### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, 

### D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### MEMORAN D UM

Date: 6/29/2015

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

### Assays

PC Code: 108801 

### Decision No.: 461243 

### Petition No.: NA 

### Ri sk Assessment Type: NA
TXR 

### No.: 0052848


### MRID No.: See Table 

### DP Barcode: D398926 

### Regi s tration No.: NA 

### Regulatory Action: NA 

### Case No.: NA 

### CAS No.: 51218-45-2
40 

### CFR: NA



### FROM: Greg Akerman, Ph.D

.

## b~ 

# tr----

Immediate Office
Health Effects Division (7509P)



### THROUGH: Jess Rowland

## ~-z.-Q-.C2.--­ 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-evaluation 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 metolachlor: Test Order# CON-108801-12.

Page I of2Page 2 of 2 

### II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for metolachlor.

III. MRID Table

Chemical: Metolachlor PC Code: 108801 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48617810
890.1150 Androgen Receptor Binding (Rat Prostate) 48617806
890.1200 Aromatase Assay (Human Recombinant) 48617805
890.1250 Estrogen Receptor Binding 48617807
890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) 48617808

890.1350 Fish Short-Term Reproduction 48617811
890.1400 Hershberger (Rat) 48617802
890.1450 Female Pubertal (Rat) 48617804
890.1500 Male Pubertal (Rat) 48617803
890.1550 Steroidogenesis (Human Cell Line – H295R) 48617809
890.1600 Uterotrophic (Rat) 48617801Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 2 of 61

DER Template Version: 22 September 2011 CITATION: Palmer, S.J., S.Z. Schneider, T.Z. Kendall, and H.O. Krueger. 2012. Metolachlor: Amphibian

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 Unpublished study performed by Wildlife

International, Ltd., Easton, MD. Laboratory report number 568A-103. Study sponsored by Metolachlor Task

Force, Stilwell, KS. Study completed January 24, 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 Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 3 of 61

DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay of metolachlor (97.3% purity) on amphibian metamorphosis of Xenopus laevis (African clawed

frog) was conducted under flow-through conditions. Amphib ian larvae at developmental Nieuwkoop-Faber (NF)
Stage 51 (80/control and treatment group; 20/replicate) were exposed to nominal concentrations of 0 (negative

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

0.075, 0.79, and 8.4 mg a.i./L. The test system was maintained at 22 ± 1 °C and a pH of 8.1 to 8.3.

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

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

Metolachlor significantly reduced (p<0.05) Day 7 wet weight by 19% and Day 7 snout-vent length by 7% at the
high treatment group relative to the negative control. Day 21 normalized (for snout-vent length) hind leg length
(HLL) was also significantly reduced (p<0.05) by 23% at the high treatment level relative to the negative control.

Metolachlor exposure had no effect on the NF developmental stage in all treatment groups relative to the negative

control on Days 7 and 21; no asynchronous development was observed. Day 21 normalized HLL at the high
treatment level was significantly reduced (p<0.05) by 23% relative to the negative control group.

Thyroid gland histopathology observations included thyroid gland hypertrophy, thyroid gland atrophy, and/or

follicular cell hypertrophy in the negative control and all treatment groups.

Analysis of Day 21 wet weight, SVL, HLL, and normalized HLL excluded late stage tadpoles ( i.e. , NF stage
=61-63); there was no pattern in the development of late stage individuals across treatment groups, with the

proportion of late stage tadpoles ranging from 2 to 6% of all tadpoles tested in the negative control and all treatment

groups.

The study met all of the performance and validity criteria.

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

Guideline 890.1100).Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 4 of 61

DER Template Version: 22 September 2011 Results Synopsis:

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

Test type: Flow-through

Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects 1,2 in the Amphibian

Metamorphosis Assay (AMA) with Metolachlor.

Treatment

(mg a.i./L)

[measured] NF Developmental

Stage Hind Limb

Length 3 Asynchronous

Dev elopment Thyroid Gross

and

Histopathology

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

0.075 No No No No No No No

0.79 No No No No No No No

8.40 No No No 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 board-certified

pathologist.

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

EPA MRID Number 48617810

Page 5 of 61

DER Template Version: 22 September 2011 

### I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guidelines outlined in the U.S.

Environmental Protection Agency Series 890- Endocrine Disruptor Screening

Program Test Guidelines, OPPTS Number 890.1100: Amphibian
Metamorphosis (Frog) ; and the OECD Guidelines for Testing of Chemicals,

Guideline 231: Amphibian Metamorphosis Assay . The following deviations were

noted:

1. The flow rate (~69 mL/min) was greater than recommended (25 mL/min), though there was

complete volume replacement approximately every 2.4 hours, which is in agreement with guideline
recommendations (complete volume replacement every 2.7 hours).

2. The reported pH (8.1-8.3) was greater than recommended (7.5 ± 0.1).

These deviations do not impact the interpretation of the study.

Compliance: Signed and dated Good Laboratory Practice and Quality Assurance statements

were provided. A No Data Confidentiality statement was not included. This

study was conducted in compliance with Good Laboratory Practice Standards
as published by the U.S. Environmental Protection Agency (40 CFR Parts 160

and 792); and OECD Principles of Good Laboratory Practice
(ENV/MC/CHEM(98) 17), with the following exceptions: periodic analyses of

water for potential contaminants were not performed according to Good

Laboratory Practice Standards; the characterization of the test substance, and

the stability of the test substance under conditions of storage at the test site,

were not determined in accordance with GLP standards; and, preliminary

analyses of water iodide concentrations were not performed according to GLP

standards.

A. Test Material MetolachlorData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 6 of 61

DER Template Version: 22 September 2011 Description: Liquid

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. : 20081115 (Lot #)

Purity: 97.3%

Impurities: None Reported

Stability of Compound: Stable. Mean-measured concentrations over the 21-day exposure yielded

recoveries of 83 to 93% of nominal.

Storage Conditions of

Test Chemicals: Stored under ambient conditions.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page7of61 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 the 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 Endocrine DisruptorScreening 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 Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page8of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained asin-house stock?YesAdult frogs for the culture wereoriginally obtained from Xenopus I ofDexter, Michigan. 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):~13 weeksDetails on parental feeding:Adults were fed an adult Xenopusdiet supplied byZeigler Brothers, Inc. ofGardners, PA at a ratesufficient to maintain thehealth of the culture.Details on parental health:Culture frogs showed no signof disease or stress prior tothe test.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page9of61 DER Template Version: 22 September 2011Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest single spawn?YesBest single spawn had 98% viability. 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 co-mixed)Number of spawns evaluated (ifapplicable):2Based on use of two breeding groupsNumber of eggs sampled perspawn:250 eggs from the bestspawn wereevaluated forviability.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:15 days post-fertilization(dpf)Mean total length at test initiation(if reported):Not ReportedRange of total length at testinitiation (if reported):Not ReportedData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page10of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas the optional size selectionmethod used?NoDetails on larval selection:All tadpoles were derivedfrom eggs spawned on thesame day. To produce eggs,two male and female adultswere injected with humanchorionic gonadotropin andeach pair was placed in aseparate spawning tankovernight.Loading rate (rearing density):2 larvae/LEstimated from 20 larvae per testvessel (10 L fill volume). 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:Sera Micron® EPA recommends Sera Micron ® throughoutData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page11of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSource of food:Sera North America,Montgomeryville, PA pre-exposure (after NF stage 45/46) and during the entire 21 - d definitive study. If another diet is used, the study report should 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:3 times/day and once on thelast day of the test EPA recommends that feeding occur at least

twice per day.C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through

system.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page12of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem (if applicable):Continuous-flow diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):~69 mL/minThe flow-through rate wascalculated based on the volumeof the test chambers (10 L) and10 volume additions per day(10). The flow-through rateresulted in complete volumereplacement approximately every2.4 hours. 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 Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page13of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems (ifapplicable):The stock solution was delivered tomixing chambers using fluid meteringpumps, and then delivered to the testvessels using rotameters andperistaltic pumps.Mean flow rate did not vary bymore than ±10% during the test. Recommended toxicant mixing for

flow - through systems: 1) Mixing chamber is recommended but not required; 2) Aeration

is not rec ommended 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%.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 is recommended to set bubblers at levels that do not cause stress on the tadpoles.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page14of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water:Dilution water was freshwaterobtained from a well approximately 40meters deep located on the WildlifeInternational, Ltd. Site. Water waspassed through a sand filter to removeparticles greater than ~25μm andaerated with spray nozzles. Prior touse, the water was filtered (0.45μm)and passed through a UV sterilizer. 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 analyzed forpesticides, heavy metals, andother contaminants?YesTOC was <1 mg/L. Nopesticides or organic weredetected. See Reviewer’sComments section for results onmetals.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page15of61 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 μ 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:Glass aquaria EPA and OECD recommend that

water - contact 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:12 LFill volume:10 LAdditional details on exposuresystem:NoneData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page16of61 DER Template Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsHardness (mg/L as CaCO3)136 144 142Weekly in the controland highesttreatment group. EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH8.18.28.1Weekly in all testvessels. EPA recommends pH 7.5 ± 1,

inter -replicate and inter-treatment differentials should not exceed 0.5.Dissolved oxygen (mg/L)7.2 8.4 7.7Weekly in all testvessels. EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration >3.5 mg/L (>40% air saturation).Temperature21.122.221.9Weekly in all testvessels.Continuously in onenegative controlreplicate. 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 Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page17of61 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsIodide (μg/L)3 6Not ReportedPeriod EPA recommends aquatic iodide range 0.5

– 10 μ g/L (supplemental iodide should not

exceed 2 μ 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 renewal. In addition, EPA recommends that DO be measured at each concentration at least weekly and that tem perature be measured continuously. OECD recommends that DO and temperature be measured at least weekly and that pH and hardness be measured at least at theFluorideNot reportedPerchlorateNot reportedChlorateNot reportedAlkalinity (mg/L as CaCO3)Conductance (μS/cm)174357182380180368Weekly in thenegative control andhighest treatmentgroup.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page18of61 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline Recommendations 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 therange-finder?Not specified 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 laevisLife stage:NF 51Test duration:96 hoursData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page19of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:A 96-hr range-findingstudy was conductedunder flow-throughconditions with nominalconcentrations of 0.13,0.41, 1.4, 4.5, 15, and50 mg a.i./L with NF 51tadpoles.The resulting LC50was 27 mg a.i./L andthe highest does in the AMA was set asone-third of the LC50to approximate themaximum tolerated dose (9.0 mg a.i./L).Two additional test concentrations wereselected with a 10X separation factor (0.9and 0.09 mg a.i./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.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page20of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in the definitivetest:One-third of thecalculated LC50from thepreliminaryrange-finding study. 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.Reference study citation (ifapplicable):None providedSeparation of test concentrations:0.1 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?YesData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page21of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Mean-measuredLimit of quantification (LOQ):0.0500 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, 14, and 21 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 EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page22of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of larvae per treatment attest initiation:80 (20/rep)Was 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?No3Hutchinson 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 onTesting and Assessment. No. 23. Paris, France.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page23of61 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs darkA 30-minute transition period was usedbetween light and dark cycles. EPA recommends photoperiod 12:12

(light:dark).Light intensity at water’s surface:0.801.68 Klux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:Test solutions reported as clear andcolorless with no evidence of precipitateData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page24of61 DER Template Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Metolachlor.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0<LOQNANone EPA and OECD recommend that test item concentrations be maintained at a coefficient of

variation (CV)≤ 20%.Treatment 10.090.0753.4NoneTreatment 20.900.793.3NoneTreatment 29.08.41.6NoneAbbreviations:CVCoefficient of variation.E. ObservationsBiological Endpoints:Development stage, hind-limb length (HLL), normalized HLL, snout-vent length (SVL), and wet weight were assessed on Days7 and 21. Histopathology was performed on tadpoles collected on Day 21. Observations for mortality and sub-lethal effects wereperformed daily.Were raw (individual) data provided?YesData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page25of61 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-based lar val selection); and thyroid gland gross pathology and histopathology (Day 21). Note the histopathology section of the test guideline also includes thyroid gross pathology observations.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 26 of 61

DER Template Version: 22 September 2011 

### II. RESULTS AND DISCUSSION

A. Results

By Day 7, a single mortality occurred in the negative control and complete survival was observed in the
remaining treatment groups (Table 9). By Day 21, no additional mortalities occurred in the control or

mean-measured 0.075 mg a.i./L treatment group. Mortality was 3.3 and 6.6% in the mean-measured

0.79 and 8.4 mg a.i./L treatment groups, respectively, on Day 21.

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 1 1.3 60 0 0

0.075 80 0 0 60 0 0

0.79 80 0 0 60 2 3.3

8.4 80 0 0 60 4 2 6.6

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

2 Two mortalities in both replicates A and D.

Median NF stage was 54 and 57 on Day 7 and 21, respectively, in the control and all treatment groups.
Individual tadpole stage ranged from 52 to 55 on Day 7 and 55 to 63 on Day 21 (Table 10). No

asynchronous development was observed in any treatment group. The 10 th and 90 th percentiles were 53

and 54, respectively, on Day 7, and 56 and 60, respectively, on Day 21.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 27 of 61

DER Template Version: 22 September 2011 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 57 0

0.075 4 54 0 4 57 0

0.79 4 54 0 4 57 0

8.4 4 54 0 4 57 0

Normalized HLL on Day 7 averaged 0.14 in the negative control and 0.16, 0.15, and 0.16 in the
mean-measured 0.075, 0.79, and 8.4 mg a.i./L treatment groups, respectively (Table 11). Normalized

HLL on Day 21 averaged 0.36 in the negative control and 0.33, 0.36, and 0.28 in the mean-measured

0.075, 0.79, and 8.4 mg a.i./L treatment groups, respectively.

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

Treatment

(mg a.i./L)

[mean-measured] Hind Limb Length (HLL)

Day 7 Day 21

n Mean

(mm) ±SD HLL:

SVL 1 n Mean

(mm) ±SD HLL:

SVL 1

Negative Control 4 2.30 0.33 0.14 4 7.90 0.47 0.36

0.075 4 2.55 0.32 0.16 4 7.53 0.39 0.33

0.79 4 2.47 0.08 0.15 4 8.00 0.97 0.36

8.4 4 2.49 0.27 0.16 4 6.10 0.84 0.28Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 28 of 61

DER Template Version: 22 September 2011 Abbreviations: SD Standard deviation.

