmol·mgprotein

− 1 

### ·min

− 1 

### at the lowest tested concentration 10

− 10 

### M and 0.232 ± 0.073 nmol·mgprotein

− 1 

### ·min

− 1 

### at the highest tested concentration 10

− 3 

### M. The mean aromatase activity of metalaxyl (expressed as %full control activity) for each concentration tested across all three runs is presented in Table 7 (above), along w ith the overall standard deviation, standard error of the mean, and % CV. Inhibition resp onse curves for metalaxyl from each run are shown in Figure 2, and the average inhibition response curve across a ll runs is shown in Figure 3.Aromatase (Human Recombinant) A ssay (2011) / Page 11 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None


### FIGURE 2. Inhibition Response Curves for Metalaxyl From Each Test Run.

FIGURE 3. Mean Inhibition Response Curve for Metalaxyl.

The effect of the positive control and meta laxyl on inhibition of aromatase activity is presented in Table 8. The response curve indi cated that the aromatase activity of metalaxyl at all test concentrations test ed was essentially equivalent ( i.e . activity was >75% across allAromatase (Human Recombinant) A ssay (2011) / Page 12 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None


### runs) to the activity observed in the full activity control. These data coul d not be fit to the 4- parameter nonlinear regressi on model; therefore, log IC 50 

### and Hill slope estimates were not determined for metalaxyl. For 4-OH ASDN, the estimated log IC 50 

### averaged

− 7.18 M and the mean Hill slope was

− 1.03. Confidence in these values is high due to the small variation (<8% CV).

TABLE 8. Effect of Metalaxyl on Aromatase Activity (as Percent of Control) From Independent Runs. a
Chemical Run 1 Run 2 Run 3 Mean SD %CV
IC 50 (M) Metalaxyl NA NA NA NA NA NA 4-OH ASDN

− 7.14

− 7.10

− 7.29

− 7.18 0.10

− 1.51 Hill Slope Metalaxyl NA NA NA NA NA NA 4-OH ASDN

− 1.00

− 1.12

− 0.98

− 1.03 0.08

− 7.94 a Data were obtained from Table 12 on page 27 of the stud y report. The Mean, SD and %CV were calculated by the reviewers based on these data. SD Standard Deviation CV Coefficient of Variance NA Not applicable; values for metalaxyl were not suitable for modeling.



### Based on the data from the average response curv e and the criteria listed above in Table 8, the results support the c onclusion that metalaxyl is a non-in hibitor of aromatase activity. Mean aromatase activity for metalaxyl was 95.45 ± 14.97% at the highest test concentration of 10

− 3 

### M.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: In three independent runs of the assay, metalaxyl tested at concentrations of 10

− 10 

### to 10
− 3 

### M resulted in essentially no decrease in aromatase activity. Based on the data interpretation criteria established for the assay, metalaxyl was classified as a non-inhibito r of the aromatase enzyme.

B. AGENCY COMMENTS: Although data demonstrating proficiency with the positive control or recommended proficiency chemicals were not included, a statement was included that proficiency of the laboratory was demonstr ated prior to analysis of metalaxyl by the conduct of three full scale test runs on each of the recommended proficiency chemicals and that all assays were performed by CeeTox pers onnel that have dem onstrated proficiency performing the assay as outlined in the test guideline.

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

− 1 

### ·min

− 1



### . The response of each full activity control wi thin a run was between 90 to 110% of the average full activity, with th e exception of the duplicate fu ll activity control responses (129% and 62%) from the end of Run 2 that were outside of the guideline recommended range. The laboratory reported that it was lik ely the sample was unevenly split between the two tubes, and had no impact on the study. Acti vity in the background controls ranged 0.43 to 0.87% and averaged 0.59% of the full activity controls. Th e response of the full activity controls and background controls were considered acceptable for each run.Aromatase (Human Recombinant) A ssay (2011) / Page 13 of 13 METALAXYL / 113501 OCSPP 890.1200 / OECD None



### 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 with in 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 mean slope was

− 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 lowest portion of the response curve was greater than 75% activity at all concentra tions of metalaxyl evaluated (10

− 10 

### to 10
− 3 

### M). Based on the data from the average response curve, meta laxyl is classified as a Non-inhibitor of aromatase activity in this assay.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The required proficiency da ta were not provided.

 Information on the stability of the test substa nce in the stock solution was not reported.

# DATA EVALUATION RECORD

METALAXYL

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012

(Revisions to MRID 48617607 to in clude Saturation binding data; Main study was originally reviewed under TA 2-46-2012)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Daniel R. Hunt, M.S. Date: 05/24/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 7/17/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/30/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/30/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).MET ALAXYL I 113501 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) I Page 1 of 18 OCSPP 890.1250/ OECD None


### Primary Reviewer: Minerva Mercado, Ph.D. 

### Health Effects Division Secondary Reviewer: Greg Akerman, Ph.D. 

### Health Effects Division

# Signal**~** Date: · · 

### Signature: ==: Date: 

# <.; h 

# rt 1 

### S--- Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC); OCSPP 890.1250



### PC CODE: 113501 

### DP BARCODE: D399227



### TXR#: 0054219 

### CAS No.: 57837-19-1



### TEST MATERIAL (PURITY): Metalaxyl (98.2% a.i.)



### SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate



### CITATION: Willoughby, J.A. (2011) Metalaxyl: Estrogen Receptor Binding (Rat Uterine Cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study No: 9143V100326ERB, December 8, 2011. MRID 48617607. Unpublished.

Willoughby, J.A. (2012). Supplemental Information

- Laboratory Proficiency Data for ER TA assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, MI. July 19 2012. MRID 48843501. Unpublished.



### SPONSOR: Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL



### TEST ORDER#: CON-113501-18



### EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48617607) for metalaxyl (98.2% purity, Batch #20100540), uterine cytosol from Sprague Dawley rats was used

as the source of ER for a competitive binding experiment, which measured the binding of a single concentration of**[3H]-17~-estradiol** 

### (1 nM) 

### in the presence of increasing concentrations 

### (10- 10 

### to 

### 10- 3 

### M) of metalaxyl. DMSO was used as the solvent vehicle at a final concentration of



### ca. 4%. A total of three runs were performed, and each run included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and**17-~-estradiol** 

### as the natural ligand reference material.

A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radio-labeled reference estrogen prior to conducting ER competitive binding experiments. Saturation binding data were not originally provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. The protein concentrations used in the saturation binding

runs varied between each run, and were approximately 3- to 6-fold greater than recommendedEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None


### (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.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Estrogen Receptor Binding Assay (OCSPP 890.1250).

COMPLIANCE: Signed and dated Data Confidentiali ty, GLP Compliance, Quality Assurance, and Certification of Authenticity statements were provided. All phases of the study were conducted in compliance with FIFRA GLP regulations except that dose concen trations of the test chemical and control chemicals were not verified using analytical methods.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc.

Location: Kalamazoo, MI Study Director: J.A. Willoughby Other Personnel: K. Rutherford, Director of Laboratory Operations D. Blakeman, Senior Scien tist/Endocrine Group Leader C. Haines, Scientist S. McColley, Scientist B. Meyer, Scientist C. Toole, Director of Project Management Study Period: May 26, 2011 to December 8, 2011



### 2. Test substance: Metalaxyl Description: Solid Source: Nation’s AG, LLC Batch #: 20100540 Purity: 98.2 ± 0.3% Solubility: Not reported (NR) Volatility: NR Stability: NR Storage conditions: Ambient CAS #: 57837-19-1 Molecular weight: 279.3 g/mol Structure:

CH 3 N O

CH 3 O CH 3 CH

3 O O CH

3



### 3. Non-labeled ligand: 17 β -estradiol Supplier: Sigma-Aldrich, St. Louis, MO Catalog #: E8875 Lot #: 110M0138V Purity: 100% CAS #: 50-28-2



### 4. Radioactive ligand: [ 3 

### H]-17 β -estradiol Supplier: Perkin-Elmer, Boston, MA Catalog #: NET517 Lot#: 650702 Radiochemical purity: >97% Specific activity: 130.2 Ci/mmol Concentration of stock: 50 nMEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### 5. Positive control: 19-Norethindrone Supplier: Sigma-Aldrich, St. Louis, MO Catalog #: N4128 Lot #: 030M1359V Purity: 99% CAS #: 68-22-4



### 6. Negative control: Octyltriethoxysilane Supplier: Sigma-Aldrich, St. Louis, MO Catalog #: 440213 Lot #: 24996KK Purity: 99.34% CAS #: 2943-75-1



### 7. Solvent/vehicle control: Dimethyl sulfoxide (DMSO)

Justification for choice of solvent: DMSO is one of the recommended solvents according to the EPA Guideline (OPPTS 890.1250) Final Concentration: ca . 4%



### B. METHODS

1. Preparation of Rat Uterine Cytosol (RUC): Female Sprague Dawley rats (number not specified) from Harlan Laboratories were ovariec tomized 7 days prior to being euthanized. Animals were 84-91 days old at the time of eu thanasia. The uteri were weighed, and placed in ice-cold TEDG (Tris, EDTA, DTT, gly cerol) buffer + PMSF (phenylmethylsulfonyl fluoride), homogenized, and centrifuged for 10 min at 2,500 × g at 4oC. The supernatants were transferred and centrif uged for 60 minutes at 105,000 × g , discarding the resulting pellets. Protein concentrati on of the cytosol was determined to be 3.22 mg/mL using a protein kit compatible with DTT in the TE DG buffer (Bradford Method). Cytosol was divided into 1- to 6-mL portions for immediate use or storage at

− 80oC until use.

2. Saturation (radioligand) Binding Experiment: A saturation binding experiment measuring total and non-specific binding of [ 3 

### H]-17 β -estradiol was performed to demonstrate that the estrogen receptor (ER) wa s present in reasonable concentrations and had the appropriate affinity for the nativ e ligand (MRID 48843501). The conditions for the saturation binding are su mmarized in Table 1.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None

TABLE 1. Summary of Conditions for Saturation Binding Experiment a Source of receptor Rat uterine cytosol Concentration of radioligand (as serial dilutions) 0.03 to 3 nM Concentration of non-labeled ligand (100X [radioligand]) 3 to 300 nM Concentration of receptor Sufficient to bind approximately 25 to 35% of radioligand at 0.03 nM Temperature 4oC Incubation time 16 to 20 hours Composition of assay buffer Tris 10 mM (pH 7.4)
EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride (PMSF) 1 mM
DTT 1 mM a Data were obtained from page 1 of the study report (MRID 48843501).



### On the day of the assay, the specif ic activity of the stock solution [ 3 

### H]-17 β -estradiol (originally 130.2 Ci/mmol as manufactured on May 6, 2011) was adjusted for decay over time (adjusted specific activities were not repo rted), and serial dilutions in TEDG+PMSF buffer were prepared to achieve the fi nal concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. Solutions of non-labeled 17 β -estradiol were prepared in a similar manner to achieve concentrations that were 100-fold greater than each re spective radiolabeled concentration to resu lt in final concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM.

For each batch of cytosol (isolated July 20, 2011), the optimal protein concentration was determined by testing serial amounts of pr otein per tube, using 0.03 nM radiolabeled estradiol, until a concentration was reached th at bound approximately 25 to 35% of the total radioactivity added. The fina l protein concentrations were 320 μg, 192 μg and 160 μg per assay tube for the first, second and third saturation binding experi ments, respectively ( Note: typically 50 ± 10 μg protein per tube) . Each assay consisted of three non-concurrent runs (conducted on August 5, 6, and 7, 2011, respectively). Each run included three replicates of each test substance at each concentration, resu lting in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment Run a Total binding b Non-specific binding c Radioligand alone d Assay Components

Tubes 1-24 Tubes 25-48 Tubes 49-72 350 μL 300 μL --- TEDG+PMSF buffer 50 μL 50 μL 50 μL [ 3 H]-17 β -estradiol (8 serial dilutions) e

--- 50 μL --- Non-labeled 17 β -estradiol (8 serial dilutions, 100x each respective labeled concentration) f 100 μL 100 μL --- Uterine cytosol (diluted to appropriate concentration) 500 μL 500 μL 50 μL Total volume in each assay tube a Data were obtained from page 2 of the study report (MRID 48843501). b Total binding = [ 3 H]-17 β -estradiol bound to ER c Non-specific binding = [ 3 H]-17 β -estradiol and 100-fold greater non-labeled bound to ER d Total [ 3 H]-17 β -estradiol alone for dpm determination at each concentration e Final concentrations of [ 3 H]-17 β -estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. f Final concentrations of non-labeled 17 β -estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 6 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### Tubes were incubated with gentle vort exing for 17.5-19 hours at approximately 4  C. To separate bound from free estradiol, hydroxyapati te (HAP) slurry was added to each tube and vortexed (3 times with 5-minute intervals). S ubsequently, the contents of each tube were washed three times as follows: 2-mL portions of TEDG+PMSF buffer were added, vortexed, centrifuged fo r 10 minutes at 1000 x g , and the supernatant decanted and discarded. After the final centrifugation, et hanol (1.5 mL) was added to the HAP pellet remaining in each tube to extract the [ 3 

### H]-17 β -estradiol, followed by vortexing, and centrifugation for 10 minutes at 1000 x g . Aliquots (1 mL) of supernatant were radioassayed by scintillation counting. The te mperature was maintain ed at approximately 4oC throughout the assay prior to extraction with ethanol.

3. Competitive Binding Experiment: A summary of the experimental conditions for the competitive binding experiment is included in Table 3.

TABLE 3. Summary of Conditions for Competitive Binding Experiment a Source of receptor Rat Uterine Cytosol Concentration of radioligand 1 nM Concentration of receptor Sufficient to bind 10-15% of radioligand b Concentration of test substance (as serial dilutions) 10

− 10 to 10

− 3 mM Temperature 4 ± 2  C Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH 7.4)
EDTA 1.5 mM Glycerol 10% Protease Inhibitor (PI) w/PMSF (unspecified) c 0.5%

DTT 1 mM a Data were obtained from pages 17-18 of the study report. b The protein concentration was 80.0 μg/assay tube (pp. 61, 64, and 67). c The recommended PI in the Guideline is 1mM PMSF, but the PI was not identified (although it was described as containing PMSF).


### No precipitation of test chemical was detected by visual observation in 100% DMSO at any of the concentrations tested. On the day of the assay, the specific activity of the stock solution [ 3 

### H]-17 β -estradiol was adjusted for decay ove r time (adjusted sp ecific activity of 128.2 Ci/mmol for Run 1; 127.8 Ci/mmol for Run 2, and 126.3 Ci/mmol for Run 3), and diluted in TEDG+PI buffer to achieve a final concentration of 1 nM. The protein concentration used in the competitive bindi ng experiment was 80.0 μg/assay tube, and was reported to be sufficient to bind 10-15% of the radioligand. Serial d ilutions of the test substance, positive control (19-norethindrone), negative control (oct yltriethoxysilane), and reference material (non-labeled 17 β -estradiol) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of th ree independent runs on three different days. Each run contained three replicates at each concentration plus six replicates of the [ 3 

### H]-17 β

estradiol only control (total radioactivity) and triplicate rep licates of the non-specific binding (NSB) and solvent controls at the beginning of each run, resulting in a total of 102 samples
per run.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 7 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None

TABLE 4. Molar (M) concentrations in Competitive Binding Assay Run a b

Metalaxyl Positive control Negative control Reference Chemical
19-Norethindrone Octyltriethoxysilane Non-labeled 17 β -estradiol Tubes 79-102 c Tubes 31-54 c Tubes 55-78 c Tubes 7-30 c
10

− 10 3.16 × 10

− 9 10

− 10 Solvent control d
10

− 9 3.16 × 10

− 8 10

− 9 10

− 11
10

− 8 10

− 7 10

− 8 10

− 10
10

− 7 3.16 × 10

− 7 10

− 7 3.16 × 10

− 10
10

− 6 10

− 6 10

− 6 10

− 9
10

− 5 3.16 × 10

− 6 10

− 5 3.16 × 10

− 9
10

− 4 3.16 × 10

− 5 10

− 4 10

− 8
10

− 3 10

− 4 10

− 3 10

− 7 a Data were obtained from pages 14-17, and 19-20 of the study report. b Each tube contains: 10μL of either the test substan ce, positive control, negative control, solvent control, or non-labeled 17 β -estradiol; 390 μL of TEDG+PI buffer with [ 3 H]-17 β -estradiol; and 100 μL of uterine cytosol (with ER), for a total of 500 μL. c Each concentration of each chemical was run in triplicate, for a total of 102 tubes per run. d Solvent is DMSO (~4%) in tubes 7-9.



### Tubes were gently vortexed and incubated for 16-20 hours at 4 ± 2  C. To separate bound from free estradiol, HAP slurry was added to each tube and vortexed once every 5 minutes for 15 minutes. Subsequently, the contents of eac h tube were washed three times as follows: TEDG buffer+PI was added, vortexe d, centrifuged for 10 min at 1000 × g , and the supernatant decanted and discarded. Ethanol wa s added to the HAP pellet remaining in each tube to extract the bound [ 3 

### H]-17 β -estradiol, followed by vortexing, and centrifugation for 10 min at 1000 × g . Aliquots of supernat ant were radioassayed by liquid scintillation counting. The temperature was maintained at 4 ± 2 oC throughout the assay prior to extraction with ethanol.

C. DATA ANALYSIS: For the saturation binding experi ment, total binding and non-specific binding data were modeled via non-linear regression by using Graph Pad Prism v. 5 (GraphPad Software, Inc., La Jolla, CA)], incorporating automatic outlier elimination according to the method of Motulsky and Brown (2006) 1 

### implemented by using the ROUT procedure in Prism v. 5 

### with a Q value of 1.0. Receptor binding data plots were corrected for ligand depletion with the method of 

### Swillens (1995) 2 

### . For the competitive binding assay, similar methods of nonlinear regression (t he Excel addition XLfit version 5.2.0.0 (IDBS, Guildford, Surrey, UK), linear regression mode l 204) were used to fit a curve for 17 β

estradiol, the positive control, and the test substance to the Hill equation formula which incorporated log IC 50 

### as a parameter to be estimated. For parameters reported from the saturation binding experiment ( 

### K d 

### and B max 

### ) and competitive binding experiment (log IC 50 

### and RBA), m ean and standard deviation were calculated for each run, and mean and standard error were calculated for the composite three runs using Microsoft Excel 2007 (v. 12.0.6557.5000;

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outlie rs when fitting data with nonlinear regression: a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics, Vol 7, pp 123-142. 2 Swillens, S. (1995) Interpretation of binding curves obtain ed with high receptor concentrations: practical aid for computer analysis. Molec. Pharmacol. 47(6):1197-1203.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 8 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### Microsoft Corporation, Redmond, WA), and mean and standard error were calculated for the composite three runs with Microsoft Excel 2010.



### 1. Definitions

a. Classification of test material: Classification of the test mate rial is based on the average of three runs. Each run was first in dividually classified as follows:

Interactive = lowest point on the fitted curve within the range of the data is less than 50% (i.e., >50% of the radiolabeled estrad iol has been displaced from the ER).

Not interactive = there are usable data points at or above 10

− 6 

### M and either the lowest point on the fitted response curve within the range of the da ta is above 75% (i.e., <25% of the radiolabeled estradiol ha s been displaced from the ER) or a binding curve cannot be fitted and the lowest average percent binding among concentration groups in the data is above 75%.

Equivocal up to the limit of concentrations tested = there are no data points at or above a test chemical concentration of 10

− 6 

### M and either a binding curve can be fit but ≤ 50% of the radiolabeled estradiol has been displaced from the ER or a binding curve cannot be f it and the lowest averag e percent binding among concentration groups in the data is >50%.

Equivocal = A run is classified as equivocal if it does not fall into any of the categories above.

The categorical classification of each run was assigned a numerical value as follows:

Run Classification Numerical Value Interactive 2 Equivocal 1 Not interactive 0 Equivocal up to the limit of concentrations tested “missing”



### The values for each run were then averaged acro ss runs and the chemical classified using the following ranges:

Test Material Classifi cation Numerical Range Interactive average ≥ 1.5 Equivocal 0.5 ≥ average <1.5 Not interactive average <0.5 Equivocal up to the limit of concentrations tested “missing”Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 9 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### b. Descriptors for receptor binding:

B max 

### : maximum specific binding number (fmol ER/1 00 μg cytosol protein) measures the concentration of active receptor sites K d 

### : dissociation constant (nM), measures the a ffinity of the receptor for its natural ligand IC 50 

### : concentration of the test substance (M) at which 50% of the radioligand is displaced from the receptor Relative Binding Affinity (RBA %): ( IC 50 

### of 17 β -estradiol ÷ IC 50 

### of test substance) × 100 Log RBA: Log 10 

### (IC 50 

### of 17 β -estradiol ÷ IC 50 

### of test substance)

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters are presented in Table 5. 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 th e prepared rat uterine cytosol. The K d 

### for each run was within the expected range of 0.03 to 1.5 nM. Although the Scatchard plots fit straight lines to the data, the concavity observed in the data sets may indicate issues with ligand depletion. Confidence in thes e numbers is high due to the goodness of fit and the small variation among runs.

TABLE 5. Saturation Binding Experiment of [ 3 H]-17 β -estradiol with Estrogen Receptor from Rat Uterine Cytosol a
Parameter Run 1 Run 2 Run 3 Mean ± SE b
R 2 (unweighted) 0.976 0.982 0.980 0.976-0.982
B max (nM) 0.148 0.102 0.080 0.110±0.035
B max (fmol/100 μ g protein) 69.25 79.76 74.65 74.55±3.03
K d (nM) 0.453 0.286 0.255 0.331±0.061 a Data were obtained from page 3 of the study report (MRID 48843501). b The range of R 2 is reported and the mean ± SEM is reported for the other parameters. R 2 Goodness of fit for curve calculated for specific binding



### Figure 1 illustrates the non-specific, sp ecific, and total binding curves for [ 3 

### H]-17 β -estradiol to the estrogen receptor for the three indepe ndent runs. The specific binding reached a plateau in each run, and non-sp ecific binding was less than 20% of total binding. Figure 2 contains the Scatchard plots that illustrate the binding of [ 3 

### H]-17 β -estradiol to the estrogen receptor.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 10 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### FIGURE 1. Binding of [ 3 

### H]-17 β -estradiol to the Estrogen R eceptor during the Saturation Binding Experiment.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 11 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### FIGURE 2. Scatchard Pl ots of the Binding of [ 3 

### H]-17 β -estradiol to the Estrogen Receptor.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 12 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### B. COMPETITIVE BINDING EXPERIMENT: The results from the competitive binding experiments are presented graphically in Figures 3-5. The parameters determined from the competitive binding curves (IC 50 

### , log IC 50 

### , and RBA) are presented in Table 6.

Metalaxyl exhibited >50% inhibition of binding at a concentration of 10

− 3 

### M in Run 2 (classified as “interactive”), however, it had <25% inhibition of binding at concentrations up to 10
− 3 

### M in Runs 1 and 3 (classified as “not in teractive”); therefore it is classified as “equivocal” in this assay (with a mean value of 0.67 across the three r uns; Table 7). There were no apparent alterations in [ 3 

### H]-17 β -estradiol binding at meta laxyl concentrations of 10
− 10 

### to 10
− 3 

### M (90.8-110.8%), with the exception of Run 2 where the specific binding of the radiolabeled ligand declined to 85.5% at a concentration of 10

− 4 

### M and to 46.8% at a concentration of 10

− 3 

### M. The estimated log IC 50 

### was

− 3.8 M for metalaxyl in Run 2, with an RBA of 0.0006%. No precip itation of metalaxyl was noted in the assay tubes, however, the study authors noted that prec ipitation in the assay tubes wa s difficult to assess visually due to the opaqueness of the cytosol solution.

The three reference chemicals generally perfor med as expected. 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 cont rol. Mean specific binding of [ 3 

### H]-17 β

estradiol in the negative c ontrol assay was 44.6% in Run 3 at a concentration of 10

− 3 

### M octyltriethoxysilane, which is lower than the tolerance limit ( ≥ 75%) for the negative control. The study author stated that bi nding of octyltriethoxysilane to [ 3 

### H]-17 β -estradiol at 10

− 3 

### M has been previously observed in othe r studies, although typically accompanied by precipitation (not observed). Exclud ing the highest concentration (10

− 3 

### M) in Run 3, the negative control, octyltriethoxysilane, ha d no effect on specific binding of the [ 3 

### H]-ligand (90.9 to 106.4%).Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 13 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None

TABLE 6. Competitive Binding Assay of Metalaxyl with Estrogen Receptor from Rat Uterine Cytosol a
Parameter Run 1 Run 2 Run 3 Mean ± SE b

R 2 (unweighted) 17 β -estradiol NR NR NR NA 19-norethindrone NR NR NR NA Metalaxyl NR NR NR NA Log IC 50 (M) 17 β -estradiol

− 9.0

− 9.0

− 9.1

− 9.0 ± 0.033 19-norethindrone

− 5.6

− 5.5

− 5.6

− 5.6 ± 0.033 Metalaxyl NA

− 3.8 NA NA IC 50 (M) b 17 β -estradiol 1.00 × 10

− 9 1.00 × 10

− 9 7.94 × 10

− 10 9.31 × 10

− 10 (± 0.69) 19-norethindrone 2.51 × 10

− 6 3.16 × 10

− 6 2.51 × 10

− 6 2.73 × 10

− 6 (± 0.22) Metalaxyl NA 1.58 × 10

− 4 NA NA Log RBA b 19-norethindrone

− 3.40

− 3.50

− 3.50

− 3.47 ± 0.033 Metalaxyl NA

− 5.20 NA NA RBA (%) b 19-norethindrone 0.0398 0.0316 0.0316 0.0344 ± 0.0027 Metalaxyl NA 0.0006 c NA NA a Data were obtained from pages 24-25 of the study report. b All values are reviewer- calculated from the log IC 50 values reported in the study report. For means expressed in scientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean value. c The study author incorrectly reported the RBA for metalaxyl in Run 2 as 0.4 on page 25 of the study report. SE Standard Error NA Not applicable. NR Not reported R 2 Goodness of fit RBA (%) Relative binding affinity

TABLE 7 Binding Classification of Metalaxyl with Estrogen Receptor a
Run 1 2 3 Mean c Binding Classification d Classification category value b 0 2 0 0.67 Equivocal a Data were obtained from pages 24-25 of the study report. b Classification category value: Interac tive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of concentrations tested (“missing”, i.e., not included in calculation of mean). c Mean of three runs expressed to the tenths place d Interactive = mean ≥ 1.5; Equivocal = 0.5 ≤ mean <1.5; Not inte ractive = mean <0.5Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### FIGURE 3. Percentage [ 3 

### H]-E2 Bound to the Estrogen R eceptor in the Presence of Unlabeled E2, 19-Norethindrone, Octylt riethoxysilane or Metalaxyl, Run 1.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### FIGURE 4. Percentage [ 3 

### H]-E2 Bound to the Estrogen R eceptor in the Presence of Unlabeled E2, 19-Norethindrone, Octylt riethoxysilane or Metalaxyl, Run 2.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 16 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### FIGURE 5. Percentage [ 3 

### H]-E2 Bound to the Estrogen R eceptor in the Presence of Unlabeled E2, 19-Norethindrone, Octylt riethoxysilane or Metalaxyl, Run 3.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 17 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None



### C. PERFORMANCE CRITERIA: To ensure that the compe titive binding assay functioned properly, each run was evaluated usi ng the criteria shown in Table 8.

TABLE 8. Criterion a Tolerance Limit(s) Value Yes No

17 β -estradiol fitted curve parameters Log e residual SD ≤ 2.35 ≤ 1.95 X Top (% binding) 94 to 111 97 to 109 X Bottom (% binding)

− 4 to 1

− 2 to 5 X c Hill Slope (Log 10 (M)

− 1 )

− 1.1 to

− 0.7

− 1.7 to

− 0.9 X d Weak Positive control (19-norethindrone) fitted curve parameters b Log e residual SD NA ≤ 1.59 NA Top (% binding) NA 98 to 100 NA Bottom (% binding) NA

− 2 to 1 NA Hill Slope (Log 10 (M)

− 1 ) NA

− 1.4 to

− 0.9 NA Solvent concentration DMSO ≤ 10% 4% X Negative control (octyltriethoxysilane) does not displace more than 25% of [ 3 H]-17 β -estradiol from the ER on average across all concentrations ≤ 25% ≤ 55.4 X e

a Data were obtained from page 28-33 and 61-69 of the study report. b The EPA Guideline does not define a set of tolerance limits for 19-norethindrone. Acceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable as they show 19-nor ethindrone to be an accepta ble weak positive control. c Bottom % binding was high in Run 2 (5%) and within the tolerance limits in the other two runs. d Hill Slope was low in Run 2 (

− 1.7) and within the tolerance limits in the other two runs. e Reviewer-calculated. The minimum specific binding of [ 3 H]-17 β -estradiol in the negative control assays was 44.6% in Run 3 at 10

− 3 M; however, excluding this va lue at the highest test concen tration, octyltriethoxysilane did not displace more than 25% of [ 3 H]-17 β -estradiol from the ER (minimum specific binding of 90.9%). NA Not applicable



### Additionally, the curve for the reference material showed that increasing concentrations of unlabeled 17 β -estradiol displaced [ 3 

### H]-17 β -estradiol in a manner consistent with one-site binding, as indicated by a descent fr om 90.4-103.8% to 7.4-9.4% binding over approximately an 81-fold increase in concentr ation of the test chemical (i.e., covering approximately 2 Log units).

Metalaxyl was tested over a concen tration range that fully define d the top of the curve. The percent binding at this top plateau 99.8-110.8% wa s within 25 percentage points of the value for the lowest concentration of the estradiol standard 95.1-107.4%.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Metalaxyl was classified as “non-interactive” in the first and third independent valid runs, and was classified as “interactive” in the second independent valid run. Although this results in metalaxyl ha ving a final classification of “equivocal,” it is important to note that the “interactive” cate gorization in the second valid independent run appears to be based on an anomaly in the data, as ER binding was >85% atEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 18 of 18 METALAXYL / 113501 OCSPP 890.1250/ OECD None


### all metalaxyl concentrations but 10

− 3 

### M in this run. Additionally, the test material was negative for binding in all other runs of the assay (including a run conducted on 01- September-11 for which the weak positive control did not meet the assay acceptance criteria). Although no specific problems or issu es could be identified with the second valid independent run to explain the inconsistency of this run with the others conducted for metalaxyl, it has been shown that when the competitive binding curve drops sharply over a single log increase in test subs tance concentration, as exhib ited by metalaxyl in the second assay run, follow up K i 

### assays generally show that the test substance is typically not a true competitive inhibitor (Laws et al, 2006) 3.



### B. AGENCY COMMENTS: The protein concentrations used in the saturation binding runs varied between each run, and were approximately 3- to 6-fold greater than recommended. The K d 

### for [ 3 

### H]-17 β -estradiol was 0.331 nM and the estimated B max 

### was 74.55 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 S catchard plots, points below 20 fmol bound ligand /100 μg reached a plateau compatible w ith ligand depletion at less than 0.3 nM [ 3 

### H]- 17 β -estradiol; this pheno mena was correctly accounted fo r in the Scatchard analysis.

For 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 we re generally met; however, octyltriethoxysilane 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 slightly outside of th e 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 (declining to 46.8% in Run 2); however, had <25% inhibition of binding at concentrations up to 10

− 3 

### M in the other two runs and is therefore classified as “e quivocal” in this assay.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The protein concentrations used in the satu ration binding runs varied between each run, and were approximately 3- to 6- fold greater than recommended.

 Cytosol preparation (July 30, 2011) and La st Saturation Assay run (August 7, 2011) occurred more than 90 days before last Competitive Assay run (November 17, 2011).

 Performance criteria were not met fo r octyltriethoxysilane in Run 3 at 10

− 3 

### M.

3 Laws, SC, Yavanhxay S, Cooper RL, Eldridge JC. Nature of the binding interaction for 50 structurally diverse chemicals with rat estrogen receptors . Toxicol Sci. 2006 Nov;94(1):46-56.

# DATA EVALUATION RECORD

METALAXYL

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012

(Revisions to MRID 48617603 to include laboratory proficiency data; Main study was originally reviewed under TA 2-46-2012)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Michelle Sharpe-Kass, M.S. Date: 6/11/2012

Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 7/16/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/24/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/24/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).METALAXYL I 113501 Estrogen Receptor Transcriptional Activation (2011) I Page 1 of 10 OCSPP 890.1300/ OECD 455



### Primary Reviewer: Sheila Healy, Ph.D. 

### Health Effects Division Secondary Reviewer: Minerva Mercado Ph.D. DABT 

### Health Effects Division

# Sign~{~e·**_____..~i.--;::.-=..,__** __ Date:**-----=~~~-=--;;r--**

Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Transcriptional Activation (Human cell Line, HeLa-9903); OCSPP 890.1300; OECD 455.



### PC CODE: 113501 

### DP BARCODE: D399227



### TXR#: 0054219 

### CAS No.: 57837-19-1



### TEST MATERIAL (PURITY): Metalaxyl (100.4% (first analysis); 98.2% (second analysis))



### SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate



### CITATION: Willoughby, J.A. (2011) Metalaxyl: Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa-9903)) Final Report. CeeTox, Inc, Kalamazoo, MI. Laboratory Project ID: 9143V-100326ERTA, December 7, 2011. MRID 48617603. Unpublished.



### SPONSOR: Willoughby, J.A. (2012). Supplemental Information

- Laboratory Proficiency Data for ER TA assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, MI. July 19, 2012. MRID 48843501. Unpublished.

Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL



### TEST ORDER#: CON-113501-18



### EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID 48617603), hERa-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 

### Min0.1% DMSO in
two independent runs. The experiments were performed using 96-well plates and each metalaxyl concentration was tested in 6 wells/plate in each run. Cells were exposed to the test agent for 24±2 hours to induce reporter (luciferase) gene products. Luciferase expression in response to activation of the estrogen receptor by metalaxyl was measured using a proprietary luciferase activity assay.