1 Summary results for snout-vent length (SVL) are presented in the next table ( Table 12 ).

On Day 7, SVL ranged from 15.6 to 16.8 mm (Table 12). By Day 21, SVL ranged from 22.0 to 22.5
mm. On Day 7, wet weight ranged from 0.262 to 0.336 g (Table 12). By Day 21, wet weight ranged

from 0.797 to 0.898 g.

Table 12: Larval Growth in Xenopus laevis

.

Treatment

(mg a.i./L)

[mean

- measured] Snout-Vent Length (SVL) Body Weight 1

Day 7 Day 21 Day 7 Day 21

n Mean

(mm) ±SD n Mean

(mm) ±SD n Mean

(g) ±SD n Mean

(g) ±SD

Negative Control 4 16.8 0.24 4 22.0 0.71 4 0.322 0.02 4 0.807 0.06

0.075 4 16.1 0.31 4 22.5 0.49 4 0.291 0.02 4 0.872 0.04

0.79 4 16.8 0.52 4 22.4 0.60 4 0.336 0.04 4 0.898 0.07

8.4 4 15.6 0.20 4 22.0 0.70 4 0.262 0.01 4 0.797 0.06

Abbreviations: SD Standard deviation.

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

There were incidences of hypertrophy and atrophy of the thyroid gland, as well as follicular cell hypertrophy
in the control and all treatment groups (Table 13). However, incidence of observation was comparable

between the control and all treatment groups

.Data Evaluation Record on the Toxicity of Metolachlor 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 17 20 17 20 17 NA NA

1 20 1 20 3 20 2 NA NA

2 20 1 20 0 20 1 NA NA

3 20 1 20 0 20 0 NA NA

0.075 0 20 15 20 17 20 19 NA NA

1 20 5 20 3 20 1 NA NA

2 20 0 20 0 20 0 NA NA

3 20 0 20 0 20 0 NA NA

0.79 0 20 16 20 19 20 20 NA NA

1 20 3 20 1 20 0 NA NA

2 20 1 20 0 20 0 NA NA

3 20 0 20 0 20 0 NA NA

8.4 0 20 14 20 20 20 15 NA NA

1 20 3 20 0 20 3 NA NA

2 20 1 20 0 20 2 NA NA

3 20 2 20 0 20 0 NA NA

Abbreviations: NA Not applicable.

1 Thyroid gland gross pathology and histopathology are 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 Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page30of61 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.Treatment(mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control0NANANANANANANANA20181NANANANANANANANA2022NANANANANANANANA2003NANANANANANANANA2000.0750NANANANANANANANA20201NANANANANANANANA2002NANANANANANANANA2003NANANANANANANANA2000.790NANANANANANANANA20201NANANANANANANANA2002NANANANANANANANA2003NANANANANANANANA2008.40NANANANANANANANA2016Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page31of61 DER Template Version: 22 September 2011Treatment(mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular Cell Height(Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence1NANANANANANANANA2042NANANANANANANANA2003NANANANANANANANA200Abbreviations: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 Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page32of61 DER Template Version: 22 September 2011Occasional observations of a few tadpoles that appeared lethargic or were surfacing were noted in the high treatment group through Day 8 of the test (Table15). However, no sublethal effects were noted throughout the remainder of the test. Beginning on Day 4, an increasing number oftadpoles in the control andall treatment groups had curved tails. By test termination, 66, 85, 98. And 48% of the surviving tadpoles had crooked tails inthe negative control andmean-measured 0.075, 0.79, and 8.4 mg a.i./L treatment groups, respectively. At test termination, tail curvature was scored (from 0-3) for each tadpole, andthere were 32, 28, 45 and 4% of tadpoles in the control, 0.075, 0.79 and 8.4 mg a.i./L that were considered to have tail curvature with a score≥2.Table 15: Clinical Signs in Xenopus laevis.Treatment(mg a.i./L)[meanmeasured]Clinical Signs1TypenIncidence2Negative ControlCrooked tail59390.075Crooked tail60510.79Crooked tail58578.4Crooked tail56271 Note that asynchronous development (unable to stage) is reported previously inTable 102 Based on Day 21 observationsData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

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

The study authors analyzed survival, developmental stage, body weight, whole body length, SVL,

normalized HLL, and incidence and severity of thyroid abnormalities. If responses appeared to be

monotonic, a step-down Jonckheere-Terpstra trend test was used to evaluate trends in the ranks of

replicate means to determine possible concentration-responsive trends among the treatment groups. All

endpoints, except for survival, development stage, and histopathology severity scores also were analyzed
by performing pair-wise comparisons using Dunnett’s multiple comparison test to determine which

treatment groups differed from the control group. Data for endpoints analyzed by Dunnett’s test were

evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test

( α = 0.01). When the data did not satisfy the assumptions, a log transformation was applied to correct

the data for non-normality or heterogeneous variances.

Survival, developmental stage, and histopathology severity scores were not amenable to the statistical

methods used for analysis of other endpoints. In particular, the most suitable unit of statistical analyses

for these endpoints was the individual animal. Therefore, survival was analyzed using the step-down

Jonckheere-Terpstra trend test and Fisher’s Exact test, and histopathology severity scores of individuals

were analyzed using step-down Jonckheere-Terpstra trend tests only. In accordance with

recommendations of OECD TG 231 and OPPTS 890.1100, analysis of metamorphic stage was performed

using the multi-quantile analyses developed by T. Springer and J. Green, and using the step-down

Jonckheere-Terpstra trend test on stage quantiles in the replicate test chambers. Statistical tests used

to evaluate treatment effects were performed at α of 0.05 with SAS software.

Treatment-related effects were limited to the mean-measured 8.4 mg a.i./L treatment group with

significant reductions in SVL on Day 7 and reductions in normalized HLL on Day 21. There were no other

significant differences in developmental or growth parameters. Further, there were no apparent

treatment-related trends in histopatholgoical observations.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 C. Reviewer’s Analysis and Conclusions

Statistical Methods: Mortality did not exceed 7% in any of the treatment groups. Therefore, the reviewer

visually determined that the test material had no effects on larval survival. All Day 7 endpoints, except

developmental stage, were non-monotonic with normal distributions and homogenous variances and were
therefore analyzed with Dunnett’s test. Day 7 developmental stage was non-monotonic but did not meet

the assumptions of parametric statistics and was analyz ed using the Mann-Whitney test. All Day 21 growth
parameters (i.e., wet weight, SVL, HLL) were non-monotonic but met the assumptions of parametric

statistics and these variables were analyzed using Dunnett’s test. Both Day 21 development parameters
(i.e., NF stage, normalized HLL) were analyzed using the Mann-Whitney test based on a lack of

monotonicity and the failure to meet the assumptions of parametric statistics. There was no asynchronous

development. There were late stage tadpoles present by Day 21 in the control and all metolachlor-treated

groups, comprising 2-6% of the total sample size, with NF stages ranging from 61-63; there was no

apparent pattern in late stage development or evidence of dose-dependency. Late stage tadpoles were

excluded from the analysis of 21-day wet weight, SVL, HLL, and normalized HLL. Unless otherwise

indicated, effects were considered statistically significant at p < 0.05.

Conclusions:

Throughout the test, no effects were observed on NF developmental stage. For the 8.4 mg a.i./L
treatment, there was a significant 23% reduction in Day 21 normalized HLL (p<0.05) from the negative
control (Table 16). Growth parameters, SVL and wet weight, were significantly reduced (p<0.05) 7 and
19%, respectively, on Day 7 and HLL was significantly reduced (p<0.05) 23% on Day 21 in the highest
treatment group relative to the negative control (Table 17). There were no clear treatment-related effects

in the histopathological observations.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page35of61 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Metolachlor4.Treatment(mg a.i./L)[mean-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 21Day 7Day 21Day 21Median pMedian p%Diff. p%Diff. p%Diff. p%Diff. pTreatment-RelatedEffects? (Yes/No)Negative Control54NA57NANANANANANANANANANo0.07554>0.9957>0.99150.25-8.30.12NANANANANo0.79540.48580.237.30.73-2.10.48NANANANANo8.4540.4857>0.99160.18-230.029NANANANANoStatistical TestMann-WhitneyMann-WhitneyDunnett’sMann-WhitneyNANANAAbbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily weightedby 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).Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page36of61 DER Template Version: 22 September 20114Several tadpoles were excluded from the analysis of Day 21 endpoints for wet weight, SVL, HLL, and norm HLL. Excluded individuals comprised 2, 4, 6, and3% of the control, 0.075, 0.79, and 8.4 mg ai/L treatment group total sample sizes, respectively. Late stage individuals rangedfrom NF 61 to 63 with noapparent pattern of occurrence or dose-dependency.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Table 17: Growth Endpoints 1,2 in the AMA with Metolachlor 3

.

Treatment

(mg a.i./L)

[mean-measured] Snout-Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

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

Negative Control NA NA NA NA NA NA NA NA

0.075 -3.9 0.052 2.0 0.63 -9.6 0.25 8.0 0.31

0.79 0.2 0.999 1.8 0.71 4.2 0.80 11.3 0.11

8.4 -7.0 0.001 -0.5 0.99 -18.7 0.02 -1.2 0.99

Statistical Test Dunnett’s Dunnett’s Dunnett’s Dunnett’s

Abbreviations: Diff. Difference. NA Not applicable.

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 Several tadpoles were excluded from the analysis of Day 21 endpoints for wet weight, SVL, HLL, and norm

HLL. Excluded individuals comprised 2, 4, 6, and 3% of the control, 0.075, 0.79, and 8.4 mg ai/L treatment

group total sample sizes, respectively. Late stage individuals ranged from NF 61 to 63 with no apparent

pattern of occurrence or dose-dependency.

E. Study Deficiencies

The high prevalence of crooked tail/tail curvature across treatments raises some concerns about the

general health and husbandry of the test specimens, although environmental conditions and feeding were

generally consistent with the guideline recommendations.

Deviations are listed in Section I. Materials and Methods of this DER. These deviations do

not impact the interpretation of the study.

F. Reviewer’s Comments

The reviewer’s analyses were similar to the study authors, however, the study author reported significant
decreases in SVL (p<0.05) at the highest treatment level on Day 21 whereas the reviewer’s analysis

did not.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Results from the periodic screening analysis of the dilution water detected the following analytes: calcium
(35.1 mg/L), chloride (3.5 mg/L), magnesium (13.0 mg/L), nitrate (0.55 mg N/L), potassium (6.77
mg/L), sodium (18.5 mg/L), and sulfate (5.6 mg/L).

Day 21 normalized HLL was significantly reduced at the top dose (23%), relative to the negative control.



### III. REFERENCES

Nieuwkoop, P.D., Faber, J. (eds.). 1994. Normal Table of Xenopus laevis (Daudin): A Systematical and

Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis . Garland

Publishing Co, New York, NY USA.

Organization for Economic Cooperation and Development. 2006. Current Approaches in the Statistical

Analysis of Ecotoxicity Data: A Guidance to Application . OECD ENV/JM/MONO(2008)18.

Personal Communication with Dr. Timothy Springer and Dr. John Green. November 30, 2010. Dr. Timothy

Springer, Wildlife International, Ltd., Easton, MD 21601, USA. Dr. John Green, E.I. DuPont de Nemours

and Company, Wilmington, DE 19898, USA.

The SAS System for Windows. 1999-2001. Version 8.2. SAS Institute, Inc., Cary, North Carolina.Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis AssayEPA MRID Number 48617810Page1of61 DER Template Version: 22 September 2011APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:EndpointMonotonic?Parametric?890.1100EFEDComments7-day wet weightNoYesDunnett’s:Dose 3 p=0.015Dunnett’s:Dose 3 p=0.015890.1100 and EFED same conclusions, significant 19%reduction (p>0.05) at the 8.4 mg ai/L level7-stage medianNoNoMann-Whitney:n.s. p>0.05Mann-Whitney:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)7-day SN-vent lengthNoYesDunnett’s:Dose 3 p=0.001Dunnett’s:Dose 3 p=0.001890.1100 and EFED same conclusions, significant 7%reduction (p>0.05) at the 8.4 mg ai/L level7-day hind-limblengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)7-day normalizedHLLNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)21-day stage medianNoNoMann-Whitney:n.s. p>0.05Mann-Whitney:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)21-day wet weightNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)21-day SN-ventlengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1100 and EFED same conclusions, no effect (p>0.05)21-day HLLNoYesDunnett’s:Dose 3 p=0.010Dunnett’s:Dose 3 p=0.010890.1100 and EFED same conclusions, significant 23%reduction (p>0.05) at the 8.4 mg ai/L level21-day normalizedHLLNoNoMann-Whitney:Dose 3 p=0.029Mann-Whitney:Dose 3 p=0.029890.1100 and EFED same conclusions, significant 23%reduction (p>0.05) at the 8.4 mg ai/L levelData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 test for amphib metamorph screen study - metolachlor 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
0.954 0.555 2.475 0.112 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.32 0.02 0.01 5.29 0.30, 0.35 Dose1 4 0.29 0.02 0.01 6.31 0.26, 0.32 Dose2 4 0.34 0.04 0.02 12.62 0.27, 0.40 Dose3 4 0.26 0.01 0.01 4.72 0.24, 0.28

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 0.32 0.31 0.35 . .
Dose1 0.29 0.28 0.32 90.38 9.62
Dose2 0.33 0.29 0.39 104.19 -4.19
Dose3 0.26 0.25 0.28 81.30 18.70

******************************************************************************** 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 6.76 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 0.32 . 0.32 . . . . . .
Dose1 0.29 0.248 0.31 0.378 . . . . .
Dose2 0.34 0.795 0.31 0.404 0.114 . . . .
Dose3 0.26 0.015 0.26 0.003 0.399 0.007 . . .

******************************************************************************** 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 9.92 0.019

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 0.32 . .
Dose1 0.29 0.154 0.041
Dose2 0.33 0.780 0.559
Dose3 0.26 0.067 0.025

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3
Jonckheere Dose3Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

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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 6.76 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 -0.32 . -0.30 . . . . . .
Dose1 -0.29 0.248 -0.30 0.911 . . . . .
Dose2 -0.34 0.795 -0.30 0.931 0.114 . . . .
Dose3 -0.26 0.015 -0.30 0.941 0.399 0.007 . . .

******************************************************************************** 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 9.92 0.019

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 -0.32 . .
Dose1 -0.29 0.154 0.959
Dose2 -0.33 0.780 0.441
Dose3 -0.26 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 amphib metamorph screen study - metolachlor 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
0.677 <.001 6.000 0.010 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS
Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 53.75 0.50 0.25 0.93 52.95, 54.55 Dose1 4 53.75 0.50 0.25 0.93 52.95, 54.55 Dose2 4 54.00 0.00 0.00 0.00 . , . Dose3 4 54.00 0.00 0.00 0.00 . , .

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 54.00 53.00 54.00 . .
Dose1 54.00 53.00 54.00 100.00 0.00
Dose2 54.00 54.00 54.00 100.47 -0.47Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Dose3 54.00 54.00 54.00 100.47 -0.47

******************************************************************************** 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 0.67 0.588

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 53.75 . 53.88 . . . . . .
Dose1 53.75 1.000 53.88 0.773 . . . . .
Dose2 54.00 0.638 53.88 0.805 0.752 . . . .
Dose3 54.00 0.638 53.88 0.822 0.752 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 2.14 0.543

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 0.500
Dose2 54.00 0.478 0.818
Dose3 54.00 0.478 0.905

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 0.67 0.588

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 -53.75 . -53.75 . . . . . .
Dose1 -53.75 1.000 -53.75 0.583 . . . . .
Dose2 -54.00 0.638 -54.00 0.215 0.752 . . . .
Dose3 -54.00 0.638 -54.00 0.222 0.752 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-valueData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 3 2.14 0.543

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 0.500
Dose2 -54.00 0.478 0.182
Dose3 -54.00 0.478 0.095

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 - metolachlor 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
0.955 0.574 0.939 0.452 USE PARAMETRIC TESTS

********************************************************************************
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 16.78 0.25 0.13 1.49 16.38, 17.17 Dose1 4 16.13 0.33 0.17 2.05 15.60, 16.65 Dose2 4 16.80 0.51 0.25 3.04 15.99, 17.61 Dose3 4 15.60 0.22 0.11 1.38 15.26, 15.94

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 16.90 16.40 16.90 . .
Dose1 16.10 15.80 16.50 96.13 3.87
Dose2 16.70 16.30 17.50 100.15 -0.15
Dose3 15.65 15.30 15.80 93.00 7.00

******************************************************************************** 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 11.08 <.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 16.78 . 16.78 . . . . . .
Dose1 16.13 0.052 16.46 0.134 . . . . .
Dose2 16.80 0.999 16.46 0.143 0.071 . . . .
Dose3 15.60 0.001 15.60 <.001 0.194 0.002 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groupsData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Degrees of Freedom TestStat P-value
3 11.50 0.009

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 16.90 . .
Dose1 16.10 0.096 0.019
Dose2 16.70 0.671 0.356
Dose3 15.65 0.062 0.007

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 11.08 <.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 -16.78 . -16.33 . . . . . .
Dose1 -16.13 0.052 -16.33 0.980 . . . . .
Dose2 -16.80 0.999 -16.33 0.987 0.071 . . . .
Dose3 -15.60 0.001 -16.33 0.989 0.194 0.002 . . .

******************************************************************************** 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 11.50 0.009

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 -16.90 . .
Dose1 -16.10 0.096 0.981
Dose2 -16.70 0.671 0.644
Dose3 -15.65 0.062 0.993

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 - metolachlor ANALYSIS RESULTS FOR VARIABLE VAR04 ( 7-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 ConclusionData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 6 of 61

DER Template Version: 22 September 2011 Test Stat P-value Test Stat P-value
0.946 0.435 1.065 0.400 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 2.30 0.33 0.16 14.20 1.78, 2.82 Dose1 4 2.55 0.32 0.16 12.53 2.04, 3.06 Dose2 4 2.47 0.08 0.04 3.16 2.34, 2.59 Dose3 4 2.49 0.27 0.14 10.97 2.06, 2.93

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 2.35 1.86 2.65 . .
Dose1 2.64 2.09 2.82 110.76 -10.76
Dose2 2.47 2.37 2.56 107.28 -7.28
Dose3 2.42 2.25 2.88 108.37 -8.37

******************************************************************************** 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 0.63 0.611

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 2.30 . 2.45 . . . . . .
Dose1 2.55 0.451 2.45 0.856 . . . . .
Dose2 2.47 0.715 2.45 0.882 0.974 . . . .
Dose3 2.49 0.630 2.45 0.895 0.991 0.999 . . .

******************************************************************************** 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 2.26 0.520

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 2.35 . .
Dose1 2.64 0.346 0.876
Dose2 2.47 0.346 0.721
Dose3 2.42 0.494 0.663

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 0.63 0.611

Dunnett - testing each trt mean signif. different than controlData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 7 of 61

DER Template Version: 22 September 2011 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 -2.30 . -2.30 . . . . . .
Dose1 -2.55 0.451 -2.50 0.184 . . . . .
Dose2 -2.47 0.715 -2.50 0.196 0.974 . . . .
Dose3 -2.49 0.630 -2.50 0.203 0.991 0.999 . . .

******************************************************************************** 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 2.26 0.520

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 -2.35 . .
Dose1 -2.64 0.346 0.124
Dose2 -2.47 0.346 0.279
Dose3 -2.42 0.494 0.337

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 - metolachlor 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
0.938 0.325 0.414 0.746 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.14 0.02 0.01 14.99 0.10, 0.17 Dose1 4 0.16 0.02 0.01 12.02 0.13, 0.19 Dose2 4 0.15 0.01 0.00 6.49 0.13, 0.16 Dose3 4 0.16 0.01 0.01 8.84 0.14, 0.18

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 0.14 0.11 0.16 . .
Dose1 0.17 0.13 0.17 114.55 -14.55
Dose2 0.15 0.14 0.16 107.27 -7.27
Dose3 0.16 0.15 0.18 116.36 -16.36

******************************************************************************** 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 1.57 0.247Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 8 of 61

DER Template Version: 22 September 2011 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 0.14 . 0.15 . . . . . .
Dose1 0.16 0.248 0.15 0.919 . . . . .
Dose2 0.15 0.727 0.15 0.938 0.824 . . . .
Dose3 0.16 0.177 0.15 0.947 0.996 0.709 . . .

******************************************************************************** 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 4.00 0.262

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 0.14 . .
Dose1 0.17 0.278 0.907
Dose2 0.15 0.555 0.701
Dose3 0.16 0.185 0.892

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 1.57 0.247

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 -0.14 . -0.14 . . . . . .
Dose1 -0.16 0.248 -0.15 0.131 . . . . .
Dose2 -0.15 0.727 -0.15 0.139 0.824 . . . .
Dose3 -0.16 0.177 -0.16 0.048 0.996 0.709 . . .

******************************************************************************** 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 4.00 0.262

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 -0.14 . .
Dose1 -0.17 0.278 0.093Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 9 of 61

DER Template Version: 22 September 2011 Dose2 -0.15 0.555 0.299
Dose3 -0.16 0.185 0.108

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose3 Jonckheere >highest dose (no sign. differences)

test for amphib metamorph screen study - metolachlor 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
0.654 <.001 13.500 <.001 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 57.00 0.00 0.00 0.00 . , . Dose1 4 57.00 0.00 0.00 0.00 . , . Dose2 4 57.75 0.96 0.48 1.66 56.23, 59.27 Dose3 4 57.00 0.00 0.00 0.00 . , .

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 57.00 57.00 57.00 . .
Dose1 57.00 57.00 57.00 100.00 0.00
Dose2 57.50 57.00 59.00 101.32 -1.32
Dose3 57.00 57.00 57.00 100.00 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
3 12 2.45 0.113

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 57.00 . 57.25 . . . . . .
Dose1 57.00 1.000 57.25 0.841 . . . . .
Dose2 57.75 0.113 57.25 0.869 0.174 . . . .
Dose3 57.00 1.000 57.00 0.636 1.000 0.174 . . .

******************************************************************************** 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 6.40 0.094

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 57.00 . .Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 10 of 61

DER Template Version: 22 September 2011 Dose1 57.00 1.000 .
Dose2 57.50 0.227 0.965
Dose3 57.00 1.000 0.743

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 2.45 0.113

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 -57.00 . -57.00 . . . . . .
Dose1 -57.00 1.000 -57.00 0.583 . . . . .
Dose2 -57.75 0.113 -57.38 0.184 0.174 . . . .
Dose3 -57.00 1.000 -57.38 0.191 1.000 0.174 . . .

******************************************************************************** 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 6.40 0.094

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 -57.00 . .
Dose1 -57.00 1.000 .
Dose2 -57.50 0.227 0.035
Dose3 -57.00 1.000 0.257

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 - metolachlor 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
0.897 0.073 0.266 0.848 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.81 0.06 0.03 7.17 0.71, 0.90 Dose1 4 0.87 0.04 0.02 4.08 0.81, 0.93Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 11 of 61

DER Template Version: 22 September 2011 Dose2 4 0.90 0.07 0.03 7.52 0.79, 1.01 Dose3 4 0.80 0.06 0.03 8.15 0.69, 0.90

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 0.80 0.75 0.89 . .
Dose1 0.87 0.84 0.91 107.99 -7.99
Dose2 0.89 0.83 0.99 111.31 -11.31
Dose3 0.77 0.75 0.89 98.76 1.24

******************************************************************************** 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 2.90 0.079

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 0.81 . 0.86 . . . . . .
Dose1 0.87 0.310 0.86 0.937 . . . . .
Dose2 0.90 0.110 0.86 0.953 0.912 . . . .
Dose3 0.80 0.989 0.80 0.527 0.311 0.115 . . .

******************************************************************************** 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 5.85 0.119

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 0.80 . .
Dose1 0.87 0.154 0.959
Dose2 0.89 0.101 0.972
Dose3 0.77 0.889 0.574

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 2.90 0.079

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 -0.81 . -0.81 . . . . . .
Dose1 -0.87 0.310 -0.86 0.154 . . . . .Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 12 of 61

DER Template Version: 22 September 2011 Dose2 -0.90 0.110 -0.86 0.164 0.912 . . . .
Dose3 -0.80 0.989 -0.86 0.169 0.311 0.115 . . .

******************************************************************************** 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 5.85 0.119

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 -0.80 . .
Dose1 -0.87 0.154 0.041
Dose2 -0.89 0.101 0.028
Dose3 -0.77 0.889 0.426

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 - metolachlor 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
0.917 0.149 0.121 0.946 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 22.05 0.70 0.35 3.20 20.93, 23.17 Dose1 4 22.50 0.49 0.24 2.18 21.72, 23.28 Dose2 4 22.45 0.58 0.29 2.58 21.53, 23.37 Dose3 4 21.95 0.72 0.36 3.27 20.81, 23.09

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 21.95 21.30 23.00 . .
Dose1 22.40 22.10 23.10 102.04 -2.04
Dose2 22.40 21.80 23.20 101.81 -1.81
Dose3 21.70 21.40 23.00 99.55 0.45

******************************************************************************** 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 0.78 0.529

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 22.05 . 22.33 . . . . . .Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 13 of 61

DER Template Version: 22 September 2011 Dose1 22.50 0.632 22.33 0.813 . . . . .
Dose2 22.45 0.705 22.33 0.844 0.999 . . . .
Dose3 21.95 0.992 21.95 0.536 0.619 0.684 . . .

******************************************************************************** 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 3.54 0.316

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 21.95 . .
Dose1 22.40 0.233 0.927
Dose2 22.40 0.494 0.880
Dose3 21.70 0.780 0.389

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 0.78 0.529

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 -22.05 . -22.05 . . . . . .
Dose1 -22.50 0.632 -22.30 0.349 . . . . .
Dose2 -22.45 0.705 -22.30 0.373 0.999 . . . .
Dose3 -21.95 0.992 -22.30 0.386 0.619 0.684 . . .

******************************************************************************** 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 3.54 0.316

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 -21.95 . .
Dose1 -22.40 0.233 0.073
Dose2 -22.40 0.494 0.120
Dose3 -21.70 0.780 0.611

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 - metolachlorData Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 14 of 61

DER Template Version: 22 September 2011 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
0.904 0.094 1.359 0.302 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 7.90 0.47 0.23 5.93 7.16, 8.65 Dose1 4 7.53 0.39 0.19 5.12 6.91, 8.14 Dose2 4 8.00 0.97 0.49 12.18 6.45, 9.55 Dose3 4 6.10 0.84 0.42 13.83 4.76, 7.44

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 7.92 7.36 8.41 . .
Dose1 7.47 7.12 8.05 95.25 4.75
Dose2 7.63 7.32 9.44 101.27 -1.27
Dose3 5.90 5.35 7.24 77.16 22.84

******************************************************************************** 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 6.12 0.009

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 7.90 . 7.90 . . . . . .
Dose1 7.53 0.800 7.77 0.467 . . . . .
Dose2 8.00 0.994 7.77 0.497 0.783 . . . .
Dose3 6.10 0.010 6.10 0.002 0.062 0.012 . . .

******************************************************************************** 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 8.81 0.032

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 7.92 . .
Dose1 7.47 0.343 0.123
Dose2 7.63 0.889 0.385
Dose3 5.90 0.067 0.010

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3
Jonckheere Dose3Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 15 of 61

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 6.12 0.009

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 -7.90 . -7.38 . . . . . .
Dose1 -7.53 0.800 -7.38 0.903 . . . . .
Dose2 -8.00 0.994 -7.38 0.924 0.783 . . . .
Dose3 -6.10 0.010 -7.38 0.934 0.062 0.012 . . .

******************************************************************************** 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 8.81 0.032

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 -7.92 . .
Dose1 -7.47 0.343 0.877
Dose2 -7.63 0.889 0.615
Dose3 -5.90 0.067 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 - metolachlor 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
0.820 0.005 1.177 0.359 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.36 0.03 0.01 7.26 0.32, 0.40 Dose1 4 0.33 0.01 0.01 3.78 0.31, 0.35 Dose2 4 0.36 0.04 0.02 10.41 0.30, 0.41 Dose3 4 0.28 0.04 0.02 12.95 0.22, 0.33

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 0.36 0.34 0.40 . .
Dose1 0.33 0.32 0.35 91.72 8.28
Dose2 0.34 0.33 0.41 97.93 2.07
Dose3 0.27 0.25 0.33 76.55 23.45Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 16 of 61

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 6.73 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 0.36 . 0.36 . . . . . .
Dose1 0.33 0.379 0.34 0.232 . . . . .
Dose2 0.36 0.969 0.34 0.248 0.711 . . . .
Dose3 0.28 0.004 0.28 <.001 0.090 0.014 . . .