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

### M. Cytotoxicity of 17 a

### methyltestosterone in the first run prevented observation of its log PCso. The mean RPCMax for metalaxyl was 0.5% for the first run and 1.1 % for the second, and the associated PCMax was 10

- 5


### M for the first run and 10- 7 

### M for the second run.Estrogen Receptor Transc riptional Activation (2011) / Page 2 of 10 METALAXYL / 113501 OCSPP 890.1300/ OECD 455


### 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.

This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor Transcriptional Activation assay (OCSPP 890.1300).

COMPLIANCE: Signed and dated GLP, Quality Assurance and Data Confidentiality statements were provided.Estrogen Receptor Transc riptional Activation (2011) / Page 3 of 10 METALAXYL / 113501 OCSPP 890.1300/ OECD 455



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc.
Location: Kalamazoo, MI Study Director: J.A. Willoughby Other Personnel: K. Rutherford (Director Laboratory Operations); D. Blakeman (Senior Scientist); C. Haines (Scientist); S. McColl ey (Scientist); B. Wallace (Lead Cell Culture Scientist); and C. Toole (Director of Project Management) Study Period: May 26, 2011 to December 7, 2011



### 2. Test Substance: Metalaxyl Description: White solid, MW 279.33 Source (Cat#): Nation's Ag. L.L.C (not reported) Lot/Batch # (Exp. Date): 20100540 (July 26, 2011 - first analysis) (June 13 2012 - second analysis) Purity: 100.4% (first analysis) 98.2% (second analysis) Solubility: Not Reported (NR) Volatility & Stability: NR Storage conditions: Ambient CAS #: 57837-19-1 Structure:

CH 3 N O

CH 3 O CH 3 CH

3 O O CH

3



### 3. Reference substances 17 β -estradiol (strong estrogen; positive control) Supplier: NR Catalog and Batch #: NR Purity: NR CAS # : 50-28-2



### 17 α -estradiol (weak estrogen) Supplier: NR Catalog and Batch #: NR Purity: NR CAS # : 57-91-0


### Corticosterone (negative compound) Supplier: NR Catalog and Batch #: NR Purity: NR CAS # : 50-22-6



### 17 α -methyltestosterone (very weak agonist) Supplier: NR Catalog and Batch #: NR Purity: NR CAS # : 58-18-4



### 4. Vehicle(s)
Solvent: DMSO Solvent control (final conc.): 0.1%Estrogen Receptor Transc riptional Activation (2011) / Page 4 of 10 METALAXYL / 113501 OCSPP 890.1300/ OECD 455



### B. METHODS

1. Cell Culture: Stably-transfected hER α -HeLa-9903 cells were obtained from the Japanese Collection of Research Bioresour ces Cell Bank and were verified to be free of mycoplasma infection using DNA fluorochrome analysis. Ce lls were maintained in Eagles Minimum Essential Medium without phenol red, supplem ented with 60 mg/L kanamycin and 10% dextran-coated charcoal-treated fetal bovine serum (source no t reported) in an incubator under 5% CO 2 

### at 37oC. Upon reaching 75-90% confluen ce, cells were subcultured at least twice prior to exposure to the test material. Th e cells used in this study were Passage 35 for the rangefinder, Passage 17 for the first run and new cells were brought up from cryopreservation and used at passage 23 for th e second run prior to seeding into plates.

2. Transcriptional Activation Assays: For each test, cells were plated at a density of 1  10 4 

### cells/100 μL medium/well in a 96-well plate and a llowed to attach for at least 3 hours. The growth media was replaced with media contai ning serial log dilutions of metalaxyl in DMSO (0.1% final concentration). Cells we re incubated for 24±2 hours at 37±1°C. Transcriptional activation of the estrogen receptor was determined as described in CeeTox Standard Operating Protocol (SOP) 2041. A lis t of reagents was provided, but the assay reagent was classified as propr ietary information, and a descri ption was not provided. Runs 1 and 2 for the main assay were conducted on June 14 and September 8, 2011, respectively.

3. Preliminary Test: A preliminary test evaluating concentrations ranging from 10

− 3 

### to 10
− 10 

### M was conducted to determine the appropri ate concentration range and to determine concentrations resulting in insolubility and/or cytotoxicity. Cytotoxicity was determined by PI uptake. An endoscope was used to assess precipitation in each sample.

4. Proficiency Chemicals: Responsiveness of the test system was evaluated on March 5, April 12 and April 28, 2011(MRID 48843501), using cells at passage 15, 25 and 28, respectively. Based on dates and passage numbe r, it is unlikely these cells are from the same frozen stock as the cells used in the main assay. The cells were tested using the following proficiency chemicals, each chemi cal tested in duplicate on separate days:

Compound CAS No. Concentration Range (M) Expected Response a Notes
Diethylstilbestrol (DES) 56-53-1 10

− 14 to 10

− 8 Positive --- 17 α -Ethynyl estradiol (EE) 57-63-6 10

− 14 to 10

− 8 Positive --- Hexestrol 84-16-2 10

− 13 to 10

− 7 Positive --- Genistein 446-72-0 10

− 12 to 10

− 5 Positive Cytotoxic at 0.01 b , 0.1, and 1 mM Estrone 53-16-7 10

− 12 to 10

− 6 Positive --- Butyl paraben 94-26-8 10

− 11 to 10

− 4 Positive Cytotoxic at 0.1 b and 1 mM 1,3,5-Tris(4- hydroxyphenyl)benzene c 15797-52-1 10

− 12 to 10

− 5 Positive Cytotoxic at 100μM. PC max approx. 50% of PC. Binds to hER α and has ER antagonistic activity Dibutyl phthalate (DBP) 84-74-2 10

− 11 to 10

− 4 Negative d Cytotoxic at 1 mM Atrazine 1912-24-9 10

− 11 to 10

− 4 Negative Cytotoxic at 1 mM b Corticosterone 50-22-6 10

− 10 to 10

− 4 Negative If not cytotoxic at 1 mM, then that should be the highest tested concentration a Positive = RPC Max ≥ 10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPC Max fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs b Cytotoxicity is expected to be close to 80% at this concentration. c Compound selected to challenge solubility and cytotoxicity d DBP is negative for ER α mediated transcriptional activation, but may not be negative for non-ER β mediated transcriptional activation. A positive result would indicate that the sy stem is detecting activity other than that due to pure ER α , and is therefore unacceptable.Estrogen Receptor Transc riptional Activation (2011) / Page 5 of 10 METALAXYL / 113501 OCSPP 890.1300/ OECD 455



### 5. Reference Chemicals: To ensure the stability of the response from the cell line, eight concentrations of each of the following reference chemicals were included in each plate in the current assay, along w ith the test chemical:

Reference Chemical CAS No. Concentration Range Class 17 β -estradiol 50-28-2 10

− 15 to 10

− 8 Strong estrogen 17 α -estradiol 57-91-0 10

− 13 to 10

− 6 Weak estrogen Corticosterone 50-22-6 10

− 11 to 10

− 4 Negative compound 17 α -methyltestosterone 58-18-4 10

− 12 to 10

− 5 Very weak agonist



### 6. Data analysis: To obtain the relative transcripti onal activity compared to the 1 nM E2 positive control (PC), the luminescence signals from the concurrent plate were analyzed by subtracting the mean value of the vehicle control from each well value to normalize the data; each normalized value was then divided by the mean value of the normalized PC. The resulting value was multiplied by 100 in order to express relative transcriptional activity as a percentage of the PC. The test material was defined as negative for inducing estrogen receptor transcriptional activation if the RPC Max 

### <PC 10 

### in at least 2 of 2 runs. Log EC 50 

### and Hill slope values were calculat ed only if a positive response wa s observed. Coefficients of variation (CV) were calculated for the lumi nescence data triplicates. Concentrations showing >20% cytotoxicity or evidence of insolubility were excluded from analyses.

7. Definitions

EC 50 

### = concentration of agonist that induces a response ha lfway between the baseline (bottom) and maximum (top) response

PC 10 

### = concentration of a test chemical at which the response is 10% of the response induced by the positive control (E2 at 1 nM) in each plate

PC 50 

### = concentration of a test chemical at which the response is 50% of the response induced by the positive control (E2 at 1 nM) in each plate

RPC Max 

### = maximum level of response induced by a test chemical, expressed as a percentage of the response induced by the positive control (1 nM E2) on the same plate

PC Max 

### = concentration of a test chemical inducing the RPC Max



### II. RESULTS

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the transcriptional activation assays, preliminary cytotoxicity and precipitation assays were conducted. Cytotoxicity was observed at 10

− 3 

### M of metalaxyl (Table 1). Based on this result, metalaxyl concentrations of 10

− 4 

### to 10
− 11 

### M were selected for the main assays.Estrogen Receptor Transc riptional Activation (2011) / Page 6 of 10 METALAXYL / 113501 OCSPP 890.1300/ OECD 455

TABLE 1. Preliminary Test for Solubility, Cytoto xicity, and Concentration-Selection for Metalaxyl a Concentration (M) % Viability Comments
10

− 3 78 Cytotoxic
10

− 4 90 10

− 5 90 10

− 6 91 10

− 7 90 10

− 8 93 10

− 9 92 10

− 10 93 a Data were obtained from page 20 of the study report.



### B. POSITIVE AND NEGATIVE REFERENCE CHEMICALS

1. Proficiency Chemicals: The laboratory proficiency assa ys using the required reference compounds were not included in th e original study report, but were provided to the Agency at a later date (MRID 48843501). The responsiven ess of cells to the required proficiency chemicals was conducted in duplicate on different days for each chemical. The reported responses are summarized in Table 2a.

TABLE 2a. Proficiency Chemicals

Compound Expected Response Lab Response
Run 1 Run 2 Run 3 Diethylstilbestrol Positive Positive Positive NA 17 α -Ethynyl estradiol Pos itive Positive Positive NA Hexestrol Positive Positive Positive NA Genistein Positive Positive Positive NA Estrone Positive Positive Positive NA Butyl paraben Positive Positive Positive NA 1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive NA Positive Positive Dibutyl phthalate Negativ e Negative Negative NA Atrazine Negative Ne gative NA Negative Corticosterone Negative Negative NA Negative
From MRID 48843501 NA = not applicable. The chemical was not tested at this time.



### 2. Reference Chemicals: Values derived from the concentration response curve ( e.g ., log PC 50 

### , log PC 10 

### , log EC 50 

### , Hill slope) for the four concurrently run reference materials are included in Table 3. The acceptance criteria we re met for all reference chemicals with the exception of 17 α -methyltestosterone in the first run, where the maximum response was 16.5%. Additionally, cytotoxicity was noted at 10

− 5 

### M of 17 α -methyltestosterone and 10
− 4 

### M of corticosterone in the fi rst run, and in the second run 17 β -estradiol (1 nM) and 17 α -estradiol (100 nM) demonstrated res ponsiveness well above the PC (143.8% and 123.6% RTA, respectively).Estrogen Receptor Transc riptional Activation (2011) / Page 7 of 10 METALAXYL / 113501 OCSPP 890.1300/ OECD 455

TABLE 3. Performance Criteria for Reference Chemicals a

Reference Chemical Parameter Acceptable Range Values Acceptable Run 1 Run 2 Yes No
17 β -estradiol Log PC 50 − 11.4 to

− 10.1

− 10.2

− 10.6 X Log PC 10 <

− 11

− 11.7

− 11.5 X Log EC 50 − 11.3 to

− 10.1

− 10.3

− 10.5 X Hill Slope 0.7 to 1.5 0.8 1.3 X Test range 10

− 14 to 10

− 8 M 10

− 15 to 10

− 8 M 10

− 15 to 10

− 8 M X
17 α -estradiol Log PC 50 − 9.6 to

− 8.1

− 8.6

− 8.7 X Log PC 10 − 10.7 to

− 9.3

− 9.8

− 9.7 X Log EC 50 − 9.6 to

− 8.4

− 8.7

− 8.7 X Hill Slope 0.9 to 2.0 1.2 1.2 X Test range 10

− 12 to 10

− 6 M 10

− 13 to 10

− 6 M 10

− 13 to 10

− 6 M X
Corticosterone Test range 10

− 10 to 10

− 4 M 10

− 11 to 10

− 4 M 10

− 11 to 10

− 4 M X
17 α -methyltestosterone Log PC 50 − 6.0 to

− 5.1 NC

− 5.1 Run 1 Log PC 10 − 8.0 to

− 6.2

− 6.6

− 6.2 X Test range 10

− 11 to 10

− 5 M 10

− 12 to 10

− 5 M 10

− 12 to 10

− 5 M X a Data were obtained from page 12 and 23 of the study report.



### C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls: Data for the vehicle and pos itive controls are included in Table 4. The mean TA values for the vehi cle control were 16658 and 22954 for Runs 1 and 2, respectively, and the mean TA values for the positive control were 1151283 and 619650 for Runs 1 and 2, respectively. The induction fo r the positive control ranged from 27- to 69- fold. The mean normalized values for the positive control were 1134625 and 596696 for Runs 1 and 2, respectively. The PC 50 

### (50% of the maximum respons e) for E2 in this assay were 567313 and 298348 for Run 1 and 2, respectively, and the PC 10 

### (10% of the maximum response) were 113463 and 59670 for Run 1 and 2, respectively. Both PC 10 

### values are greater than respective vehi cle control values + 2SD.

TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a

Sample Vehicle Control Positive Control b Normalized Positive Control b
Runs Mean SD Mean SD Fold Induction c Mean SD
1 16658 4730 1151283 353650 69 1134625 353650 2 22954 4536 619650 55512 27 596696 55512 a Data were calculated by the reviewer from da ta obtained on pages 31-32 of the study report. b Positive control was 17 β -estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC)



### 2. Test Material: Relative (to the PC) transcriptional activation at each concentration of metalaxyl during the two assay runs is presen ted in Table 5. The concentration-response curves depicting fold induction of relative transcriptional activation are presented in Figure 1. The mean RPC Max 

### 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. Because theEstrogen Receptor Transc riptional Activation (2011) / Page 8 of 10 METALAXYL / 113501 OCSPP 890.1300/ OECD 455



### RPC Max 

### < PC 10 

### in both runs, metalaxyl was consider ed negative for estrogen receptor transcriptional activation in this test system.

TABLE 5. Relative Transcriptional Activation (RTA) of Metalaxyl a

Parameter RTA (mean ± SD); % of Positive Control (PC) Run 1 Run 3 Conc. (M) Mean SD Mean SD
10

− 4 0.4 0.2

− 0.3 1.1 10

− 5 0.5 0.4 0.9 1.0 10

− 6 0.4 0.5 0.4 0.8 10

− 7 0.5 0.6 1.1 0.9 10

− 8

− 0.2 0.2 0.1 1.3 10

− 9

− 0.2 0.4 0.8 1.2 10

− 10

− 0.1 0.2

− 0.2 0.9 10

− 11

− 0.1 0.3 0.4 1.1 Log EC 50 b NA NA Hill Slope b NA NA RPC Max 0.5 1.1 PC Max 10

− 5 10

− 7
PC 50 NA NA PC 10 NA NA a Data were obtained from pages 21-22 of the study report. NA = Not ApplicableEstrogen Receptor Transcriptional Activation (2011) / Page 9 of 10 METALAXYL / 113501 OCSPP 890.1300/ OECD 455



### FIGURE 1. Fold Induction of Relative Tr anscription Activation (RTA) of Metalaxyl Compared to the Positive Control.

3. Performance Criteria: The acceptance criteria were met fo r all reference chemicals with the exception of 17 α -methyltestosterone in the first run, where the maximum response was 16.5%. For 17 α -methyltestosterone, the mean RPC Max 

### was 16.5% for the first run and 52.8% for the second run. Additionally, cytotoxicity was noted at 10

− 5 

### M of 17 α -methyltestosterone and 10

− 4 

### M of corticosterone in the fi rst run, and in the second run 17 β -estradiol and 17 α -estradiol demonstrated resp onsiveness well above the PC (123.6-143.8%). Cytotoxicity should not be observed at any level for the reference chemicals, and the maximum responsiveness sh ould be observed in the positive control, 1 nM 17 β -estradiol. Due to this, the ability of th is test system to observe transcriptional changes in response to chemical exposure was not considered valid.Estrogen Receptor Transc riptional Activation (2011) / Page 10 of 10 METALAXYL / 113501 OCSPP 890.1300/ OECD 455



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The suitable top concentration of metalaxyl for use in the transcriptional activation assays was 10

− 4 

### M. There was no significant cytotoxicity at any of the concentrations tested in both independent runs. In two independent runs of the transcriptional activ ation assay, metalaxyl did not result in an increase in luciferase activity at any other concentrations test ed; metalaxyl is not an agonist of the human estrogen receptor alpha (hER α ) in the HeLa-9903 model system.

B. AGENCY COMMENTS: Metalaxyl was tested up to the limit of cytotoxicity 10

− 4 

### M. The mean RPC Max 

### 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. Because the RPC Max 

### < PC 10 

### in both runs, metalaxyl was considered ne gative for estrogen receptor transcriptional activation in this test system. However cytot oxicity was observed at higher concentrations of 17 α -methyltestosterone in the first r un, preventing observation of its log PC 50



### . Although the conditions of this assay were not optimal to detect very weak activity, metalaxyl responses were similar to those of the negative control corticosterone and not comparable to the responses of 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 concentration possi ble 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.

C. STUDY DEFICIENCIES: The following deficiencies were noted:

 In both assays, cytotoxicity was observed in cells treated with corticosterone and 17 α -methyltestosterone.

 Certificates of Analyses were not pr ovided for the reference chemicals.

 The source of the fetal bovine serum was not provided.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 1 of 75 Data Requirement: EPA DP Barcode 401220

OECD Data Point 229

EPA MRID 48685802
EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test material: Metalaxyl Purity: 100.4%

Common name Chemical name: IUPAC: Methyl N-(methoxyacetyl)-N-2,6-xylyl)-DL-alaninate
CAS name: Methyl 2-{[2,6-dimethylphenyl)methoxyacetyl]amino}propionate

CAS No.: 57837-19-1
Synonyms: Metalaxyl TC.

EPA PC Code: 113501

Primary Reviewer: Joan Gaidos Signature:
Senior Scientist, Cambridge Environmental, Inc. Date: 12/6/2012

Secondary Reviewer: Teri S. Myers Signature:
Senior Scientist, Cambridge Environmental, Inc Date: 12/20/2012

Primary and Final Additional Reviewer: Robin Sternberg Signature:
Wildlife Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 02/27/2014, 05/27/2015

Additional Reviewer: Anita Ullagaddi Signature: No longer with EPA
Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date:

Date Evaluation Completed: 05/27/2015 Digitally signed by ROBIN STERNBERG DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN STERNBERG, dnQualifier=0000039126 Date: 2015.06.01 16:36:07 -04'00'

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 2 of 75 CITATION: York, D.O. 2012. Metalaxyl - Short-term Reproduction Assay with the Fathead Minnow ( Pimephales

promelas ). Performed by Smithers Viscient, Wareham, Massachusetts, Laboratory Project No. 13694.6143.

Submitted by Metalaxyl EDSP Consortium, Burr Ridge, Illinois. Completion date March 2, 2012.

The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven
screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the

strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within
the assay, where available, and redundancy across the battery. Thus, the results of each individual assay

should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has
the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay
results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

Disclaimer: The guideline recommendations in this DER template are offered as a general reference to aid in
preparation of the DER. The purpose of these recommendations is not to serve as substitute for the Test Guidelines,
nor to provide any guidance on how the study should be conducted.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 3 of 75 EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay of metalaxyl with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions. Adult fish (16 spawning groups; 2 males and 4 females in each
group; 21 weeks old) were exposed to metalaxyl (100. 4% purity) at nominal concentrations of 0 (negative
control), 1.0, 10.0, and 100 mg a.i./L; mean-measured concentrations were <LOQ (<0.066, negative control),

1.1, 10.0, and 104 mg a.i./L. The test system was maintained at 25 to 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, andhigh treatment groups, respectively. Male body weight exhibited a significant (p<0.05), generally concentrationdependent reduction of 7 to 16% across all treatment levels compared to the negative control. There were no
notable differences in secondary sex characteristics or clinical signs for any treatment group compared to the
negative control.

In the negative control, fecundity was 19 eggs/female/day/replicate, and fertilization success averaged 96%.
Fecundity and fertility were not significantly affected at the low and mid treatment levels. However, there were
no eggs available for assessment at the high treatment level.

At the high treatment level, there was a 96% decrease in female plasma vitellogenin (VTG) concentrations
that was not statistically significant (p=0.092). However upon visual observation of the data, this reduction in

VTG was considered to be biologically significant. Female gonado-somatic index (GSI) was significantly reduced
(p<0.05) by 23% at the high treatment level compared to the negative control. Male tubercle score was
significantly reduced (p<0.05) at the high treatment level compared to the negative control.

There were no notable differences in gonadal stage. In male fish, there was an increased incidence of
increased percentage of spermatogonia at the mid and high treatment levels and asynchronous gonad
development at the high treatment level. In female fish, there was decreased yolk synthesis at the high
treatment level. There was an increase in the incidence of atretic oocytes and granulomatous inflammation in
females in the high treatment group. However, given that these findings were occasionally coincident with the presence of protozoal spores and were also observed in negative control females (at a lower incident rate),

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 4 of 75 these lesions were not considered to be treatment related. Plasma testosterone and 17ß-estradiol were not
measured in this study.

All of the performance criteria were met with the exception of the following: the dissolved oxygen concentrations

fell to ca. 42% of saturation (3.3 mg/L) on Day 6 which is less than the recommended ≥ 4.9 mg/L ( ≥ 60% saturation); however, gentle aeration was added such that dissolved oxygen concentrations remained above

60% of saturation for the remainder of the test. This deviation did not impact the interpretation of the study.

This assay satisfies the EDSP Tier 1 Test Order requirements for a Fish Short-Term Reproduction Assay
(OCSPP Guideline 890.1350).

Results Synopsis

Test Organism age at test initiation: 21 weeks Mean body weight at test initiation (if measured): Male 3.4 g; Female 1.6 g
Mean length at test initiation (if measured): Not reported.
Test Type (Flow-through, Static, Static Renewal): Flow-throughData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 5 of 75Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Metalaxyl.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF1.1NoNoNoNoNoNoNoNoNoNoNANANANA10NoNoNoNoNoNoYesNoNoNoNANANANA104Yes3Yes3YesNoNoYesYesYesNoNoNANANANAAbbreviations:Conc.Concentration.Diff.Difference.E217ß-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.3No eggs were produced at the highest test concentration. Therefore fecundity was 0, and there were no fertility data to analyze.

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Page 6 of 75 I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP 890.1350: “Fish Short-Term Reproductive Assay” and OECD 229 (2009). The

following deviations were noted:

1. The physico-chemical properties of the test material were not reported.

2. The total organic carbon, unionized ammonia, and residual chlorine in the test water were not reported.
The OCSPP 890.1350 performance criteria establish maximum levels for these values, and it is

unclear if the maximum recommendations were exceeded.

3. Dissolved oxygen concentrations in the test solutions remained at or above 60% of saturation (4.9
mg/L) for most of the exposure period. However, dissolved oxygen concentrations fell to ca. 42% of saturation (3.3 mg/L) on Day 6, and gentle aeration was added. Dissolved oxygen concentrations

remained above 60% of saturation for the remainder of the test. OCSPP 890.1350 guideline requirements recommend dissolved oxygen (DO) ≥ 4.9 mg/L ( ≥ 60% air saturation).

These deviations do not impact on the interpretation of the study.

COMPLIANCE: Signed and dated No Data Confidentiality, GLP, and Quality Assurance

statements were provided. This study was conducted in compliance with all
pertinent U.S. EPA Good Laboratory Practice regulations with the following
exceptions: routine dilution water and food contaminant screening analyses
were conducted following standard validated methods, this exception has no
impact on the study results.

A. TEST MATERIAL: Metalaxyl; CAS# 57837-19-1

Description: Not reported

OECD recommends describing water solubility, melting/ boiling point stability in water and light, pKa, Pow or
Kow, vapor pressure of test compound, expiration date.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

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Page 7 of 75 Lot No./Batch No. : 20100540

Purity: 100.4% (tested as 100%)

Impurities: None reported

Stability of Compound: Analytical verification of the test material from Days 0, 7, 14, and 21 yielded

recoveries of 100% to 110% of nominal concentrations. Stock solution (FETAX) fortified with metalaxyl at concentrations of 0.005, 1.0, and 100

mg a.i./L yielded recoveries of 94.3 to 105%. There was no pattern of
decline during the study period, indicating the test material was stable under

the test conditions.

Storage Conditions of

Test Chemicals: Stored at room temperature in dark, ventilated cabinet.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 8 of 75B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead Minnow EPA recommends fathead minnow (Pimephales promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):Not reportedWere fish obtained from asingle laboratory stock?Yes EPA recommends that fish be from a single laboratory stock.Were acclimation conditionssame as definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive

test.Acclimation period:16 days (pre-exposure) EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 9 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:No mortalities were reported during the7 daysprior to pre-exposure period.Fish did not receive any treatment fordiseasein the two weeks prior tospawning (pre-exposure period).Behavioral abnormalities or clinical signswere not reported. EPA recommends that mortality during the 7 days prior to the pre - exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA recommends that the fish be rejected. If mortality is between 5 -10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur

durin g this extended acclimation period, EPA recommends that the fish not be used.Type of food:Frozen brine shrimp EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body

condition.Source of food:Not reportedRepresentative samples of the food sourcewere periodically analyzed for pesticides,PCBs, and toxic metals by GeoLabs, Inc.and analyte levels were below LOC specifiedby ASTM (2005).Frequency of feeding:2 times/dayDetails on feeding:NoneData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 10 of 75Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation: Ca. 21 weeks EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months

old.Mean weight of males at testinitiation (if determined):3.4 gBased on 20 males from the testpopulationused to stock aquaria forpre-exposure phase EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for each sex.Range of individual weights(males) at test initiation (ifdetermined):3.0 to 4.0Individual weights within ±20% of theestimated meanMean weight of females at testinitiation (if determined):1.6 gBased on 20 females from the testpopulationused to stock aquaria forpre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):1.4 to 1.8Individual weights within ±20% of theestimated meanMean length of males at testinitiation (if determined):Not reportedData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 11 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase:16 days EPA recommends a minimum of 14 days.Were pre-exposure conditionsidentical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:36Extra replicates were established toaccount for a potential lack of spawninginsome chambers and/or mortalityduring this phase. EPA recommends that additional tanks set up at the beginning of pre -exposure will ensure that sufficient replicates with the correct sex ratio are available for the definitive test.Number of males per tank:2Number of females per tank:4Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 12 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsPre-exposure fecundity:>15 eggs/female/reproductive day/replicate EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Number of spawns during preexposure:>two times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Details on pre-exposure:NoneData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 13 of 75C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-throughEPA recommends the use of a flowthrough system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this assay.Type of flow-through dilutionsystem:Intermittent-flowproportional diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate: Ca. 45 mL/minute or 2.7L/hr; 6.5 volumereplacements/dayCalculated by the reviewer based on 0.5L/cycle, 130 cycles/24 hrs and 60minutes/hr Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.1U.S. Environmental Protection Agency (EPA). (2011). Correctionsand Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 14 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems:The mixing chamber of the diluter waspositioned over a magnetic stir plate andmixed with a magnetic stir bar.Thecontinuous stirring aided the solubilizationof the test substance with the dilutionwater.Flow-splitting chambers were used topromote further mixing and to equallydistribute the test solutions through glassdelivery tubes to4 replicate test vessels.Flow splitting accuracy not reported.Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy
is within 10%.Aeration?YesOn test day 6, oxygen levels fell to 42%of saturation. Aeration was added and thedissolved oxygen levels increased to>60% of saturation within a few hours. EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (<60%
saturation).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 15 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water:Well waterAerated prior to use EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?YesTest vessel type/materials:Glass aquaria withsilicone adhesiveEPA and OECD recommend that watercontact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:20 L; 20 cm wide x 39cm long x 25 cm high;solution depth 13 cm EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 16 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsFill volume:10 L EPA recommends 10 L solution per tank.Spawning substrate material:Aged PVC pipe EPA recommends that each tank contain

three semi - circular spawning substrates, e.g., aged PVC pipe, 10 - 20 cm in length, split lengthwise.Spawning substrate size:4 inch diameter PVC cutlengthwisein half(arches) and into 7 cmsectionsAdditional details on exposuresystem:NoneTable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMean1Measurement IntervalGuideline RecommendationsTemperature(°C) 25 26 25.5Continuously (upper (control replicate B)and lower (control replicate D) waterbaths). Weekly(each concentration andcontrol aquarium at exposure initiation andsubsequently in alternating replicates) EPA recommends temperature 25±1 o C;

inter -replicate and inter-treatment differentials should not exceed 1 o C.pH6.97.67.25 EPA recommends pH 6.5 to 9.0.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 17 of 75ParameterMinimumMaximumMean1Measurement IntervalGuideline RecommendationsDissolved oxygen(mg/L)3.3 (42%)8.3 (100%)5.8(71%)Weekly (each concentration and controlaquarium at exposure initiation andsubsequently in alternating replicates) EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity(mg/L as CaCO3)22 28 25Weekly(one replicate of one treatment level andthe control alternatingbetween treatmentlevels and replicate aquaria) EPA recommends total alkalinity >20 mg/L as CaCO 3.Hardness(mg/L as CaCO3)52 64 58Total organic carbon(mg/L)0.21 (July)0.33 (June)NotreportedTwice (June and July), dilution water EPA recommends that total organic carbon in dilution water be <2 mg/L.Unionized ammonia(Pg/L)NotreportedNot reportedNotreportedNot reported EPA recommends that unionized ammonia in the dilution water be <1 P g/L.Residual chlorine(Pg/L)NotreportedNot reportedNotreportedNot reported EPA recommends that residual chlorine in dilution water be <10 P g/L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 18 of 75ParameterMinimumMaximumMean1Measurement IntervalGuideline RecommendationsConductivity(Pmhos/cm)280360 320Weekly(each concentration and controlaquarium at exposure initiation andsubsequently in alternating replicates) General recommendations for frequency of measurements: EPA recommends that temperature, pH, and dissolved oxygen be measured in all test tanks at least weekly and that hardness and alkalinity be measured in controls and in one tank at

the high est test concentration at least weekly. In addition, continuous temperature monitoring of at least one tank is encouraged.Abbreviations:NANot applicable.1Calculated by reviewer as mean of minimum and maximum values.D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes EPA recommends conducting a range-finder

if 96 -hour LC

50 data for the fathead minnow are unavailable.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 19 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsIf yes, what was the method fordetermining the highest testconcentration in the rangefinder?96-hr LC50rangefinding studyNominal test concentrations of 6.3, 13,25, 50 and 100 mg/L EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is appropriate.Species: Pimephales promelasLife stage:Approximatelythe same age and sizeas those used in thedefinitive exposure EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in the

test.Test duration:96-hr EPA recommends a 96-hour exposure.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 20 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:One test vessel,containing two malesand four females, wasestablished for eachconcentration and thecontrol.At test termination (96 hours), nomortality or adverse effects wereobserved among fish exposed to any ofthe treatment levels tested or thecontrol. The LC50 value for thisexposure was empirically estimated tobe >100 mg/L for combined survival. EPA recommends conducting a range-finder with five test concentrations plus a control

(six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentration -response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the

derived 96 -hr LC

50, may be selected as the highest exposure concentration in the 21-

day test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 21 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in thedefinitive test:The acute toxicity 96-hr LC50value wasestimated to be >100mg/L. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96 -hour LC

50 ), whichever concentration is lowest.Reference study citation (ifapplicable):NASeparation of testconcentrations:0.1 (ten-fold):0 (negative control),1.0, 10, and 100mga.i./L EPA suggests that a concentration separation of between 0.33 (or threefold) and 0.1 (or ten - fold) is scientifically

acceptable 1 .Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 22 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Mean measuredLimit of quantification (LOQ):0.053-0.066 mga.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reportedFrequency of measurement:0, 7, 14, and 21 daysSamples were collected from twoalternating replicates (A, C and B, D,respectively) on each sampling day. It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two replicates per treatment level.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 23 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsWas the randomized completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks during preexposure. Tanks are then ranked according to pre - exposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Number of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per testitem treatment level:4 EPA recommends 4 replicates.Number of male fish perreplicate at test initiation:2 EPA recommends 2 males per replicate.Number of female fish perreplicate at test initiation:4 EPA recommends 4 females per replicate.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 24 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsWas a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA EPA recommends that the solvent not exceed 0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1

ml/L4 .Was a positive control used?NoPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8
(light:dark).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing andAssessment. No. 23. Paris, France.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 25 of 75ParameterValue(s)Details or RemarksGuideline RecommendationsLight intensity at water’ssurface:720-1100 luxFluorescent bulbs EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:Following overnightmixing with anoverhead mixer, thestock solutions wereobserved to be clearand colorless with novisible undissolved testsubstance followingpreparation.Throughout the study period, all exposuresolutions were observed to be clear andcolorless. No undissolved test substancewas observed in the diluter system.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 26 of 75Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Metalaxyl.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0<LOQNA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV)≤ 20%.Treatment 11.01.16.6Treatment 210105.1Treatment 310010414.6Abbreviations:CVCoefficient of variation.LOQ=0.066 mg a.i./L.1The study author reported a mean measured concentration of 100 mg a.i./L; however, this was not consistent with the data provided, and thereviewer’s mean-measured concentration was used.

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Page 27 of 75 E. Observations

Biological Endpoints: Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination (Day 21), secondary sex characterization (body
color, pattern, body shape), body weight, length, tubercle score, gonadal

staging and histopathology, and plasma vitellogenin were evaluated.

Were raw (individual) data provided? Yes

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical signs occur at least daily. At test termination (Day 21), additional observations include body

weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma VTG, and plasma sex
steroids (testosterone and 17 β -estradiol, if measured). GSI is calculated using a ratio of gonad weight to
body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination.

Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other

abnormal behavior.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 28 of 75II. RESULTS AND DISCUSSIONA.ResultsMale survival values were 100, 88, 100 and 88% and female survival values were 100, 100, 100, and 94% in the mean-measured 0 (negativecontrol), 1.1, 10, and 104 mg a.i./L treatment levels, respectively (Table 9).Table 9: Adult Fish Survival in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]MalesFemalesn1# Surviving% Survivaln1# Surviving% SurvivalNegative control (<LOQ)8810016161001.187881616100108810016161001048788161594Abbreviations:NANot applicable.LOQ=0.066 mg a.i./L.1Total number of fish at test initiation.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 29 of 75Mean male body weight values were 3.13, 2.89, 2.63, and 2.623 g and female body weight values were 1.28, 1.44, 1.34 and 1.16 gin the meanmeasured 0 (control), 1.1, 10 and 104 mg a.i./L treatment levels, respectively (Table 10). Mean male body length values were 63.0, 60.1, 60.1,and 61.4 mm and female body length values were 47.8, 49.2, 49.4and 49.0 mm in the mean-measured 0 (control), 1.1, 10 and 104 mg a.i./Ltreatment levels, respectively.Table 10: Size at Test Termination in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDNegative control (<LOQ)43.130.1241.280.09463.01.3447.80.901.142.890.2041.440.03460.12.3449.21.51042.630.3041.340.09460.11.7449.41.210442.630.1641.160.12461.41.9449.02.2Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.066 mg a.i./L.

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Page 30 of 75 Mean fecundity values were 19, 19, 18 and 0 eggs/female/day and fertilization success was 96, 96, 97 and 0% in the mean-measured 0 (control), 1.1, 10 and 104 mg a.i./L treatment levels, respectively (Table
11).

Table 11: Fecundity and Fertilization Success in Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[mean-measured] Fecundity 1 Fertilization Success (%) 2

Negative control (<LOQ) 19 96

1.1 19 96

10 18 97

104 0 0

Abbreviations: NA Not applicable. ND Not determined.
LOQ=0.066 mg a.i./L.

1 Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate.

2 Fertilization success (%) is calculated as the number of embryos divided by the number of eggs,

multiplied by 100.

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Page 31 of 75 Median male tubercle scores were 31.5, 34.5, 32 and 6.25 in the mean-measured 0 (control), 1.1, 10 and
104 mg a.i./L treatment levels, respectively (Table 12). None of the surviving females were found to have

tubercles.

Table 12: Nuptial Tubercle Score in Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[mean-measured] Males Females

n Median

Tubercle Score 1 n Median

Tubercle Score

Negative control (<LOQ) 4 32 4 0

1.1 4 35 4 0

10 4 32 4 0

104 4 6 4 0

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.
LOQ=0.066 mg a.i./L.

1 Mean tubercle scores: 32, 35, 32, and 9 for the negative control and mean-measured 1.1, 10, and 104 mg a.i./L
treatment levels, respectively.

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Page 32 of 75 Mean male GSI was 1.3, 1.3, 1.3 and 1.4% and mean female GSI was 11, 11, 12 and 8.4% in the mean-measured 0 (control), 1.1, 10 and 104 mg a.i./L treatment levels, respectively (Table 13).

Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[mean-measured] Males Females

n Me an GSI 1

(%) ±SD n Mean GSI 1

(%) ±SD

Negative control (<LOQ) 4 1.3 0.08 4 11 0.82

1.1 4 1.3 0.32 4 11 1.8

10 4 1.3 0.17 4 12 1.3

104 4 1.4 0.49 4 8.4 0.75

Abbreviations: NA Not applicable.
LOQ=0.066 mg a.i./L.

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x

100.

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Page 33 of 75 Median male gonadal stage was 2, 2, 2 and 3 and median female gonadal stage was 3, 3, 3 and 3 in the mean-measured 0 (control), 1.1, 10 and 104 mg a.i./L treatment levels, respectively (Table 14).

Table 14: Gonadal Staging in Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[mean-measured] Males Females

n Median Stage 1 n Median Stage 2

Negative control (<LOQ) 4 2 4 3

1.1 4 2 4 3

10 4 2 4 3

104 4 3 4 3

Abbreviations: J Juvenile. NA Not applicable. ND Not determined. UTS Unable to stage.
LOQ=0.066 mg a.i./L.

1 The guideline recommends the following gonadal staging scale for male fathead minnow: 0=undeveloped,
1=early spermatogenic, 2=mid-spermatogenic, 3=late spermatogenic, 4=spent.

2 The guideline recommends the following gonadal staging scale for female fathead minnow: 0=undeveloped,
1=early development, 2=mid-development, 3=late development, 4=late development/hydrated, 5=postovulatory.

In male fish, there was an increase in incidence of increased percentage of spermatogonia at the 10 and 104 mg a.i./L treatment levels and asynchronous gonad development in the 104 mg a.i./L treatment (Tables 15-
16). In female fish, increased oocyte atresia was present in all groups, although a larger percentage of females

from the 104 mg a.i./L treatment group were affected. Also, there was decreased yolk synthesis in females at the 104 mg a.i./L treatment (Tables 17-18). Females throughout the study had minimal to mild

granulomatous inflammation associated with oocyte atresia and/or protozoal spores. There were no significant
findings observed in the gonadal stage.

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Page 34 of 75 Table 15: Gonadal Histopathology in Male Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Increased
Proportion of
Spermatogonia Presence of

Testis-Ova Increased

Testicular
Degeneration Duct

Mineralization Interstitial cell
(Leydig)
hypertrophy/hype
rplasia

n Incidence n Incidence n Incidence n Incidence n Incidence

Negative

control
(<LOQ) 0 8 7 NA NA 8 8 NA NA NA NA

1 8 0 NA NA 8 0 NA NA NA NA

2 8 0 NA NA 8 0 NA NA NA NA

3 8 1 NA NA 8 0 NA NA NA NA

4 8 0 NA NA 8 0 NA NA NA NA

1.1 0 7 7 NA NA 7 7 NA NA NA NA

1 7 0 NA NA 7 0 NA NA NA NA

2 7 0 NA NA 7 0 NA NA NA NA

3 7 0 NA NA 7 0 NA NA NA NA

4 7 0 NA NA 7 0 NA NA NA NA

10 0 9 6 NA NA 9 8 NA NA NA NA

1 9 2 NA NA 9 1 NA NA NA NA

2 9 1 NA NA 9 0 NA NA NA NA

3 9 0 NA NA 9 0 NA NA NA NA

4 9 0 NA NA 9 0 NA NA NA NA

104 0 7 1 NA NA 7 7 NA NA NA NA

1 7 4 NA NA 7 0 NA NA NA NA

2 7 2 NA NA 7 0 NA NA NA NA

3 7 0 NA NA 7 0 NA NA NA NA

4 7 0 NA NA 7 0 NA NA NA NA

Abbreviations: NA Not applicable.

LOQ=0.066 mg a.i./L.

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,
2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 35 of 75Table 16: Additional Gonadal HistopathologyObservations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceNegative control(<LOQ)0NANANANA88NANANANA1NANANANA80NANANANA2NANANANA80NANANANA3NANANANA80NANANANA4NANANANA80NANANANA1.10NANANANA77NANANANA1NANANANA70NANANANA2NANANANA70NANANANA3NANANANA70NANANANA4NANANANA70NANANANA100NANANANA99NANANANA1NANANANA90NANANANA2NANANANA90NANANANA3NANANANA90NANANANA4NANANANA90NANANANAData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 36 of 75Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence1040NANANANA72NANANANA1NANANANA73NANANANA2NANANANA72NANANANA3NANANANA70NANANANA4NANANANA70NANANANAAbbreviations:NANot applicable.LOQ=0.066 mg a.i./L.1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.

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Page 37 of 75 Table 17: Gonadal Histopathology in Female Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell
Hyperplasia/
Hypertrophy Decreased Yolk

Formation Aggregates of
macrophages,

multifocal

n Incidence n Incidence n Incidence n Incidence

Negative control
(<LOQ) 0 15 10 NA NA 15 13 NA NA

1 15 4 NA NA 15 0 NA NA

2 15 1 NA NA 15 2 NA NA

3 15 0 NA NA 15 0 NA NA

4 15 0 NA NA 15 0 NA NA

1.1 0 16 12 NA NA 16 15 NA NA

1 16 3 NA NA 16 1 NA NA

2 16 1 NA NA 16 0 NA NA

3 16 0 NA NA 16 0 NA NA

4 16 0 NA NA 16 0 NA NA

10 0 15 8 NA NA 15 13 NA NA

1 15 6 NA NA 15 2 NA NA

2 15 1 NA NA 15 0 NA NA

3 15 0 NA NA 15 0 NA NA

4 15 0 NA NA 15 0 NA NA

104 0 14 1 NA NA 14 0 NA NA

1 14 5 NA NA 14 8 NA NA

2 14 8 NA NA 14 3 NA NA

3 14 0 NA NA 14 2 NA NA

4 14 0 NA NA 14 1 NA NA

Abbreviations: NA Not applicable.

LOQ=0.066 mg a.i./L

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,
2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

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Page 38 of 75 Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( Pimephales
promelas)

.

Treatment
(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Interstitial fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

Negative

control
(<LOQ) 0 NA NA 15 14 15 10 NA NA

1 NA NA 15 1 15 5 NA NA

2 NA NA 15 0 15 0 NA NA

3 NA NA 15 0 15 0 NA NA

4 NA NA 15 0 15 0 NA NA

1.1 0 NA NA 16 14 16 13 NA NA

1 NA NA 16 1 16 2 NA NA

2 NA NA 16 1 16 1 NA NA

3 NA NA 16 0 16 0 NA NA

4 NA NA 16 0 16 0 NA NA

10 0 NA NA 15 11 15 13 NA NA

1 NA NA 15 2 15 2 NA NA

2 NA NA 15 1 15 0 NA NA

3 NA NA 15 1 15 0 NA NA

4 NA NA 15 0 15 0 NA NA

104 0 NA NA 14 13 14 8 NA NA

1 NA NA 14 0 14 6 NA NA

2 NA NA 14 1 14 0 NA NA

3 NA NA 14 0 14 0 NA NA

4 NA NA 14 0 14 0 NA NA

Abbreviations: NA Not applicable.

LOQ=0.066 mg a.i./L

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,
2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

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Page 39 of 75 Mean male VTG was 247, 99, 33 and 73 and female VTG was 1.97x10 6 , 1.14x10 6 , 1.63x10 6 and 7.48x10 4
ng/mL in the mean-measured 0 (control), 1.1, 10 and 10 4 mg a.i./L treatment levels, respectively (Table
19).

Table 19: Plasma Vitellogenin in Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[mean-measured] Plasma Vitellogenin (VTG)

Males Females

n Mean
(ng/mL plasma) ±SD n Mean
(ng/mL plasma) ±SD

Negative control
(<LOQ) 4 247 431 4 1.97 x 10 6 1.83 x 10 6

1.1 4 99 101 4 1.14 x 10 6 8.75 x 10 5

10 4 33 10 4 1.63 x 10 6 1.07 x 10 6

104 4 73 96 4 7.48 x 10 4 8.95 x 10 4

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.
LOQ=0.066 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 40 of 75Plasma testosterone and plasma 17β-estradiol in male and females was not measured (Table 20).Table 20: Plasma Sex Steroids in Fathead Minnow ( Pimephales promelas). Not measured.Treatment(mg a.i./L)[mean-measured]Plasma Testosterone (T)Plasma 17β-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNegative control (<LOQ)NANANANANANANANANANANANA1.1NANANANANANANANANANANANA10NANANANANANANANANANANANA100NANANANANANANANANANANANAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.066 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 41 of 75There were no notable observations with regards to behavior or changes in appearance (coloration/banding, presence of ovipositor and dorsal napepad), including secondary sex characteristics in the control or treated groups (Table 21). Two males were mis-sexed as femalesand inadvertentlyadded to the control replicate B and 10 mg a.i./L replicate C.Table 21: Secondary Sex Characteristicsand Clinical Signs in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceat TestTerminationTypenIncidenceat TestTerminationNegative control (<LOQ)Deformed81None 4 0Color bars811.1Color bars71Bloated16210Color bars81None40104None40None40LOQ=0.066 mg a.i./L.

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Page 42 of 75 B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, tubercle score, GSI, fertility, fecundity, and VTG. Data
were gender specific and analyzed in comparison to the control.

Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant
effects were detected for p<0.05 with the exception of Shapiro-Wilk’s and Bartlett’s Tests, which were
based on p<0.01 (CETIS, version 1.8.5, 2011). Survival data were analyzed using a Fisher’s Exact

Test/Bonferroni-Holm Test. Fecundity, fertility, female weight and GSI, and male VTG were analyzed
using ANOVA and Dunnett’s Multiple Comparison test. Male tubercle scores, male weight and GSI

were analyzed by ANOVA and Jonckheere-Terpstra Step–Down test. Female VTG was analyzed by
Tamhane-Dunnett Step-Down test. Male and female length was recorded but statistical analysis was
not reported. Prior to Dunnett’s, data were analyzed by Shapiro-Wilk’s test and Bartlett’s to test for
normality and homogeneity of variances, respectively. If normality and homogeneity tests passed (p>0.01), a parametric analysis was performed. If non-normality or unequal variance were indicated
(p<0.01), a non-parametric analysis was performed on the ranks of the data. These methods appear

to be consistent with the methods recommended in the guideline.

Statistical analysis (Fisher Exact/Bonferroni-Holm Test) determined no significant difference in male,

female, or combined survival. At the 104 mg a.i./L treatment level, fecundity was 0
eggs/female/reproductive day, and fertility was not reported since there was no spawning therefore
this treatment level was excluded from statistical analysis. With the highest treatment removed, there were no significant differences in fecundity or fertility compared to the negative control (p>0.05;
Dunnett Multiple Comparison Test). There was a significant reduction in median male tubercle score
at the 104 mg a.i./L treatment level compared to the negative control (p<0.05; Jonckheere-Terpstra
Step-Down test). There were no tubercles observed in females. There was a significant decrease in

mean male weight at the 10 and 104 mg a.i./L treatment levels compared to the negative control (p<0.05; Jonckheere-Terpstra Step-Down test). There was also a significant increase in mean female
body weight at the 1.1 mg a.i./L treatment level compared to the negative control (p<0.05; Dunnett’s
Multiple Comparison test). Statistical analysis (Jonckheere-Terpstra Step-Down Test) determined no
significant decrease (p>0.05) in mean male GSI score among fish exposed to any of the treatment

levels tested, compared to the negative control. There was a significant decrease in mean female GSI

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Page 43 of 75 score at the 104 mg a.i./L treatment level compared to the negative control (p<0.05; Dunnett’s
Multiple Comparison test). One male in the 10 mg/L concentration was empirically determined to be
an outlier and was removed from the statistical analysis. Statistical analysis (Dunnett’s Multiple
Comparison Test) determined no significant difference (p >0.05) for male vitellogenin concentration

in any of the treatment levels compared to the negative control. There was also a significant differencein mean female VTG at the 104 mg a.i./L treatment level compared to the control (p<0.05; TamhaneDunnett Step-Down test).

In male fish, there were an increased incidence of increased percentage of spermatogonia at the 10
and 104 mg a.i./L treatment levels and asynchronous gonad development in the 104 mg a.i./L
treatment level. In female fish, there was decreased yolk synthesis at the 104 mg a.i./L treatment
level. There were no significant findings observed in the gonadal stage. Ninety-one out of 96 fish were
examined. Four of the fish were not examined due to mortality. The fifth fish was not examined due
to the size of the ovaries being too small for examination.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via

Toxstat 3.5. Male body weight was consistent with a monotonic concentration-response; female
vitellogenin no other endpoints were associated with a monotonic response. All data were tested for
normality using Shapiro-Wilks test and for homogeneity of variance using Levene’s test. Data which
met the assumptions of normality and homogeneity of variance were then analyzed using the parametric
Dunnett’s test. Data which did not meet the parametric assumptions were analyzed using the nonparametric Mann-Whitney U test.

Male VTG and fecundity were neither monotonic nor parametric and were analyzed using MannWhitney. Male body weight exhibited monotonic trends and was analyzed using the non-parametric
Jonckheere-Terpstra test, as recommended by the OCSPP 890.1350 guideline. None of the surviving
females were found to have tubercles. Unless otherwise indicated, effects were considered statistically
significant at p<0.05. These analyses were conducted using SAS ® (SAS Institute, Cary, NC; version
8.1). Both the study author and reviewer relied on the mean-measured concentrations to discuss

effects in this study.

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Page 44 of 75 Conclusions:

There were no significant differences in either male or female survival at any treatment levels compared to the negative control (Table 9). Male body weight (Table 24) exhibited a significant (p<0.05)
reduction of 7 to 16% at all treatment levels compared to the negative control. Female GSI (Table
23) was significantly (p<0.05) reduced 23% of control at the 104 mg a.i./L treatment level. Although

the study author found a significant reduction in female plasma VTG at the 104 mg a.i./L treatment level, the reviewer did not find this effect to be significant using Dunnett’s test (SAS) or Dunnett’s T3
(CETIS). Median male tubercle score was also significantly reduced (p<0.05) 80% compared to the

control at the 104 mg a.i./L treatment level. There was no significant reduction in fecundity or fertility (Table 23) at the low and middle treatment levels; there were no eggs available for assessment at

the highest treatment level. Therefore, that treatment level was excluded from data analysis, but the
lack of fecundity at the highest treatment level is considered a significant effect. There were no other
significant effects on other endpoints. Although not analyzed statistically, in male fish, there were an
increased percentage of spermatogonia at the 10 and 104 mg a.i./L treatment levels and asynchronous
gonad development in the 104 mg a.i/L treatment. In female fish, there was decreased yolk synthesis
at the 104 mg a.i./L treatment. There were no significant findings observed in the gonadal stage, and
no notable observations secondary sex characteristics or clinical signs for any treatment group

compared to the controls.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 45 of 75Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with Metalaxyl.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.p% Diff.pNegative control(<LOQ)32 NA0 NANo 0 NANA NANANA1.1350.689-1.920.999No-59.720.780NANANANA10321.000-1.920.999Yes-86.540.491NANANANA1046<0.00110.960.847Yes-70.650.780NANANANAStatistical TestDunnett’s3Dunnett’sNAMann-WhitneyNANAAbbreviations:Conc.Concentration.Diff.Difference.E217β-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.LOQ=0.066 mg a.i./L.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusionsregarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Mean tubercle scores: 32, 35, 32, and 9 for the negative control and mean-measured 1.1, 10, and 104 mg a.i./L treatment levels,respectively.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 46 of 75Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with Metalaxyl.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.PMedianp% Diff.pEffect?(Yes/No)% Diff.p% Diff.p%Diff.pNegative control(<LOQ)0NA0NA0NA0NANo 0NANANANANA1.14.190.400-0.260.9820NA0.001.00No-42.040.625NANANANA10-4.050.65710.780.8540NA11.360.373No-17.330.952NANANANA104-100.030.0286–3–30NA-23.410.030Yes-96.200.092NANANANAStatistical TestMann-WhitneyDunnett’sNADunnett’sNADunnett’sNANAAbbreviations:Conc.Concentration.Diff.Difference.E217β-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.LOQ=0.066 mg a.i./L.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusionsregarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3No eggs were produced at the highest test concentration; therefore fecundity was 0, and there were no fertility data to analyze.

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Page 47 of 75 Table 24: Growth Endpoints 1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Metalaxyl.

Treatment
(mg a.i./L)

[mean-measured] Body Weight Length

Males Females Males Females

% Diff. p % Diff. p % Diff. p % Diff. p

Negative control
(<LOQ) 0 NA 0 NA 0 NA 0 NA

1.1 -7.44 0.042 12.08 0.071 -4.57 0.112 2.98 0.446

10 -15.95 0.003 4.85 0.639 -4.57 0.112 3.51 0.327

104 -15.92 0.001 -9.34 0.183 -2.50 0.501 2.72 0.514

Statistical Test Jonckheere Dunnett’s Dunnett’s Dunnett’s

Abbreviations: Diff. Difference. NA Not applicable.
LOQ=0.066 mg a.i./L.

1 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative
(clean water) control.

2 Unless otherwise specified, effects are considered statistically significant at p<0.05.

E. Study Deficiencies

There were a couple of deviations described in Section I. Materials and Methods of this DER regarding
failure to report certain water characteristics and meet environmental criteria. These deviations do not
impact the interpretation of the study. The remaining validity and performance criteria for OCSPP

890.1350 were met.

F. Reviewer’s Comments

The reviewer’s and the study authors’ results were in general agreement. Both the study author’s and reviewer’s analysis detected statistically significant reductions (p<0.05) in male tubercle scores and female

GSI at the 104 mg a.i./L. The study author found a significant decrease (p<0.05) in mean male body

weight at the 10 and 104 mg a.i./L, while the reviewer found significant decreases at all treatment levels

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Page 48 of 75 compared to the negative control. The study author also reported a significant decrease in female VTG at
the 104 mg a.i./L treatment level and a significant increase in mean female weight at the 1.1 mg a.i./L
treatment level compared to the negative control. The reviewer’s conclusions based on the OCSPP
890.1350 flowchart are presented in the Executive Summary and Conclusions sections of this DER.

During the study, two males were mis-sexed as females and inadvertently added to the negative control replicate B and the mid concentration (10 mg a.i./L) replicate C. The study author did not specify which

males were mis-sexed and used the data for all three males to calculate descriptive statistics for the
affected replicates. Since the study report did not explicitly identify the mis-sexed fish, the reviewer used
data for all three males in the affected replicates for statistical analysis even though the mis-sexed males could probably be identified by their tubercles scores ( i.e. , 0).

There is a discrepancy in the statistical test used to analyze mean male body weight. The data table (Table 5, p. 39) reports the significant differences in mean male body weight at the 10 and 104 mg a.i./L
treatment levels compared to the control were determined using Dunnett’s Multiple Comparison test, while
the “Results and Discussion” text reported the differences were determined using Jonckheere-Terpstra
Step-Down test. The reviewer reported Jonckheere-Terpstra Step-Down test was used.

III. REFERENCES

Ankley, G.T., Jensen, K.M., Kahl, M.D., Korte, J.J., and Makynen E.A., 2001. Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environ Toxicol Chem

20:1276-1290.

APHA, AWWA, WPCF, 1995. Standard Methods for the Examination of Water and Wastewater. 19th
Edition, Washington, DC, 2-23-25.

ASTM, 2005. Committee E-47 on Biological Effects and Environmental Fate. Standard Guide for
Conducting Early Life-Stage Toxicity Tests with Fishes. ASTM Designation E: 1241-05. American Society
for Testing and Materials, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, Pennsylvania.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 49 of 75 Ives, Michael, 2011. CETIS, Comprehensive Environmental Toxicity Information System TM . User’s Guide.
Tidepool Scientific Software, McKinleyville, California.

Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water

Research 1: 21-29.

Sprague, J.B., 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity.

Water Res. 3: 793-821.

OECD (2009). Fish Short Term Reproduction Assay. OECD Guideline for the testing Chemicals: Test No.

229. Paris, France. 40 pp

U.S. EPA, 1989. 40 CFR, Part 160. Federal Insecticide, Fungicide, and Rodenticide Act. Good Laboratory
Practices Standards; Final Rule. Office of the Federal Register, National Archives and Records
Administration. U.S. Government Printing Office, Washington, D.C.

U.S. EPA. 1996. Office of Prevention, Pesticides and Toxic Substances. Ecological Effects Guideline,
OCSPP 850.1000. Special Consideration for Conducting Aquatic Laboratory Studies. “Public Draft”. EPA
712-C-96-113. April 1996. U.S. Environmenta l Protection Agency, Washington, D.C.

U.S. EPA, 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters
to marine and estuarine organisms. Third edition. Office of Water, U. S. Environmental Protection Agency,
Washington, DC 20460. EPA/821/R-02/014.

U.S. EPA, 2009. Office of Prevention, Pesticides and Toxic Substances. Endocrine Disruptor Screening
Program Test Guideline, OCSPP 890.1350. Fish Short-Term Reproduction Assay. EPA 740-C-09-007.
October 2009. U.S. Environmental Protection Agency. Washington, D.C.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 50 of 75APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:Reviewer’s statistical verification tableEndpointMonotonic?Parametric?890.1350EFEDCommentsFemale body weightNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectMale body weightYesYesJonckheere:Dose 1 p=0.042Dose 2 p=0.003Dose 3 p=0.001Jonckheere:Dose 1 p=0.042Dose 2 p=0.003Dose 3 p=0.001890.1350 and EFED same conclusions; Jonckheere’sreveals significant 7 to 16% reduction in male body weightat all treatment levels, relative to the negative controlFemale body lengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectMale body lengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectFemale VTGNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectMale VTGNoNoMannWhitney:n.s. p>0.05MannWhitney:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale GSINoYesDunnett’s:Dose 3 p=0.030Dunnett’s:Dose 3 p=0.030890.1350 and EFED same conclusions, significant 23%reduction at the highest level.Male GSINoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectFemale tuberclescoreNANANANANo scoreData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetalaxylEPA MRID Number 48685802Page 51 of 75EndpointMonotonic?Parametric?890.1350EFEDCommentsMale tubercle scoreNoYesDunnett’s:Dose 3 p<0.001Dunnett’s:Dose 3 p<0.001890.1350 and EFED same conclusions, significant 81 %reduction (median values) at the highest level.FecundityNoNoMannWhitney:Dose 3 p=0.052MannWhitney:Dose 3 p=0.052890.1350 and EFED same conclusions; no eggs producedat the high dose level.FertilityNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effect at lowand middle doses (no data for high dose).F testosteroneNANANANANAM testosteroneNANANANANAF estradiolNANANANANAM estradiolNANANANANA

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Page 52 of 75 test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.976 0.920 1.729 0.214 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.28 0.09 0.05 7.10 1.14, 1.43 Dose1 4 1.44 0.03 0.01 1.92 1.39, 1.48 Dose2 4 1.34 0.09 0.04 6.44 1.21, 1.48 Dose3 4 1.16 0.12 0.06 10.36 0.97, 1.35

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.29 1.18 1.37 . . Dose1 1.44 1.40 1.47 112.08 -12.08 Dose2 1.33 1.27 1.45 104.85 -4.85 Dose3 1.18 1.00 1.28 90.66 9.34

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 6.84 0.006

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.28 . 1.36 . . . . . . Dose1 1.44 0.071 1.36 0.934 . . . . . Dose2 1.34 0.639 1.34 0.919 0.473 . . . . Dose3 1.16 0.183 1.16 0.050 0.004 0.054 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.66 0.022

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 1.29 . . Dose1 1.44 0.067 0.990 Dose2 1.33 0.494 0.810 Dose3 1.18 0.346 0.112

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3

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Page 53 of 75 Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 6.84 0.006

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.28 . -1.28 . . . . . . Dose1 -1.44 0.071 -1.31 0.365 . . . . . Dose2 -1.34 0.639 -1.31 0.389 0.473 . . . . Dose3 -1.16 0.183 -1.31 0.403 0.004 0.054 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.66 0.022

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -1.29 . . Dose1 -1.44 0.067 0.010 Dose2 -1.33 0.494 0.190 Dose3 -1.18 0.346 0.888

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.967 0.790 1.390 0.293 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3.13 0.12 0.06 3.81 2.94, 3.31 Dose1 4 2.89 0.20 0.10 6.78 2.58, 3.20 Dose2 4 2.63 0.30 0.15 11.32 2.15, 3.10 Dose3 4 2.63 0.16 0.08 6.09 2.37, 2.88

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 3.12 3.01 3.25 . . Dose1 2.95 2.62 3.06 92.56 7.44

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 54 of 75 Dose2 2.62 2.29 2.98 84.05 15.95 Dose3 2.62 2.46 2.80 84.08 15.92

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 5.53 0.013

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 3.13 . 3.13 . . . . . . Dose1 2.89 0.295 2.89 0.078 . . . . . Dose2 2.63 0.012 2.63 0.003 0.301 . . . . Dose3 2.63 0.013 2.63 0.003 0.305 1.000 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.64 0.022

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 3.12 . . Dose1 2.95 0.156 0.042 Dose2 2.62 0.067 0.003 Dose3 2.62 0.067 0.001

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose1

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 5.53 0.013

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -3.13 . -2.82 . . . . . . Dose1 -2.89 0.295 -2.82 0.989 . . . . . Dose2 -2.63 0.012 -2.82 0.993 0.301 . . . . Dose3 -2.63 0.013 -2.82 0.995 0.305 1.000 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 55 of 75 Degrees of Freedom TestStat P-value 3 9.64 0.022

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -3.12 . . Dose1 -2.95 0.156 0.958 Dose2 -2.62 0.067 0.997 Dose3 -2.62 0.067 0.999

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.953 0.536 1.268 0.329 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 47.75 0.90 0.45 1.88 46.32, 49.18 Dose1 4 49.18 1.50 0.75 3.05 46.79, 51.56 Dose2 4 49.43 1.23 0.61 2.48 47.47, 51.38 Dose3 4 49.05 2.22 1.11 4.53 45.51, 52.59

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 47.95 46.50 48.60 . . Dose1 49.50 47.10 50.60 102.98 -2.98 Dose2 49.70 47.70 50.60 103.51 -3.51 Dose3 49.35 46.20 51.30 102.72 -2.72

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.95 0.448

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 47.75 . 48.85 . . . . . . Dose1 49.18 0.446 48.85 0.899 . . . . . Dose2 49.43 0.327 48.85 0.921 0.995 . . . . Dose3 49.05 0.514 48.85 0.931 0.999 0.985 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 56 of 75 Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 2.53 0.470

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 47.95 . . Dose1 49.50 0.235 0.926 Dose2 49.70 0.233 0.939 Dose3 49.35 0.494 0.897

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.95 0.448

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -47.75 . -47.75 . . . . . . Dose1 -49.18 0.446 -49.18 0.128 . . . . . Dose2 -49.43 0.327 -49.24 0.124 0.995 . . . . Dose3 -49.05 0.514 -49.24 0.128 0.999 0.985 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 2.53 0.470

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -47.95 . . Dose1 -49.50 0.235 0.074 Dose2 -49.70 0.233 0.061 Dose3 -49.35 0.494 0.103

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 57 of 75 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.950 0.496 0.303 0.823 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 62.98 1.29 0.65 2.06 60.92, 65.03 Dose1 4 60.10 2.29 1.15 3.81 56.46, 63.74 Dose2 4 60.10 1.68 0.84 2.79 57.43, 62.77 Dose3 4 61.40 1.92 0.96 3.13 58.34, 64.46

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 62.75 61.90 64.50 . . Dose1 60.80 56.90 61.90 95.43 4.57 Dose2 59.75 58.70 62.20 95.43 4.57 Dose3 60.90 59.80 64.00 97.50 2.50

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 2.22 0.138

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 62.98 . 62.98 . . . . . . Dose1 60.10 0.112 60.53 0.049 . . . . . Dose2 60.10 0.112 60.53 0.052 1.000 . . . . Dose3 61.40 0.501 60.53 0.053 0.750 0.750 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 5.39 0.145

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 62.75 . . Dose1 60.80 0.096 0.019 Dose2 59.75 0.154 0.028 Dose3 60.90 0.233 0.112

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 2.22 0.138

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 58 of 75 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -62.98 . -61.06 . . . . . . Dose1 -60.10 0.112 -61.06 0.958 . . . . . Dose2 -60.10 0.112 -61.06 0.970 1.000 . . . . Dose3 -61.40 0.501 -61.40 0.955 0.750 0.750 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 5.39 0.145

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -62.75 . . Dose1 -60.80 0.096 0.981 Dose2 -59.75 0.154 0.972 Dose3 -60.90 0.233 0.888

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.875 0.033 3.372 0.055 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1969500 1831612 915805.8 93.00 -945003, 4884003 Dose1 4 1141509 874488.3 437244.2 76.61 -249998, 2533015 Dose2 4 1628250 1071015 535507.3 65.78 -75973.2, 3332473 Dose3 4 74800.00 89463.85 44731.92 119.60 -67556.9,217156.9

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1125000 918000.0 4710000 . . Dose1 1343000 34.00 1880000 57.96 42.04 Dose2 1295000 753000.0 3170000 82.67 17.33 Dose3 37700.00 15800.00 208000.0 3.80 96.20

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 2.07 0.158

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 59 of 75 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1969500 . 1969500 . . . . . . Dose1 1141509 0.625 1384879 0.290 . . . . . Dose2 1628250 0.952 1384879 0.310 0.930 . . . . Dose374800.00 0.092 74800.00 0.023 0.572 0.273 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 6.42 0.093

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 1125000 . . Dose1 1343000 0.678 0.282 Dose2 1295000 1.000 0.442 Dose3 37700.00 0.067 0.025

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 2.07 0.158

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1969500 . -1203515 . . . . . . Dose1-1141509 0.625 -1203515 0.887 . . . . . Dose2-1628250 0.952 -1203515 0.910 0.930 . . . . Dose3-74800.0 0.092 -1203515 0.921 0.572 0.273 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 6.42 0.093

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -1125000 . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 60 of 75 Dose1 -1343000 0.678 0.718 Dose2 -1295000 1.000 0.558 Dose3 -37700.0 0.067 0.975

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.758 <.001 5.989 0.010 USE NON-PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 247.00 430.96 215.48 174.48 -438.76, 932.76 Dose1 4 99.50 101.43 50.72 101.94 -61.91, 260.91 Dose2 4 33.25 9.84 4.92 29.61 17.59, 48.91 Dose3 4 72.50 95.66 47.83 131.95 -79.72, 224.72

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 41.00 13.00 893.00 . . Dose1 64.00 22.00 248.00 40.28 59.72 Dose2 28.50 28.00 48.00 13.46 86.54 Dose3 31.00 13.00 215.00 29.35 70.65

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.68 0.581

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 247.00 . 247.00 . . . . . . Dose1 99.50 0.689 99.50 0.224 . . . . . Dose2 33.25 0.432 52.88 0.158 0.975 . . . . Dose3 72.50 0.580 52.88 0.163 0.998 0.995 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 1.80 0.616

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 61 of 75 Ctrl 41.00 . . Dose1 64.00 0.780 0.668 Dose2 28.50 0.491 0.209 Dose3 31.00 0.780 0.141

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.68 0.581

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -247.00 . -113.06 . . . . . . Dose1 -99.50 0.689 -113.06 0.864 . . . . . Dose2 -33.25 0.432 -113.06 0.890 0.975 . . . . Dose3 -72.50 0.580 -113.06 0.903 0.998 0.995 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 1.80 0.616

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -41.00 . . Dose1 -64.00 0.780 0.332 Dose2 -28.50 0.491 0.791 Dose3 -31.00 0.780 0.859

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.952 0.523 2.768 0.088 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 11.00 0.82 0.41 7.42 9.70, 12.30

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 62 of 75 Dose1 4 11.00 1.78 0.89 16.19 8.17, 13.83 Dose2 4 12.25 1.26 0.63 10.27 10.25, 14.25 Dose3 4 8.43 0.75 0.38 8.90 7.23, 9.62

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 11.00 10.00 12.00 . . Dose1 10.80 9.40 13.00 100.00 0.00 Dose2 12.00 11.00 14.00 111.36 -11.36 Dose3 8.80 7.30 8.80 76.59 23.41

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 6.91 0.006

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 11.00 . 11.42 . . . . . . Dose1 11.00 1.000 11.42 0.767 . . . . . Dose2 12.25 0.373 11.42 0.799 0.497 . . . . Dose3 8.43 0.030 8.43 0.007 0.049 0.004 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.79 0.020

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 11.00 . . Dose1 10.80 1.000 0.442 Dose2 12.00 0.214 0.870 Dose3 8.80 0.061 0.059

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 6.91 0.006

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -11.00 . -10.67 . . . . . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 63 of 75 Dose1 -11.00 1.000 -10.67 0.733 . . . . . Dose2 -12.25 0.373 -10.67 0.767 0.497 . . . . Dose3 -8.43 0.030 -10.67 0.785 0.049 0.004 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.79 0.020

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -11.00 . . Dose1 -10.80 1.000 0.558 Dose2 -12.00 0.214 0.130 Dose3 -8.80 0.061 0.941

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.944 0.404 2.603 0.100 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.30 0.08 0.04 6.28 1.17, 1.43 Dose1 4 1.28 0.31 0.15 24.28 0.78, 1.77 Dose2 4 1.28 0.17 0.09 13.39 1.00, 1.55 Dose3 4 1.44 0.49 0.24 33.91 0.66, 2.22

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.30 1.20 1.40 . . Dose1 1.20 1.00 1.70 98.08 1.92 Dose2 1.25 1.10 1.50 98.08 1.92 Dose3 1.35 0.97 2.10 110.96 -10.96

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.28 0.839

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 64 of 75 Ctrl 1.30 . 1.32 . . . . . . Dose1 1.28 0.999 1.32 0.628 . . . . . Dose2 1.28 0.999 1.32 0.663 1.000 . . . . Dose3 1.44 0.847 1.32 0.682 0.863 0.863 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 0.38 0.945

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 1.30 . . Dose1 1.20 0.671 0.277 Dose2 1.25 0.775 0.354 Dose3 1.35 1.000 0.500

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.28 0.839

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.30 . -1.28 . . . . . . Dose1 -1.28 0.999 -1.28 0.616 . . . . . Dose2 -1.28 0.999 -1.28 0.650 1.000 . . . . Dose3 -1.44 0.847 -1.44 0.344 0.863 0.863 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 0.38 0.945

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -1.30 . . Dose1 -1.20 0.671 0.723 Dose2 -1.25 0.775 0.646 Dose3 -1.35 1.000 0.500

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 65 of 75 test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value
. . . . NO DATA FOR TEST

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.00 0.00 0.00 . . , . Dose1 4 0.00 0.00 0.00 . . , . Dose2 4 0.00 0.00 0.00 . . , . Dose3 4 0.00 0.00 0.00 . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.00 0.00 0.00 . . Dose1 0.00 0.00 0.00 . . Dose2 0.00 0.00 0.00 . . Dose3 0.00 0.00 0.00 . .