******************************************************************************** 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 9.71 0.021

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 0.36 . .
Dose1 0.33 0.123 0.029
Dose2 0.34 0.484 0.206
Dose3 0.27 0.067 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 6.73 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 -0.36 . -0.33 . . . . . .
Dose1 -0.33 0.379 -0.33 0.957 . . . . .
Dose2 -0.36 0.969 -0.33 0.969 0.711 . . . .
Dose3 -0.28 0.004 -0.33 0.974 0.090 0.014 . . .

******************************************************************************** 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 9.71 0.021Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 17 of 61

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 -0.36 . .
Dose1 -0.33 0.123 0.971
Dose2 -0.34 0.484 0.794
Dose3 -0.27 0.067 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 Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 18 of 61

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



### Obs **conc endpoint** 

**1** 

### 0 0.34

**2** 

### 0 0.40

**3** 

### 0 0.35

**4** 

### 0 0.36

**5** 

### 1 0.33

**6** 

### 1 0.33

**7** 

### 1 0.32

**8** 

### 1 0.35

**9** 

### 2 0.41

**10** 

### 2 0.34

**11** 

### 2 0.33

**12** 

### 2 0.34

**13** 

### 3 0.27

**14** 

### 3 0.33

**15** 

### 3 0.25

**16** 

### 3 0.26Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 19 of 61

DER Template Version: 22 September 2011 

### The NPAR1WAY

Procedure

**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 51.50 34.0 8.154753 12.8750

**1** 

### 4 29.50 34.0 8.154753 7.3750

**2** 

### 4 42.50 34.0 8.154753 10.6250

**3** 

### 4 12.50 34.0 8.154753 3.1250

**Average scores were used for ties.**

**Kruskal-Wallis Test**

**Chi-Square** 

### 9.7105

**DF** 

### 3

**Asymptotic Pr > Chi-Square** 

### 0.0212

**Exact Pr >= Chi-Square** 

### 0.0065Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 20 of 61

DER Template Version: 22 September 2011 

### The NPAR1WAY

Procedure

**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 24.50 18.0 3.422614 6.1250

**1** 

### 4 11.50 18.0 3.422614 2.8750

**Average scores were used for ties.**

**Wilcoxon Two-Sample Test**

**Statistic (S)** 

### 24.5000

**Normal Approximation**

**Z** 

### 1.7530

**One-Sided Pr > Z** 

### 0.0398

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

### 0.0796

**t Approximation**

**One-Sided Pr > Z** 

### 0.0615

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

### 0.1230

**Exact Test**

**One-Sided Pr >= S** 

### 0.0429

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

### 0.0857

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

**Kruskal-Wallis Test**

**Chi-Square** 

### 3.6067

**DF** 

### 1

**Pr > Chi-Square** 

### 0.0575Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 21 of 61

DER Template Version: 22 September 2011 

### The NPAR1WAY

Procedure

**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 21.0 18.0 3.380617 5.250

**2** 

### 4 15.0 18.0 3.380617 3.750

**Average scores were used for ties.**

**Wilcoxon Two-Sample Test**

**Statistic (S)** 

### 21.0000

**Normal Approximation**

**Z** 

### 0.7395

**One-Sided Pr > Z** 

### 0.2298

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

### 0.4596

**t Approximation**

**One-Sided Pr > Z** 

### 0.2418

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

### 0.4837

**Exact Test**

**One-Sided Pr >= S** 

### 0.2571

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

### 0.5143

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

**Kruskal-Wallis Test**

**Chi-Square** 

### 0.7875

**DF** 

### 1

**Pr > Chi-Square** 

### 0.3749Data Evaluation Record on the Toxicity of Metolachlor to Amphibians, Metamorphosis Assay

EPA MRID Number 48617810

Page 22 of 61

DER Template Version: 22 September 2011 

### The NPAR1WAY

Procedure

**Wilcoxon Scores (Rank Sums) for Variable endpoi**
**nt Classified by Variable conc** 

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

**0** 

### 4 26.0 18.0 3.464102 6.50

**3** 

### 4 10.0 18.0 3.464102 2.50

**Wilcoxon Two-Sample Test**

**Statistic (S)** 

### 26.0000

**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** 

### 0.0209

# DATA EVALUATION RECORD

METOLACHLOR

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

EPA Contract No. EP10H001452 Task Assignment No. 2-49-2012 (MRID 48617806)

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: 05/04/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/10/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/21/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/21/2012

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).METOLACHLOR/108801 Androgen Receptor Binding (Rat Prostate Cytoso l) (2012) I Page 1of16
OCSPP 890.1150/ OECD None



### Primary Reviewer: Ayaad Assaad, D .V .M, Ph.D. 

### Health Effects Division Signature: 

# A

- '/}0h 

# cJ**Date: b;~**  Signature:
Date: c:;;(t.:- /, 

# rSecondary Reviewer:**_G_re_.,g..._A_k_er_m_a_n~,_P_h_.D_.** 

### ___ _ 

### Health Effects Division Template version 08/2011



### DATA EVALUATION RECORD

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



### PC CODE: 108801 

### DP BARCODE: D398926



### TXR#: 0052848 

### CAS No.: 51218-45-2



### TEST MATERIAL (PURITY) : Metolachlor (97.3%)



### SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide



### CITATION: Thomas, 

### I.A. (2012). 

### In Vitro Rat Prostate Androgen Receptor Competitive Binding Assay. WIL Research Laboratories, LLC, Ashland, OH. Laboratory Study No .: WIL-786007, January 4, 2012. MRID 48617806. Unpublished.



### SPONSOR: Metolachlor Task Force, 16160 Nall, Stilwell, KS 66085



### TEST ORDER#: CON108801-12



### EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48617806),
ventral prostate cytosol from previously castrated (23-24 hours prior to collection) Sprague Dawley rats, was used as the source of AR to conduct saturation and competitive binding experiments. The saturation binding experiment was conducted to demonstrate that the AR in
the rat prostate was present in reasonable numbers and was functioning with appropriate affinity
for the radio-labeled reference androgen ([3H]-Rl 881) prior to conducting the competitive binding experiments. The competitive binding assay measured the binding of a single
concentration of [3H]-Rl881 (1 

### nM) in the presence of increasing concentrations 

### (10

- 10 

### to 

### 10

- 3 

### M) of metolachlor (97.3% purity, Lot# 20081115). Ethanol was used as the solvent vehicle at a final assay concentration of 2

. 6%. A total of three runs were performed, and each run included
dexamethasone as a weak positive control, and RI 881 as the ligand reference standard.

In the saturation binding experiment, the maximum binding capacity (Bmax) was 14 .1 fmol/l 00

μg protein and the dissociation constant (Kct) was 0.83 nM. The Kct and Bm ax values were within
the range reported in the EPA validation program. Confidence in these numbers is high according to the goodness of fit (R 2 

## = 0.985) ; however

, only one run was performed. The specific binding reached a plateau, and non-specific binding (NSB) was :S 8.8% of total bindin g The Scatchard plot for the saturation assay was not linear for the entire [3H]-Rl 881 concentration

range tested; however

, it was linear over the range from 0.5 to 10 nM

, which encompasses the ligand concentration used in the competition binding experiment.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 2 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None



### In the competitive binding experiment, the estimated mean log IC 50 

### s for R1881 (

− 8.8 M) and the weak positive control, dexamethasone (

− 4.2 M), were within the expe cted ranges. The mean relative binding affinity (RBA) for the weak positive control was 0.0026%. The solvent control responses indicated no drift in the study assay. All performance criteria were generally met.

In each run, there were no a pparent reduction in average [ 3 

### H]-R1881 binding at metolachlor concentrations from 10

− 10 

### to 10

− 4 

### M (87.7-106.8%), but average bi nding of the radiolabeled ligand declined to 48.96-50.99% at 10

− 3 

### M. The average binding was approximately 50% in Runs 2 and 3 (50.94 and 50.99%), the average binding curve for metolachlor across all three runs indicates that ~50% displacement (com petitive binding to the AR) occurred at 10

− 3 

### M. The average log IC 50 

### and RBA for metolachlor were

− 2.994 M and 0.00016%, respectively.

Based on the results from the three runs, metolach lor is classified as a 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: An unsigned Data Confidentiality st atement, partially signed and dated GLP Compliance statement, and signed and dated Qu ality Assurance statements were provided.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 3 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH Study Director: Jennifer A. Thomas
Other Personnel: Justin L. Godsey, Group Supervisor, Metabolism Janet M. House, Data Coordinator, Metabolism Stephanie L. Ledyard, Quality Control Coordinator, Metabolism Celeste M. MacElrevey, Research Scientist, Metabolism Aimee P. Mahoney, Project Specialist, Metabolism Timothy B. Riehl, Jr., Group Supervisor, Metabolism Katie A. Alonso, Publishing Specialist, Reporting & Technical Support Services Melissa A. Hull, Group Manager, Re porting & Technical Support Services Robert A. Wally, Operations Manager, Reporting & Technical Support Services Study Period: October 26, 2010 to January 4, 2012



### 2. Test substance: Metolachlor

Description: Dark brown clear liquid
Source: Sipcam, Valdosta, GA
Lot #: 20081115 Purity: 97.3% Solubility: Not reported (NR) Volatility: NR Stability: NR Storage conditions: Room temperature CAS #: 51218-45-2 Molecular weight: 283.8 g/mol
Structure:

CH 3

CH 3 N
O CH 3 O Cl

CH 3



### 3. Non-labeled ligand: R1881 Supplier: PerkinElmer, Waltham, MA Catalog #:
Lot #: NLP005020MG
626128 Purity: >97%
CAS #: 965-93-5



### 4. Radioactive ligand: [ 3 

### H]-R1881 Supplier: PerkinElmer, Waltham, MA Catalog (Lot #): NET-590 (614814) Date of production: NR
Date of use: December 8, 2010 to December 20, 2010 Radiochemical purity: >97% Specific activity: 85.1 Ci/mmol
Concentration of stock: 1 mCi/mLAndrogen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 4 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None



### 5. Positive control: Dexamethasone
Supplier: Sigma-Aldrich Catalog #
Lot #: Purity: ≥ 98%
CAS # : 50-02-2



### 6. Solvent/vehicle control: Ethanol

Justification for choice of solvent: Not provided

Final Concentration: 2.6% (reviewer-calculated from the raw data for the solvent control tubes; the solvent concentration in the test substance and control tubes were not reported but typically would be equivalent to the solvent control tubes)



### B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: A total of 30 male Sprague Dawley rats (Charles River Laboratories, Ra leigh, NC) were castrated at ca . 79 days of age and were euthanized after ca . 23-24 hours. The ventral prostate s were excised immediately after termination, weighed, and placed in ice-cold TEDG (Tris, EDTA, DTT, glycerol) + PMSF (phenylmethylsulfonyl fluoride) buffer, hom ogenized, and centrifuged for 30 min at 30,000

× g at 4oC. Supernatant fracti ons were pooled, and the result ing pellets were discarded. Protein concentration of the cytosol was determined to be 4.33 mg/mL initially with a protein kit compatible with DTT in the TEDG buffer (Quick Start Bradford Protein Assay kit, Bio-Rad Laboratories, Inc.). Protein concentrations ranged from 3.67 to 4.60 mg/mL over the course of the assays. Cytosol was divided into 5-mL aliquots and stored at

− 70o C until use.

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

### H]-R1881 was performed to demonstrate that the AR was present in reasonable concentra tions and had the appropriate affinity for the R1881 ligand. The conditions for the saturation binding experiment are summarized in Table 1.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 5 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None

TABLE 1. Summary of Conditions for Saturation Binding Experiment a Source of receptor Rat ventral prostate cytosol Concentration of radioligand (as serial dilutions) 0.25-10 nM Concentration of non-labeled ligand (100X [radioligand]) 25-1000 nM Optimization of receptor concentration Sufficient to bind 25-35% of radioligand at 0.25 nM b Temperature 4o C
Incubation time 20 hours Composition of assay buffer (TEDG) Tris 10 mM (pH 7.4)

EDTA 1.5 mM Glycerol 10% Sodium molybdate 1 mM Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM a Data were obtained from pages 16-19, of the study report. Triamcinolone acetonide was added to all assay tubes to saturate the progesterone receptor present in the rat prostate homogenate. b The protein concentration used was 0.88 mg/tube (2.6 mg/mL).



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

### H]-R1881 was adjusted for decay over time, and serial dilutions in TEDG buffer were prepared to achieve the final concentrations in cytosol of 0.25, 0.5, 0.7, 1, 1.5, 2.5, 5, and 10 nM to determine total binding. To determine NSB, solutions of non- labeled R1881 were pr epared in a similar manner to achieve concentrations that we re 100-fold greater than each respective radiolabeled concentration, resulting in fi nal concentrations in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1,000 nM. In the absence of cytosol, the 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 op timal protein concentration was determined by calculating specific binding to differing am ounts of protein per tube, using 0.25 nM radiolabeled R1881. The optimal protein concentration was determined to be 2.6 mg protein/mL, which resulted in the binding of 34.30% of the total radioactivity added. A single run contained three conc urrent replicates at each c oncentration, resulting in the 72 samples depicted in Table 2.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 6 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None

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
1.00 1.00 100 10 30
1.50 1.50 150 10 45
2.50 2.50 250 100 7.5
5.00 5.00 500 100 15
10.00 10.00 1000 100 30 a Data were obtained from pages 19, and 92-94 of the study report. b Each concentration was run in triplicate for a total of 72 samples. c Tubes 25-48 contained 50 μL of triamci nolone acetonide and 7.5-45 μL of 10 nM [ 3 H]-R1881 or 7.5-30 μL of 100 nM of [ 3 H]-R1881. Samples were dried, and 300 μl of prostate cytosol were added. d Tubes 1-24 contained 50 μL of triamcinolone acetonide and 7.5-45 μL of 10 nM [ 3 H]-R1881 or 7.5-30 μL of 100 nM of [ 3 H]-R1881. R1881 was added in a 100-fold molar excess of [ 3 H]-R1881 in a volume of 7.5- 45 μL of 1 μM R1881 or 7.5 to 30 μL of 100 μM R1881. 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, or 30 μL of 100 nM [ 3 H]-R1881 without cytosol or other components to determine the total counts added.