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value . . . 1 . . . . . . .

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.00 . . . . . . . . Dose1 0.00 . . . . . . . . Dose2 0.00 . . . . . . . . Dose3 0.00 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 0.00 1.000

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.00 . . Dose1 0.00 1.000 . Dose2 0.00 1.000 . Dose3 0.00 1.000 .

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 66 of 75 Jonckheere Dose1

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value . . . 1 . . . . . . .

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.00 . . . . . . . . Dose1 0.00 . . . . . . . . Dose2 0.00 . . . . . . . . Dose3 0.00 . . . . . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 0.00 1.000

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.00 . . Dose1 0.00 1.000 . Dose2 0.00 1.000 . Dose3 0.00 1.000 .

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose1 Jonckheere Dose1

test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.921 0.172 2.258 0.134 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 31.75 4.11 2.06 12.95 25.21, 38.29 Dose1 4 34.50 3.87 1.94 11.23 28.34, 40.66 Dose2 4 32.00 0.82 0.41 2.55 30.70, 33.30 Dose3 4 8.75 6.22 3.11 71.14 -1.16, 18.66

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 31.50 27.00 37.00 . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 67 of 75 Dose1 34.50 30.00 39.00 108.66 -8.66 Dose2 32.00 31.00 33.00 100.79 -0.79 Dose3 6.25 4.50 18.00 27.56 72.44

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 32.64 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 31.75 . 33.13 . . . . . . Dose1 34.50 0.689 33.13 0.760 . . . . . Dose2 32.00 1.000 32.00 0.653 0.836 . . . . Dose3 8.75 <.001 8.75 <.001 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.29 0.026

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 31.50 . . Dose1 34.50 0.494 0.807 Dose2 32.00 0.775 0.559 Dose3 6.25 0.067 0.015

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 32.64 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -31.75 . -26.75 . . . . . . Dose1 -34.50 0.689 -26.75 0.972 . . . . . Dose2 -32.00 1.000 -26.75 0.981 0.836 . . . . Dose3 -8.75 <.001 -26.75 0.984 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 68 of 75 Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.29 0.026

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -31.50 . . Dose1 -34.50 0.494 0.193 Dose2 -32.00 0.775 0.441 Dose3 -6.25 0.067 0.985

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.845 0.012 5.092 0.017 USE NON-PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 18.50 4.04 2.02 21.85 12.07, 24.93 Dose1 4 19.28 8.17 4.09 42.39 6.27, 32.28 Dose2 4 17.75 2.87 1.44 16.18 13.18, 22.32 Dose3 4 0.00 0.00 0.00 . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 18.50 15.00 22.00 . . Dose1 23.00 7.10 24.00 104.19 -4.19 Dose2 18.00 14.00 21.00 95.95 4.05 Dose3 0.00 0.00 0.00 0.00 100.00

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 15.07 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 18.50 . 18.89 . . . . . . Dose1 19.28 0.991 18.89 0.631 . . . . . Dose2 17.75 0.992 17.75 0.520 0.968 . . . . Dose3 0.00 <.001 0.00 <.001 <.001 0.001 . . .

********************************************************************************

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 69 of 75 NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.66 0.022

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 18.50 . . Dose1 23.00 0.478 0.816 Dose2 18.00 0.673 0.328 Dose3 0.00 0.052 0.005

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 15.07 <.001

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -18.50 . -13.88 . . . . . . Dose1 -19.28 0.991 -13.88 0.948 . . . . . Dose2 -17.75 0.992 -13.88 0.962 0.968 . . . . Dose3 0.00 <.001 -13.88 0.968 <.001 0.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.66 0.022

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -18.50 . . Dose1 -23.00 0.478 0.184 Dose1 -23.00 0.478 0.184 Dose1 -23.00 0.478 0.184 Dose2 -18.00 0.673 0.672 Dose2 -18.00 0.673 0.672 Dose2 -18.00 0.673 0.672 Dose3 0.00 0.052 0.995 Dose3 0.00 0.052 0.995 Dose3 0.00 0.052 0.995

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 70 of 75 test for fish screen study - Metalaxyl ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.848 0.035 1.294 0.321 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 96.00 2.71 1.35 2.82 91.69, 100.31 Dose1 4 95.75 0.96 0.48 1.00 94.23, 97.27 Dose2 4 96.75 2.63 1.31 2.72 92.57, 100.93 Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 97.00 92.00 98.00 . . Dose1 95.50 95.00 97.00 99.74 0.26 Dose2 97.50 93.00 99.00 100.78 -0.78 Dose3 . . . . .

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value . . . 9 . . 2 . . . . .

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 96.00 . . . . . . . . Dose1 95.75 0.982 . . . . . . . Dose2 96.75 0.854 . . 0.808 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 1.58 0.455

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value Ctrl 97.00 . . Dose1 95.50 . 0.186 Dose2 97.50 . 0.673 Dose3 . . .

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere >highest dose (no sign. differences)

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 71 of 75 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value . . . 9 . . 2 . . . . .

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -96.00 . . . . . . . . Dose1 -95.75 0.982 . . . . . . . Dose2 -96.75 0.854 . . 0.808 . . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 1.58 0.455

MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -97.00 . . Dose1 -95.50 . 0.814 Dose2 -97.50 . 0.327 Dose3 . . .

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose1 Jonckheere >highest dose (no sign. differences)

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 72 of 75 Fecundity (Mann-Whitney exact test)



### Obs **conc endpoint**  **1** 0 15.0 **2** 0 22.0 **3** 0 15.0 **4** 0 22.0 **5** 1 24.0 **6** 1 24.0 **7** 1 22.0 **8** 1 7.1 **9** 2 21.0 **10** 2 14.0 **11** 2 18.0 **12** 2 18.0 **13** 3 0.0 **14** 3 0.0 **15** 3 0.0 **16** 3 0.0

**Wilcoxon Scores (Rank Sums) for Variable endpoint Classified by Variable conc** 

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score**  **0** 4 41.0 34.0 8.142481 10.250 **1** 4 49.0 34.0 8.142481 12.250 **2** 4 36.0 34.0 8.142481 9.000 **3** 4 10.0 34.0 8.142481 2.500 **Average scores were used for ties.**

**Kruskal-Wallis Test Chi-Square**  9.6606
**DF** 3 **Asymptotic Pr > Chi-Square** 0.0217 **Exact Pr >= Chi-Square** 0.0071

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 73 of 75 

### Wilcoxon Scores (Rank Sums) for Variable endpoint **Classified by Variable conc**

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score**  **0** 4 15.0 18.0 3.338092 3.750 **1** 4 21.0 18.0 3.338092 5.250 **Average scores were used for ties.**

**Wilcoxon Two-Sample Test Statistic (S)**  15.0000

**Normal Approximation**
**Z** -0.7489 **One-Sided Pr < Z** 0.2269 **Two-Sided Pr > |Z|** 0.4539

**t Approximation One-Sided Pr < Z**  0.2392 **Two-Sided Pr > |Z|** 0.4783

**Exact Test One-Sided Pr <= S**  0.2000 **Two-Sided Pr >= |S - Mean|** 0.4000 **Z includes a continuity correction of 0.5.**

**Kruskal-Wallis Test Chi-Square**  0.8077
**DF** 1 **Pr > Chi-Square** 0.3688

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 74 of 75 

### Wilcoxon Scores (Rank Sums) for Variable endpoint **Classified by Variable conc**

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score**  **0** 4 20.0 18.0 3.401680 5.0 **2** 4 16.0 18.0 3.401680 4.0 **Average scores were used for ties.**

**Wilcoxon Two-Sample Test Statistic (S)**  20.0000

**Normal Approximation**
**Z** 0.4410 **One-Sided Pr > Z** 0.3296 **Two-Sided Pr > |Z|** 0.6592

**t Approximation One-Sided Pr > Z**  0.3363 **Two-Sided Pr > |Z|** 0.6725

**Exact Test One-Sided Pr >= S**  0.3286 **Two-Sided Pr >= |S - Mean|** 0.6571 **Z includes a continuity correction of 0.5.**

**Kruskal-Wallis Test Chi-Square**  0.3457
**DF** 1 **Pr > Chi-Square** 0.5566

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metalaxyl

EPA MRID Number 48685802

Page 75 of 75 

### Wilcoxon Scores (Rank Sums) for Variable endpoint **Classified by Variable conc**

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score**  **0** 4 26.0 18.0 3.207135 6.50 **3** 4 10.0 18.0 3.207135 2.50 **Average scores were used for ties.**

**Wilcoxon Two-Sample Test Statistic (S)**  26.0000

**Normal Approximation**
**Z** 2.3385 **One-Sided Pr > Z** 0.0097 **Two-Sided Pr > |Z|** 0.0194

**t Approximation One-Sided Pr > Z**  0.0260 **Two-Sided Pr > |Z|** 0.0520

**Exact Test One-Sided Pr >= S**  0.0143 **Two-Sided Pr >= |S - Mean|** 0.0286 **Z includes a continuity correction of 0.5.**

**Kruskal-Wallis Test Chi-Square**  6.2222
**DF** 1 **Pr > Chi-Square** 0.0126

# DATA EVALUATION RECORD

METALAXYL

Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-46-2012 (MRID 48617604)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Sandra Hastings Date: 4/19/2012

Secondary Reviewer: Signature: Kelly Luck, M.S. Date: 5/15/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 6/05/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 6/05/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).METALAXYL I PC Code 113501 In vivo Hershberger Assay (2011) I Page 1of16 QCSPP 890.140Q/ OECD 441
J

--- 

### Primary Reviewer: Vincent Chen. M.S. 

### Signature:

## ~~~-=:::=V'~~Z::::=-- Risk Assessment Branch III, Health Effects Division (7509P) Date: z.4 :s'v'N Z.e>JS 

### Secondary Reviewer: Greg Akerman, Ph.D. 

### Signature: 

# .aa**.~**

# 1 Health Effects Division (7509P) Date: Cb:r:J3 Template version 08/2011 

### DATA EVALUATION RECORD

STUDY TYPE: In Vivo 

### Hershberger Assay (Rat); OCSPP 890.1400; OECD 441



### PC CODE: 113501 

### DP BARCODE: D399227

TXR#: 0054219 

### CAS#: 57837-19-1



### TEST MATERIAL (PURITY): Metalaxyl (98

. 2% a.i.)



### SYNONYMS: Alanine, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-, methyl ester



### CITATION: Carlson, M

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

. Charles River Laboratories, Preclinical Services, Horsham, PA. Laboratory Project ID number: 20012407, December 9, 2011. MRID 48617604

. Unpublished

.



### SPONSOR: Metalaxyl EDSP Consortium, 20 Ambriance Drive

, Burr Ridge, IL



### TEST ORDER#: CON-113501-18



### EXECUTIVE SUMMARY: In a Hershberger Assay (MRID 48617604) screening for androgenic activity, metalaxyl (98.2% a.i., Batch/lot# 20100540) in 2.0% aqueous carboxymethylcellulose (CMC) was administered daily via oral gavage to groups of eight 57-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 125, or 375 mg/kg/day. An 

### androgenic positive control group consisted of eight castrated rats exposed to 0.4 mg/kg/day of

testosterone propionate (TP) by subcutaneous (s.c.) injection.

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

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

, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 40, 125, or 375 mg/kg/d a y in conjunction with

a daily dose ofreference androgen TP at 0.4 mg/k g/ day by s.c. injection. The anti-androgenic positive control group consisted of eight castrated rats exposed to 0.4 mg/kg/day TP (s.c. injection) and 3 mg/kg/day flutamide (FT, via oral gavage). TP alone was used as the anti-androgenic negative control.

F or both components of t he assay , body weights and food consumption w e r e determined daily. Th e ani mals were dos e d for 10 cons e cutive days and t e rminated appro x imately 24 hour s after the final dose administration. At necropsy , the five androgen-dependent tissues

, liver, and kidneys

were collected and weighed.

All animals survived until scheduled termination. No adverse clinical signs of toxicity were obs e rv e d in the androg e n ago nist assay. In the anti

- andro ge n assay, treatm e n t r e la te d clinicalIn vivo Hershberger Assay (201 1) / Page 2 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441


### signs of toxicity (urine-stain ed abdominal fur) were observe d in three rats from the 375 mg/kg/day group.

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.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for a Hershberger assay (OCSPP 890.1400).In vivo Hershberger Assay (201 1) / Page 3 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441



### COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance and Quality Assurance statements were provided.In vivo Hershberger Assay (201 1) / Page 4 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories, Preclinical Services, Pennsylvania (PCS-PA)
Location: Horsham, PA Study Director: M.B. Carlson (June 29, 2011); A.M. Hoberman (previous) Other Personnel: K. Strauss (Principal Investigator) Study Period: June 24, 2011 to December 9, 2011



### 2. Test Substance: Metalaxyl Description: White to beige powder Source: Nation’s Ag, LLC (Knoxville, TN) Lot/Batch #: 20100540 (expiry date: June 13, 2012) Purity: 98.2% a.i. Stability: Stable in vehicle at room temperat ure for 25 hours and under refrigerated conditions for 10 days CAS #: 57837-19-1 Structure:

CH 3 N O

CH 3 O CH 3 CH

3 O O CH

3



### 3. Reference Androgen: Testosterone propionate (TP) Supplier: PCCA USA (Houston, TX) Item #: Lot/Batch #: 23-1029 C141154 (expiry date: May 19, 2013) Purity: 99.7% CAS #: 57-85-2



### 4. Reference Anti-androgen: Flutamide (FT) Supplier: Product #: Tokyo Chemical Industry (TC I), Co., LTD (Tokyo, Japan) F0663 Lot/Batch #: 7EDOD (expiry date: January 12, 2012) Purity: 99.8% CAS #: 1311-84-7



### 5. Solvent/Vehicle Control (test substance): Carboxymethylcellulose (CMC)

Supplier: Product #: Sigma Aldrich (St. Louis, MO) C5678 (sodium salt, low viscosity) Lot/Batch #: 060M0079V (expiry (retest) date: June 30, 2012) Rationale (if other than water): Use of an aqueous solution/suspension is recommended and an analytical method is available. Final concentration: 2.0% in reverse osmosis memb rane-processed deionized waterIn vivo Hershberger Assay (201 1) / Page 5 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441



### Solvent/Vehicle Control (reference substances): Corn oil

Supplier: Charkit Chemical Corporation Lot/Batch #: M-631 (expiry date: March 18, 2012) Rationale (if other than water): Not applicable Final concentration: Not applicable



### 6. Test Animals: Species: Rat (castrated peripubertal males) Strain: Sprague Dawley, Crl:CD(SD) Age/weight at dose initiation: Post-natal day (PND) 54-60; 213.4 – 271.8 g Source: Charles River Laborat ories (Portage, MI) Housing: 2-3 rats/cage in clear plas tic nesting boxes with heat-treated laboratory-grade pine shavings Diet: Harlan’s Teklad 2018c meal food or Certified RodentDiet® 5002 (PMI® Nutrition International, St. Louis, MO), ad libitum ; total genistein compounds were ≤ 213 ppm Chewable Nylabones® were supplied during the study. Water: Reverse-osmosis treated tap water , ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 19-25oC 30-70% >10/hr 12 hrs light/12 hrs dark Acclimation period: 7 days at facility (androgen a ssay); 12 days post-castration 6 days at facility (anti-androg en assay); 11 days post-castration



### B. STUDY DESIGN

1. In-life dates: Start: June 28, 2011 End: July 21, 2011

2. Study Design: In a Hershberger Assay conducted to screen for pot ential androgenic activity, metalaxyl was administered daily via oral gavage to castrated male rats. A positive control group received testosterone propionate (TP). Positive androgenic activity is defined as a significant increase in two or more target organ weights compared to the vehicle control group. To screen for the potential anti-androgenic activity, metalaxyl was administered daily via oral gavage to gr oups of castrated male rats in conjunction with TP at 0.4 mg/kg/day by s.c. injection. The positiv e control group received FT plus TP. Anti-androgenic activity is indicated by a statis tically significant decrease in two or more target organ weights of the treated groups (t est substance + TP) comp ared to the TP-only control group. For both assa ys, the animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the fi nal dose administration for measurement of target organ weights.

3. Study Schedule: Castrated sexually mature male rats (castrated by the supplier on PND 43, 45, or 49) were received at th e laboratory at PND 50 (androge n assay) or PND 48 or 54 (anti-androgen assay). Dosing was initiated fo llowing a 6- or 7-day acclimation period. The dose administration period was from PND 57 to 66 (androgen assay), or PND 54 to 63 or PND 60 to 69 (anti-androgen assay). Ra ts were euthanized on PND 67, 64, or 70, respectively, approximately 24 hours after the last dose and necropsied for organ weight
measurements.

4. Animal Assignment: Animals were assigned, stratified by body weight, to the test groups noted in Table 1 using computer-generated (weight-ordered) rando mization procedures.In vivo Hershberger Assay (201 1) / Page 6 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441


### Statistical analysis indicated that there were no significant differences in group means at study initiation. Furthermore, the body weight of each animal was within  20% of the
group mean.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Males Androgen Agonist Assay Vehicle control (2% aqueous CMC) 0 8 Metalaxyl Low 125 8 Metalaxyl High 375 8 Testosterone propionate (TP), positive control 0.4 8 Anti-Androgen Assay Vehicle control (2% aqueous CMC) + TP 0 + 0.4 8 Corn Oil Vehicle + TP 0 + 0.4 8 Metalaxyl Low (+TP) 40 + 0.4 8 Metalaxyl Mid (+TP) 125 + 0.4 8 Metalaxyl High (+TP) 375 + 0.4 8 Flutamide + TP, positive control 3 + 0.4 8 a Data were obtained from Text Tables 9 and 10 on pages 25-26 of the study report.



### 5. Dose Selection Rationale: The dose levels were selected based on the results from rangefinding studies (Protocols 20012405 and 20013495); citations for the studies were not provided. Clinical signs of systemic toxicity were observed in adult male and female rats and weanlings at 500 mg/kg/day; mortality was s een in adult female rats at >500 mg/kg/day and in weanlings of both sexes at 750 mg/kg/ day. Body weight gain was decreased at the two highest dose levels in adult male rats (750 and 1000 mg/kg/day) a nd liver weights were increased at all dose levels in the adult male rats (250-1000 mg /kg/day) and in weanlings of both sexes (250 and 500 mg/kg/day). Due to the severity of the clinical signs observed in adult rats at >500 mg/kg/day, the high dose selected for the Hers hberger assay was 375 mg/kg/day. The next lower dose of 125 mg/kg/day represents one-third of the high dose, and the low dose for the anti-androgen assay ( 40 mg/kg/day) equals one-third of the 125 mg/kg/day dose.

6. (a) Dose Preparation: Metalaxyl dose formulations were prepared weekly by mixing appropriate amounts of test substance with 2% aqueous CMC; daily aliquots were dispensed and stored refrigerated until use. Prepared doses were allowed to return to room temperature and stirred for at least 30 minutes prior to dosing. Reference materials (TP and FT) were prepared daily in corn oil.

Homogeneity and stability of metalaxyl formulations (0.5 and 100 mg/mL) in 2% aqueous CMC were previously evaluated in conjunc tion with Protocol 20012404 (report attached as Appendix 5 of the subject study) . During the Hershberger st udy, duplicate samples of dose formulations (8, 25, and 75 mg/mL) were collected on the first and last days of preparation, and analyzed for achieved concentration.In vivo Hershberger Assay (201 1) / Page 7 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441


### ( b) Dose Analysis:

Results

Homogeneity (% RSD): 0.4% and 1.5%

Stability (% of Day 0): 101.3% and 101.8% following storag e protected from light at room temperature (20-25°C) for 25 hours; 99.7% and 100% following storage under refrigerated conditions (2-8°C) for 10 days

Concentration (% of nominal): 99.1-106.7%

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

7. Dosage administration: Metalaxyl formulations were administered to the animals daily via oral gavage (5 mL/kg) for 10 da ys. TP was given via s.c. inje ction at 0.5 mL/kg; injection sites were rotated between six areas on th e dorsum to minimize ir ritation. FT was administered via oral gavage at 5 mL/kg. Do se volumes were adjusted daily based on the most recently recorded body weight and admini stered at approximately the same time each day to each animal.

8. Statistics: Means, standard deviations, coeffici ents of variance, and percentages were calculated. Statistical analyses were perfor med using SAS System, version 6.12 or Quattro Pro 8, version 8.0. Clinical observations and other proportional data were analyzed using the variance test for homogeneity of the binomial distribution.

Continuous data (e.g., body wei ghts, body weight changes, fo od consumption values, and organ weight data) were analyzed using Ba rtlett’s test of hom ogeneity of variances and the analysis of variance when appropriate [i.e., Bartlett’s test was not significan t (p>0.001)]. If the analysis of vari ance was significant (p ≤ 0.05), Dunnett’s test was used to identify the statistical significance of the individual groups . If the analysis of variance was not appropriate [i.e., Bartlett ’s test was significant (p ≤ 0.001)], the Kruskal-Wallis test was used ( ≤ 75% ties). In cases where the Kruskal-Wa llis test was statistically significant (p ≤ 0.05), Dunn’s method of multiple comparisons was used to identify the statistical significance of the individual groups. If there we re >75% ties, Fisher’s Exact test was used to analyze the data. Data from the evaluation for preputial separation were analyzed using Fisher’s Exact test. Absolute organ weights were also an alyzed by analysis of covariance (ANCOVA), using the initial body weight from the first day of treatment as the covariate. Relative organ weight comparisons were not made. The stat istical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Animals were assessed for vi ability twice daily. General appearances were observed on the day of recei pt, at least weekly during the acclimation period and daily before administration during the dosing period. Additionally post-dose observations were recorded w ithin 2 hours of dosing and at the end of the normal working day. Preputial separation was recorded once on the day of termination.In vivo Hershberger Assay (201 1) / Page 8 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441



### 2. Body Weight: Animals were weighed weekly during the acclimation period, at randomization, daily throughout the dos ing period, and at termination.

3. Food Consumption: Food consumption was measured daily during dosing and at termination. Values were reported as group mean daily food consumption (g/animal/day).

4. Serum Hormone Measurements (Optional): At study termination, each animal was euthanized by decapitation and blood samples were collected from trunk blood for potential future serum hormone analyses; however, seru m hormone analyses we re not conducted for this study.

5. Dissection and Measurement of Tissue and Organ Weights: On PND 64, 67, or 70 (approximately 24 hours after the final administ ration of the test s ubstance), all animals were euthanized by decapitation and subjected to a gross necropsy. The five mandatory androgen-dependent organs (ven tral prostate, seminal vesicl es, LABC, Cowper’s gland, and glans penis) were excised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400). Additionally, the liver and kidneys were collected, trimmed of any adhering fascia or fat, and weighed. Representative samples of Cowper’s glands, seminal vesicles, ventral prostate, kidneys, and liver were preserved in 10% neutral buffered formalin for possible future histopathological evaluation. In addition, a portion of the liver was flash frozen for possible future analysis.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived unt il scheduled termination.

2. Clinical signs of toxicity: No adverse clinical signs of toxicity were observed in the androgen agonist assay. In the anti-androgen assay, three rats from the 375 mg/kg/day group had urine-stained abdominal fur, which was increased (p ≤ 0.01) in comparison with the 2% CMC vehicle control group (0 occurrence s). Other clinical signs observed in the corn oil vehicle group (red fur on back; ungroomed coat; and chromorhinorrhea) and in the 125 mg/kg/day metalaxyl group (red fur and s cabbing on back) were considered to be unrelated to treatment because a dose-depende nt pattern of effect was not observed, the clinical sign was attributed to the TP injec tion site, and/or the c linical sign was only observed in the corn oil vehicle control group.

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data for the androgen agonist assay are pres ented in Table 2. Mean body weights and body weight gains were comparable to the vehicle c ontrols throughout the duration of the assay.

Mean body weights were increased (p ≤ 0.05) by 7-9% on Days 7-11 in the TP positive control group compared to the vehicle controls, resulting in an increase (p ≤ 0.01) of 31% in overall (Days 1-11) body weight gains.In vivo Hershberger Assay (201 1) / Page 9 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

TABLE 2. Selected Group Mean Body Weights and Cumu lative Body Weight Gains (g) in the Androgen Agonist Assay a

Study Day Dose (mg/kg/day) Vehicle Control TP (0.4) Metalaxyl (125) Metalaxyl (375) N Mean SD N Mean SD N Mean SD N Mean SD
1 8 229.8 12.2 8 237.6 18.0 8 232.5 14.2 8 233.4 15.1 4 8 248.7 16.0 8 260.8 19.1 8 252.3 15.7 8 249.4 16.1 7 8 266.1 16.6 8 285.2* ( ↑ 7) 18.5 8 271.4 15.3 8 271.3 17.8

8 8 270.1 16.3 8 294.4** ( ↑ 9) 19.7 8 276.5 16.7 8 275.8 18.2

9 8 276.5 16.5 8 300.6** ( ↑ 9) 18.3 8 283.5 15.8 8 282.0 19.6

10 8 281.3 15.6 8 307.4** ( ↑ 9) 19.7 8 288.1 16.7 8 286.6 19.1

11 8 293.0 15.6 8 320.6** ( ↑ 9) 20.4 8 298.7 16.1 8 299.7 20.1

B ody Weight Gain (Days 1-11) 8 63.2 7.4 8 83.0** ( ↑ 31) 7.7 8 66.2 5.9 8 66.3 7.2

a Data were obtained from Tables 2 and 3 on pages 52 and 54 of the study report. Percent differences from controls (calculated by the reviewers) are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01



### Selected body weight and body weight gain data for the anti-androgen assay are presented in Tables 3a and 3b. Mean body weights of the metalaxyl treatment groups were comparable to the vehicle controls throughout the durat ion of the assay. Significant reductions (p ≤ 0.05) in mean body weight gains occurred on Days 1 to 2 in the 375 mg/kg/day group and on Days 4 to 5 in the 40 and 375 mg/kg/da y groups, and a signif icant increase (p ≤ 0.01) occurred on Days 3 to 4 in the 375 mg/kg/ day group. These fluctuations in mean body weight gain contributed to non- statistically signifi cant body weight gain reductions (90% to 91% of the metalaxyl vehicle control group va lue) during the overall study period in the 40, 125, and 375 mg/kg/day groups.

Mean body weights of the FT positive contro l group and overall mean body weight gains were comparable to the co rn oil vehicle controls.In vivo Hershberger Assay (201 1) / Page 10 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

TABLE 3a. Selected Group Mean Body Weights and Cu mulative Body Weight Gains (g) in the AntiAndrogen Assay a

Study Day Dose (mg/kg/day) Vehicle Control + TP (0.4) Metalaxyl (40) + TP (0.4) Metalaxyl (125) + TP (0.4) Metalaxyl (375) + TP (0.4) N Mean SD N Mean SD N Mean SD N Mean SD
1 8 245.0 11.7 8 244.6 12.0 8 243.3 14.3 8 245.3 14.4 4 8 271.8 12.6 8 273.5 14.6 8 271.3 16.1 8 275.4 16.1 8 8 306.2 13.5 8 301.5 13.7 8 300.6 20.0 8 302.0 18.0 11 8 342.4 19.4 8 332.6 17.3 8 332.3 21.0 8 333.4 20.9
B ody Weight Gain (Days 1-11) 8 97.4 11.7 8 88.0 8.8 8 89.0 7.9 8 88.1 10.2

a Data were obtained from Tables 8 and 9 on pages 64 and 66 of the study report. N Number of animals in the group SD Standard Deviation

TABLE 3b. Selected Group Mean Body Weights and Cu mulative Body Weight Gains (g) in the AntiAndrogen Assay a

Study Day Dose (mg/kg/day) Corn Oil Vehicle Control + TP (0.4) Flutamide (3) + TP (0.4) N Mean SD N Mean SD
1 8 245.8 11.0 8 243.4 10.9 4 8 272.8 13.9 8 270.6 13.0 8 8 304.8 18.0 8 296.2 15.2 11 8 338.5 22.5 8 326.3 16.7
B ody Weight Gain (Days 1-11) 8 92.7 12.7 8 82.9 8.2 a Data were obtained from Tables 8 and 9 on pages 64 and 66 of the study report. N Number of animals in the group SD Standard Deviation



### C. FOOD CONSUMPTION: Select food consumption values for the androgen agonist assay are presented in Table 4. Although increased (p ≤ 0.05) food consumption was observed on Days 4-5, Days 5-6, and Days 7- 8 for the 375 mg/kg/day group ( ↑ 8-12%) and fluctuations (p ≤ 0.05) in food consumption were observed on Days 4-5 ( ↑ 6%), Days 6-7 ( ↓ 6%), and Days 10-11 ( ↓ 5%) in the 125 mg/kg/day group, overall (Day s 1-11) average food consumption for both metalaxyl treatment groups were co mparable to the vehicle controls.

Food consumption in the TP positive control group was increased (p ≤ 0.05) starting on Day 4 through the dosing period ( ↑ 6-18%), for an increase (p ≤ 0.01) of 10% in average overall food consumption compared to the vehicle control.In vivo Hershberger Assay (201 1) / Page 11 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

TABLE 4. Selected Food Consumption (g/r at/day) in the Androgen Agonist Assay a

Study Day Dose (mg/kg/day) Vehicle Control TP (0.4) Metalaxyl (125) Metalaxyl (375) N Mean SD N Mean SD N Mean SD N Mean SD 4-5 8 19.0 1.2 8 22.4** ( ↑ 18) 1.0 8 20.2* ( ↑ 6) 1.3 8 20.8** ( ↑ 9) 1.1

5-6 8 21.0 1.4 8 22.8* ( ↑ 9) 1.8 8 20.5 1.1 8 23.6* ( ↑ 12) 3.2

6-7 8 21.4 1.2 8 24.1** ( ↑ 13) 1.4 8 20.1* ( ↓ 6) 1.0 8 21.4 1.0

7-8 8 20.5 1.6 8 24.1** ( ↑ 18) 0.7 8 20.6 0.9 8 22.2* ( ↑ 8) 1.4

9-10 8 22.9 1.0 8 26.5** ( ↑ 16) 0.8 8 22.1 1.2 8 22.9 1.5

10-11 8 21.4 0.4 8 22.6* ( ↑ 6) 1.3 8 20.4* ( ↓ 5) 0.7 8 21.6 1.3

(Days 1-11) 8 20.6 0.7 8 22.7** ( ↑ 10) 0.8 8 20.1 0.3 8 20.9 1.0

a Data were obtained from Table 4 on page 56 of the study report. Percent differences from controls (calculated by the reviewers) are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01



### Select food consumption values for the anti-and rogen assay are presented in Tables 5a and 5b. Decreased (p ≤ 0.05) food consumption was observed on Days 1-2, Days 4-5, and Days 8-9 for the 125 mg/kg/day group ( ↓ 6-28%); and Days 4-5 and Days 8-9 for the 40 mg/kg/day group ( ↓ 7% and 8%). In the 375 mg/kg/ day group, food consumption was increased (p ≤ 0.05) on Days 3-4 and 9-10 ( ↑ 9% and 11%) and decreased (p ≤ 0.01) on Days 1-2 and 4-5 ( ↓ 33% and 8%). Overall average food consumption was decreased (p ≤ 0.01) only in animals treated with metalaxyl at 125 mg/kg/day ( ↓ 6%) compared to the vehicle control.

Food consumption in the FT positive control group was decreased (p ≤ 0.05) starting on Day 2 through the dosing period ( ↓ 6-14%), for a decrease (p ≤ 0.01) of 7% in overall average food consumption compared to the corn oil vehicle control.In vivo Hershberger Assay (201 1) / Page 12 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

TABLE 5a. Selected Food Consumption (g/rat/day) in the Anti-Androgen Assay a

Study Day Dose (mg/kg/day) Vehicle Control + TP (0.4) Metalaxyl (40) + TP (0.4) Metalaxyl (125) + TP (0.4) Metalaxyl (375) + TP (0.4) N Mean SD N Mean SD N Mean SD N Mean SD 1-2 8 24.9 6.5 8 20.4 1.9 8 17.9* ( ↓ 28) 1.4 8 16.6** ( ↓ 33) 1.2

3-4 8 21.2 0.8 8 21.8 1.9 8 20.6 1.1 8 23.1* ( ↑ 9) 1.2

4-5 8 25.1 1.0 8 23.4* ( ↓ 7) 1.4 8 22.2** ( ↓ 12) 1.7 8 23.0** ( ↓ 8) 1.2

8-9 8 25.4 0.6 8 23.4** ( ↓ 8) 1.2 8 23.9** ( ↓ 6) 0.4 8 25.2 1.3

9-10 8 24.9 0.9 8 25.8 0.6 8 25.6 1.5 8 27.6** ( ↑ 11) 1.4

(Days 1-11) 8 24.6 1.2 8 23.7 1.0 8 23.2** ( ↓ 6) 0.6 8 23.9 0.6

a Data were obtained from Table 10 on page 68 of the study report. Percent differences from controls (calculated by the reviewers) are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01

TABLE 5b. Selected Food Consumption (g/rat/day) in the Anti-Androgen Assay a

Study Day Dose (mg/kg/day) Corn Oil Vehicle Control + TP (0.4) Flutamide (3) + TP (0.4) N Mean SD N Mean SD
2-3 8 21.6 0.8 8 19.2** ( ↓ 11) 0.5

4-5 8 21.6 1.9 8 19.5** ( ↓ 10) 1.0

6-7 8 24.0 1.0 8 22.4** ( ↓ 7) 0.4

7-8 8 22.8 1.3 8 19.0** ( ↓ 17) 1.0

8-9 8 23.1 1.4 8 19.8** ( ↓ 14) 1.2

9-10 8 24.4 1.5 8 21.9** ( ↓ 10) 0.7

10-11 8 22.1 1.4 8 20.8* ( ↓ 6) 0.8

(Days 1-11) 8 21.9 0.2 8 20.3** ( ↓ 7) 0.5

a Data were obtained from Table 10 on page 68 of the study report. Percent differences from controls (calculated by the reviewers) are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01In vivo Hershberger Assay (201 1) / Page 13 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441



### D. SERUM HORMONE CONCE NTRATIONS (Optional): Serum hormone concentrations were not determined.

E. ORGAN WEIGHTS: Accessory sex organ, liver, and kidney weights for the androgen agonist assay are presented in Ta ble 6. There were no treatment-r elated effects in any of the accessory sex organs or kidneys of animals dos ed with metalaxyl. However, mean liver weights were increased (p ≤ 0.01) by 17% in the 375 mg/kg/ day group compared to the vehicle controls. The study author reported th at increased liver wei ghts were consistent with results from other studies conducted with metalaxyl. No gross lesions were observed at necropsy, but one animal in the 375 mg/kg/ day group did not achieve preputial separation by necropsy.

Animals in the positive contro l (TP) group had increased (p ≤ 0.01) accessory sex organ weights as follows: 500% in seminal vesicles ; 691% in ventral prostate; 177% in LABC; 645% in Cowper’s glands; and 71% in glans pe nis. Liver and kidney weights in the positive control group were similar to the vehicle controls.

The %CV values for each accessory sex organ of the vehicle control and metalaxyl high dose group were less than the maximum Guideline-specified %CV values.

TABLE 6. Accessory Sex Organ, Liver, and Kidney We ights (g) from Androgen Agonist Assay in Sprague Dawley Rats a

Organ Dose (mg/kg/day) Vehicle Control Metalaxyl (125) Metalaxyl (375) TP (0.4)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Seminal vesicles 8 0.0853 0.0173 20 8 0.0729 0. 0168 23 8 0.0925 0.0122 13 8 0.5121** ( ↑ 500) 0.1130 22

Ventral prostate 8 0.0242 0.0063 26 8 0.0276 0. 0070 25 8 0.0232 0.0081 35 8 0.1914** ( ↑ 691) 0.0271 14

LABC 8 0.2247 0.0298 13 8 0.2003 0. 0357 18 8 0.2092 0.0248 12 8 0.6224** ( ↑ 177) 0.0549 9

Cowper’s glands 8 0.0074 0.0022 30 8 0.0074 0. 0025 34 8 0.0070 0.0012 17 8 0.0551** ( ↑ 645) 0.0337 61

Glans penis 8 0.0631 0.0085 14 8 0.0573 0. 0083 15 8 0.0618 0.0056 9 8 0.1079** ( ↑ 71) 0.0097 9

Liver 8 11.7 1.6 14 8 12.3 1.2 10 8 13.7** ( ↑ 17) 1.3 10 8 13.1 1.8 14

Kidneys 8 2.02 0.16 8 8 1.87 0.14 7 8 2.04 0.16 8 8 2.13 0.11 5 a Data were obtained from Table 6 on page 60 of the study report. Percent differences from controls (calculated by the reviewers) are included in parentheses. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p<0.05 ** Significantly different from controls at p<0.01



### Accessory sex organ, liver, and kidney weight s for the anti-androgen assay are presented in Tables 7a and 7b. There were no treatment-re lated effects in any of the accessory sex organs or kidneys of animals dosed with metalaxyl. However, mean liver weights were increased (p ≤ 0.01) by 13% in the 375 mg/kg/day group co mpared to the vehicle controls.In vivo Hershberger Assay (201 1) / Page 14 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441


### 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. A ll animals achieved preputial separation by
necropsy.

Animals in the FT group had decreased (p ≤ 0.01) accessory sex organ weights as follows: 78% in seminal vesicles; 70% in ventral pros tate; 44% in LABC; 53% in Cowper’s gland; and 19% in glans penis.

The %CV values for each accessory sex organ of the vehicle control and metalaxyl high dose group were less than the maximum guide line-specified %CV values, except for the %CV for glans penis of the vehicle control group (CV = 18%; maximum permissible CV = 17%).

TABLE 7a. Accessory Sex Orga n, Liver, and Kidney Weights (g) from Anti-Androgen Assay in Sprague Dawley Rats a

Organ Dose (mg/kg/day) Vehicle Control + TP (0.4) Metalaxyl (40) + TP (0.4) Metalaxyl (125) +TP (0.4) Metalaxyl (375) + TP (0.4)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Seminal vesicles 7 b 0.5628 0.0984 18 8 0.4942 0.1168 24 8 0.4598 0.0914 20 8 0.5547 0.0769 14

Ventral prostate 8 0.2494 0.0777 31 8 0.1985 0.0356 18 8 0.1799 0.0428 24 8 0.2119 0.0612 29

LABC 8 0.7214 0.0782 11 8 0.6827 0. 0859 13 8 0.6885 0.0338 5 8 0.6900 0.0562 8 Cowper’s glands 7 b 0.0513 0.0097 19 7 c 0.0510 0.0072 14 8 0.0496 0.0075 15 8 0.0530 0.0123 23

Glans penis 8 0.1401 0.0254 18 d 8 0.1412 0.0167 12 8 0.1406 0.0122 9 8 0.1315 0.0186 14

Liver 8 13.4 0.8 6 8 12.9 0.8 6 8 12.8 1.0 8 8 15.1** ( ↑ 13) 1.6 11

Kidneys 8 2.34 0.18 8 8 2.28 0.20 9 8 2.27 0.18 8 8 2.29 0.16 7 a Data were obtained from Table 12 on page 72 of the study re port. Percent differences from controls (calculated by the reviewers) are included in parentheses. b Excluded values that appeared incorrectly recorded. c Excluded values from one rat which only had the right Cowper’s gland. d Percent CV exceeded the maximum allowable limit. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01In vivo Hershberger Assay (201 1) / Page 15 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441

TABLE 7b. Accessory Sex Organ, Live r, and Kidney Weights (g) from Anti-Androgen Assay in Sprague Dawley Rats a

Organ Dose (mg/kg/day) Corn Oil Control + TP (0.4) FT (3) + TP (0.4)

N Mean SD CV (%) N Mean SD CV (%)

Seminal vesicles 8 0.5063 0.0728 14 8 0.1125** ( ↓ 78) 0.0383 34

Ventral prostate 8 0.1980 0.0443 22 8 0.0597** ( ↓ 70) 0.0107 18

LABC 8 0.6567 0.0554 8 8 0.3699** ( ↓ 44) 0.0467 13

Cowper’s glands 8 0.0475 0.0055 12 8 0.0221** ( ↓ 53) 0.0054 24

Glans penis 8 0.1504 0.0148 10 8 0.1218** ( ↓ 19) 0.0185 15

Liver 8 13.5 1.3 10 8 12.6 1.0 8 Kidneys 8 2.26 0.21 9 8 2.14 0.09 4 a Data were obtained from Table 12 on page 72 of the study re port. Percent differences from controls (calculated by the reviewers) are included in parentheses. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: On the basis this bioassay, administration of metalaxyl at dose levels as high as 375 mg/ kg/day did not result in an androgenic or antiandrogenic response.

B. AGENCY COMMENTS: All animals survived until scheduled termination. No adverse clinical signs of toxicity were observed in the androgen a gonist assay. In the anti-androgen assay, treatment related clini cal signs of toxicity (urine -stained abdominal fur) were observed in three rats from the 375 mg/kg/day group.

In the androgen agonist assay, mean body wei ghts and body weight gains in the metalaxyl treatment groups were comparable to the ve hicle controls throughout the duration of 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. Although significant fluctuations in food consumption was obser ved on several days for the 375 and 125 mg/kg/day groups, overall average food consum ption for both metala xyl treatment groups was comparable to the vehicle controls. Overall average food consumption in the positive control group was increased (p ≤ 0.01) by 10%. No treatment-related effects were observed in any of the accessory sex organs or kidneys of metalaxyl-dosed animals. However, mean liver weights were increased (p ≤ 0.01) by 17% in the 375 mg/kg/day group compared to the vehicle controls, which is reportedly consiste nt with results from other studies conducted with metalaxyl. No gross lesions were obser ved at necropsy, but one animal in the 375 mg/kg/day group did not achieve preputial sepa ration by necropsy. Animals in the TP groupIn vivo Hershberger Assay (201 1) / Page 16 of 16 METALAXYL / PC Code 113501 OCSPP 890.1400/ OECD 441


### had the expected increases in accessory se x organ weights. Th e performance criteria indicated that this assay wa s performing as expected.

In the anti-androgen assay, mean body weight s and overall body weight gains of the metalaxyl treatment groups were comparable to the vehicle controls. Animals dosed with TP + FT (positive control) had mean body we ights and overall mean body weight gains comparable to the corn oil vehi cle controls. Al though significant (p ≤ 0.05) decreases in food consumption were observed on several days for the 40, 125, and 375 mg/kg/day groups, overall average food consumption was decreased (p ≤ 0.01) only in animals treated with metalaxyl at 125 mg/kg/day ( ↓ 6%). Overall average food c onsumption in the TP + FT group was decreased (p ≤ 0.01) by 7% compared to the corn oil vehicle control. No treatment-related effects were observed in any of the accessory sex organs or kidneys of metalaxyl-dosed animals. However, mean liver weights were increased (p ≤ 0.01) by 13% in the 375 mg/kg/day group compared to the vehicle controls. No gross lesions were observed at necropsy, but the left Cowpe r’s gland was not present in one animal in the 40 mg/kg/day group. Animals in the TP + FT group had the expected decreases in accessory sex organ weights.

The %CV values for each accessory sex organ of the vehicle control and metalaxyl high dose group were less than the maximum guide line-specified %CV values, except for the %CV for glans penis of the vehicle control group (CV = 18%; maximum permissible CV = 17%).

The high dose adequately challenged the animal s based on data from earlier studies showing clinical signs of system ic toxicity at doses ≥ 500 mg/kg/day and the increased liver weights observed at 375 mg/kg/day in the subject assay.

No statistically significant changes were seen in two or more of the five androgen sensitive tissue weights. Glyphosate was negative for an drogenicity and anti-androgenicity in the Hershberger assay.

C. STUDY DEFICIENCIES: None

# DATA EVALUATION RECORD

METALAXYL

Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-19-2012 (MRID 48701301)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 9/18/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 9/27/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 10/02/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 10/02/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).METALAXYL/ 113501 Female Pubertal Assay (2012) I Page 1 of 17 OCSPP 890.1450/ OECD None



### Primary Reviewer: Vincent Chen. M.S.

## Signature:~....!::::::....!:""""""=::..._-~ __ Risk Assessment Branch III, Health Effects Division (7509P)**Date::=~'Z...~Lt~~ijl~~** 

### Secondary Reviewer: Elizabeth Mendez, Ph.D. 

### Signaturlt! Health Effects Division (7509P) Date:**-- --"'** **+-"~ "'---** 


### ! DATAEVALUATIONRECORD

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None.



### PC CODE: 113501 

### DP BARCODE: D404681



### TXR#: 0054219 

### CAS #: 57837-19-1



### TEST MATERIAL (PURITY): Metalaxyl (>98%)



### SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate



### CITATION: Carlson, M.B. (2012). A Pubertal Development and Thyroid Function Study ofMetalaxyl by Oral Gavage in Peripubertal Male and Female Rats. Charles River Laboratories Preclinical Services, Pennsylvania; Horsham, PA. Laboratory Study No.: 20012408, July 17, 2012. MRID 48701301. Unpublished.



### SPONSOR: Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL



### TEST ORDER#: CON-113501-18



### EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48701301), 15 female Sprague-Dawley (Crl:CD(SD)) rats/dose group were treated daily via oral gavage with metalaxyl (>98% a.i., Lot# 20100540) in 2.0% aqueous carboxymethylcellulose at doses of 0, 125, 250, or 350 mg/kg/day from postnatal day (PND) 22 to 42 or 43. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment were recorded. Following sacrifice on PND 42 or 43, blood was collected for clinical chemistry analyses, and total thyroxine (T 4) and thyroid stimulating hormone (TSH) levels were determined using radioimmunoassays. Adrenal, liver, pituitary, thyroid, and urogenital organs

were weighed and examined macroscopically. The uterus, ovary, thyroid, and kidney were examined microscopically.

No treatment-related related effects were observed on survival, clinical signs, body weights, body weight gains, or gross pathology. No treatment-related histological lesions were noted in the thyroid or uterus, and the colloid area and follicular cell height in the thyroids of the treated
groups were generally similar to control. Age of attainment of VO, body weight at VO, and proportion/incidence of unopened vagina in the treated groups were similar to control animals. No treatment-related effect was observed on the mean age of first vaginal estrus, mean cycle length,% cycling,% regularly cycling, and the cycle status at necropsy. Serum T4 and TSH levels in the treated groups were similar to the control values.Female Pubertal Assay (2012) / Page 2 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None



### 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.

The assay satisfies the EDSP Tier 1 Test Order require ments for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE: Signed and dated Data Confidentiali ty, GLP Compliance, Quality Assurance, and Flagging statements were provided.Female Pubertal Assay (2012) / Page 3 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories Prec linical Services, Pennsylvania

Location: Horsham, PA Study Directors: M.B. Carlson Other Personnel: Julian Gulbinski, III,(dose formulation analys is and laboratory manager), Dr. Saroj Das (hormone analysis), Carol S. Sloan (horm one analysis), Rebecca M. Lucke (clinical chemistry), Carol J. Detrisac (histology and histopathology) and Frederick T. Les (ovarian follicle counts) Study Period: June 17, 2011 through July 17, 2012



### 2. Test Substance: Metalaxyl Description: White to beige powder Source: Nation’s Ag, LLC, Knoxville, TN Lot #: 20100540 (expires June 13, 2012 and July 26, 2011) Purity: 98.2±0.3% and 100.4±0.7% Stability: Protected from light, the test compound was stable in the vehicle at 20-25°C for at least 25 hours and at 2-8°C for 10 days. CAS #: 57837-19-1 Structure:

CH 3 N O

CH 3 O CH 3 CH

3 O O CH

3



### 3. Vehicle:

Source: Catalog #: Lot #: 

### 2.0% aqueous carboxymethylcellulose in reverse osmosis membrane-processed deionized water Sigma Aldrich, St. Louis, MO C5678 060M0079V (retest date: June 30, 2012)



### 4. Test Animals: Species: Rat (females) Strain: Sprague-Dawley (Crl:CD(SD)) Age/ Mean Weight at Study Initiation: PND 22 / 59.6-60.2 g

Source: Charles River Laboratories International, Inc. (Portage, MI) Housing: Parental dams were individual ly housed in clear plastic nest ing boxes (n=2/box) with heattreated, laboratory-grade, pine shaving beddi ng; each dam was housed with her litter until weaning on PND 21. After weaning, 2 females were housed per cage. Diet: Harlan Teklad 2018c meal feed (Teklad Diets, Madison, WI), ad libitum . It was stated that the total genistein equivalents (aglycone) or daidzein were ≤ 300 ppm. Water: Reverse osmosis-purified and deionized tap water, ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 69.0-80.2°F 42.5-76.7% ≥ 10/hour 14 h light / 10 h darkFemale Pubertal Assay (2012) / Page 4 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None



### STUDY DESIGN

1. In-Life Dates: Start: July 6, 2011 End: Approximately July 27, 2011

2. Mating: Time-mated, pregnant nulliparous Spra gue Dawley Crl:CD(SD) dams (age not reported) were received from the supplier on Gestation Day 8 and were allowed to deliver naturally and rear their pups to PND 21. On PND 4, litters with 9 or more pups were standardized to 8 pups (4 males and 4 female s whenever possible) through removal of pups with weights furthest from the population mean for the appropriate sex within each litter. Litters with fewer than 8 pups were not retained.

3. Animal Assignment: Following weaning on PND 21, an equal number of rats from the heavy and light ends of the body weight distribu tion were eliminated, and the remaining rats were randomly assigned to the dose groups, stra tified by dose weight. Littermates were not placed in the same dose group. The te st groups are provided in Table 1.

TABLE 1. Study Design a
Test group Dose (mg/kg/day) # of Females

Control 0 15

Low 125 15

Medium 250 15 High 350 15
a Data were obtained from Text Table 5 on page 26 of the study report.



### 4. Dose Selection Rationale: The doses were selected based on the results from dose-range studies conducted in rats (Protocols 20012405 and 20013495) and clinical signs observed in the uterotrophic study on metala xyl (MRID 48617606). Clinical si gns of systemic toxicity were observed in adult male and female rats and weanlings at ≥ 500 mg/kg/day; mortality was seen in adult female rats at ≥ 500 mg/kg/day and in weanlings of both sexes at 750 mg/kg/day. Body weight gain was decreased at the two highest dose le vels in adult male rats (750 and 1000 mg/kg/day), and liver weights were increased at all metalaxyl dose levels in the adult male rats (250, 500, 750, and 1000 mg /kg/day) and in weanlings of both sexes (100, 250, 500, and 750 mg/kg/day). Based on thes e results, 350 mg/kg/day was selected as the high dose. The Sponsor utilized three dose levels to facilitate the interpretation of results. Minimal or no toxicity was expect ed at the low dose leve l of 125 mg/kg/day, and the midrange dose of 250 mg/kg/day was select ed per Guideline recommendation for a twofold increase from the low dose.

5. Dose Preparation and Analysis: Metalaxyl dose formulati ons were prepared weekly by adding appropriate amounts of the test com pound to 2.0% aqueous carboxymethyl-cellulose in deionized water. The dose formulations we re dispensed into dail y aliquots (volume not reported), which were stored refrigerated until use. Samples from the first and last preparation of each dose group were collected an d shipped on ice packs to the analytical laboratory. The samples were shipped on the date prepared or one day after preparation and were analyzed upon receipt or stored in a re frigerator (2-8°C) until analysis. DuplicateFemale Pubertal Assay (2012) / Page 5 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None



### samples collected from the middle stratum of the formulations were analyzed for concentration. Single sets of samp les were analyzed for density.

Stability and homogeneity analyses were previously performed in conjunction with Protocol 20012404. Stability was evaluated for 0.5 mg/mL and 100 mg/mL dose formulations. The dose formulations were analyzed before and af ter storage (while prot ected from light) at 20-25°C for at least 25 hours and at 2-8°C for 10 days. The relative standard deviation (RSD) of the mean for each of the average concentration values for the top, middle, and bottom of each dose formulation was calculated to assess homogeneity.

Results of Dose Analysis

Homogeneity (RSD): 0.4-1.5%

Stability (% of time zero): 99.7-101.8%

Concentration (% of nominal): 96.9-102.6%

The analytical data indicate d that the mixing procedure wa s adequate and the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: The dose formulations were allowed to warm to room temperature, and then stirred co ntinuously for at least 30 minut es prior to administration and throughout dosing. Dose formulations were ad ministered once daily by oral gavage in a dose volume of 5 mL/kg body weight. The dose fo rmulations were administered for 21 or 22 consecutive days from PND 22-42/43. Dose s were adjusted based on the daily body weight recorded before administration and we re administered at a pproximately the same time each day, between 0700 hours and 0900 hours, except on one occasion (no details were provided for this exception).

7. Statistics: Mean, standard deviation, and coefficien ts of variation (CVs) were calculated. The following statistical analyses were performed.Female Pubertal Assay (2012) / Page 6 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None

Classification Procedure
Body weights Body weight changes Age at VO Bodyweight at VO Absolute organ weights Relative liver, kidney, adrenal, and pituitary weights
Serum hormone values Clinical chemistry data Data were analyzed with Ba rtlett’s Test of Homogeneity of Variances and Analysis of Variance (ANOVA), when appropriate [i .e., Bartlett’s Test was not significant ( p >0.001)]. If the ANOVA was significant ( p ≤ 0.05), Dunnett’s Test was used to identify the statistically significant diff erences between the control group and each treated group. If the ANOVA was not appropriate [i.e., Bart lett’s Test was significant ( p ≤ 0.001)], the Kruskal-Wallis Test was used ( ≤ 75% ties). In cases where the Kruskal-Wallis Test was statistically significant ( p ≤ 0.05), Dunn’s Method of Multiple Comparisons was used to identify the stat istically significant differences between the control group and each treated group.

Age at VO Bodyweight at VO Organ weight Data were also analyzed by Analysis of Covariance (ANCOVA), with the body weight recorded at weaning on PND 21 as the covariate. If the ANCOVA results were significant ( p ≤ 0.05), ANOVA was used to identify the statistically significant differences between the control group a nd each treated group. A modified Bonferroni correction was used for each pairwise comparison. A pairwise comparison was deemed statistically significant if its corresponding p -value was lower than or equal to the considered significance level (0.05 or 0.01) divided by the square root of the number of performed pairwise comparisons. In cases where sexual maturation did not occur prior to eu thanasia, the last day of observation +1 day was considered the age of sexual maturation. Cycling status Percent of animals cycling regularly Data were analyzed with the Variance Test for Homogeneity of the Binomial Distribution.



### The statistical analyses were considered appropriate.

C. METHODS

1. Mortality and Clin ical Examinations: Animals were observed at least twice daily for mortality. Rats were observed for general a ppearance daily prior to dose administration. Postdose observations were reco rded within 2 hours of dose ad ministration and at the end of the normal working day.

2. Body Weight: Body weights were collected prior to allocation, daily before dose administration, and at termination. Over all (PND 22-42) body weight gain was also reported.

3. Food Consumption: Food consumption was recorded daily.

4. Vaginal Opening (VO): Female rats were evaluated fo r the age of vaginal patency daily beginning on PND 22. The appearance of a small "pin hole", a vaginal thread, and complete VO was recorded on the days observed. The body weight on the day of complete VO was also reported.

5. Estrous Cyclicity: Estrous cycling were evaluate d daily by examination of vaginal cytology beginning on the day complete VO was observed, and continued until the day before euthanasia. The stage of estrous at necropsy was confirmed by the pathologist by evaluation of the fixed tissues. Vaginal smears were classified as diestrus (presence of leukocytes and/or cornified/irregularly shaped nucleated epithelial cells), proestrus (presence of nucleated epithelial cells), or estrus (presence of cornified epithelial cells) and the stage recorded daily. Metestrus was classified as an early part of diestrus, rather than aFemale Pubertal Assay (2012) / Page 7 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None



### late part of estrus. Age at first estrus was recorded, and ra ts that stopped cycling were noted. All estrous smears were taken at appr oximately the same time each day (after dose administration). The overall pattern of estrous cycling for each female was characterized as regular cycling (recurring 4- to 5-day cycles), irregular cycli ng (diestrus longer than 3 days or a period of cornification longer than 2 days), or not cycling (prolonged periods of either cornification or leukocytic smears). In cas es where there were too few days between vaginal opening and the end of the study to obser ve more than one cycle, classification was based on the available data. The default assump tion was that animals we re cycling regularly if the partial data fit the definition, and were irregularly cycling if the study ended without being able to distinguish between irregular cycling and not cycling.

6. Sacrifice and Pathology: Animals were moved to the necropsy holding room on the day prior to the final dose admini stration. Doses were administered in the holding room between 0700 and 0900 hours. Euthanasia by decapitation began 2 hours later and was completed by 1300 hours. Dose administration and termination was performed across dose groups. After euthanasia and prior to tissue collection, trunk blood was collected from all animals, and serum was obtained by centrifuga tion of whole blood samples (1.5 and 2 mL, respectively) in serum separator tubes. Blood samples for thyroid hormone and testosterone analyses (1.5 mL) were centrifuged fo r approximately 30 min at 4°C and 3000 × g ; samples for clinical chemistry evaluation were allowed to clot for 30 min at room temperature and centrifuged for approximately 10 mi n at room temperature and 3500 × g . The two sets of serum samples were immediately frozen on dry ice, and shipped on dry ice to AniLytics, Inc. (Gaithersburg, MD) for hor mone analysis or Charles Ri ver Laboratories, Preclinical Services, Ohio for clinical chemistry analysis . Serum samples for hormone analyses were maintained at ≤− 80°C until analysis, and clinical chemistry samples were maintained at ≤− 20°C. Necropsies were performed for each an imal, gross lesions were collected (not evaluated microscopically), and images we re generated for illustration of gross observations.

a. Hormone Analysis: Total T 4 

### and TSH were measured in serum using radioimmunoassays (RIAs). Total T 4 

### was determined with a RIA kit from Siemens (Cat # TKT41) and TSH was determined with an in-house-deve loped assay specific for rat TSH.

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.Female Pubertal Assay (2012) / Page 8 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None

ELECTROLYTES OTHER

X Calcium X Albumin
X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol
X Potassium X Globulins
X Sodium X Glucose

ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) X Triglycerides Creatine phosphokinase X Albumin /globulin ratio (calculated) Lactic acid dehydrogenase (LDH) Bile acids X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase (SDH) X Gamma-glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in fe male rats based on guideline 890.1450.



### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed and grossly observed fr om all study animals. The (XX) organs, in addition, were subjected to histological examination.

UROGENITAL OTHER XX Ovaries (paired, without oviducts)*+ XX Thyroid a * + XX Uterus*+ (with cervix, wet and blotted) X Liver* XX Kidneys (paired)*+ X Adrenals (paired)* X Pituitary* a Thyroid was weighed post-fixation. * Weights required base d on guideline 890.1450. + Histopathological examination re quired based on guideline 890.1450.



### All collected organs, except the thyroid/trachea , were weighed prior to fixation. Paired organs (kidneys, adrenals, and ovaries) were we ighed together. The ut erus and cervix were separated from the vagina and weighed. The uterus was weighed again following removal of the fluid in the lumen (blotted weight).

The right kidney, both ovaries, a nd uterus were fixed in neutral buffered, 10% formalin for at least 24 hours, and rinsed and retained in 70% ethanol until histolog ical processing. After the initial fixation of the thyroid in 10% fo rmalin, the thyroid/para thyroid was dissected from the trachea, blotted, weighed, and re tained in 70% ethanol until histological processing. Tissues were embedded in para ffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Tissues were shipped under ambient conditions to Charles River Laboratories, Pathology Associat es (MD). Other tissues were collected and retained, but not examined microscopically; thes e tissues included the ad renal, gross lesions, liver, and pituitary.Female Pubertal Assay (2012) / Page 9 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None



### Thyroid sections were evaluated for follicular ce ll height and colloid ar ea using a five-point grading scale (1 = shortest/sma llest; 5 = tallest/largest), and any abnormalities/lesions were noted. A minimum of two sections of each of the two lobes of the thyroid were evaluated.

Ovarian histopathology included evaluation of follicular development (including presence/ absence of tertiary/antral follicles, presence/absence of corpora lutea, changes in corpus luteum development, changes in number of both primary and atretic follicles), in addition to any abnormalities/lesions (such as ovarian atro phy). Five random sections of each ovary were evaluated. Any lesions or atrophy within the ovary and any changes in corpora lutea were evaluated histopathologically by the Principal Investigator for histopathology at Charles River Laboratories, Pathology Associat es, IL. The ovarian fo llicular counts were conducted by the Principal Investigator at Ch arles River Laboratories, Pathology Associates (MD).

Uterine histopathology documented cases of uterine hyper- or hypotrophy (as characterized by changes in uterine horn diameter and my ometrial, stromal, or endometrial gland development). On the last day of dose administ ration, female rats did not receive an estrous cycle evaluation; therefore, th e pathologist evaluated the reta ined uterus and cervix of each female rat to determine the final stage of es trous on the day of necropsy. When possible, a longitudinal section including a portion of th e remaining vaginal epithelium was obtained for evaluation.

II. RESULTS

A. MORTALITY: No mortality was observed.

B. CLINICAL SIGNS OF TOXICITY: Incidental adverse clinical signs in the female rats were limited to one animal in the 250 mg/kg/ day dose group that pres ented with ptosis on PND 22, and one animal in the 350 mg/kg/ day dose group that presented with chromodacryorrhea on PND 31-42 and lacrimation on PND 32-34.

C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Body weights, body weight gains, age of attainment of VO, body weight at VO, and proportion/incidence of unopened vagina are presented in Table 2, and all values in the treated gr oups were similar to controls. No effect was noted on daily body weight measurements or feed consumption.

The Guideline performance criteria were me t for age and weight at VO and final body weight, except for two differences: CV for age at VO (7.9%) was greater than the maximum acceptable value (6.52%); and CV for body weight at VO (14.8%) was greater than the maximum acceptable value (13.97%).Female Pubertal Assay (2012) / Page 10 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None

TABLE 2. General Growth and Preputial Separation (PPS) a

Parameter Evaluated Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial Body Weight (PND 22; g) U 15 59.6 3.8 6.4 15 59.9 4.4 7.3 15 59.8 4.1 6.9 15 60.2 4.8 8.0

A 15 59.8 --- --- 15 59.8 --- --- 15 59.8 --- --- 15 60.1 --- ---
Body Weight at VO (g) U 15 119.6 17.7 14.8 15 121.4 16.0 13.2 15 119.4 14.3 12.0 15 128.8 13.7 10.6

A 15 119.6 --- --- 15 121.4 --- --- 15 119.4 --- --- 15 128.8 --- --- Final Body Weight (PND 42; g) U 15 166.8 9.2 5.5 15 171.7 10.3 6.0 15 170.5 13.2 7.7 15 172.5 9.4 5.4

A 15 166.9 --- --- 15 171.7 --- --- 15 170.5 --- --- 15 172.4 --- ---
Final Body Weight (% of control) U --- 102.9 102.2 103.4

Body Weight Gain (PND 22- 42; g) U 15 107.2 8.6 8.0 15 111.8 9.0 8.1 15 110.7 13.0 11.7 15 112.3 8.9 7.9

A 15 107.2 --- --- 15 111.8 --- --- 15 110.7 --- --- 15 112.3 --- ---
Age at Incomplete VO (PND) U 15 30.7 2.5 8.1 15 32.0 2.9 9.1 15 30.6 2.3 7.5 15 31.4 3.1 9.9

A 15 30.7 --- --- 15 32.0 --- --- 15 30.6 --- --- 15 31.4 --- ---
Age at VO (PND) U 15 33.0 2.6 7.9 15 33.1 2.6 7.9 15 33.1 2.3 6.9 15 34.3 2.2 6.4

A 15 33.0 --- --- 15 33.1 --- --- 15 33.1 --- --- 15 34.3 --- --- Proportion unopened (#/N) 0/15 0/15 0/15 0/15
a Data were obtained from Table 5 on pages 79-80 and Appendi x 15 on pages 374-380 of the study report. No statistical differences were detected between the treated groups and controls. b Calculated by the reviewers U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 N Number of animals examined SD Standard Deviation CV Coefficient of Variation --- Not applicable



### D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3. 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 controls. All ot her organ weights in th e treated groups were similar to controls. All adju sted weights in the treated gr oups were similar to controls.