### Following addition of triamcinolone acetonide, [ 3 

### H]-R1881, and/or R1881, the samples were dried, dissolved in diluted prostate cytoso l (300 μL), and incubated in the refrigerator for 20 hours at 4°C. Samples were maintain ed on ice except during whole rack vortexing. To separate bound from free R1881, hydroxyapatite (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 aspirated and discarded. The samples were washed 3 times in 50 mM Tris buffer. Following the last wash and decanting of the Tris buffer, the pellets were then extracted by additional of 2 ml ethanol. Th e samples were vortexed 3 times at 5 minute intervals. Portions of each ethanol supernatant were then de canted into scintillation vials, and the radiation was quantifie d by liquid scintilla tion counting. A total of 3 runs were performed.

3. Competitive Binding Experiment: A summary of the assay conditions for the competitive binding experiment is included in Table 3.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 7 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None

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 25-35% of 1 nM radioligand b Concentration of test substance (as serial dilutions) 10

− 10 to 10

− 3 M Incubation Temperature 4o C
Incubation time 20 hours Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM Glycerol 10% Sodium molybdate 1 mM Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM a Data were obtained from pages 16-17, 20, of th e study report. Triamcinolone acetonide was added to all assay tubes to saturate the progesterone receptor present in the rat prostate homogenate. b The protein concentration used was 0.77-0.84 mg protein/tube (2.6 mg/mL).



### The competitive binding experiment was performed according to the protocol provided in the EPA Test Guidelines OCSPP 890.1150, except that the receptor concentration was higher than specified in the guideline (sufficien t to bind 10-15% of 1 nM radioligand). The competitive binding experiment measures the binding of a single concentration of [ 3 

### H]- R1881 (adjusted specific activity of 82.9 Ci/mmol) to the AR in the presence of increasing concentrations of a test substance.

Ethanol was used as a vehicle. Solubility of metolachlor in ethanol and assay buffer was not reported. Results from the preliminary activ ity assay demonstrated that 2.6 mg/mL (0.8 mg/tube) of cytosolic protein contains enough receptor to bind 21.68% of the [ 3 

### H]-R1881 at 1 nM.

Dilutions of the test subs tance, reference standard (R1881), weak positive control (dexamethasone), and solvent control (ethanol) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of th ree independent runs on three different days. Each run included triplicate sets of [ 3 

### H]-R1881 only (for total bi nding calculations) at the beginning of each run, duplicate sets of solvent blank and 1 μ M R1881 (NSB) at the beginning and end of each run, along with duplicat e tubes of the refere nce standard and the weak positive control at each concentration and tr iplicate tubes of the test chemical at each concentration, resulting in a total of 61 samples per run.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 8 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None

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 Ethanol R1881 Dexamethasone Metolachlor
Tubes 4-5 and 58-59 Tubes 6-17 and 60-61 c Tubes 18-33 Tubes 34-57 Tubes 1-3

-- 1×10

− 3 1×10

− 3
-- 1×10

− 4 1×10

− 4

-- 1×10

− 5 1×10

− 5

1×10

− 7 1×10

− 6 1×10

− 6

1×10

− 8 1×10

− 7 1×10

− 7

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 20, and 95-106 of the study report. b Each concentration of each chemical was run in duplicate (R1881 and dexamethasone) or triplicate (metolachlor) for a total of 61 tubes pe r run. Tubes 4-61 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 4-61 also contained 10 μL of the solvent control, reference standard (non-radiolabe led R-1881), weak positive control, or test substance, with the exception of Tubes 6-7 and 60-61 that contained 30 μL of non-radiolabeled R1881 (used to evaluate NSB). Tubes 1-3 contained only 30 μL of [ 3 H]-R1881. c Tubes 6-7 and 60-61 were used to evaluate NSB by adding 100X of cold (non-radiolabeled) R1881.



### The procedures for assay incubation, separa tion of bound from free R1881 (washing with buffer and extraction with ethanol), fo llowed by scintillation counting of bound [ 3 

### H]-R1881 were similar to those used in the saturation bi nding experiment. A total of three runs were performed as described.

4. Data Analysis: The maximal binding capacity (B max 

### ), dissociation constant (K d 

### ), and inhibition concentration (IC 50 

### ) were calculated using nonlin ear regression analysis with Graph Pad Prism v. 5.02 (GraphPad Software, Inc ., La Jolla, CA). A Scatchard plot was also plotted for the binding data. Mean and standard error were calculated (IC 50 

### ) for the composite three runs of the competitive binding experiment with Microsoft Excel.



### 5. Definitions

a. Classification of test material

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

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 curve falls outside the range fo r 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.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 9 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None



### 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

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters are presented in Table 5. The K d 

### for [ 3 

### H]-R1881 was 0.83 nM, and the estimated B max 

### was 14.1 fmol/100 μ g protein for the single batch of prosta te cytosol that was prepared. Only one saturation binding run was performed, rather than the three runs recommended in the guideline. The K d 

### and B max 

### were within the range reported in the EPA validation program. The Scatchard plot for the single satura tion assay was not linear for the entire [ 3 

### H]-R1881 concentration range tested; however, it was li near over the range from 0.5 to 10 nM, which encompasses the ligand concentration used in the competition binding experiment and demonstrates that the androgen receptor was f unctioning with the appropriate affinity for the reference ligand R1881 at the ligand c oncentration used in the assay.

TABLE 5. Saturation Binding Experiment of R-1881 wi th Androgen Receptor from Rat Prostate Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SEM

R 2 0.985 NA NA NA

B max (nM) 0.377 NA NA NA

B max (fmol/100 μ g protein) 14.1 NA NA NA

K d (nM) 0.83 NA NA NA a Data were obtained from pages 27, and 45-46 of the study report. Only one run was performed.
R 2 Goodness of fit for curve calculated for specific binding NA Not applicable



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

### H]-R1881 to the androgen receptor. The specific binding reac hed a plateau, and NSB was less than 20% of total binding (2.5-8.8%). Figur e 2 is a Scatchard plot that illustrates the binding of [ 3 

### H]-R1881 to the androgen receptor. The data fit results in a linear plot over a test concentration of 0.5 to 10 nM [ 3 

### H]-R1881.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 10 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None



### FIGURE 1. Binding of [ 3 

### H]-R1881 to the Androgen Receptor during the Saturation Binding Experiment.

FIGURE 2. Scatchard Pl ot of the Binding of [ 3 

### H]-R1881 to the Androgen Receptor.

B. COMPETITIVE BINDING EXPERIMENT: Competitive binding experiment parameters are presented in Table 6 and s hown graphically in Figures 3 through 5.

The estimated average log IC 50 

### was

− 8.803 M for R1881 and

− 4.194 M for the weak positive control (dexamethasone). Compared to R1881, the mean RBA for dexamethasone was 0.0026%. Confidence in these numbers is high due to the small variation. No precipitation of the test com pound was reported. The solvent control responses indicated no drift in the study assay.

There were no apparent alterations in radiolabeled R1881 binding at metolachlor concentrations of 10

− 10 

### to 10

− 4 

### M (81.73-124.73%), with the specific binding of the radiolabeled ligand declining to 47. 82-52.43% at a concentration of 10

− 3 

### M. The estimated log IC 50 

### was -3.013 M for metolachlor in Run 1. The study authors calculated the log IC 50 

### and RBA for metolachlor in these runs as the mean binding in both runs was essentially atAndrogen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 11 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None



### 50% (50.94% for Run 2 and 50.99% for Run 3) at 10

− 3 

### M. The estimated log IC 50 

### in Runs 2 and 3 were

− 2.982 and

− 2.987 M, respectively. Across all th ree runs the estimate log IC 50 

### and RBA for metolachlor were

− 2.994 M and 0.00016%, respectivel y. Confidence in these numbers is high due to the small variation.

TABLE 6. Competitive Binding Assay of Metola chlor with AR from Rat Prostate Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SEM b

R 2 (unweighted) R1881 1.000 0.9978 0.9990 0.9978-1.000
Dexamethasone 0.9958 0.9988 0.9926 0.9926-0.9988
Metolachlor 0.9682 0.9907 0.9697 0.9682-0.9907 Log IC 50 (M) R1881

− 8.918

− 8.604

− 8.888

− 8.803 ± 0.100 Dexamethasone

− 4.165

− 4.208

− 4.209

− 4.194 ± 0.015 Metolachlor

− 3.013

− 2.982 c

− 2.987 c

− 2.994 ± 0.010
IC 50 (M) R1881 1.208 x 10

− 9 2.489 x 10

− 9 1.294 x 10

− 9 1.664 x 10

− 9 (± 0.413)
Dexamethasone 6.839 x 10

− 5 6.194 x 10

− 5 6.180 x 10

− 5 6.405 x 10

− 5 (± 0.217)
Metolachlor 9.705 x 10

− 4 1.042 x 10

− 3 c 1.030 x 10

− 3 c 1.014 x 10

− 3 (± 0.022) Log %RBA b Dexamethasone

− 2.753

− 2.396

− 2.679

− 2.609 ± 0.109 Metolachlor

− 3.905

− 3.622

− 3.901

− 3.809 ± 0.094 RBA (%) b Dexamethasone 0.0018 0.0040 0.0021 0.0026 ± 0.0007
Metolachlor 0.00012 0.00024 0.00013 0.00016 ± 0.00004 a Data were obtained from page 42 of the study report. b The range of R 2 is reported and the mean ± SE is reported for the other parameters. %RBA, log %RBA, and mean ± SEM values are reviewer-calcu lated. 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 Hill equation could not be fit to the data, therefore the reported IC 50 was interpolated from the formula for estimating log(EC50): Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) NR Not reported NA Not applicable
R 2 Goodness of fit RBA Relative binding affinityAndrogen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 12 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None



### FIGURE 3. Percentage [ 3 

### H]-R1881 Bound in the Presen ce of Radioinert R1881, Dexamethasone, and Metolachlor.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 13 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None



### FIGURE 4. Mean % [ 3 

### H]-R1881 Bound in the Presence of Metolachlor in Three Competitive Binding Assays.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 14 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None



### FIGURE 5. Mean % [ 3 

### H]-R1881 in the Presence of Metolachlor a



### .

a Average curve was calculated by the reviewers using GraphPad Prism.



### C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay was functioning properly, each run was evaluated using the criteria show n in Table 7. The assays generally met the performance criter ia, although the % ligand depletion (20-23%) was above the guideline specified 15% for a 1 nM concentration of [ 3 

### H]-R1881, and the Hill slope was out of range for R1881 in Run 2.

However, the curve for the reference material showed that increasing concentrations of unlabeled R1881 displaced [3H]-R1881 in a mann er consistent with one-site binding, as indicated by a Hill slope of -1.2027. Examina tion across the runs indicated consistency of the Hill slope, placement along the X-axis, and top and bottom plateaus.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 15 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None

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% 20.23-23.20% X

Test chemical Top (% binding) 80 to 115 95.6 to 101.5 X
R1881 fitted curve parameters Top (% binding) 82 to 114 89.49 to 101.5 X Bottom (% binding)

− 2.0 to 2.0 -1.18 to 0.671 X Hill Slope

− 1.2 to

− 0.8 -1.588 to -1.003 X c Weak positive control (dexamethasone) fitted curve parameters Top (% binding) 87 to 106 96.0 to 101.1 X Bottom (% binding)

− 12 to 12 -3.219 to 4.265 X Hill Slope

− 1.4 to

− 0.6 -1.057 to -0.9262 X Saturation Binding Experiment K d (nM) 0.685 to 1.57 0.83 X d Non-specific binding (%) ≤ 10.0 2.5 to 8.8% X a Data were obtained from pages 42, 97-98, 101-102, and 105-106 of the study report. b These values represent ranges from the validation study. c The Hill Slope was low (-1.588) in Run 2, but within the recommended range in the other two runs. d Based on a single run.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Rat prostate cytosol was successfully prepared and characterized, then employed in competition assays with the goal of investigating the potential of metolachlor to act as an endocri ne disruptor. Results from these experiments suggest that metolachlor is not a viable subs trate for the rat AR and does not bind to the active site in a competitive manner with R1881. However, metolachlor displaced <50% of the reference androgen when present in million-fold excess versus the radiolabeled ligand and must therefore be classifi ed as an equivocal binder.

B. AGENCY COMMENTS: The saturation binding experi ment demonstrated that the androgen receptor was present in a reasona ble concentration and functioning with appropriate affinity for the R1881 ligand. The K d 

### for [ 3 

### H]-R1881 was 0.83 nM, and the estimated B max 

### was 14.1 fmol/100 μ g protein for the single batch of prostate cytosol that was prepared. The K d 

### and B max 

### values were within the range reported in the EPA validation program. Confidence in these numbers is high according to the goodness of fit (R 2 

### = 0.985); however, only one run was performed. The specific binding reached a plateau, and NSB

was ≤ 8.8% of total binding. The Scatchard plot for the single saturation assay was not linear for the entire [ 3 

### H]-R1881 concentration range tested ; however, it was linear over the range from 0.5 to 10 nM, which encompasse s the ligand concentr ation used in the competitive binding experiment.

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

### s for R1881 (

− 8.8 M) and the weak positive control, dexamethasone (

− 4.2 M), were within the expected ranges. The mean RBA for the weak positive control was 0.0026%. Confidence in these numbers is high due to the small variation. The solvent c ontrol responses indicated no drift in the study assay. The performance criter ia were generally met.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 16 of 16 METOLACHLOR/108801 OCSPP 890.1150/ OECD None



### The percent [ 3 

### H]-R1881 binding was approximately 50% in Runs 2 and 3 (50.94% and 50.99%, respectively), and the average binding cu rve for metolachlor ac ross all three runs indicates that ~50% displacement of the radioligand occurred at 10

− 3 

### M which is the threshold for indicating a competitive binder to th e AR; therefore, metolachlor is considered to be an AR binder under the conditions of this assay. The average log IC 50 

### and RBA for metolachlor were

− 2.994 M and 0.00016%, respectively.