The unadjusted values for all organ weights in the control group were within the acceptable range of the Guideline performance criteria, w ith the exception of the mean paired adrenal weight of 54.5 mg (acceptable range: 38.34-48.84 mg) and the mean fixed thyroid weight of 27.26 mg (acceptable range: 6.20-22.20 mg).Female Pubertal Assay (2012) / Page 11 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Liver (g) U 15 8.61 1.06 12.3 15 9.51* ( ↑ 10) 0.86 9.0 15 10.21** ( ↑ 19) 1.11 10.9 15 10.61** ( ↑ 23) 0.95 9.0

A 15 8.62 --- --- 15 9.50 --- --- 15 10.20 --- --- 15 10.61 --- ---

R 15 5.07 0.37 7.3 15 5.46** ( ↑ 8) 0.28 5.2 15 5.86** ( ↑ 15) 0.35 5.9 15 6.06** ( ↑ 19) 0.38 6.3

Kidneys (paired; g) U 15 1.66 0.16 9.6 15 1.66 0.12 7.2 15 1.67 0.12 7.2 15 1.67 0.12 7.2

A 15 1.66 --- --- 15 1.66 --- --- 15 1.67 --- --- 15 1.67 --- ---

R 15 0.981 0.055 5.6 15 0.953 0.056 5. 9 15 0.962 0.043 4.5 15 0.955 0.064 6.7 Pituitary (mg) U 15 12.0 2.3 19.2 15 11.1 2.2 19.8 15 12.8 2.4 18.8 15 11.8 1.5 12.7

A 15 12.0 --- --- 15 11.1 --- --- 15 12.8 --- --- 15 11.8 --- ---

R 15 7.09 1.26 17.7 15 6.36 1.11 17.5 15 7.34 1.39 18.9 15 6.74 0.90 13.3
Adrenals (paired; mg) U 15 54.5 9.1 16.7 15 53.5 5.4 10.1 15 55.3 7.1 12.8 15 51.7 5.8 11.2

A 15 54.5 --- --- 15 53.5 --- --- 15 55.3 --- --- 15 51.7 --- ---

R 15 0.033 0.005 15.2 15 0.031 0.002 6. 5 15 0.032 0.004 12.5 15 0.029 0.004 13.8
Ovaries (paired; mg) U 15 79.0 9.8 12.4 15 73.8 9.2 12.5 15 76.6 11.8 15.4 15 76.3 14.0 18.3
A 15 79.0 --- --- 15 73.8 --- --- 15 76.6 --- --- 15 76.3 --- ---
Uterus with cervix, wet (mg) U 15 407.2 185.7 45.6 15 498.6 225.0 45.1 15 348.1 101.3 29.1 15 421.1 151.1 35.9

A 15 407.1 --- --- 15 498.8 --- --- 15 348.2 --- --- 15 420.9 --- ---

Uterus with cervix, blotted (mg) U 15 330.8 85.9 26.0 15 352.3 98.4 27.9 15 304.8 69.5 22.8 15 347.3 79.7 22.9

A 15 330.3 --- --- 15 352.4 --- --- 15 305.0 --- --- 15 347.6 --- --- Thyroid, fixed (mg) U 15 27.26 8.05 29.5 15 24.18 7.51 31.1 15 26.45 5.12 19.4 15 25.46 8.05 31.6
A 15 25.20 --- --- 15 24.19 --- --- 15 26.47 --- --- 15 25.49 --- --- a Data were obtained from Table 6 on pages 87-89 of the study report, and were sometimes rounde d by the reviewers. Percent difference from control (cal culated by reviewers from unrounded cited data) is included in parentheses. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 R Organ-to-body weight ratio (relative to body weight) N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



### E. ESTROUS CYCLICITY: Estrous cycle data are provi ded in Table 4. No treatmentrelated effect was observed on the mean age of first vaginal estrus, mean cycle length, % cycling, % regularly cycling, and the cycle status at necropsy.Female Pubertal Assay (2012) / Page 12 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None

TABLE 4. Estrous Cyclicity a

Treatment Groups N b Mean Age at First Vaginal Estrus (PND) Mean Cycle Length (days) Cycling (%) Not Cycling

(%) Regularly Cycling

(%) Cycle Status at Necropsy (# Females) Diestrus c Proestrus Estrus
Vehicle 15 33.8 4.4 100 0 86.7 5 8 2 Metalaxyl 125 mg/kg/day 15 34.5 5.1 73.3 26.7 53.3 7 8 0
Metalaxyl 250 mg/kg/day 15 34.5 4.3 86.7 13.3 73.3 8 7 0
Metalaxyl 350 mg/kg/day 15 35.4 4.1 73.3 26.7 46.7 6 7 2
a Data were obtained from Table 5 on page 81 an d Appendix 26 on page 499 of the study report. b In the dose groups 0, 125, 250, and 350 mg /kg/day, the number of animals evaluated for the first day of vaginal estrus was 15, 13, 13, and 14, respectively, and was 13, 8, 10, and 4, respectively for cycle length. c Includes diestrus and metestrus N Number of animals



### F. HORMONE LEVELS AND CLINICAL CHEMISTRY: Mean hormone levels and clinical chemistry values and are presented in Table 5. Nominal ranges (provided from testing facility historical control data; n=20) for the clinical chemistry values were provided

Serum T 4 

### and TSH levels in the treated groups we re similar to control values. Gammaglutamyl transpeptidase levels were increased (p ≤ 0.01) by 175% and 305% at 250 and 350 mg/kg/day, respectively; however, the serum levels were reported to be within the historical control range. The fo llowing clinical chemistry parameters differed (p ≤ 0.05 or 0.01) in the treated groups compared to cont rols: (i) albumin decreased by 3-6% in all treated groups; (ii) albumin/globulin ratio decreased by 5-9% in all treated groups; (iii ) sodium increased by 1-2% in all treated groups; and (iv) chlori de increased by 2% at 350 mg/kg/day. These differences were minor in magnitude, without clear dose dependence, and were within the historical control range. All other values in the treated gr oups were similar to control values.

Serum T 4 

### mean and CV values in the control group were within the acceptable range of the Guideline performance criteria.Female Pubertal Assay (2012) / Page 13 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None

TABLE 5. Serum Hormone Levels and Clinical Chemistry a

Parameter Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Thyroid Hormones Total T4 (μg/dL) 15 3.84 1.08 28.0 15 4.30 1. 24 28.7 15 3.69 0.58 15.8 15 3.87 1.02 26.3 TSH (ng/mL) 15 1.79 0.94 52.4 15 2.23 0.90 40.3 15 2.06 0.95 45.8 15 2.01 0.77 38.5 Clinical Chemistry Creatinine (mg/dL) 15 0.23 0.02 6.9 15 0.24 0. 02 9.4 15 0.23 0.02 8.6 15 0.23 0.01 4.4 BUN (mg/dL) 15 17.3 3.4 19.7 15 15.9 1. 8 11.3 15 16.5 3.5 21.2 15 15.8 2.8 17.7 AST (IU/L) 15 205 41 19.8 15 207 39 18.8 15 224 41 18.5 15 203 31 15.4 ALT (IU/L) 15 53.9 6.2 11.5 15 57.3 8.1 14.1 15 60.5 6.5 10.7 15 57.5 5.9 10.3 ALP (IU/L) 15 347 86 24.7 15 377 125 33.3 15 303 71 23.5 15 309 64 20.6

GGT (IU/L) 15 0.22 0.22 100 15 0.22 0.24 112 15 0.61** ( ↑ 175) 0.37 60.3 15 0.90** ( ↑ 305) 0.56 62.5

Total Bilirubin (mg/dL) 15 0.14 0.01 8.8 15 0.14 0.02 14.8 15 0.14 0.02 11.3 15 0.13 0.01 10.2

Cholesterol (mg/dL) 15 94.7 11.9 12.6 15 93. 7 9.8 10.5 15 97.8 12.8 13.1 15 95.5 9.2 9.6 Triglycerides (mg/dL) 15 68.1 18.8 27.6 15 61.1 19.0 31.1 15 71.1 26.9 37.8 15 66.2 15.5 23.4

Total Protein (g/dL) 15 5.69 0.15 2.6 15 5.54 0.26 4.7 15 5.61 0.19 3.3 15 5.60 0.32 5.6

Albumin (g/dL) 15 3.45 0.11 3.2 15 3.29** ( ↓ 5) 0.15 4.6 15 3.34* ( ↓ 3) 0.14 4.1 15 3.26** ( ↓ 6) 0.15 4.5

Globulin (g/dL) 15 2.24 0.11 4.9 15 2.25 0. 14 6.1 15 2.27 0.10 4.5 15 2.34 0.18 7.6

Albumin/Globulin 15 1.54 0.09 5.9 15 1.47** ( ↓ 5) 0.06 4.4 15 1.47* ( ↓ 5) 0.08 5.6 15 1.40** ( ↓ 9) 0.06 4.2

Glucose (mg/dL) 15 132 10 7.7 15 139 6.3 4.5 15 136 11 8.0 15 138 7.7 5.6 Calcium (mg/dL) 15 10.8 0.3 2.6 15 10.8 0.4 3.3 15 10.9 0.3 3.1 15 10.8 0.3 2.5 Phosphorus (mg/dL) 15 10.3 0.8 7.6 15 10.9 0.8 7.6 15 11.0 0.8 7.1 15 10.9 0.9 8.0

Sodium (mg/dL) 15 136 1.3 1.0 15 138* ( ↑ 1) 1.8 1.3 15 138** ( ↑ 2) 1.8 1.3 15 139** ( ↑ 2) 1.7 1.2

Potassium (mg/dL) 15 6.60 0.44 6.7 15 6.21 0. 41 6.6 15 6.38 0.41 6.4 15 6.27 0.46 7.3

Chloride (mg/dL) 15 97.5 1.3 1.3 15 97.7 1.0 1.0 15 97.5 1.6 1.6 15 99.1** ( ↑ 2) 1.6 1.6
a Data were obtained from Table 7 on page 91 and Table 8 on pa ges 93-96 of the study report. Percent differences from controls (calculated by reviewers) ar e included in parentheses. N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different fro m controls at p<0.05. ** Significantly different fro m controls at p<0.01.



### G. GROSS PATHOLOGY: There were no treatment-related macroscopic findings. The only gross observations reported were a dilated pelvis of the right kidney in a single 350 mg/kg/day rat and dilatation of the right kidney in a single 250 mg/kg/day rat.

H. HISTOPATHOLOGY: The incidences of histopathologi cal findings of the thyroid gland are presented below in Table 6; follicular ce ll shape was not reported. The colloid area andFemale Pubertal Assay (2012) / Page 14 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None



### follicular cell height in the treated groups were generally similar to the controls, without a clear dose-dependent effect.

TABLE 6. Thyroid Colloid Area and Follicular Cell Height a

Treatment Groups Parameter Evaluated

Colloid Area Follicular Cell Height (Increase)

Grade b Incidence Grade b Incidence O E O E

Vehicle Control 1 0 15 1 7 15 2 0 15 2 8 15 3 0 15 3 0 15 4 1 15 4 0 15 5 14 15 5 0 15

125 mg/kg/day Metalaxyl 1 0 15 1 9 15 2 0 15 2 6 15 3 0 15 3 0 15 4 5 15 4 0 15 5 10 15 5 0 15

250 mg/kg/day Metalaxyl 1 0 15 1 9 15 2 0 15 2 6 15 3 0 15 3 0 15 4 2 15 4 0 15 5 13 15 5 0 15

350 mg/kg/day Metalaxyl 1 0 14 1 11 14
2 0 14 2 3 14
3 0 14 3 0 14
4 4 14 4 0 14
5 10 14 5 0 14 a Data were obtained from Appendix 26 on page 499 of the study report. b A 5-point grading scale (1 = shortest/s mallest; 5 = tallest/largest) was used. O No. Observed E No. Examined



### The incidences of histopathological findings of the ovaries are presented in Table 7. Total primary follicle counts were increased (p ≤ 0.05 or ≤ 0.01) by 43 and 57% at 250 and 350 mg/kg/day, respectively. Atretic follicle coun ts in the treated groups were similar to controls.Female Pubertal Assay (2012) / Page 15 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None

TABLE 7. Number of Ovarian Follicles a

Parameter Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Primary Follicle Count

Left 15 11.4 4.9 43.0 15 11.5 4.8 41.7 15 14.7 7.0 47.6 15 17.7** ( ↑ 55) 3.3 18.6

Right 15 10.2 5.3 52.0 15 12.1 5.4 44.6 15 16.1* ( ↑ 58) 7.1 44.1 15 16.3* ( ↑ 60) 8.4 51.5

Total 15 21.6 8.8 40.7 15 23.6 9.0 38.1 15 30.8* ( ↑ 43) 11.0 35.6 15 34.0** ( ↑ 57) 9.7 28.5

Atretic Follicle Count

Left 15 1.0 1.6 160.0 15 0.3 0.6 200. 0 15 1.0 0.8 80.0 15 1.3 1.3 100.0 Right 15 0.5 0.6 120.0 15 0.3 0.6 200. 0 15 0.9 1.1 122.2 15 1.2 1.6 133.3
Total 15 1.5 1.8 120.0 15 0.6 0.8 133. 3 15 1.9 1.5 78.9 15 2.5 2.3 92.0 a Data were obtained from Table 9 on page 98 of the study report. SD Standard Deviation CV Coefficient of Variation



### The incidences of histopathological findings of the kidneys are presented in Table 8, and no treatment-related finding was observed. No treatm ent-related histological lesions were noted in the thyroid or uterus.

TABLE 8. Histopathological Findings in the Kidney a

Parameter Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day Infiltration, mixed cell, minimal 0 1 1 2 Dilatation Minimal 0 0 1 0 Mild 0 0 0 0 Moderate 0 0 0 1 a Data (n=15) were obtained from Appendi x 26 on page 498 of the study report.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Metalaxyl resulted in increases in absolute liver weights and/or ratios of liver to terminal body weight, concurrent with reductions in albumin and albumin/globulin ratio and increase s in sodium, as well as increases in gamma glutamyl transferase at 250 and 350 mg/kg/day and in chloride at 350 mg/kg/day. No other treatment related effect wa s observed in this study.

Based on these data, administration of metala xyl under the tested c onditions resulted in effects indicative of systemic/liver target orga n toxicity, but no effects on endocrine activity or indications of potential interactions with th e estrogen or thyroid pa thways were apparent.Female Pubertal Assay (2012) / Page 16 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None



### B. AGENCY COMMENTS: No adverse, treatment-relate d related effects were observed on survival, clinical signs, body we ights, body weight gains, or gross pathology. No treatmentrelated histological lesions were noted in the thyroid, or uterus , and thyroid colloid area and follicular cell height in the treated groups were generally similar to the control. Age of attainment of VO, body weight at VO, and pr oportion/incidence of unopened vagina in the treated groups were similar to control animals. No treatmen t-related effect was observed on the mean age of first vaginal estrus, mean cycle length, % cycling, % regularly cycling, and the cycle status at necropsy. Serum T 4 

### and TSH levels in the treat ed groups were similar to control.

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 values. This slight effect is considered treatment related. Gamma-glutamyl transpeptidase levels were increased (p ≤ 0.01) by 175% at 250 mg/kg/day and by 305% at 350 mg/kg/da y; however, the serum levels were reported to be within the historical control range. The following clinical chemistry parameters differed (p ≤ 0.05 or 0.01) in the treated groups compared to controls: (i) albumin decreased by 3-6% in all treated groups; (i i) albumin/globulin ration decr eased by 5-9% in all treated groups; (iii) sodium increased by 1-2% in all treated groups; and (iv) chloride increased by 2% at 350 mg/kg/day. These differences were relatively minor in magnitude, lacked clear dose dependence, and were within the historic al control range. Cons idered together, the increases in liver weight and serum GG T levels and decreases in albumin and albumin/globulin levels suggest liver toxicity; however, the degree of toxicity achieved is modest considering the magnit ude of these changes and natu ral variation as evidenced by historical controls.

Total primary follicle counts were increased (p ≤ 0.05) at 250 mg/kg/day by 43% and increased (p ≤ 0.01) at 350 mg/kg/day by 57% . Atretic follicle counts in the treated groups were similar to controls. The significance of the increases in total primary follicle counts and their relation to treatment is questionabl e because no other major pathological findings were observed in the ovaries and the presence of different follicle types is normal for nulliparous, young, female rats.

The high dose (350 mg/kg/day) was determin ed to be adequate based on generalized summaries from several ranging- finding studies and findings in the previously reviewed uterotrophic study (MRID 48617606), which showed decreased motor activity, ptosis, ataxia, lacrimation, and hunched posture in fema le rats in the 300 mg/kg/day dose group.

Under the conditions of this study, there wa s no treatment-related effects on pubertal development or thyroid function in the juvenile female rat.

C. STUDY DEFICIENCIES: The following minor deficienci es were noted that are not considered to have had an adverse impact on th e results, interpretation or conclusions of this study:

 Control CV for age at VO (7.9%) was grea ter than the performance criteria maximum of 6.52%Female Pubertal Assay (2012) / Page 17 of 17 METALAXYL / 113501 OCSPP 890.1450/ OECD None



###  Control CV for body weight at VO (14.8%) wa s greater than the performance criteria maximum of 13.97%

 Control mean paired adrenal weight (54.5 mg) was outside the performance criteria acceptable range (38.34-48.84)

 Control mean fixed thyroid weight (27.26 mg) was outside the performance criteria acceptable range (6.20-22.20)

# DATA EVALUATION RECORD

METALAXYL

Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-19-2012 (MRID 48701301)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 9/18/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 9/27/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 10/03/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 10/03/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).Male Pubertal Assay (2012) I Page 1 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None 

### Primary Reviewer: Vincent Chen, M.S.

# Signature:~ Risk Assessment Branch III, Health Effects Division (7509P) Date: z..4 'Jv.r.Jo::?z.o.l-S 

### Secondary Reviewer: John Liccione, Ph.D. 

### Signature:

## ~ , Health Effects Division (7509P) Date:

## ~;. 

# '?ft f
Template version 08/2011



### .

# ~'**_D_A_T_A_E_V_AL_U_A_T_IO_N_RE_c_o_RD_~**

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500; OECD None.



### PC CODE: 113501 

### DP BARCODE: D404681

TXR 

### #: 0054219 

### CAS #: 57837-19-1



### TEST MATERIAL (PURITY): Metalaxyl (>98% a.i.)



### SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate



### CITATION: Carlson, M.B. (2012). A Pubertal Development and Thyroid Function Study of Metalaxyl by Oral Gavage in Peripubertal Male and Female Rats. Charles River Laboratories Preclinical Services, Pennsylvania, Horsham, PA. Laboratory Study No.: 20012408, July 17, 2012. MRID 48701301. Unpublished.



### SPONSOR: Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL



### TEST ORDER#: CON-113501-18



### EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48701301), 15 male SpragueDawley (Crl:CD(SD)) rats/dose group were treated daily via oral gavage with metalaxyl (>98% a.i., Lot# 20100540) in 2

. 0% aqueous carboxymethylcellulose at doses of 0, 125, 250, or 350 mg/kg/day from postnatal day (PND) 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 wa.S collected for clinical chemistry analyses, and testosterone, total thyroxine (T 

### 4), and thyroid stimulating hormone (TSH) levels

were determined using radioimmunoassays. Adrenal, liver, pituitary, thyroid

, and urogenital
organs were weighed and examined macroscopically. The testes, epididymides, right kidney

, and thyroid were examined microscopically

.

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

, body weight gains, organ weights, clinical chemistry, or gross pathology. No adverse histological lesion was observed in the testes, epididymides, or kidneys. Mean serum testosterone, T 4, 

### and TSH levels in the treated groups were similar to control, and the thyroid follicular cell heights in the treated g roups were generally similar to control. Although colloid

area in the thyroid was slightly reduced in a dose-related manner, there were no concomitant effects noted in thyroid weights or hormone concentrations

.Male Pubertal Assay (2012) / Page 2 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None


### 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 male 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.

The assay satisfies the EDSP Tier 1 Test Or der requirements for a Male Pubertal Assay (OCSPP 890.1500).

COMPLIANCE: Signed and dated Data Confidentiali ty, GLP Compliance, Quality Assurance, and Flagging statements were provided.Male Pubertal Assay (2012) / Page 3 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories Prec linical Services, Pennsylvania
Location: Horsham, PA Study Directors: M.B. Carlson Other Personnel: Julian Gulbinski, III (dose formulation analysis and senior laboratory manager), Dr. Saroj Das (hormone analysis), Carol S. Sloan (horm one analysis), Rebecca M. Lucke (clinical chemistry), Carol J. Detrisac (histology and histopathology)

Study Period: June 17, 2011 through July 17, 2012



### 2. Test Substance: Metalaxyl Description: White to beige powder Source: Nation’s Ag, LLC, Knoxville, TN Lot #: 20100540 (expires June 13, 2012 and July 26, 2011) Purity: 98.2±0.3% and 100.4±0.7% Stability: Protected from light, the test compound was stable in the vehicle at 20-25°C for at least 25 hours and at 2-8°C for 10 days. CAS #: 57837-19-1 Structure:

CH 3 N O

CH 3 O CH 3 CH

3 O O CH

3



### 3. Vehicle:

Source: Catalog #: Lot #: 

### 2.0% aqueous carboxymethylcellulose in reverse osmosis membrane-processed deionized water Sigma Aldrich, St. Louis, MO C5678 060M0079V (retest date: June 30, 2012)



### 4. Test Animals: Species: Rat (males) Strain: Sprague-Dawley (Crl:CD(SD)) Age/ Mean Weight at Study Initiation: PND 23 / 67.9-68.8 g

Source: Charles River Laboratories International, Inc. (Portage, MI) Housing: Parental dams were individual ly housed in clear plastic nest ing boxes (n=2/box) with heattreated, laboratory-grade, pine shaving beddi ng; each dam was housed with her litter until weaning on PND 21. After weani ng, 2 males were housed per cage. Diet: Harlan Teklad 2018c meal feed (Teklad Diets, Madison, WI), ad libitum . It was stated that the total genistein equivalents (aglycone) or daidzein were ≤ 300 ppm. Water: Reverse osmosis-purified and deionized tap water, ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 69.0-80.2°F 42.5-76.7% ≥ 10/hour 14 h light / 10 h darkMale Pubertal Assay (2012) / Page 4 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None



### B. STUDY DESIGN

1. In-Life Dates: Start: July 7, 2011 End: August 7, 2011

2. Mating: Time-mated, pregnant nulliparous Spra gue Dawley Crl:CD(SD) dams (age not reported) were received from the supplier on ge station day 8 and were allowed to deliver naturally and rear their pups to PND 21. On PND 4, litters with 9 or more pups were standardized to 8 pups (4 males and 4 female s whenever possible) through removal of pups with weights furthest from the population mean for the appropriate sex within each litter. Litters with fewer than 8 pups were not retained.

3. Animal Assignment: Following weaning on PND 21, an equal number of rats from the heavy and light ends of the body weight distribu tion were eliminated, and the remaining rats were randomly assigned to the dose groups, stra tified by dose weight. Littermates were not placed in the same dose group. The te st groups are provided in Table 1.

TABLE 1. Study Design a
Test group Dose (mg/kg/day) # of Females

Control 0 15

Low 125 15

Medium 250 15 High 350 15
a Data were obtained from Text Table 5 on page 26 of the study report.



### 4. Dose Selection Rationale: The doses were selected based on the results from dose-range studies conducted in rats (Protocols 20012405 and 20013495) and clinical signs observed in the uterotrophic study on metalaxyl (48617606.DER, previously reviewed). Clinical signs of systemic toxicity were observed in adult male and female rats and weanlings at ≥ 500 mg/kg/day; mortality was seen in adult female rats at ≥ 500 mg/kg/day and in weanlings of both sexes at 750 mg/kg/day. Body weight gain was decreased at the two highest dose levels in adult male rats (750 and 1000 mg/kg/da y), and liver weights we re increased at all metalaxyl dose levels in the adult male rats (250, 500, 750, and 1000 mg/kg/day) and in weanlings of both sexes (100, 250, 500, and 750 mg/kg/day). Based on these results, 350 mg/kg/day selected as the maximum dose. The Sponsor utilized three dose levels to facilitate the interpretation of results. Minima l or no toxicity was expected at the low dose level of 125 mg/kg/day; a midrange dose of 250 mg/kg/day was selected per Guideline recommendation for a two-fold increase from the low dose.

5. Dose Preparation and Analysis: Metalaxyl dose formulati ons were prepared weekly by adding appropriate amounts of the test com pound to 2.0% aqueous carboxymethyl-cellulose in deionized water. The dose formulations we re dispensed into dail y aliquots (volume not reported), which were stored refrigerated until use. Samples from the first and last preparation of each dose group were collected an d shipped on ice packs to the analytical laboratory. The samples were shipped on the date prepared or one day after preparation and were analyzed upon receipt or stored in a re frigerator (2-8°C) until analysis. DuplicateMale Pubertal Assay (2012) / Page 5 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None


### samples collected from the middle stratum of the formulations were analyzed for concentration. Single sets of samp les were analyzed for density.

Stability and homogeneity analyses were previously performed in conjunction with Protocol 20012404. Stability was evaluated for 0.5 mg/mL and 100 mg/mL dose formulations. The dose formulations were analyzed before and af ter storage (while prot ected from light) at 20-25°C for at least 25 hours and at 2-8°C for 10 days. The relative standard deviation (RSD) of the mean for each of the average concentration values for the top, middle, and bottom of each dose formulation was calculated to assess homogeneity.

Results of Dose Analysis

Homogeneity (RSD): 0.4-1.5%

Stability (% of time zero): 99.7-101.8%

Concentration (% of nominal): 96.9-102.6%

The analytical data indicate d that the mixing procedure wa s adequate and the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: The dose formulations were allowed to warm to room temperature and were then stirred conti nuously for at least 30 minutes prior to administration and throughout dosing. Dose fo rmulations were administered once daily by oral gavage in a dose volume of 5 mL/kg body weight. Th e dose formulations were administered for 31 or 32 consecutive days fr om PND 23-53/54. Doses were adjusted based on the daily body weight recorded before ad ministration and were administered at approximately the same time each day, between 0700 hours and 0900 hours, except on one occasion (no details were provided for this exception).

7. Statistics: Mean, standard deviation, and coefficien ts of variation (CVs) were calculated. The following statistical analyses were performed.Male Pubertal Assay (2012) / Page 6 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None

Classification Procedure
Body weights Body weight changes Age at PPS Bodyweight at PPS Absolute organ weights Relative liver, kidney, adrenal, and pituitary weights
Serum hormone values Clinical chemistry data Data were analyzed with Ba rtlett’s Test of Homogeneity of Variances and Analysis of Variance (ANOVA), when appropriate [i .e., Bartlett’s Test was not significant ( p >0.001)]. If the ANOVA was significant ( p ≤ 0.05), Dunnett’s Test was used to identify the statistically significant diff erences between the control group and each treated group. If the ANOVA was not appropriate [i.e., Bart lett’s Test was significant ( p ≤ 0.001)], the Kruskal-Wallis Test was used ( ≤ 75% ties). In cases where the Kruskal-Wallis Test was statistically significant ( p ≤ 0.05), Dunn’s Method of Multiple Comparisons was used to identify the stat istically significant differences between the control group and each treated group.

Age at PPS Bodyweight at PPS Organ weight Data were also analyzed by Analysis of Covariance (ANCOVA), with the body weight recorded at weaning on PND 21 as the covariate. If the ANCOVA results were significant ( p ≤ 0.05), ANOVA was used to identify the statistically significant differences between the control group a nd each treated group. A modified Bonferroni correction was used for each pairwise comparison. A pairwise comparison was deemed statistically significant if its corresponding p -value was lower than or equal to the considered significance level (0.05 or 0.01) divided by the square root of the number of performed pairwise comparisons. In cases where sexual maturation did not occur prior to eu thanasia, the last day of observation +1 day was considered the age of sexual maturation.



### The statistical analyses were considered appropriate.

C. METHODS

1. Mortality and Clin ical Examinations: Animals were observed at least twice daily for mortality. Rats were observed for general a ppearance daily prior to dose administration. Postdose observations were reco rded within 2 hours of dose ad ministration and at the end of the normal working day.

2. Body Weight: Body weights were collected prior to allocation, daily before dose administration, and at termination. Over all (PND 23-53) body weight gain was also reported.

3. Food Consumption: Food consumption was recorded daily.

4. Preputial Separation (PPS): Male rats were evaluated for the occurrence of PPS daily beginning on PND 30. The appearance of partial and complete PPS, or a persistent thread of tissue between the glans and prepuce, was recorded. The body weight recorded on the day of complete PPS was used for subsequent evaluation of this endpoint.

5. Sacrifice and Pathology: Animals were moved to the necropsy holding room on the day prior to the final dose admini stration. Doses were administered in the holding room between 0700 and 0900 hours. Euthanasia by decapitation began 2 hours later and was completed by 1300 hours. Dose administration and termination was performed across dose groups. After euthanasia and prior to tissue collection, trunk blood was collected from all animals, and serum was obtained by centrifuga tion of whole blood samples (1.5 and 2 mL, respectively) in serum separator tubes. Blood samples for thyroid hormone and testosterone analyses (1.5 mL) were centrifuged fo r approximately 30 min at 4°C and 3000 × g ; samples for clinical chemistry evaluation were allowed to clot for 30 min at room temperature andMale Pubertal Assay (2012) / Page 7 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None


### centrifuged for approximately 10 mi n at room temperature and 3500 × g . The two sets of serum samples were immediately frozen on dry ice, and shipped on dry ice to Ani Lytics, Inc. (Gaithersburg, MD) for hor mone analysis or Charles Ri ver Laboratories, Preclinical Services, Ohio for clinical chemistry analysis .. Serum samples for hormone analyses were maintained at ≤− 80°C until analysis, and clinical chemistry samples were maintained at ≤− 20°C. Necropsies were performed for each an imal, gross lesions were collected (not evaluated microscopically), and images we re generated for illustration of gross observations.

The single male rat (#349) in the high dose group that died before scheduled termination was examined for the cause of death or condi tion as soon as possible after the observation was made. The rat was examined for gross le sions. The lungs, trachea, and esophagus were perfused with neutral buffered 10% formali n. Tissue dissection and weighing procedures were not conducted, and blood samples for hormone analysis and clinical chemistry were not obtained from this animal.

a. Hormone Analysis: Total testosterone, T 4 

### , and TSH were measured in serum using radioimmunoassays (RIAs). Total testosterone and T 4 

### were determined with RIA kits from Siemens (Cat # TKTT1 and TKT41, respectivel y), and TSH was determined with an inhouse-developed assay specific for rat TSH.

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER
X Calcium X Albumin X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol X Potassium X Globulins X Sodium X Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) X Triglycerides Creatine phosphokinase X Albumin /globulin ratio (calculated) Lactic acid dehydrogenase (LDH) Bile acids X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase (SDH) X Gamma-glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in fe male rats based on guideline 890.1450.



### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed and grossly observed fr om all study animals. The (XX) organs, in addition, were subjected to histological examination.Male Pubertal Assay (2012) / Page 8 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None

UROGENITAL OTHER XX Testes (left and right separately)* + XX Thyroid a * + XX Epididymides (left and right separately)* + X Liver* X Seminal vesicle plus coagulating glands (with and without fluid)* X Adrenals (paired)* X Ventral prostate* X Pituitary* X Dorsolateral prostate* X Levator ani-bulbocavernosus (LABC) muscle complex* XX Kidneys (paired)* + a Thyroid was weighed post-fixation. * Weights required based on Guideline 890.1500 + Histopathological examination re quired based on Guideline 890.1500



### All collected organs, except the thyroid/trachea , were weighed prior to fixation. Paired organs (kidneys and adrenals) were weighed to gether, but individual we ights were recorded for each testis and epididymis.

The right kidney was fixed in neutral buffere d, 10% formalin for at least 24 hours, and rinsed and retained in 70% ethanol until histolog ical processing. After the initial fixation of the thyroid in 10% formalin, the thyroid/parath yroid was dissected from the trachea, blotted, weighed, and retained in 70% ethanol until histological processing. The epididymis and testis were retained in Bouin's fluid for no l onger than 24 hours and rinsed and retained in 70% ethanol. All other organs were fixed in neutral buffered, 10% formalin. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Tissues were shipped under ambient conditions to Charles River Laboratories, Pathology Associates, MD. Other tissues were collected and retained, but not examined microscopically; these tissues included the ad renal, gross lesions, liver, and pituitary.

Thyroid sections were evaluated for follicular ce ll height and colloid ar ea using a five-point grading scale (1 = shortest/sma llest; 5 = tallest/largest), and any abnormalities/lesions were noted. A minimum of two sections of each of the two lobes of the thyroid were evaluated.

For each male rat, both testes, both epididym ides, and the right kidney were evaluated for pathologic abnormalities and potential treatment-related effects.

Besides gross lesions such as atrophy or tumors , testicular histopathol ogical evaluation was conducted in order to identify tr eatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated gi ant cells, or sloughing of spermatogenic cells into the lumen. Examination of the intact ep ididymis included the caput, corpus, and cauda, which was accomplished by evaluation of a longi tudinal section. This examination was conducted in order to identify such lesions as sperm granulomas, leukocytic infiltration (inflammation), aberrant cell types within the lumen, or the absence of clear cells in the caudal epididymal epithelium.Male Pubertal Assay (2012) / Page 9 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None



### II. RESULTS

A. MORTALITY: One 350 mg/kg/day male (#349) was found dead on PND 40. Although the cause of death was not determined, it was not due to gavage error (perforation of lungs, trachea, or esophagus). All tissu es appear normal at necropsy, and no adverse clinical signs were noted in this animal on any day during th e study. Therefore, this death is considered incidental.

B. CLINICAL SIGNS OF TOXICITY: No adverse, clinical signs were reported.

C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Body weights, body weight gains, age of attainment of PPS, body weight at PPS, and proportion/incidence of separated prepuce are presented in Table 2. All values in the treated groups were similar to control with the exception of minor decreases (p ≤ 0.05) in the adjusted body weight at PPS at 125 mg/kg/day ( ↓ 6%) and unadjusted and adjusted body weights (p ≤ 0.01) at PPS at 250 and 350 mg/kg/day ( ↓ 6-8%). No effect was noted on daily body weight measurements or feed consumption.

The Guideline performance criteria were met for age and weight at PPS and weaning and final body weight, except for the following: (i) CV for body weight at PPS (9.1%) was greater than the maximum acceptable value (7.5 7%); (ii) mean final body weight of 332.5 mg (acceptable range: 259.235-332.059 g); and (iii ) mean body weight at weaning of 60.5 g (acceptable range: 45.472-59.812 g).Male Pubertal Assay (2012) / Page 10 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None

TABLE 2. General Growth and Preputial Separation (PPS) a

Parameter Evaluated Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial Body Weight (PND 23; g) U 15 68.5 4.7 6.9 15 68.4 4.9 7.2 15 67.9 5.4 8.0 15 68.8 4.0 5.8

A 15 68.9 --- --- 15 68.2 --- --- 15 68.2 --- --- 15 68.4 --- ---
Body Weight at PPS (g) U 15 236.2 21.5 9.1 15 223.9 20.0 8.9 15 217.7* ( ↓ 8) 17.2 7.9 14 b 221.4* ( ↓ 6) 13.0 5.9

A 15 236.8 --- --- 15 223.4* ( ↓ 6) --- --- 15 218.2** ( ↓ 8) --- --- 14 b 220.7* ( ↓ 7) --- --- Final Body Weight (PND 53; g) U 15 332.5 18.4 5.5 15 330.9 23.9 7.2 15 321.3 21.4 6.7 14 b 319.6 19.8 6.2

A 15 332.9 --- --- 15 330.6 --- --- 15 321.6 --- --- 14 b 319.2 --- ---
Final Body Weight (% of control) U --- 99.5 96.6 96.1

Body Weight Gain (PND 23-53; g) U 15 264.0 18.2 6.9 15 262.5 21.8 8.3 15 253.3 19.5 7.7 14 b 250.8 20.4 8.1

A 15 264.0 --- --- 15 262.5 --- --- 15 253.3 --- --- 14 b 250.7 --- ---
Age at Incomplete PPS (PND) U 15 40.9 0.9 2.2 15 40.3 1.2 3.0 15 40.5 1.2 3.0 14 b 40.5 1.4 3.5

A 15 40.9 --- --- 15 40.3 --- --- 15 40.5 --- --- 14 b 40.5 --- ---
Age at PPS (PND) U 15 43.2 2.3 5.3 15 41.9 1.2 2.9 15 41.7 1.1 2.6 14 b 42.3 1.4 3.3

A 15 43.2 --- --- 15 41.9 --- --- 15 41.8 --- --- 14 b 42.3 --- --- Proportion unseparated (#/N) 0/15 0/15 0/15 1/15 b

a Data were obtained from Table 1 on pages 59-60 and Appendix 7 on pages 342-343 of the study report. b Animal #349 was found dead on PND 40 prior to PPS U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 N Number of animals examined SD Standard Deviation CV Coefficient of Variation --- Not applicable * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



### D. ORGAN WEIGHTS: Organ weights at necropsy are pr esented in Table 3, and all values in the treated groups were simila r to controls, except for a mi nor increase in relative liver weight at 350 mg/kg/day (5.38%) co mpared to controls (4.89%).

The unadjusted values for all organ weights in the control group were within the acceptable range of the Guideline performance criteria, w ith the following exceptions: (i) mean liver weight of 16.55 g (acceptable range: 9.990-15.350 g); (ii) CV for pituitary weight (23.5%) was greater than the maximum acceptable valu e (15.98%); and (iii) CV for fixed thyroid weight (27.2%) was greater than th e maximum acceptable value (23.63%).Male Pubertal Assay (2012) / Page 11 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N b Mean SD CV (%) Liver (g) U 15 16.6 1.66 10.0 15 17.0 2.20 12.9 15 16.6 1.26 7.6 14 17.5 1.58 9.0

A 15 16.6 --- --- 15 17.0 --- --- 15 16.6 --- --- 14 17.5 --- ---

R 15 4.89 0.233 4.8 15 5.06 0. 318 6.3 15 5.10 0.268 5.3 14 5.38** ( ↑ 10) 0.269 5.0

Kidneys (paired; g) U 15 2.84 0.25 8.8 15 2.90 0.27 9.3 15 2.81 0.27 9.6 14 2.81 0.19 6.8

A 15 2.84 --- --- 15 2.90 --- --- 15 2.81 --- --- 14 2.81 --- ---

R 15 0.839 0.043 5.1 15 0.865 0.053 6.1 15 0.861 0.052 6.0 14 0.867 0.044 5.1 Pituitary (mg) U 15 11.5 2.7 23.5 15 12.3 2.2 17.9 15 11.5 2.3 20.0 14 11.0 3.1 28.2

A 15 11.5 --- --- 15 12.3 --- --- 15 11.5 --- --- 14 10.9 --- ---

R 15 3.40 0.724 21.3 15 3.66 0.487 13.3 15 3.52 0.666 18.9 14 3.37 0.911 27.0
Adrenals (paired; mg) U 14 c 56.0 7.3 13.0 15 55.4 7.8 14.1 15 53.7 8.0 14.9 14 56.3 6.9 12.3

A 14 c 56.1 --- --- 15 55.3 --- --- 15 53.8 --- --- 14 56.2 --- ---

R 14 c 0.018 0.004 22.2 15 0.018 0.004 22.2 15 0.017 0.004 23.5 14 0.018 0.004 22.2
Seminal vesicle + coagulating gland, with fluid (mg) U 15 759.1 168.9 22.3 15 706.8 116.3 16.5 15 753.1 146.4 19.4 14 746.4 142.7 19.1

A 15 759.4 --- --- 15 706.5 --- --- 15 753.4 --- --- 14 746.1 --- ---

Seminal vesicle + coagulating gland, without fluid (mg) U 15 430.1 88.4 20.5 15 406.9 54.7 13.4 15 445.0 89.0 20.0 14 441.0 86.5 19.6

A 15 431.6 --- --- 15 406.3 --- --- 15 445.5 --- --- 14 440.3 --- ---

Ventral prostate (mg) U 15 261.5 45.2 17.3 15 243.0 66.9 27.5 14 d 245.9 67.9 27.6 14 238.9 68.2 28.5

A 15 261.6 --- --- 15 242.9 --- --- 14 d 246.0 --- --- 14 238.8 --- ---

Dorsolateral prostate (mg) U 15 203.8 44.2 21.7 15 221.2 56.2 25.4 14 d 187.6 51.2 27.3 14 222.9 46.2 20.7

A 15 204.0 --- --- 15 220.9 --- --- 14 d 188.0 --- --- 14 222.5 --- ---

LABC (mg) U 15 75.53 11.50 15.2 15 74.06 82.9 11.2 15 73.16 85.7 11.7 14 67.97 85.2 12.5
A 15 754.5 --- --- 15 741.3 --- --- 15 730.8 --- --- 14 680.7 --- --- Epididymis, left (mg) U 15 238.1 35.4 14.9 15 245.1 25.5 10.4 15 245.6 33.5 13.6 14 236.6 27.4 11.6

A 15 238.8 --- --- 15 244.2 --- --- 15 246.2 --- --- 14 235.9 --- --- Epididymis, right (mg) U 15 256.5 27.6 10.8 15 254.5 33.9 13.3 15 260.1 39.6 15.2 14 252.3 23.5 9.3

A 15 256.8 --- --- 15 254.2 --- --- 15 260.4 --- --- 14 252.0 --- --- Testis, left (mg) U 15 1508.5 148.5 9.8 15 1454. 4 188.5 13.0 15 1489.3 111.1 7.5 14 1468.1 93.1 6.3

A 15 1510.2 --- --- 15 1453. 0 --- --- 15 1490.7 --- --- 14 1466.3 --- ---
Testis, right (mg) U 15 1508.2 140.9 9.3 15 1449. 7 239.3 16.5 15 1480.0 107.4 7.3 14 1461.0 89.0 6.1

A 15 1510.4 --- --- 15 1447. 9 --- --- 15 1481.9 --- --- 14 1458.9 --- ---Male Pubertal Assay (2012) / Page 12 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N b Mean SD CV (%) Thyroid, fixed (mg) U 15 34.59 9.41 27.2 15 36.04 10.25 28.4 15 36.54 10.64 29.1 14 29.87 7.66 25.6

A 15 0.34.67 --- --- 15 35.97 --- --- 15 36.61 --- --- 14 29.78 --- --- a Data were obtained from Table 2 on pages 67-70 of the study report, and were sometimes rounde d by the reviewers. Percent difference from control (cal culated by reviewers from unrounded cited data) is included in parentheses. b Organ weights were not measured for Rat #349, which was found dead on PND 40. c Excludes values for Rat # 304, because th e left adrenal was lo st during processing. d Excludes values for Rat #339, which had a mass on the prostate. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 R Organ-to-body weight ratio (relative to body weight) N Number of animals examined SD Standard Deviation CV Coefficient of Variation ** Significantly different from controls at p ≤ 0.01.



### E. HORMONE LEVELS AND CLINICAL CHEMISTRY: Mean hormone levels and clinical chemistry values and are presented in Table 5. Nominal ranges (provided from testing facility historical control data; n=20) for the clinical chemistry values were provided in the study report.

Mean serum testosterone, T 4, 

### and TSH levels in the treated groups were similar to the control values. An increase (p ≤ 0.01) of 7% was noted in gl obulin at 350 mg/kg/day, and minor increases (p ≤ 0.05 or 0.01) of 1% in sodium were noted in each dose group. All other clinical chemistry values in the treate d groups were similar to control values.

Control group means and CVs for testosterone, T 4, 

### and TSH were within the acceptable range of the Guideline performance criteria.

TABLE 5. Serum Hormone Levels and Clinical Chemistry a

Parameter Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N b Mean SD CV (%) Thyroid Hormones Total T4 (μg/dL) 15 5.171 0.790 15.3 15 4.863 0.672 13.8 15 4.631 1.001 21.6 14 4.760 0.908 19.1 TSH (ng/mL) 15 5.472 2.296 42.0 15 4.766 2. 453 51.5 15 5.490 2.194 40.0 14 5.812 2.432 41.8
Testosterone (ng/mL) 14 c 2.644 2.246 85.0 15 4.113 2.258 54.9 15 4.430 2.289 51.7 14 3.872 2.085 53.9

Clinical Chemistry Creatinine (mg/dL) 12 d 0.231 0.024 10.4 14 d 0.243 0.018 7.4 15 0.231 0.025 10.8 13 d 0.240 0.019 7.9 BUN (mg/dL) 12 d 16.6 1.7 10.2 14 d 17.4 1.7 9.8 15 15.5 2.6 16.8 13 d 15.1 1.5 9.9 AST (IU/L) 12 d 188.8 31.5 16.7 14 d 225.7 57.2 25.3 15 188.9 35.8 19.0 13 d 187.5 40.4 21.5 ALT (IU/L) 12 d 56.6 5.2 9.2 14 d 59.6 5.8 9.7 15 56.1 8.0 14.3 13 d 52.8 8.5 16.1Male Pubertal Assay (2012) / Page 13 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None

TABLE 5. Serum Hormone Levels and Clinical Chemistry a

Parameter Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N b Mean SD CV (%) ALP (IU/L) 12 d 409.1 84.9 20.8 14 d 417.3 74.6 17.9 15 395.4 91.6 23.2 13 d 373.4 67.0 17.9 GGT (IU/L) 12 d 0.140 0.334 239 14 d 0.040 0.094 235 15 0.008 0.031 388 13 d 0.057 0.205 360
Total Bilirubin (mg/dL) 12 d 0.161 0.035 21.7 14 d 0.169 0.028 16.6 15 0.161 0.034 21.1 13 d 0.157 0.029 18.5

Cholesterol (mg/dL) 12 d 90.8 12.0 13.2 14 d 93.6 10.4 11.1 15 87.4 12.5 14.3 13 d 90.0 16.2 18.0 Triglycerides (mg/dL) 12 d 126.6 24.2 19.1 14 d 143.7 48.1 33.5 15 148.1 37.4 25.3 13 d 133.9 32.3 24.1

Total Protein (g/dL) 12 d 5.609 0.166 3.0 14 d 5.737 0.193 3.4 15 5.720 0.193 3.4 13 d 5.777 0.146 2.5 Albumin (g/dL) 12 d 3.243 0.100 3.1 14 d 3.296 0.086 2.6 15 3.281 0.118 3.6 13 d 3.235 0.236 7.3

Globulin (g/dL) 12 d 2.366 0.109 4.6 14 d 2.441 0.122 5.0 15 2.439 0.095 3.9 13 d 2.542** ( ↑ 7) 0.233 9.2

Albumin/Globulin 12 d 1.372 0.067 4.9 14 d 1.351 0.048 3.6 15 1.345 0.043 3.2 13 d 1.286 0.164 12.8 Glucose (mg/dL) 12 d 139.7 11.4 8.2 14 d 133.2 17.5 13.1 15 128.5 16.0 12.5 13 d 131.5 16.1 12.2 Calcium (mg/dL) 12 d 10.50 0.342 3.3 14 d 10.53 0.400 3.8 15 10.62 0.411 3.9 13 d 10.60 0.400 3.8 Phosphorus (mg/dL) 12 d 9.929 0.516 5.2 14 d 10.35 0.626 6.1 15 10.41 0.632 6.1 13 d 10.53 0.564 5.4

Sodium (mg/dL) 12 d 139.6 1.2 0.9 14 d 140.8** ( ↑ 1) 1.0 0.7 15 140.8** ( ↑ 1) 1.1 0.8 13 d 140.8* ( ↑ 1) 0.9 0.6

Potassium (mg/dL) 12 d 6.526 0.499 7.6 14 d 6.482 0.396 6.1 15 6.537 0.379 5.8 13 d 6.540 0.468 7.2 Chloride (mg/dL) 12 d 99.2 1.2 1.2 14 d 99.1 0.7 0.7 15 99.3 1.5 1.5 13 d 99.2 1.1 1.1 a Data were obtained from Table 3 on page 72 and Table 4 on pages 74-77 of the study report. Percent differences from controls (calculated by reviewers) are included in parentheses. b Serum was not analyzed for Rat #349, which was found dead on PND 40. c Excludes values for Rat # 304, because sample quantity was insufficient. d Excludes rats with severely hemolyzed serum that were not analyzed. N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different fro m controls at p<0.05. ** Significantly different fro m controls at p<0.01.



### G. GROSS PATHOLOGY: There were no treatment-relate d macroscopic findings. The following gross observations were observed: (i) moderate dilation in the pelvis of the right kidney in 1/15 males at 125 mg/ kg/day; (ii) dilation in the right kidney in 0/15, 1/15, 2/15, and 1/14 males in the 0, 125, 250, and 350 mg/kg/ day dose groups, respectively; and (iii) a firm mass on the ventral and dorso lateral prostate occurred in 1/15 males at 250 mg/kg/day.

H. HISTOPATHOLOGY: The incidences of histopathologi cal findings of the thyroid gland are presented below in Table 6; follicular cell shape was not reported. Compared to the control, thyroid colloid area was slightly redu ced in a dose-dependent manner, but there was no clear effect on follicular cell height.Male Pubertal Assay (2012) / Page 14 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None

TABLE 6. Thyroid Colloid Area and Follicular Cell Height a

Treatment Groups Parameter Evaluated

Colloid Area Follicular Cell Height (Increase)

Grade b Incidence Grade b Incidence O E O E

Vehicle Control 1 0 15 1 7 15 2 0 15 2 8 15 3 0 15 3 0 15 4 5 15 4 0 15 5 10 15 5 0 15

125 mg/kg/day Metalaxyl 1 0 15 1 9 15 2 0 15 2 6 15 3 0 15 3 0 15 4 4 15 4 0 15 5 11 15 5 0 15

250 mg/kg/day Metalaxyl 1 0 15 1 11 15 2 0 15 2 4 15 3 0 15 3 0 15 4 8 15 4 0 15 5 7 15 5 0 15

350 mg/kg/day Metalaxyl 1 0 14 1 5 14
2 0 14 2 9 14
3 2 14 3 0 14
4 6 14 4 0 14
5 6 14 5 0 14 a Data were obtained from Appendix 26 on page 499 of the study report. b A 5-point grading scale (1 = shortest/s mallest; 5 = tallest/largest) was used. O No. Observed E No. Examined



### The incidence of histopathological findings of the testes, epididymides, and kidneys are presented in Table 7. No adverse histol ogical lesion was observed in the testes, epididymides, or kidneys.Male Pubertal Assay (2012) / Page 15 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None

TABLE 7. Incidence of Histopathological Lesi ons of the Testes, Epididymides, and Kidney a

Findings Vehicle Control Metalaxyl 125 mg/kg/day Metalaxyl 250 mg/kg/day Metalaxyl 350 mg/kg/day

# Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined

Testis Seminiferous tubule degeneration, mild 0 15 1 15 0 15 0 14
Spermatocyte degeneration, minimal 0 15 0 15 0 15 1 14
Decreased spermatid elongation, moderate 0 15 1 15 0 15 0 14
Epididymidis Granuloma, minimal 1 15 0 15 0 15 0 14 Cauda hypospermia minimal 7 15 4 15 1 15 2 14

mild 1 15 0 15 0 15 0 14 marked 0 15 1 15 0 15 0 14 Mononuclear cell infiltration, minimal 0 15 0 15 2 15 0 14
Kidney Mixed cell infiltration, minimal 0 15 0 15 1 15 1 14

Dilation, minimal 0 15 2 15 2 15 0 14 mild 2 15 2 15 1 15 2 14 a Data were obtained from Appendix 26 on page 498-499 of the study report.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Metalaxyl resulted in reductions in mean body weight at the age of pr eputial separation (but no change in the timing of preputial separation initiation or completion) and increases in abso lute liver weights and/ or ratios of liver to terminal body weight, concurrent with a slig ht increase in globulin in the 350 mg/kg/day dose group and slight increases in sodium in each of the groups administered the test substance. A single mortality among the male rats at 350 mg/kg/day was considered likely to be exposure related. No other treatment related effect was observed in this study.

Based on these data, administration of metala xyl under the tested c onditions resulted in effects indicative of systemic/liver target orga n toxicity, but no effects on endocrine activity or indications of potential interactions with th e androgen or thyroid pa thways were apparent.

B. AGENCY COMMENTS: No adverse, treatment-relate d related effects were observed on survival, clinical signs, body wei ghts, body weight gains, organ weights, clinical chemistry, or gross pathology. No adverse histological le sion was observed in the testes, epididymides, or kidneys. Mean serum testosterone, T 4, 

### and TSH levels in the treated groups were similar to the control values. The thyroid follicular cell heights in the treated groups were generally similar to the controls, without a clear dose-dependent effect. Although colloid area in the thyroid was slightly reduced in a dose-related manner, there were no concomitant effects noted in fixed thyroid weights or thyroid hormone concentrations.Male Pubertal Assay (2012) / Page 16 of 16 METALAXYL / 113501 OCSPP 890.1500/ OECD None


### Body weights, body weight gains, age of a ttainment of PPS, body weight at PPS, and proportion/incidence of separated prepuce in th e treated groups were similar to control animals, with the excepti on of minor decreases (p ≤ 0.05 or 0.01) in the adjusted body weight at PPS at 125 mg/kg/day and unadjusted and adjusted body we ights at PPS at 250 and 350 mg/kg/day. These minor decreases were not dose-dependent in magnitude and were not considered a treatment-related effect. No effect was noted on daily body weight
measurements.

All organ weight values in th e treated groups were similar to control values, except for a slight increase (p ≤ 0.01) of 10% in relative liver wei ght at 350 mg/kg/day compared to control. This effect was minor, without a correl ated effect on absolute or adjusted weight, and without corroborating gross pa thology or clinical chemistry findings of hepatotoxicity. Consequently, this effect was considered incidental.

An increase (p ≤ 0.01) of 7% was noted in globulin concentration at 350 mg/kg/day, and minor increases in sodium were noted in each dose group. All other clinical chemistry values in the treated groups were similar to control.

One 350 mg/kg/day male was found dead on PND 40. Although the study authors considered this death to be treatment rela ted, no data were pres ented to support that conclusion. The cause of death was not dete rmined, but was not due to gavage error (perforation of lungs, trachea, or esophagus). All tissues appear nor mal at necropsy, and no adverse clinical signs were noted in this anim al on any day during the study. Therefore, this death is considered incidental by the reviewers.

A clear adverse effect was not observed in this study; however, based on generalized summaries from several rangi ng-finding studies and clini cal signs observed in the previously reviewed uterotrophic assa y (MRID 48617606) at 300 mg/kg/day and liver weight increases in male rats in the He rshberger assay (MRID 48617604) at 375 mg/kg/day, the selected high dose (350 mg /kg/day) for the male pube rtal study appears to be appropriate.

Under the conditions of this study, there was no evidence to support an effect of metalaxyl on pubertal development or thyroid f unction of juvenile male rats.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 CV for body weight at PPS (9.1%) was gr eater than the maximum acceptable value (7.57%);

 Mean final body weight of 332.5 mg (acceptable range: 259.235-332.059 g);

 Mean weaning body weight of 60.5 g (acceptable range: 45.472-59.812 g);

 Mean liver weight of 16.55 g (acceptable range: 9.990-15.350 g);

 CV for pituitary weight (23.5%) was gr eater than the maximum acceptable value (15.98%);

 CV for fixed thyroid weight (27.2%) was greater than the maximum acceptable value (23.63%).

# DATA EVALUATION RECORD

METALAXYL

Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-46-2012 (MRID 48617605)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Michelle Sharpe-Kass, M.S. Date: 5/25/2012

Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 5/31/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 6/06/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 6/06/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).Steroidogenesis Assay (2011) I Page 1 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None


### Primary Reviewer: Vincent Chen, M.S.

# Signature:~ Risk Assessment Branch III, Health Effects Division (7509P)**Date:~** 

### z.o.LS 

### Secondary Reviewer: John Liccione, Ph.D. 

### Signature: 

## -= - Health Effects Division (7509P) Date:

## ~ 

# '?(!(
Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550



### PC CODE: 113501 

### DP BARCODE: D399227

TXR#: 0054219 

### CAS No.: 57837-19-1



### TEST MATERIAL (PURITY): Metalaxyl (98.2 ± 

### 0.3%)



### SYNONYMS: Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate



### CITATION: Wagner, H. (2011) Metalaxyl: H295R Steroidogenesis Assay. CeeTox, Kalamazoo, MI. Study ID: 9143V-100326STER, December 7, 2011. MRID 48617605. Unpublished.



### SPONSOR: Metalaxyl EDSP Consortium 20 Ambriance Drive, Burr Ridge, IL 60527, USA



### TESTORDER#: CON-113501-18



### EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48617605), H295R cells cultured 

### in vitro in 24-well plates were incubated with metalaxyl (98.2% a.i., Batch# 20100540)
at log concentrations from 0.0001 to 100 μMin triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle, at final assay concentration of 0.05%.

Testosterone and estradiol levels were measured using HPLC/MS/MS using internal standards. Four independent experiments were performed; however, only Runs 2 through 4 were deemed successful. A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at
two concentration levels; positive controls included a known inhibitor (prochloraz) and known inducer (forskolin) of estradiol and testosterone production.

All guideline acceptability recommendations and requirements were met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low coefficients of variation, %CV), and appropriate induction and inhibition with positive controls.

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

or in Run 4 at**~10** 

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

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

, but the increase at 100 μM was not significant (1.09-fold, NS). For estradiol, there were noSteroidogenesis Assay (2011) / Page 2 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None


### 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.

The assay satisfies the EDSP Tier 1 Test Order requi rements for a Steroidogenesis assay (OCSPP 890.1550).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Steroidogenesis Assay (2011) / Page 3 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox
Location: Kalamazoo, MI Study Director: Hilary Wagner Other Personnel: Toole, C., Director of Project Manager Rutherford, K., Director of Laboratory Operations Blakeman, D., Senior Scientis t/ Endocrine Group Leader Wallace, B., Lead Cell Culture Scientist McColley, S., Scientist Haines, C., Scientist Wong, F., Scientist Burgam, J., Associate Scientist. Blakeman, L., Associate Scientist Study Period: May 26 to December 7, 2011



### 2. Test Substance: Metalaxyl Description: Solid Lot / Batch #: (Expiration Date) 20100540 (June 13, 2012) Purity: 98.2 ± 0.3% Solubility (in Solvent): At least 200 mM Volatility: Not reported Stability: Not reported Storage conditions: Ambient CAS #: 57837-19-1 Molecular weight: 279.3 Structure:

CH 3 N O

CH 3 O CH 3 CH

3 O O CH

3



### 3. Positive Control (Inducer): Forskolin Description: White powder Source: Sigma Aldrich (St. Louis, MO) Lot / Batch #: (Expiration Date): 109K50571V (July 2016) Purity: 98% Solubility (in Solvent): Not reported Storage conditions: Ambient CAS #: 66575-29-9



### 4. Positive Control (Inhibitor): Prochloraz Description: White powder Source: Sigma Aldrich (St. Louis, MO) Lot / Batch #: (Expiration Date) SZE6220X (Aug 8, 2012) Purity: 99.1% Solubility (in Solvent): Not reported Storage conditions: Ambient CAS #: 67747-09-5Steroidogenesis Assay (2011) / Page 4 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None



### 5. Solvent/Vehicle Control: DMSO Description: Not provided Source: Sigma Aldrich (St. Louis, MO) Lot #: (Expiration Date) RNBB7617 (expiry date not provided) Purity: Not provided Storage conditions: Not provided CAS #: 67-68-5 Justification for choice of solvent: One of the solvents recommended in the Guideline Final concentration: (% volume in assay) 0.05%



### 6. Other Materials: 22R-Hydroxycholesterol Description (molecular weight): White powder (402.65) Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): 089K4132, 060M4098 (not provided) Purity: 99.0% Storage conditions: Room temperature Solvent used: Ethanol, final concentratio n in assay, 0.025% v/v CAS #: 17954-98-2



### 7. Stock Medium: Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture
Source: Sigma Aldrich Lot / Batch #: (Expiration Date) Lot #021M8304 Sodium bicarbonate: Sigma Aldrich (Lot # 068K0105) Nu-Serum: Lot number 81515; tested for background hormone concentrations by performing laboratory ITS+ premix: Lot numbers 05245, 09233



### 8. Test Cells: H295R human adrenocortical carcin oma cells (ATCC CLR-2128; lot 7635054)

. Cells were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for seven or eight additional passages prior to use in the assay. The culture medium was supplemented with 10 μM 22R-hydroxycholestero l at the time of plating, dosing and harvest. The H295R cells were maintained in supplemented media containing DMEM:F12 media base with ITS + Premix and Nu -Serum. Cells were incubated at 37 ° C in an atmosphere of 5% CO 2



### . The following performance criteria were met (indicated by an “x”): NR Cell passage identifier. Cell Passage #: not reported X Cells frozen down at passage 5 X Frozen cells cultured for 4 additional passages X Total number of passages does not exceed 10



### B. METHODS

1. Pre-Test Information

a. Hormone Assay Interference Test: Analysis of the production of testosterone and 17 β -estradiol by H295R cells was conducted by HP LC/MS/MS using internal standards to measure extraction and quantitati on efficiency. No interference would be anticipated with this analytical methodology.Steroidogenesis Assay (2011) / Page 5 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None



### b. Hormone Extraction: Testosterone and estradiol were extracted from H295R supplemented medium by liquid-liquid extract ion using methyl tert -butyl ether (MTBE) after the addition of [ 2 

### H 5 

### ]-testosterone and [ 2 

### H 5 

### ]-estradiol as internal standards. Extracts were analyzed by HPLC-MS/MS using pos itive ion multiple reaction monitoring.

c. Laboratory Proficiency Test: It was stated that all assays were performed by personnel that demonstrated proficiency performing the assay as outlined in the test guideline. Proficiency records are referenced in the study records, but were not included in the study report.

2. Test Solutions: A 200 mM stock solution was prepar ed by dissolving an appropriate amount of metalaxyl in DMSO. Six serial 1: 10 dilutions were then prepared, yielding a range of 200 mM to 0.2 μM. Forskolin and proc hloraz solutions were prepared in a similar manner. All dilutions were then d iluted 1:2000 in 10 μM 22R hydroxycholesterolcontaining supplemented medium to yield the concentrations presented in Tables 1 and 2.

3. Cell Plating and Preincubation: H295R cells (ATCC CLR-2128) were grown for five passages, frozen in liquid nitrogen, then thaw ed and cultured for seven or eight additional passages. The cells were then seeded in 24- well plates at a dens ity of 300,000 cells/mL. The plates were incubated for 24 hours at 37°C in a 5% CO 2 

### atmosphere yielding approximately 50-60% confluency.

4. Exposure: The cells were checked microscopi cally for good attachment and proper morphology, and the medium was removed and re placed with 1 mL of medium containing 10 μM 22R-hydroxycholesterol and the concentrati ons of metalaxyl in triplicate according to the schematic presented in Table 1.

TABLE 1. Dosing Schematic for the Exposure of H295 R Cells to Metalaxyl (Final Concentration in μM) a
1 2 3 4 5 6
A DMSO DMSO DMSO 0.1 0.1 0.1
B 100 100 100 0.01 0.01 0.01 C 10 10 10 0.001 0.001 0.001
D 1 1 1 0.0001 0.0001 0.0001 a Data were obtained from page 16 of the study report. Dosing was calculated based on a to tal volume of 1 mL per well.



### A concurrent QC plate was included with each of the four independent runs of the test chemical plates to demonstrate the assay’s res ponse to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testoste rone and estradiol production). The QC plate was prepared and dosed in the same manner w ith either forskolin or prochloraz according to the schematic presented in Table 2.Steroidogenesis Assay (2011) / Page 6 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None

TABLE 2. Dosing Schematic for the QC Plate for Po sitive Controls (Final Concentrations in μM) a
1 2 3 4 5 6
A Blank b Blank Blank Background c Background Background
B DMSO DMSO DMSO DMSO + MeOH d DMSO + MeOH DMSO + MeOH C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochlor az 0.1μM Prochloraz 0.1μM Prochloraz 0.1μM
D Forskolin 10μM Forskolin 10μM Forskolin 10μM Proc hloraz 1μM Prochloraz 1μM Prochloraz 1μM a Data were obtained from page 16 of the study report. Dosing was calculated based on a to tal volume of 1 mL per well. b Blank wells received medium contai ning 10 μM 22R-hydroxycholesterol. c Background wells received medium without 22R-hydroxycholesterol). d MeOH = 70% methanol was added to these wells for 30 mi nutes at room temperature following medium removal.



### Following dosing, the plates were incubated for 48 hours under the conditions previously described. The medium from each well was rem oved, split into equal volume aliquots, and frozen at

− 80°C until hormone measurements.

5. Cell Viability/Cytotoxicity Assay: Cell viability was determined by MTT assay after the 48±2 hour exposure. Media was removed a nd 0.5 mL of 0.5 mg/mL MTT solution in supplemented medium containing 10μM 22R-hydroxycholesterol was added to each well of the test chemical and QC plates. Plates were incubated at 37±2 ° C for 3 hours, after with the MTT solution was removed and replaced with 0.5 mL of isopropanol. Plates were incubated at room temperature for 20 minutes , and absorbance at 570 nm and 650 nm were measured.

6. Hormone Measurement System: The concentration of each hormone was determined in triplicate in the culture medium following liqui d-liquid extraction with MTBE. Prior to extraction, [ 2 

### H 5 

### ]-testosterone and [ 2 

### H 5 

### ]-estradiol were added to the medium samples as internal standards to account for hormone r ecovery. The extracts were analyzed by HPLCMS/MS using positive ion multiple reaction moni toring. The lower limit of quantification (LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol using a 300 μ L aliquot of H295R supplemented medium.

The following performance criteria were met (indicated by an “x”):
X Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol)

X Spiked sample recovery acceptable fo r two concentrations of testosterone and estradio l (mean measured amount from triplicate samples within 30% of nominal concentration) NA Hormone cross-reactivity (antibody-based assays only; ≤ 30% of basal production of the respective hormone) X Solvent control within 75% range be low maximum response on standard curve NA Test compound tested for interf erence with meas urement system



### C. DATA ANALYSIS: Mean values (pg/mL) and standard deviations for concentrations of testosterone and estradiol were calculated for each test level of the reference chemicals and the solvent controls, as well as for the bla nk and background wells. Relative changes in hormone production were calculated using the following equation:

Relative Change = [Hormone] in each well ÷ [Hormone] of mean solvent control

For forskolin induction of testosterone, the background hormone production was subtracted from the forskolin-treated wells and blank and solvent control wells before calculating the relative change. This was done because of the high backgrou nd production of testosterone and the small dynamic range for forskolin induction. Background hormone production wasSteroidogenesis Assay (2011) / Page 7 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None



### calculated from three wells with cells on the QC plate that received no 22Rhydroxycholesterol.

All concentrations that exhibi ted greater than 20% cytotoxicity in the MTT cell viability assay were excluded from further analysis . Concentrations where precipitation was observed were also excluded from further analysis.

Normality of the data was evaluated using Shapiro-Wilk’s test. Homogeneity of the variances between the tr eatment groups was evaluated using Levene’s test. If the p-values were greater than 0.05 in both tests, statisti cal significance between each treatment group and the control group was evaluated using Dunnett’s test. If the p-values were less than or equal to 0.05 in either the normality or th e homogeneity test, a log transformation was performed on the data to attempt to approxi mate a normal distribution. If, following the transformation, p-values were greater than 0.05 in both the normality and homogeneity tests, Dunnett’s test was performed on the transformed data to evaluate st atistical significance between the each treatment group and th e control group. If following the log transformation, p-values were less than or equal to 0.05 in either the normality or the homogeneity test, the non-tr ansformed data set was anal yzed using the nonparametric Kruskal-Wallis test followed by Dunn’s test to evaluate statistical significance between each treatment group and the control group.