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:

 Only a single run was performed in the satura tion binding experiment. Test Guidelines recommend that a series of sa turation radioligand binding as says be performed (typically three) prior to conducting competitive binding assays.

 The amount of cytosol used in the co mpetitive binding assay bound 20.2-23.2% of the radioactive R1881, which is higher th an the Guideline specified 10-15%.

 Tubes of unlabeled R1881 and dexamethas one were prepared in duplicate at each concentration. EPA’s protocol specifies th at there are three replicates assayed simultaneously at each concentration.

# DATA EVALUATION RECORD

METOLACHLOR

Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-49-2012 (MRID 48617805)

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: 5/1/2012

Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 5/2/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/2/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/2/2012

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).METOLACHLOR/108801 Aromatase (Human Recombinant) Assay (2012) I Page I of 11
OCSPP 890.1200 I OECD None



### Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D.


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


### Health Effects Division Signature: 

# £;z · (j ?za-=» 

# J.

# Signa~:::~**~J?** Date:

__ _....

co

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## _,,/.._,1'-" a= «--- Tem plate version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200



### PC CODE: 108801 

### DP BARCODE: D398926



### TXR#: 0052848 

### CAS No.: 51218-45-2



### TEST MATERIAL (PURITY): Metolachlor (97.3% a.i.)



### SYNONYMS: 2-chloro-N-(2-ethyl-6-methy lphenyl)- N-(2-methoxy-l-methy leth yl)acetamide



### CITATION: Thomas

, J.A. (2012) Jn vitro 

### inhibition assay with human recombinant

aromatase. WIL Research Laboratories, LLC, Ashland, OH. Laboratory Report
No. : WIL-786005

, January 4, 2012. MRID 48617805. Unpublished



### SPONSOR: Metolachlor Task Force, Chemical Consultants International, Inc., 16160 Nall, Stilwell, KS



### TEST ORDER# : CON-108801-12



### EXECUTIVE SUMMARY: In an in vitro 

### aromatase (CYP19) assay (MRID 48617805),
metolachlor (97.3% a

. i., Lot# 20081115) was incubated with human recombinant aromatase and
tritiated androstenedione 

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

- 1 0 to 10

- 3 

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

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Three runs were conducted and each run included a full activity control

, a background activity control

, a positive control series (10

- 10 to 10

- 5 

### M) using a known inhibitor ( 4-hydroxyandrostenedione; 4-0H ASDN)

, and the test chemical series (I 

### 0

- 1 0 

### to 10

- 3 

### M) with three repetitions per concentration.

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

- 1·min

- 1 for the 3 test runs, with a mean and standard deviation of 0.3924±0.0224 nmol·mg-protein

- 1 ·min

- 1 • 

### Activity in the background controls ranged from 0.23 to 1.17% and averaged 0.49% of the full activity controls. The responses of the full activity controls and background controls were acceptable for each run.

For the positive control substance (4-0H ASDN) aromatase activity was within the
recommended ranges for the top and bottom of the curve, Hill slope, log IC 

### so and %CV forAromatase (Human Recombinant) A ssay (2012) / Page 2 of 11 METOLACHLOR/108801 OCSPP 890.1200 / OECD None



### replicates of each concentration within runs. The estimated log IC 50 

### averaged

− 7.3 M and the Hill slope averaged

− 0.93.

For metolachlor, aromatase activity averaged 87.86±6.2% of the full ac tivity control at the lowest tested concentration of 10

− 10 

### M and 41.63±3.0% of the full activ ity control at the highest tested concentration of 10

− 3 

### M. Aromatase activity at the se cond highest tested concentration (10

− 4 

### M) averaged 84.53±2.9% of the full activity cont rol. For Runs 1 and 3, the report indicated that the log IC 50 

### values were "ambiguous" but values of

− 3.79 and

− 3.81 M, respectively, as well as Hill slope values of –8.05 and –8.31, re spectively, were reported. A log IC 50 

### value and Hill slope were not determined for Run 2 as the concentration response curve did not fit the 4- parameter model.

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

− 3 

### M) in all three runs, the Hill slope (-8.4) for the average res ponse curve was steeper than -2. The unusually steep slope may indicate an issue with protein denaturation or solubility at this test concentration. Therefore, metolachlor is classified as Equivoc al for aromatase inhibition in this assay.

The assay satisfies the EDSP Tier 1 Test Order requireme nts 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 (2012) / Page 3 of 11 METOLACHLOR/108801 OCSPP 890.1200 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Metolachlor
Description: Dark brown clear liquid, technical
Source: Sipcam (Valdosta, GA) Lot/Batch # (Exp. Date): 20081115 (Nov 15, 2013) Purity: 97.3% Volatility: Vapor pressure: 1.3×10 -5 mm Hg at 20 °C Storage conditions: Room temperature Stability: Stable after storage at 0±1°C for 7 days , and stable after storage at 54±2°C for 14 day
Solvent: Ethanol Solubility (in test solvent): At least 0.1 M Highest Concentration Tested: 0.001 M Stock Solution Preparation: Not reported Molecular weight: MW 283.8
CAS #: 51218-45-2
Structure:

CH 3

CH 3 N
O CH 3 O Cl

CH 3



### 2. Non-Labeled Substrate: Androstenedione (ASDN)

CAS # : 63-05-8 Source (catalog #): AK Scientific (H320) Lot/Batch # (expiration date): 90330HC (not reported) Purity: 98%



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

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

### H]ASDN) Source (catalog #): Perkin Elmer Life Sciences (NET926001MC) Lot/Batch # (expiration date): 619344 (not reported) Radiochemical Purity (Supplier): >97% Specific activity: 26.3 Ci/mmol (on August 6, 2010) Radiochemical Purity (In-lab determination): 94.1% (recovery via radio-HPLC)



### 4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN)

CAS # 566-48-3 Source (catalog #): Sigma-Aldrich (not reported) Lot/Batch # (expiration date): 081K2133 Purity: Not reported



### 5. Solvent (Vehicle Control): Ethanol Lot/Batch # (expiration date): Not reported
Justification for choice of solvent Not reported
Concentration (% of total volume in assays) 1%Aromatase (Human Recombinant) A ssay (2012) / Page 4 of 11 METOLACHLOR/108801 OCSPP 890.1200 / OECD None



### 6. Test Microsomes: Human recombinant aromatase (CYP19) microsomes

Source (catalog #): BD Biosciences(not reported) Lot/Batch # (expiration date): Not reported
Protein concentration: 0.598-0.776 mg protein per mL Cytochrome C reductase activity: Not reported Aromatase activity: Not reported



### 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 100 nM, and the amount of tritiu m added to each incubation tube was 0.1 μ Ci in a volume of 100 μ L.

Test chemical(s) stock solutions were prepar ed such that the total volume of each test chemical formulation used per assay was no mo re than 1% of the total assay volume to minimize the potential for the solvent to inhibi t the enzyme. Rationale for selection of the solvent (ethanol) was not reported.

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

− 10 

### to 10

− 5 

### M; Table 4) were achieved by the addition of 20 μ L of the dilution for a fina l assay volume of 1 mL.

Three vials of human recombinant microsomes were purchased from BD Biosciences, and stored at ≤− 70oC. Before use of the microsomes , the vials were thawed, pooled, and aliquoted into individual vials before being refrozen to minimize freeze-thaw cycles to no more than one.

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

TABLE 1. Assay Components and Conditions 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



### 2. Suitability Assessments: The nominal protein concentration of the microsomes (lot # not reported) was supplied by the vendor as 7.0 mg/m L. A six-point standard curve prepared with bovine serum albumin (BSA) was analyzed with a standard protei n assay kit, and theAromatase (Human Recombinant) A ssay (2012) / Page 5 of 11 METOLACHLOR/108801 OCSPP 890.1200 / OECD None



### protein concentration of the microsome prepar ation was determined on each day of use of the microsomes in the aromatase assay.

Aromatase activity in each lot of human recombinant microsomes was determined to demonstrate the presence of sufficient activity for analysis of metolachlor. The minimumaromatase activity in the three acceptable runs was determined to be 0.2747 nmol·mgprotein

− 1 

### ·min

− 1 

### , which was greater than the minimu m recommended aromatase activity of 0.1 nmol·mg-protein

− 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 of each control were run at the end of the assay. A full concentration curve in duplicate for the positiv e control, and a full concentration curve in triplicate for the test substa nce were established. The 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 pmol·mg-protein

− 1 

### ·min

− 1



### .
4. Demonstration of Proficiency: No data were provided on prof iciency testing of the CYP19 aromatase assay.

a. Positive Control

(1) Initial Demonstration of Laboratory Proficiency: The positive control data 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.

 Coefficient of variation (CV) for replicates within each sample type and concentration of 4-OH ASDN was <15%.

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

(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.

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

− 0.96

− 0.90 Top (%) 90 110 93.1 97.5 Bottom (%)

− 5 +6

− 0.3 0.21 Log IC 50 (M)

− 7.3

− 7.0

− 7.36

− 7.31 a Data were obtained from pages 24 and 56 of the study report.



### b. Proficiency Chemicals: No data were provided on te sting of proficiency chemicalsAromatase (Human Recombinant) A ssay (2012) / Page 6 of 11 METOLACHLOR/108801 OCSPP 890.1200 / OECD None

TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations

Econazole 24169-02-6 Inhibitor Not reported
Fenarimol 60168-88-9 Inhibitor Not reported
Nitrofen 1836-75-5 Inhibitor Not reported
Atrazine 1912-24-9 Non-inhibitor Not reported



### 5. Determination of Aromatase Ac tivity with Test Chemical(s): The response of aromatase activity to the presence of eight concentrations of metol achlor per run, in triplicate, was tested during three independent runs. No problems with insolubi lity were reported.

The full enzymatic activity was obtained at the tw o lowest concentrations of metolachlor to define the top of the concentration-response curve.

TABLE 4. Test Chemical Study Design for each Test Run

Sample Type Repetitions (Tubes) Description Reference or Chemical (M) Full Activity Control 4 All test components a plus solvent vehicle 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
Metolachlor Conc 1 b 3 All test components plus metolachlor 1×10

− 3

Metolachlor Conc 2 b 3 All test components plus metolachlor 1×10

− 4
Metolachlor Conc 3 b 3 All test components plus metolachlor 1×10

− 5
Metolachlor Conc 4 b 3 All test components plus metolachlor 1×10

− 6
Metolachlor Conc 5 b 3 All test components plus metolachlor 1×10

− 7
Metolachlor Conc 6 b 3 All test components plus metolachlor 1×10

− 8
Metolachlor Conc 7 b 3 All test components plus metolachlor 1×10

− 9
Metolachlor Conc 8 b 3 All test components plus metolachlor 1×10

− 10 a The complete assay contained buffer, propylene glycol, microsomal protein, [ 3 H]ASDN, and NADPH. b Data were obtained from pages 15-16 of the study report.



### C. DATA ANALYSIS

1. Raw Data: Raw data were converted to arom atase activity (nmol·mg protein

− 1 

### ·min

− 1 

### ) and percent control for the positive control, proficiency chemicals and test chemical(s). The following raw data and calculated endpoints fo r each run were included in the report (Table 5).Aromatase (Human Recombinant) A ssay (2012) / Page 7 of 11 METOLACHLOR/108801 OCSPP 890.1200 / OECD None

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: Statistical analyses and displays such as summary tables, graphical displays, analysis of variance, and multiple comparisons were conducted using Microsoft Excel ® 

### spreadsheets using full floating decimal point calculations. Analysis of variance (ANOVA) was not performed.



### Response curves were fit using Prism 5.02 (Graph Pad Software, Inc., San Diego, CA). Data from each assay were recorded into an assay-specific worksheet for the analysis of aromatase inhibition data. Data recorded incl uded the assay date, technician, chemical and log chemical concentration, total DPM minus background DPM, and percent activity. The spreadsheet calculated DPM/mL for each aliquot of extracted aqueous incubation mixture and average DPM/mL and total DPM for each aque ous portion after extraction. Total initial DPM was calculated by the multiplication of the volume of substrate solution added to the incubation by the average substrate solution radiochemical content (DPM/mL) and yielded the total DPM present in the a ssay tube. Background DPM was calculated as th e average of the DPMs present in the aqueous portion for th e background tubes and was subtracted from Total DPMs for all samples to provide DPMs for calculating aromatase activity. This corrected DPM was then converted to nmol product formed by dividing by the substrate specific activity (DPM/nmol). The activity of the enzyme reaction is expressed in nmol (mg protein)

− 1 

### min

− 1 

### and is calculated by dividing the am ount of estrogen formed (nmol) by the product of mg microsomal protein used and the incubation time.

Assay drift was assessed by comparing the % fu ll activity and background activity at values at the beginning of each run to the end of each run.

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.Aromatase (Human Recombinant) A ssay (2012) / Page 8 of 11 METOLACHLOR/108801 OCSPP 890.1200 / OECD None

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 0.2747 to 0.4910 nmol·mg-protein

− 1 

### ·min

− 1 

### for the 3 test runs, with a mean and standard deviation of 0.3924±0.0224 nmol·mg-protein

− 1 

### ·min

− 1 

### . Activity in the background controls ranged from 0.23 to 1.17% and averaged 0.49% of the full activity controls. The responses of the full activity controls and background c ontrols were acceptable for each run.

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 93.76± 1.97 nmol·mg-protein

− 1 

### ·min

− 1 

### at the lowest tested concentration (10

− 10 

### M) and 0.71±0.22 nmol·mg-protein

− 1 

### ·min

− 1 

### at the highest tested concentration (10

− 5 

### M). The mean aromatase activity of the positive control (expressed as % full control activity) for each concentration tested across a ll three runs is presented in Table 7, along with the overall standard devi ation and % CV. Individual inhi bition response curves for the positive control and for metolachlor from all three runs are shown in Figure 1.