II. RESULTS

A. TEST COMPOUND: The hormone concentrations afte r exposure to metalaxyl, and the fold difference change relative to the SC and the mean ± SD for the three successful assays are presented in Table 3 and shown graphically in Figures 1 and 2. No results were reported for Run 1 as it was not successful. Precipitati on was not observed at any tested metalaxyl concentration. The %CVs based on absolute ho rmone concentrations within each plate for the SC were 1.8-5.7% for testosterone and 0.8-8. 7% for estradiol. The between-plate %CVs for the SC based on absolute concentrations (calculated by reviewer) were 24% and 29% for testosterone and estradiol, respectively. These values are below the Guideline limit of ≤ 30%.

For testosterone, there were no signifi cant changes in production in Run 2 at any concentration tested or in Run 4 at ≤ 10 μ M of metalaxyl, although there was a slight decrease in testosterone pr oduction (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). The statistical significance noted in Run 3 was due to lower control values in this run compared to Runs 2 and 4.

For estradiol, there were no si gnificant changes in production in Runs 2 or 4 at any tested concentration, and there was onl y a slight increase (1.18- to 1.19-fold; p<0.05) in estradiol production in Run 3 at the three lowe st tested concentrations (0.0001 – 0.01 μ M). The statistical significance noted in Run 3 was due to lower control values in this run compared to Runs 2 and 4.Steroidogenesis Assay (2011) / Page 8 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None

TABLE 3. Mean (±SD) Hormone Concentrations Follo wing Treatment with Metalaxyl for 48 Hours.

Nominal Concentration (μM) Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b ± SD b Statistical Significance

Testosterone (pg/mL) a Fold Difference b
DMSO 2569  46 1585  87 2391  137 -- --- -- -- -- -- 0.0001 2562  104 1854  26 2272  46 1.00 1.17 0.95 1.04 0.12 Run 3 0.001 2607  92 1837  40 2310  95 1.01 1.16 0.97 1.05 0.10 Run 3 0.01 2599  190 1850  121 2376  87 1.01 1.17 0.99 1.06 0.10 Run 3 0.1 2566  97 1819  64 2438  51 1.00 1.15 1.02 1.06 0.08 Run 3 1 2604  80 1843  85 2327  107 1.01 1.16 0.97 1.05 0.10 Run 3 10 2456  28 1831  22 2212  76 0.96 1.16 0.93 1.01 0.12 Run 3 100 2372  56 1729  38 2105  68 0.92 1.09 0.88 0.96 0.11 Run 4 Estradiol (pg/mL) a Fold Difference b
DMSO 255  2.0 140  12.2 216  5.8 -- -- -- -- -- -- 0.0001 242  10.3 166  1.4 221  4.4 0.95 1.19 1.02 1.05 0.12 Run 3 0.001 250  7.7 166  3.5 216  2.0 0.98 1.18 1.00 1.05 0.11 Run 3 0.01 245  15.3 165  6.6 222  4.2 0.96 1.18 1.03 1.06 0.11 Run 3 0.1 253  6.7 162  5.7 219  6.1 0.99 1.15 1.01 1.05 0.09
1 260  10.7 159  4.4 226  9.6 1.02 1.13 1.05 1.07 0.06
10 248  5.8 159  0.9 214  6.6 0.97 1.14 0.99 1.03 0.09
100 247  10.4 154  0.6 202  6.3 0.97 1.10 0.94 1.00 0.09 a Data were obtained from pages 30-31 and 57-59 of the study report. b Calculated by reviewerSteroidogenesis Assay (2011) / Page 9 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None



### FIGURE 1. Change in Testosterone Producti on Relative to Metalaxyl Concentration.Steroidogenesis Assay (2011) / Page 10 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None



### FIGURE 2. Change in Estrogen Production Relative to Metalaxyl Concentration.

B. CYTOTOXICITY: Metalaxyl was not cytotoxic to the H295R cells (>80% viability, Table 4). Viability across all of the test plates ranged from 91.9-109.3%.Steroidogenesis Assay (2011) / Page 11 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None

TABLE 4. Mean (±SD) MTT Cell Viability Results af ter Treatment with Forskolin, Prochloraz, or Metalaxyl for 48 Hours. a

Compound Concen. (μM) Cell Viability – Run 2 Cell Viabilit y – Run 3 Cell Viability – Run 4 Mean SD Mean SD Mean SD
Blank NA 104.3 2.69 104.3 2.81 100.7 3.98 Background NA 95.4 4.14 102.5 1.79 98.9 0.94 SC + Methanol NA 5.6 2. 51 11.4 1.99 5.1 0.14 Forskolin 1 b 107.8 1.52 109.3 1.17 105.0 1.55 Forskolin 10 107.0 2.43 108.5 4.17 102.4 2.58 Prochloraz 0.1 98.3 2.20 101.2 0.47 100.7 0.86 Prochloraz 1 99.4 1.19 101.5 0.78 99.5 2.37 Metalaxyl 0.0001 95.9 2.00 93.3 4.04 96.0 0.75 Metalaxyl 0.001 95.2 2.18 95.0 1.51 98.1 1.15 Metalaxyl 0.01 96.9 0.99 94.8 2.73 96.2 2.35 Metalaxyl 0.1 97.4 0.79 98.0 0.87 98.1 0.94 Metalaxyl 1 91.9 0.73 97.1 4.49 94.9 2.07 Metalaxyl 10 95.4 2.70 98.3 6.07 97.2 1.23 Metalaxyl 100 93.4 2.56 101.8 2.57 97.8 1.40 a Data were obtained from pages 24-25 of the study report. NA Not applicable SC Solvent control



### C. QC PLATE: The hormone concentrations after expos ure to the reference chemicals, SC, and blank samples, as well as the fold difference change relative to SC (individual and mean ± SD) for the three assay runs are presente d in Table 5. The minimum basal hormone production levels for testosterone (500 mg/mL) and estradiol (40 pg/mL) were met in both blank and SC wells. Testosterone production in the presence of 10 μM forskolin was 1.6- to 1.9-fold, which is below the Guideline criteria of ≥ 2-fold; however, when the background is subtracted, 10 μM forskolin induced testos terone an average of 2.5-fold.. Estradiol production in the presence of 10 μM forskolin was greater than the ≥ 7.5-fold requirement. Compared to SC, 1 μM prochloraz on average inhibited the synthesi s of testosterone 0.41- fold and estradiol 0.32-fold. Thus, the guideli ne requirements were met indicating that the assay was sensitive to induction and inhibition of testosterone and estradiol.

The variability (%CV) between the runs (calculated by the re viewer) based on the absolute hormone concentrations in the SC were 11% for estradiol and 15% for testosterone, and within the Guideline recommended limit of ≤ 30%. The %CVs for the SC within each run for the QC plates were 0.8-5.4% for te stosterone and 2.9-6.2 % for estradiol.Steroidogenesis Assay (2011) / Page 12 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None

TABLE 5. Hormone Concentrations Following Treat ment with Forskolin or Prochloraz for 48 Hours. a

Compound Conc. (μM) Run 1 Run 2 Run 4 Run 1 Run 2 Run 4 Mean b ± SD b Testosterone (pg/mL) Fold Difference Background NA 1135 1073 1262 0.45 0.57 0.54 0.52 0.06 Blank NA 2581 1873 2304 1.03 1.00 0.99 1.01 0.02 DMSO NA 2511 1874 2339 1.00 1.00 1.00 1.00 -- Forskolin 1 3764 2595 3120 1.50 c 1.38 c 1.33 c 1.40 c 0.09 Forskolin 10 4751 3019 3780 1.89 d 1.61 d 1.62 d 1.71 d 0.16 Prochloraz 0.1 1816 1149 1818 0.72 0.61 0.78 0.70 0.09 Prochloraz 1 1188 501 1153 0.47 0.27 0.49 0.41 0.12 Estradiol (pg/mL) Fold Difference Background NA 41 56 77 0.21 0.31 0.35 0.29 0.07 Blank NA 208 182 228 1.04 1.01 1.02 1.02 0.02 DMSO NA 200 179 223 1.00 1.00 1.00 1.00 -- Forskolin 1 1343 1254 1538 6.72 7.01 6.91 6.88 0.15 Forskolin c 10 2116 1661 2477 10.60 9.28 11.13 10.34 0.95 Prochloraz 0.1 133 104 171 0.67 0.58 0.77 0.67 0.10 Prochloraz 1 57 52 83 0.29 0.29 0.37 0.32 0.05 a Data were obtained from pages 26-27 of the study report. b Calculated by the reviewers. c When the background is subtracted, the valu es are 1.91-, 1.90-, and 1.74-fold difference for Runs 2, 3, and 4, respectively, with a mean of 1.85-fold change. d When the background is subtracted, the valu es are 2.63-, 2.43-, and 2.34-fold difference for Runs 2, 3, and 4, respectively, with a mean of 2.47-fold change.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: For estradiol, no statistically significant effects were observed in two of the three runs of the steroidogenesis assay. Fo r testosterone, in one run, no statistically significant effects were id entified. In anothe r run, a statistically significant reduction was observed at the highest concentration tested. Although statistically significant effects were identified for testosterone and estradiol in the remaining run of the assay, the run was considered an anomaly as it differed from the other two runs. Overall there was no consistent evidence that meta laxyl caused an increase or decrease in testosterone or estradiol bios ynthesis in the H295R cell line.

B. AGENCY COMMENTS: Precipitation was not observe d at any tested metalaxyl concentration, and metalaxyl was not overtly cy totoxic to the H295R cells. Viability for metalaxyl on the test plates was ≥ 91.9%. Four independent test runs of the steroidogenesis assay were conducted with metalaxyl. Run 1 was not analyzed because one of the forskolin concentrations on the QC plates was 3.33μM instead of 1 μM.

All guideline acceptability recomm endations and requirements we re met, including lack of cytotoxicity, adequate production of testoster one and estradiol, acceptable reproducibility (low %CV), and appropriate induction a nd inhibition with positive controls.

There were no significant change s in testosterone production in Run 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 testos terone 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-Steroidogenesis Assay (2011) / Page 13 of 13 METALAXYL / 113501 OCSPP 890.1550/ OECD None


### fold, NS). For estradiol, there were no signi ficant changes in producti on 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). As there were no reproducible, concentration-dependent effect s on either testosterone or estradiol production, metalaxyl does not appear to affect testosterone or estradiol production under the conditions of this assay.

C. STUDY DEFICIENCIES: The following deficiency was no ted that is not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 Although the steroidogenesis assa y proficiency and validati on tests for the performing laboratory were reported as completed and su fficient, no data were included in the study report to s upport this claim.

# DATA EVALUATION RECORD

METALAXYL

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-46-2012 (MRID 48617606)

Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer: Signature: Sandra Hastings Date: 4/25/2012

Secondary Reviewer: Signature: Kelly Luck, M.S. Date: 5/11/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 6/05/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 6/05/2012

This Data Evaluation Record may have been alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).METALAXYL/ 113501 In vivo Uterotrophic Assay (2011) I Page I of 10 OCSPP 890.1600/ OECD 440


### Primary Reviewer: Minerva Mercado 

### Ph.D. DABT

# Signatu**~** Health Effects Division

## Date**~** Secondary Reviewer: ---'J"-'e=s=-s ""'R=o-'-w'""'lan=d"--

________ 

### Signature: 

# d=-.,...,_**~** 

### Health Effects Division Date: 

## W't=[ 

## 1:;,- Template version 09/2011 

### DATA EVALUATION RECORD

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440



### PC CODE: 113501 

### DP BARCODE: D399227



### TXR#: 0054219 

### CAS#: 57837-19-1



### TEST MATERIAL (PURITY): Metalaxyl (98.2% a.i.)



### SYNONYMS: Alanine, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-, methyl ester



### CITATION: Hoberman, A.M. (2011) Oral (Gavage) Uterotrophic Bioassay ofMetalaxyl in Female Rats. Charles River Laboratories, Preclinical Services, Horsham, PA. Laboratory Project ID: 20012406, December 9, 2011. MRID 48617606. Unpublished.



### SPONSOR: Metalaxyl EDSP Consortium, 20 Ambriance Drive, Burr Ridge, IL



### TEST ORDER#: CON-113501-18



### EXECUTIVE SUMMARY: In a Uterotrophic Assay (MRID 48617606) conducted to screen for potential estrogenic activity, metalaxyl (98.2% a.i., Batch/lot# 20100540) in 2.0% aqueous carboxymethylcellulose (CMC) was administered daily via oral gavage to groups of six ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 100 or 300/200 mg/kg/day on post-natal days (PND) 57-59 or 63-65. A positive control group was treated with

a daily dose of 17a-ethynyl estradiol (EE) at 5 μg/kg/day by subcutaneous (s.c.) injection. Body weights and food consumption were determined daily. All animals were terminated and necropsied on PND 60 or PND 66 approximately 24 hours after the final dose administration to determine wet and blotted uterine, and liver weights.

All animals survived until scheduled termination. Following administration of metalaxyl at 300 mg/kg/day on Day 1, adverse clinical signs were observed (decreased motor activity, ptosis, ataxia, lacrimation, hunched posture, intermittent body jerks, chromodacryorrhea, and slight

excess salivation), as well as reductions in food consumption. Therefore, the 300 mg/kg/day dose level was reduced to 200 mg/kg/day on Study Days 2 and 3. Following the dose reduction, food consumption in this group increased and no other adverse clinical signs were observed.

Body weights throughout the dosing period and overall (Days 1-4) body weight gain (BWG) in the metalaxyl treated groups were comparable to controls. Overall (Days 1-4) food consumption for both metalaxyl treatment groups was decreased (p::S0.05) by 6-8% compared to the vehicle controls. Uterine weights in the metalaxyl treated groups were comparable to the vehicle controls. Absolute and relative liver weights in the 300/200 mg/kg/day group were increasedIn vivo Uterotrophic Assay (2011) / Page 2 of 10 METALAXYL / 113501 OCSPP 890.1600/ OECD 440


### (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.

The assay satisfies the EDSP Tier 1 Test Order require ments for a uterotrophic assay (OCSPP 890.1600).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.In vivo Uterotrophic Assay (2011) / Page 3 of 10 METALAXYL / 113501 OCSPP 890.1600/ OECD 440



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories, Preclinical Services, Pennsylvania (PCS-PA)
Location: Horsham, PA Study Director: A.M. Hoberman Other Personnel: K. Strauss (Principal Investigator) Study Period: June 14, 2011 to December 9, 2011



### 2. Test Substance: Metalaxyl Description: White to beige powder Source: Nation’s Ag, LLC (Knoxville, TN) Lot/Batch #: 20100540 (expiry date: June 13, 2012) Purity: 98.2% a.i. Stability: Stable in vehicle at room temperat ure for 25 hours and under refrigerated conditions for 10 days CAS #: 57837-19-1 Structure:

CH 3 N O

CH 3 O CH 3 CH

3 O O CH

3



### 3. Reference Estrogen: 17 α -ethynyl estradiol (EE) Supplier: Sigma Aldrich (St. Louis, MO) Lot/Batch #: 011M1252V (expiry date: June 8, 2012) Purity: 99% CAS #: 57-63-6



### 4. Solvent/Vehicle Control (test substance): Carboxymethylcellulose (CMC)

Supplier: Product #: Sigma Aldrich (St. Louis, MO) C5678 (sodium salt, low viscosity) Lot/Batch #: 060M0079V (expiry (retest) date: June 30, 2012) Rationale (if other than water): Use of an aqueous solution/suspension is recommended and an analytical method is available. Final concentration: 2.0% in reverse osmosis memb rane-processed deionized water



### Solvent/Vehicle Control (reference estrogen): Corn oil

Supplier: Spectrum Chemical Lot/Batch #: YR1134 (expiry date: June 30, 2012) Rationale (if other than water): None provided Final concentration: Not applicableIn vivo Uterotrophic Assay (2011) / Page 4 of 10 METALAXYL / 113501 OCSPP 890.1600/ OECD 440



### 5. Test Animals: Species: Rat (ovariectomized females only) Strain: Sprague Dawley, Crl:CD(SD) Age/weight at dose initiation: 57 or 63 days old/ 198.2-242.6 g Source: Charles River Laborat ories (Portage, MI) Housing: 2 rats/cage in clear plastic nesting boxes with heat-tre ated laboratory-grade pine shavings. Diet: Harlan’s Teklad 2018c meal food, ad libitum ; concentration of genistein equivalents (aglycone) or daidzein was ≤ 300 ppm per lot. Chewable Nylabones® were supplied during the study. Water: Reverse-osmosis treated tap water , ad libitum from individual, non-plastic water bottles attached to the nesting boxes Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 19-25oC 30-70% >10/hr 12 hrs light/12 hrs dark Acclimation period: 6 days



### B. STUDY DESIGN

1. In-Life Dates: Start: June 20, 2011 End: June 23, 2011

2. Study Design: Sexually mature ovariectomized rats (PND 51 or 57) were received from Charles River Laboratories (ovariectomized on PND 43 or 49, respectively). Animals were acclimated for 6 days prior to initiation of dosing. Vaginal smears were taken daily during the last five days of the acclimation period a nd examined microscopically to verify complete removal of the ovaries as indicated by a lack of estrous cycling. The dose administration period was from PND 57 th rough 59 or PND 63 through 65. Rats were euthanized approximately 24 hours later on PND 60 or PND 66, respectively, and necropsied for uterine and liver weight measurements.

3. Animal Assignment: Animals were assigned, stratified by body weight, to the test groups noted in Table 1 using computer-generated (weight-ordered) rando mization procedures. Statistical analysis indicated that there were no significant differences in group means at study initiation. Furthermore, the body weight of each animal was within 20% of the group

mean.

TABLE 1. Study Design a Test Group Dose (mg/kg/day) # of Females Estrogen Agonist Assay Vehicle Control (2% CMC) 0 6 Metalaxyl Low 100 6 Metalaxyl High 300/200 b 6 17 α -ethynyl estradiol (EE), Reference Estrogen 5 μg/kg/day 6 a Data were obtained from Text Table 5 on page 23 of the study report. b Following the first day of dosing at the high dose (300 mg/kg/day) adverse clinical signs in the animals were observed, so the dose volume was reduced from 5 mL/kg to 3.33 mL/kg to achieve a dose of approximately 200 mg/kg/day (199.8 mg/kg/day) for the next two days of dosing.In vivo Uterotrophic Assay (2011) / Page 5 of 10 METALAXYL / 113501 OCSPP 890.1600/ OECD 440



### 4. Dose Selection Rationale: The dose levels were selected based on the results from rangefinding studies (Protocols 20012405 and 20013495); citations for the studies were not provided. Clinical signs of systemic toxicity were observed in adult male and female rats and weanlings at >500 mg/kg/da y; mortality was seen in adult female rats at >500 mg/kg/day and in weanlings of both sexes at 750 mg/kg/day. Due to the severity of the clinical signs observed in adult female rats at >500 mg/kg/day, the high dose selected for the uterotrophic assay was 300 mg/ kg/day. The low dose of 100 mg/kg/day represents one-half log interval of the high dose.

5. Dose Preparation and Analysis: Metalaxyl dose formulations were prepared once prior to the start of dosing by mixing appropriate amount s of the test substance with 2% aqueous CMC; daily aliquots were dispensed and stored refrigerated until use. Prepared doses were allowed to return to room temperature and stir red for at least 30 minutes prior to dosing. EE was prepared daily in corn oil.

Homogeneity and stability of metalaxyl formulations (0.5 and 100 mg/mL) in 2% aqueous CMC were previously evaluated in conjunc tion with Protocol 20012404 (report attached as Appendix 5 of the subject study) . During the uterotrophic st udy, duplicate samples of dose formulations (20 and 60 mg/mL) were collected on the day of preparation, and analyzed for achieved concentration.

Results of Dose Analysis

Homogeneity (% RSD): 0.4% and 1.5%

Stability (% of Day 0): 101.3% and 101.8% following storag e protected from light at room temperature (20-25°C) for 25 hours; and 99.7% and 100% following storage under refrigerated c onditions (2-8°C) for 10 days

Concentration (% of nominal): 101.1%-102.1%

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: Metalaxyl formulations were administered to the animals daily via oral gavage (5 mL/kg) for 3 days. The or al route of administration was selected for the current study because it is a likely potential r oute of human exposure. It is noted that the dose volume of the high dose concentration (300 mg/kg/day) was reduced after Day 1 administration to 3.33 mL/kg to achieve a lowe r dose concentration (200 mg/kg/day) for the next two doses. EE was given dail y via two s.c. injections at 2.5 μ g/kg in 2.5 mL/kg/injection; injection sites were rotate d between six areas on the dorsum to minimize irritation. Dose volumes were adjusted daily based on the most recently recorded body weight and administered at approximately the same time each day to each animal.In vivo Uterotrophic Assay (2011) / Page 6 of 10 METALAXYL / 113501 OCSPP 890.1600/ OECD 440



### 7. Statistics: Means, standard deviations, and per centages were calculated. Statistical analyses were performed using SAS System, version 6.12 or Quattro Pro 8, version 8.0. Clinical observations and other proportional data were analyzed using the vari ance test for homogeneity of the bi nomial distribution.

Continuous data (e.g., body wei ghts, body weight changes, and food consumption values) were analyzed using Bartlett’s te st of homogeneity of variances and the analysis of variance when appropriate [i.e., Bartlett’s test was not significant (p>0.001)]. If the analysis of variance was significant (p ≤ 0.05), Dunnett’s test was used to identify the statistical significance of the individual groups. If the an alysis of variance was not appropriate [i.e., Bartlett’s test was significant (p ≤ 0.001)], the Kruskal-Wallis test was used ( ≤ 75% ties). In cases where the Kruskal-Wallis test was statistically significant (p ≤ 0.05), Dunn’s method of multiple comparisons was used to identify the statistical significance of the individual groups. If there were >75% ties, Fisher’s Exact test was used to analyze the data. Wet and blotted uterine weights were analyzed by analysis of covariance (ANCOVA), using the terminal body weight as the covariate. The stat istical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Animals were assessed for vi ability twice daily. General appearances were observed on the day of recei pt, weekly during the acclimation period and daily before administration during the dosing period. Additionally post-dose observations were recorded within 2 hours of dosing and at the end of th e normal working day. Vaginal patency was recorded once on the day of termination.

2. Body Weight: Animals were weighed weekly during the acclimation period, daily throughout the dosing period, and at termination.

3. Food Consumption: Food consumption was measured daily during dosing and at termination. Values were reported as group mean daily food consumption (g/animal/day).

4. Necropsy and Measurement of Uterine Weight: On PND 60 or PND 66 (approximately 24 hours after final administration of the test substance), all animals were euthanized by carbon dioxide asphyxiation and subjected to a gr oss necropsy. Dissection of the uterus was performed according to the U.S. EPA Guidelin e. Briefly, the vagina was removed just below the cervix in order to retain the lumina l fluid in the uterus. The “wet” uterus (i.e., containing the luminal fluid) was weighed. S ubsequently, both uterine horns were pierced or cut longitudinally and the uterus place on lightly moistened filter paper and gently pressed with a second piece of lightly moiste ned filter paper to completely remove the luminal fluid. The blotted uterus was weighe d. After weighing, the uterus was fixed in neutral buffered 10% formalin and retained for possible future histopathological evaluation. In addition, the liver was exci sed and weighed, and portions pr eserved in neutral buffered 10% formalin or flash frozen for possible future evaluation.In vivo Uterotrophic Assay (2011) / Page 7 of 10 METALAXYL / 113501 OCSPP 890.1600/ OECD 440



### 5. Microscopic Examination (Optional): Microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination.

2. Clinical Signs of Toxicity: Decreased motor activity, pt osis, ataxia, lacrimation, and hunched posture occurred in a significantly increased (p ≤ 0.01) number of female rats in the 300 mg/kg/day dose group on Study Day 1, in comp arison with the vehicle control group. Intermittent body jerks, chromodacryorrhea, and s light excess salivation were also observed in female rats in this dose group on Day 1. All of the adverse clinical signs were attributed to treatment with the test substance. The 300 mg/kg/day dose was reduced to 200 mg/kg/day on Day 2 of study and these adverse clinical signs were not observed on Days 2 and 3. No other adverse clini cal signs of toxicity were obs erved for any of the dose groups.

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data are presented in Table 2. Body weight s throughout the dosing pe riod and overall (Days 1-4) BWG in the metalaxyl treated gr oups were comparable to controls.

The body weights for the positive cont rol (EE) group were decreased (p ≤ 0.05) by 5% on Day 4, and overall BWG for the EE animals was decreased (p ≤ 0.01) by 54% in comparison with the vehicle controls.

TABLE 2. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a

Study Day Dose (mg/kg/day)

Vehicle Control Metalaxyl (100) Metalaxyl (300/200 b ) Reference Estrogen EE (5 μ g/kg/day) N Mean SD N Mean SD N Mean SD N Mean SD
1 6 222.5 5.0 6 219.7 11.3 6 225.4 9.9 6 220.3 9.2 2 6 228.4 6.6 6 224.0 11.0 6 229.4 10.7 6 226.3 11.4 3 6 234.7 6.8 6 229.4 12.6 6 234.8 11.0 6 226.5 10.0
4 (termination) 6 243.2 8.4 6 238.8 12.6 6 243.5 9.7 6 229.9* ( ↓ 5) 10.3

Body Weight Gain (Days 1-11) 6 20.7 4.5 6 19.2 3.1 6 18.1 2.9 6 9.6** ( ↓ 54) 2.7

a Data were obtained from Table 2 on page 41 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. b Following the first day of dosing at the high dose (300 mg/kg/day) adverse clinical signs in the animals were observed, so the dose volume was reduced from 5 mL/kg to 3.33 mL/kg to achieve a dose of approximately 200 mg/kg/day (199.8 mg/kg/day) for the next two days of dosing. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01In vivo Uterotrophic Assay (2011) / Page 8 of 10 METALAXYL / 113501 OCSPP 890.1600/ OECD 440



### C. FOOD CONSUMPTION: Food consumption values are presented in Table 3. Food consumption was decreased (p ≤ 0.01) on Days 1-2 in both metalaxyl treated groups ( ↓ 16% and 23% for the low and high doses, respectively) compared to the controls. Food consumption was then increased (p ≤ 0.01) on Days 2-3 in the 300/200 mg/kg/day group ( ↑ 13%), due to the reduction of the dose from 300 mg/kg/day to 200 mg/kg/day. Overall average (Days 1-4) food consumption for bot h metalaxyl treatment groups was decreased (p ≤ 0.05) by 6-8% compared to the vehicle controls.

Food consumption in the EE positive control group was decreased (p ≤ 0.01) throughout the study ( ↓ 14-26%), for a decrease (p ≤ 0.01) of 19% in overall average food consumption compared to the vehicle control.

TABLE 3. Food Consumption (g/rat/d ay) in the Estrogen Agonist Assay a

Study Day Dose (mg/kg/day)

Vehicle Control Metalaxyl (100) Metalaxyl (300/200 b ) Reference Estrogen EE (5 μ g/kg/day)
N Mean SD N Mean SD N Mean SD N Mean SD
1-2 6 21.2 0.9 6 17.8** ( ↓ 16) 0.9 6 16.3** ( ↓ 23) 0.5 6 18.3** ( ↓ 14) 1.0

2-3 6 20.5 1.2 6 19.8 1.1 6 23.2** ( ↑ 13) 0.2 6 16.7** ( ↓ 19) 1.0

3-4 6 20.2 1.8 6 19.2 1.0 6 18.3 1.7 6 15.0** ( ↓ 26) 3.1 Overall Average (Days 1-4) 6 20.6 1.2 6 19.0** ( ↓ 8) 1.0 6 19.3* ( ↓ 6) 0.5 6 16.7** ( ↓ 19) 1.6
a Data were obtained from Table 3 on page 43 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. b Following the first day of dosing at the high dose (300 mg/kg/day) adverse clinical signs in the animals were observed, so the dose volume was reduced from 5 mL/kg to 3.33 mL/kg to achieve a dose of approximately 200 mg/kg/day (199.8 mg/kg/day) for the next two days of dosing. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01



### D. PATHOLOGY

1. Uterine Weights: Uterine and liver weight data are presented in Ta ble 4. Uterine weights in the metalaxyl treated groups were comparable to the vehicle controls. Absolute liver weights in the 300/200 mg/kg/ day group were increased (p ≤ 0.05) by 24% compared to vehicle controls. Relative liver weights ( not reported in Table 4) were increased (p ≤ 0.05) in the 100 mg/kg/day and 300/200 mg/kg/day group, by 13% and 25%, respectively.

Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p ≤ 0.01) by 547% and 207%, respectively, as expect ed; liver weights were comparable to the vehicle controls.In vivo Uterotrophic Assay (2011) / Page 9 of 10 METALAXYL / 113501 OCSPP 890.1600/ OECD 440


### All females selected for the assay were noted to be in persistent diestr us. At necropsy it was observed that adipose tissue on the right ut erine horn had adhered to a portion of the duodenum in one animal from the vehicle control group and one animal from the 100 mg/kg/day group. The mean blotted uterine weight for the vehicle control group was 0.042% of terminal body weight, which was generally within the performance criteria (<0.04%) of the test guideline.

TABLE 4. Uterine and Liver Weights from Estrogen Agonist Assay in SD Rats a

Parameter Dose (mg/kg/day)

Vehicle Control Metalaxyl (100) Metalaxyl (300/200 b ) Reference Estrogen EE (5 μ g/kg/day) N Mean SD N Mean SD N Mean SD N Mean SD
Terminal BW 6 243.2 8.4 6 238.8 12.6 6 243.5 9.7 6 229.9 10.3 Wet, absolute (g) 6 0.1177 0.0215 6 0.1162 0.0147 6 0.1134 0.0123 6 0.7610** ( ↑ 547) 0.2686

Wet, relative (%) 6 0.052 0.008 6 0.048 0.008 6 0.045 0.005 6 0.333** 0.129 Blotted, absolute (g) 6 0.1061 0.0179 6 0.1052 0.0121 6 0.1033 0.0088 6 0.3264** ( ↑ 207) 0.0345

Blotted, relative (%) 6 0.042 0.008 6 0.043 0.005 6 0.042 0.004 6 0.143** 0.021 Liver, absolute (g) 6 9.9 1.3 6 11.0 1.0 6 12.3** ( ↑ 24) 0.6 6 -- --

a Data were obtained from Table 5 on page 47 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. b Following the first day of dosing at the high dose (300 mg/kg/day) adverse clinical signs in the animals were observed, so the dose volume was reduced from 5 mL/kg to 3.33 mL/kg to achieve a dose of approximately 200 mg/kg/day (199.8 mg/kg/day) for the next two days of dosing. BW Body weight N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01



### 2. Microscopic Examination (Optional): Microscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Increases in the mean absolute and relative weights of the liver were observed in th e 100 and 300/200 mg/kg/da y dose groups; these increases were consistent with the resu lts of previous dose range-finding studies.

There were no significant increases in either the absolute or bl otted weights of the uteri in either the 100 or the 300/200 mg/kg/day dos e group, in comparison with the respective vehicle control group values. Th erefore, the results of this study indicate that there was no positive response for estrogenic activity as a result of administration of metalaxyl at dose levels as high as 300/200 mg/kg/day.In vivo Uterotrophic Assay (2011) / Page 10 of 10 METALAXYL / 113501 OCSPP 890.1600/ OECD 440



### B. AGENCY COMMENTS: All animals survived until scheduled termination. Following administration of metalaxyl on Day 1 at 300 mg /kg, adverse clinical signs were observed, including decreased motor activity, ptosis, at axia, lacrimation, hunche d posture, intermittent body jerks, chromodacryorrhea, and slight exces s salivation, as well as a reduction in food consumption. Therefore, the 300 mg/kg/day do se level was reduced to 200 mg/kg/day on Days 2 and 3. Following the dose reduction, fo od consumption in this group increased, and no adverse clinical signs were observed.

Body weights throughout the dosing period and ov erall (Days 1-4) body weight gain in the metalaxyl treated groups were comparable to controls. The body weights for the positive control (EE) group were decreased (p ≤ 0.05) by 5% on Day 4, and overall BWG for the EE animals was decreased (p ≤ 0.01) by 54% in comparison wi th the vehicle controls.

Overall (Days 1-4) food consumption for bot h metalaxyl treatment groups was decreased (p ≤ 0.05) by 6-8% compared to the vehicle cont rols. Food consumption in the EE positive control group was decreased (p ≤ 0.01) throughout the study, for an overall decrease (p ≤ 0.01) of 19% in average food consumption compared to the vehicle control.

All females selected for the assay were noted to be in persistent diestr us. Uterine weights in the metalaxyl treated groups were comparable to the vehicle controls. Absolute wet and blotted uterus weights for the positiv e control (EE) group were increased (p ≤ 0.01) by 547% and 207%, respectively, as expected. The mean blotted uterine wei ght for the vehicle control group was 0.042% of terminal body we ight, which was gene rally within the performance criteria (<0.04 %) of the test guideline.

Absolute and relative liver weights in the 300/200 mg/kg/ day group were increased (p ≤ 0.05) compared to vehicle controls ( ↑ 24-25%); relative liver weights were increased (p ≤ 0.05) in the 100 mg/kg/day group ( ↑ 13%). Liver weights in the EE group were comparable to the vehicle controls.

The high dose used in the study was adequate based on the adverse clin ical signs observed following a single dose of metala xyl at 300 mg/kg, and the increas es in 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.

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The EE dose of 5 μg/kg/day via s.c. injection was 17 times higher than the 0.3 ug/kg/day s.c. dose recommended by Guidance (OCSPP 890.1600)

 No rational was given for use of a different vehicle for EE (corn oil) than the test chemical and vehicle control (2.0% a queous carboxymethylcellulose). OCSPP 890.1600 recommends test substances be dissolved in a minimal amount of 90% ethanol or other suitable solvent before dissolving in vehicle.