TABLE 7. Effect of Metolachlor on Aromatase Acti vity (as percent of control) from Independent Runs a

Chemical Concent. (Log M) # Runs Overall Mean Overall SD Overall SEM Overall %CV

4-OH ASDN (positive control) − 5 3 0.71 0.22 0.13 31.0

− 6 3 6.09 0.07 0.04 1.1

− 6.5 3 15.50 0.19 0.11 1.2

− 7 3 31.69 1.93 1.12 6.1

− 7.5 3 59.72 1.49 0.86 2.5

− 8 3 78.81 2.11 1.22 2.7

− 9 3 93.17 1.48 0.85 1.6

− 10 3 93.76 1.97 1.14 2.1
Metolachlor

− 3 3 41.63 3.04 1.75 7.3

− 4 2 84.53 2.87 2.03 3.4

− 5 3 93.31 4.98 2.88 5.3

− 6 3 91.64 3.80 2.20 4.1

− 7 3 90.23 7.31 4.22 8.1

− 8 3 90.23 6.78 3.91 7.5

− 9 3 88.49 3.72 2.15 4.2

− 10 3 87.86 6.20 3.58 7.1 a Data were obtained from Tables 1 and 2 on pages 35-36 of the study report. Overall means, SD, SEM, and %CV were calculated by the reviewers. SD Standard Deviation SEM Standard Error of the Mean CV Coefficient of VarianceAromatase (Human Recombinant) Assay (2012) / Page 9 of 11 METOLACHLOR/108801 OCSPP 890.1200 / OECD None



### FIGURE 1. Inhibition Response Curves for 4-OH ASDN and Metolachlor.

C. TEST SUBSTANCE: For metolachlor, aromatase activ ity averaged 87.86±6.2% of full activity control at the lowest tested concentration (10

− 10 

### M) and 41.63±3.0% of full activity control at the highest te sted concentration (10

− 3 

### M). The mean aromatase activity of metolachlor (expressed as % full activity) for ea ch concentration is presented in Table 7, along with the overall standard deviation a nd % CV. Average inhibition response curve across all runs is shown in Figure 2.

FIGURE 2. Mean Inhibition Response Cu rves for 4-OH ASDN and Metolachlor From Each Test Run.

The effect of metolachlor on inhi bition of aromatase activity is presented in Table 8. As the data for Run 2 could not be fit into the 4- parameter nonlinear regression model, a log IC 50 

### and Hill slope were not determined for this run. For 4-OH ASDN, the estimated log IC 50Aromatase (Human Recombinant) A ssay (2012) / Page 10 of 11 METOLACHLOR/108801 OCSPP 890.1200 / OECD None



### averaged

− 7.3 M and the Hill slope averaged

− 0.93. The variation in the positive control values was acceptable (<15 % CV).

TABLE 8. Effect of Metolachlor on Aromatase Acti vity (as Percent of Contro l) From Independent Runs a

Chemical Run 1 Run 2 Run 3 Mean b SEM b %CV b Log IC 50 (M)
Metolachlor –3.79 NC –3.81

− 3.8 NA NA
4-OH ASDN

− 7. 36

− 7.32

− 7.31

− 7.3 0.03

− 0.01 Hill Slope
Metolachlor –8.05 NC –8.31

− 8.4 NA NA
4-OH ASDN

− 0.90

− 0.92

− 0.96

− 0.93 0.02 1.00 a Data were obtained from Figure 4 on page 41 of the study report. b Calculated by the reviewers from data presented in this table. SD Standard Deviation NC Not calculable NA Not applicable



### Based on the data from the average response curv e and the criteria listed above in Table 7, the results support the c onclusion that metolachlor is equivo cal as an inhibitor of aromatase activity in this assay. Alt hough mean aromatase activity was 41.63% at the highest tested concentration of 10

− 3 

### M, the calculated Hill slope (-8.4) was steeper than -2. The unusually steep slope indicates a potential issue with protein denaturation or solubility at this concentration. The overall respons e is considered equivocal.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: Enzyme activity was above the minimum acceptable level. The test system correctly responded to the positive control compound, 4-OH ASDN. Evaluation of the effect of meto lachlor on aromatase ac tivity indicated an effect only at the highest con centration of metolachlor, 10

− 3 

### M, but the overall evaluation of the data indicated that the effect was not a result of competitive bi nding. In accordance with the Test Guidelines, the interaction between meto lachlor and aromatase is labeled equivocal.

B. AGENCY COMMENTS: Data demonstrating proficiency with the positive control and recommended proficiency chemicals were not in cluded, and no statement was included as to the proficiency of the laboratory with recommended proficiency chemicals.

Aromatase activity in the fu ll activity controls ranged from 0.2747 to 0.4910 nmol·mgprotein

− 1 

### ·min

− 1 

### for the 3 test runs, with a mean and standard deviation of 0.3924±0.0224 nmol·mg-protein

− 1 

### ·min

− 1 

### . Activity in the background controls ranged from 0.23 to 1.17% and averaged 0.49% of the full activity controls. The responses of the full activity controls and background controls were acceptable for each run.

For the positive control substance (4-OH ASDN) aromatase activity was within the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC 50 

### and % CV for replicates of each concentration within runs. The estimated log IC 50 

### averaged

− 7.3 M and the Hill slope averaged

− 0.93.Aromatase (Human Recombinant) A ssay (2012) / Page 11 of 11 METOLACHLOR/108801 OCSPP 890.1200 / OECD None



### For metolachlor, aromatase activity averaged 87.86±6.2% of the full ac tivity control at the lowest tested concentration of 10

− 10 

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

− 3 

### M. Aromatase activity at the second highest tested concentration (10

− 4 

### M) averaged 84.53±2.9% of the full ac tivity control. For Runs 1 and 3, the report indicated that the log IC 50 

### values were "ambi guous" but values of

− 3.79 and

− 3.81 M, respectively, as well as Hill slope valu es of –8.05 and –8.31, respectively, were reported. Values for the log IC 50 

### and Hill slope could not be calculated for Run 2 as the concentration response curve did not fit the 4-parameter model.

Although there appears to be an e ffect on aromatase activity at 10

− 3 

### M metolachlor, the Hill slope was steeper than

− 2 (average

− 8.4) indicating a potential issue with protein denaturation or solubility at this test con centration. Therefore the classification for metolachlor in this aromatase assay is equivocal.

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:

 No data were provided on test ing of proficiency chemicals

# DATA EVALUATION RECORD

METOLACHLOR

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-49-2012 (MRID 48617807)

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: 05/02/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 05/07/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/17/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/17/2012

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).METOLACHLOR/ 1 08801 Estroge n R eceptor Bind i ng (Rat Uter i ne Cytoso l ) (2012) I Page I of 17
OCSPP 890.1250/ OECO None



### Primary Reviewer: Ayaad Assaad, D

. V.M., Ph.D. 

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

. D. 

### Health Effects Division S i g n a tu re: 

# fJ 

# fj 55 & 

# J**Date: ,:_~**  Sig n a tur e:

## =~ D a t e: G- ''

..**[J** 

### ,,-- Template version 08/2011



### DATA EVALUAT IO N REC ORD

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



### PC CODE: 108801 

### DP B A R C OD E: D398926



### TXR#: 0052848 

### CAS No.: 51218-45-2



### TEST MATERIAL (PURITY): Metolachlor (97.3%)



### SYN 0 NYMS: 2-chloro-N-(2-ethyl-6-methy 1 phenyl )-N-(2-methoxy- l -methy I ethyl )acetamide



### CITATION: Thomas, J.A. 

### (2012). 

### In Vitro Rat Uterine Estrogen Receptor Competitive Binding Assay. WIL Research Laboratories, LLC, Ashland, OH. Laboratory Study No.: WIL-786006, January 4, 2012. MRID 48617807. Unpublished.



### SPONSOR: Metolachlor Task Force, 16160 Nall, Stilwell, KS 66085



### TEST ORDER#: CON-108801-12



### EXECUTIVE SUMMARY: In 

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

, Lot# 20081115), uterine cytosol from previously ovariectomized Sprague Dawley rats was used as the source of ER to conduct saturation binding and competitive binding experiments. The saturation binding experiment was conducted to demonstrate that the
ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radiolabeled reference estrogen prior to conducting the competitive binding experiments. The competitive binding experiment measured the binding of a single
concentration of[ 3 

### H]- l n-estradiol (I 

### nM) in the presence of increasing concentrations (logarithmic increase from 10

- 1 0 

### to 10

- 3 

### M) of metolachlor. Ethanol was used as the solvent
vehicle at a final concentration of 2%. A total of 5 runs were performed, and each run included
19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control

, and**17-~­** 

### estradiol as the natural ligand reference material.

In the saturation binding experiments, the mean maximum binding capacity (Bmax) was 31. 7
fmol/l 00 μg protein and the mean dissociation constant (Kct) was 0.075 nM. The Bm ax value was within the recommended range and demonstrated that the estrogen receptor was present in a reasonable concentration. The Scatchard plots for the three Saturation assays were linear. The specific binding appeared to reach a plateau

, and NSB was generally Jess than 20% of total binding (1.97 to 21.51%)

.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 2 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



### For the competitive binding experiment, the estimated mean log IC 50 

### was

− 8.675 M for 17 β

estradiol and

− 5.254 M for the weak positive control. The mean RBA was 0.045% for the weak positive control. Confidence in these numbers is high due to the small variation. All performance criteria were generally met; however , parameters for 19-noret hindrone were outside of the tolerance limits for a weak positive contro l in Run 5, and a technical error occurred with the negative control, octy ltriethoxysilane, in Run 2.

Metolachlor had no significant e ffect on specific binding of the [ 3 

### H]-ligand to the ER in runs 2-5; therefore, a log IC 50 

### and RBA could not be calculated fo r metolachlor (speci fic binding of [ 3 

### H]- 17 β -estradiol was ≥ 72.28% in Run 1).

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

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

COMPLIANCE: An unsigned Data Confidentiality st atement, partially signed and dated GLP Compliance statement, and signed and dated Qu ality Assurance statements were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 3 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH Study Director: Jennifer A. Thomas
Other Personnel: Justin L. Godsey, Group Supervisor, Metabolism Janet M. House, Data Coordinator, Metabolism Stephanie L. Ledyard, Quality Control Coordinator, Metabolism Celeste M. MacElrevey, Research Scientist, Metabolism Aimee P. Mahoney, Project Specialist, Metabolism Timothy B. Riehl, Jr., Group Supervisor, Metabolism Gregory A. Hawks, Group Supervisor, Re porting & Technical Support Services Melissa A. Hull, Group Manager, Re porting & Technical Support Services Robert A. Wally, Operations Manager, Reporting & Technical Support Services Study Period: October 20, 2010 to January 4, 2012



### 2. Test substance: Metolachlor
Description: Dark brown clear liquid
Source: Sipcam (Valdosta, GA)
Lot #: 20081115 Purity: 97.3% Solubility: Not reported (NR) Volatility: NR Stability: NR Storage conditions: Stored at room temperature
CAS #: 51218-45-2 Molecular weight: 283.793 g/mol
Structure:

CH 3

CH 3 N

O CH 3 O Cl

CH 3



### 3. Non-labeled ligand: 17 β -estradiol
Supplier: Sigma-Aldrich Catalog #: E8875
Lot #: 060M0149V Purity: ≥ 98%
CAS #: 50-28-2



### 4. Radioactive ligand: [ 3 

### H]-17 β -estradiol
Supplier: American Radiolabeled Chemicals, St. Louis, MO Catalog #: ART 1549
Lot #: 110114 Radiochemical purity: 99% Specific activity: 162.9 Ci/mmol
Concentration of stock: Not reportedEstrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 4 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



### 5. Positive control: 19-Norethindrone
Supplier: Sigma-Aldrich Catalog #: NR
Lot #: NR Purity: ≥ 98%
CAS #: 68-22-4



### 6. Negative control: Octyltriethoxysilane
Supplier: Sigma-Aldrich Catalog #: NR
Lot #: NR Purity: ≥ 97.5%
CAS #: 2943-75-1



### 7. Solvent/vehicle control: Ethanol (EtOH)

Justification for choice of
solvent: Not provided

Final Concentration: 2% (reviewer-calculated from the raw data for the solvent control tubes; the solvent concentration in the test substance and control tubes were not reported but typically would be equivalent to the solvent control tubes)



### B. METHODS

1. Preparation of Rat Uterine Cytosol (RUC): A total of 25 female Sprague Dawley rats (Charles River Laboratories, Stone Ridge, NY) were ovariectomized 9 days prior to being euthanized. Animals were 87-99 days old at the time of euthanasia. The uteri were weighed, placed in ice-cold TEDG (Tris, EDTA, DTT, glycerol) + PMSF (phenylmethylsulfonyl fluoride) buffer and used immediately. Uteri were homogenized in buffer, centrifuged for 30 min at 2500 × g at 4oC. Supernatan t was transferred and centrifuged for 60 minutes at 100,000 × g , and the resulting pellets were discarded. Protein concentration of the cytosol was determined w ith a protein kit compatible with DTT in the TEDG buffer (Quick Start Bradfo rd Protein Assay kit, Bio-Ra d Laboratories, Inc.). The protein concentration of the cy tosol preparation was determin ed to be 3.276 mg/mL initially, and the cytosol protein concentration ranged fr om 2.57 to 3.33 mg/mL over the course of the assay runs. Cytosol was divided into 2- mL aliquots and stored at approximately

− 70oC until

use.

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

### H]-17 β -estradiol was performed to demonstrate that the estrogen receptor (ER) was present in reason able concentrations and had the appropriate affinity for the native ligand. The conditions for the saturation binding are summarized in Table 1.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 5 of 17 METOLACHLOR/108801 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) 0.03-3.0 nM Concentration of non-labeled ligand (100X [radioligand]) 3-300 nM Concentration of receptor Sufficient to bind 25-35% of radioligand at 0.03 nM b Temperature 4oC
Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM a Data were obtained from pages 11 and 16-19 of the study report. b The protein concentration used was 40 μg protein/tube (0.341-0.391 mg/mL, measured; p. 42).



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

### H]-17 β -estradiol was not adjusted for decay over time, and serial dilu tions in TEDG + PMSF buffer were prepared to achieve the final 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 respective radi olabeled concentration to result in final concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM. For each batch of cytosol, the optimal protein concentration was determined by testing serial amounts of protein per tube, using 0.03 nM radiolabeled estradiol, until a concentration was reached that bound 25-35% of the total radioactivity added (40 μg protein per tube). Each assay consisted of three nonconcurrent runs. Each run included three re plicates of each test substance at each concentration, resulting 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 se rial 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 conc.) 500 μL 500 μL 50 μL Total volume in each assay tube a Data were obtained from pages 104-112 of the study report. b Total binding = [ 3 H]-17 β -estradiol bound to ER c NSB = [ 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.



### Tubes were incubated with gen tle vortexing for 16-20 hours at 4  C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and vortexed onceEstrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 6 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



### every 5 minutes for 15 minutes. Subsequently, th e contents of each tube were washed three times as follows: TEDG + PMSF buffer was added, vortexed, centrifuged for 10 min at 1000 x g , and the supernatant decanted and discarded. EtOH was then added to the HAP pellet remaining in each tube to extract the [ 3 

### H]-17 β -estradiol, followed by vortexing three times (once every five minutes), and centrifugation for 10 min at 1000 x g . An aliquot of each supernatant was radioassayed by scinti llation counting. The temperature was maintained at 4oC throughout the as say prior to extraction with EtOH.

3. Competitive Binding Experiment: A summary of the experiment 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 M Temperature 4  C
Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM a Data were obtained from pages 11, 16-18, and 22 of the study report. b The protein concentration used was 90 μg protein/tube (0.725-0.891 mg/mL, measured; p. 42).



### Solubility of metolachlor in EtOH and assay buffer was not reported specifically but the metolachlor stock solution c oncentration (in EtOH) was 100 mM and the highest assay concentration was 10

− 3 

### M. On the day of the assay, the specific activity of the stock solution [ 3 

### H]-17 β -estradiol was not adjusted for decay over time, and diluted in TEDG + PMSF buffer to achieve a final concentration of 1 nM. For each batch of cytosol, the optimal protein concentration was determined by testi ng serial amounts of prot ein per tube, using 1.0 nM radiolabeled estradiol, unt il a concentration was reached that bound 10-15% of the total radioactivity added (90 μg protein per tube). Se rial dilutions of the test substance, positive control (19-norethindrone), nega tive control (octyltriethoxysilane ), and reference material (non-labeled 17 β -estradiol) were prepared to achieve the concentrations shown in Table 4. The competitive binding experiment consisted of five independent runs on five different days. Each run included duplicate sets of 0.1 μ M estradiol (NSB) at the beginning and triplicate sets at the end of each run, along with duplicate tubes of the reference standard, the weak positive control, and nega tive control at each concentr ation, and triplicate sets of metolachlor at each concentrati on, resulting in a total of 81 sa mples per run (including three replicates of [ 3 

### H]-17 β -estradiol only, for total binding calculations).Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 7 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None

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

Metolachlor Positive control Negative control Reference Chemical 19-norethindrone Octyltriethoxysilane Non-labeled 17 β -estradiol
Tubes 52-75 c Tubes 36-51 c Tubes 20-35 c Tubes 4-19 and 76-81 c

10

− 10 10

− 8.5 10

− 10 Solvent control d

10

− 9 10

− 7.5 10

− 9 10

− 11

10

− 8 10

− 7 10

− 8 10

− 10

10

− 7 10

− 6.5 10

− 7 10

− 9.5

10

− 6 10

− 6 10

− 6 10

− 9

10

− 5 10

− 5.5 10

− 5 10

− 8.5

10

− 4 10

− 4.5 10

− 4 10

− 8

10

− 3 10

− 4 10

− 3 10

− 7 a Data were obtained from pages 21-22, and 113-142 of the study report. b Each tube contains: 10μL of either the test substance, positive control, negative control, solvent control, or non-labeled 17 β -estradiol; 390 μL of TEDG + PMSF 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 dupli cate except metolachlor (trip licate), for a total of 81 tubes per run. d Solvent is EtOH in tubes 4-5 and 76-78.



### The procedures for assay incubation, separati on of bound from free estradiol (washing with buffer and extraction with EtOH), followed by scintillation counting of bound [ 3 

### H]-17 β

estradiol were similar to those used in the saturation binding experiment.

C. DATA ANALYSIS: For the saturation binding experi ment, total binding and NSB data were modeled via non-linear regression usi ng Graph Pad Prism v. 5.02 (GraphPad Software, Inc., La Jolla, CA), incorporating automatic outlier elimination according to the method of Motulsky and Brown (2006) 1 

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

### Swillens (1995) 2 

### . For the competitive binding assay, similar methods of nonlinear regr ession 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 

### ), mean values were calculated for the composite three runs using Microsoft Excel. For the competitive binding experiment, the composite mean was reported for RBA and the composite m ean and standard error was reported for log IC 50 

### using Microsoft Excel

.



### 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).

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) (2012) / Page 8 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



### 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 has 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”



### b. Descriptors for receptor binding:

B max 

### : maximum specific binding number (fmol ER/ 100 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

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters are presented in Table 5. The mean Kd for [ 3 

### H]-17 β -estradiol was 0.075 nM, and the mean estimated Bmax was 31.7 fmol/100 μg protein for th e prepared rat uterine cytosol. The Kd for each run was within the expected range of 0.03 to 1.5 nM. The data produced consistent andEstrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 9 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



### linear Scatchard plots. Confid ence in these numbers is high due to the small variation among runs. The goodness of fit (R 2 

### ) was not reported.

Figure 1 illustrates the non-specific and total binding curves for [ 3 

### H]-R1881 to the estrogen receptor (specific binding was not presented graphically). Based on the raw data, the specific binding reached a plateau in Run 1 and Run 3, and NSB was generally less than 20% of total binding (1.97 to 21.51%). Figure 2 contains the Scatchard plots th at illustrate the binding of [3H] R1881 to the estrogen recepto r across the three runs: the data fits resulted in linear plots.

TABLE 5. Saturation Binding Experiment of 17 β -estradiol with Estrogen Receptor from Rat Uterine Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SEM b

R 2 (unweighted) NR NR NR NA

B max (nM) 0.023 0.028 0.025 0.025 ± 0.001

B max (fmol/100 μ g protein) 29.2 34.8 31.2 31.7 ± 1.64

K d (nM) 0.063 0.083 0.078 0.075 ± 0.006 a Data were obtained from page 43 of the study report. b SEM values were calculated by the study reviewer.
R 2 Goodness of fit NR Not reported NA Not applicableEstrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 10 of 17 METOLACHLOR/108801 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) (2012) / Page 11 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



### FIGURE 2. Scatchard Plot s of the Binding of [ 3 

### H]-17 β -estradiol to the Estrogen Receptor.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 12 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



### B. COMPETITIVE BINDING EXPERIMENT: The results from the competitive binding experiments are presented in Table 6 and shown graphically in Figures 3 to 4. A total of five independent assay runs were conducted. The three reference chemicals generally performed as expected; however, parameters for 19-norethindrone we re outside of the tolerance limits for a weak positive control (developed for norethynodrel) in Run 5, and the negative control octyltriethoxys ilane displaced >25% of [ 3 

### H]-17 β -estradiol at the highest concentration tested in Run 1 and the two highest concentrations in Run 2. Octyltriethoxysilane had no effect on specific binding of the [ 3 

### H]-ligand in Runs 3-5 (77.95 to 116.13%, excluding one outlier at 20.61%); however, minimum total binding of [ 3 

### H]- 17 β -estradiol in the negative cont rol assay was 47.24% in Run 1 at the highest concentration tested, indicating that octyltriet hoxysilane appeared to have some interaction w ith the ER in Run 1 (data for Run 2 were compromised due to a technical error). The estimated mean log IC 50 

### was

− 8.675 M for 17 β -estradiol and

− 5.254 M for the weak posi tive control, and the mean RBA was 0.045% for the weak positive control. Confidence in these numbers is high due to the small variation. Overall, the results for 17 β -estradiol were acceptable for all five runs, with four and three acceptable runs for 19-norethindr one and octyltriethoxysilane, respectively.

Metolachlor had no signifi cant effect on specific bindi ng (74.86-114.57%, excluding one replicate at 62.24% in Run 1) of the [ 3 

### H]-ligand; therefore, a log IC 50 

### and RBA could not be calculated for metolachlor. Based on the results of the five runs, metolachlor is classified as “not interactive” with the estrogen receptor und er the conditions of this assay (Table 7).Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 12 of 17METOLACHLOR/108801OCSPP 890.1250/ OECD NoneTABLE 6. Competitive Binding Assay of Metolachlor with Estrogen Receptor from RatUterine CytosolaParameter Run1Run 2 Run 3Run 4 Run 5 Mean ± SEMR2(unweighted) 17β-estradiol 0.9988 0.9899 0.9980 0.9985 0.9994 NA19-norethindrone 0.9980 0.9680 0.9970 0.9994 0.9657 NAMetolachlor NA NA NA NA NA NALog IC50(M) 17β-estradiol−8.954−8.524−8.607−8.643−8.647−8.675 ± 0.073b19-norethindrone−5.05−4.97−5.282−5.535−5.435−5.254 ± 0.108bMetolachlor NA NA NA NA NA NAIC50(M) 17β-estradiol 1.11 x 10−92.99 x 10−92.47 x 10−92.28 x 10−92.25 x 10−92.22 x 10−9(± 0.31)b19-norethindrone 8.913 x 10−61.0715 x 10−55.224 x 10−62.917 x 10−63.673 x 10−66.288 x 10−6(± 1.514)bMetolachlor NA NA NA NA NA NALog %RBAb19-norethindrone−1.905−1.554−1.325−1.107−1.213−1.421 ± 0.142bMetolachlor NA NA NA NA NA NARBA (%)b19-norethindrone 0.012 0.028 0.047 0.078 0.061 0.045 ± 0.012bMetolachlor NA NA NA NA NA NAa Data were obtained from page 44 of the study report.b Values are reviewer-calculated. For meanvalues reported in scientific notation, the SEM are reported in the same exponential.R2Goodness of fitRBA (%) Relative binding affinityNA Not applicableEstrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 13 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None

TABLE 7 Binding Classification of Me tolachlor with Es trogen Receptor a

Run 1 2 3 4 5 Mean Binding Classification c Classification category value b 1 0 0 0 0 0.2 Not interactive a Binding classifications were determined by the reviewer. 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 Interactive = mean ≥ 1.5; Equivocal = 0.5 ≤ mean <1.5; Not inte ractive = mean <0.5



### FIGURE 3. Percentage E2 Bound to the Estrog en Receptor in the Presence of Unlabeled E2, 19-Norethindrone, Octyltriethoxysilane or Metolachlor.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 14 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD NoneEstrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 15 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



### FIGURE 4. Mean Percentage [ 3 

### H]-E2 Bound to the Estrogen Receptor in the Presence of Metolachlor in Five Competitive Assays.

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 NR Top (% binding) 94 to 111 98.5 to 106.6 X Bottom (% binding)

− 4 to 1

− 3.600 to

− 1.060 X Hill Slope (Log 10 (M)

− 1 )

− 1.1 to

− 0.7

− 1.1260 to

− 0.9208 X c Weak Positive control (19-norethindrone) fitted curve parameters b Log e residual SD NA NR Top (% binding) NA 92.63 to 103.8 X d Bottom (% binding) NA

− 10.41 to 2.589 X d Hill Slope (Log 10 (M)

− 1 ) NA

− 1.13 to

− 0.79 X d

Solvent concentration
Ethanol ≤ 3% 2% 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% ≤ 25% X e

a Data were obtained from page 44 of the study report. b The EPA Guideline does not define a set of tolerance li mits for 19-norethindrone. A cceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable for Runs 1-4 as they show 19-norethindrone to be an acce ptable weak positive control. c The Hill slope was low (

− 1.1260) in Run 3, but within the recommended range in the other four runs. d Excludes Run 5, which exhibited an ambiguous curve that was not consistent with one-site competitive binding behavior. e The minimum total binding of [ 3 H]-17 β -estradiol in the negative control assays was 47.24% in Run 1 and 5.01% in Run 2, however, the average binding across all concentrations was >75% for all runs. NA Not applicable NR Not reportedEstrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 16 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



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

### H]-17 β -estradiol in a manner consiste nt with one-site binding, as indicated by a mean Hill slope of

− 1.0091.

Metolachlor was tested over a concentration rang e that fully defined the top of the curve. The percent binding at this top plateau 76.14- 107.70% was within 25 percentage points of the value for the lowest concentration of the estradiol standard 95.43-109.03%.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Rat uterine cytosol was successfully prepared and characterized, then employed in competition assays with the goal of investigating the potential of metolachlor to act as an endocri ne disruptor. Results from these experiments demonstrate that metolachlor was not a viable substrate for the rat ER and did not bind to the receptor in a manner that disrupts 3 

### H-E 2 

### binding.

B. AGENCY COMMENTS: For the saturation binding experiment, the mean maximum binding capacity (B max 

### ) was 31.7 fmol/100 μg protein and the mean dissociation constant (K d 

### ) was 0.075 nM. The B max 

### value was within the recommended range (10 to 150 fmol/100 μg) and demonstrated that the estr ogen receptor was present in a reasonable concentration. The K d 

### for each run was within the exp ected range of 0.03 to 1.5 nM. The Scatchard plots for the three Saturation assays were linear. The specific binding reached a plateau in Run 1 and Run 3, and NSB was genera lly less than 20% of total binding (1.97 to 21.51%).

For the competitive binding experiment, the estimated mean log IC 50 

### was

− 8.675 M for 17 β

estradiol and

− 5.254 M for the weak positive control. The mean RBA was 0.045% for the weak positive control. Performance criteria were generally met; however, parameters for 19- norethindrone were outside of the tolerance lim its for a weak positive control in Run 5 and the negative control, octyltriethoxysilane, appeared to show some activity against the estrogen receptor at the highe st concentrations in Run 1 (activity at the two highest concentrations in Run 2 was attr ibuted to a technical error).

The estimated mean IC 50 

### and RBA were not calculated fo r metolachlor as it did not show competitive binding ( i.e . displacement of the radiolabel ed estradiol from the ER). Metolachlor is classified as non-interactive in this assay as the mean binding classification from the five runs was <0.5 (mean specific radiolabeled ligand ER binding was >75% at metolachlor concentrations up to 10 –3 

### M in Runs 2-5, and ≥ 72.28% in Run 1).

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

 Specific binding of [ 3 

### H]-17 β -estradiol to the ER during th e saturation binding experiment was not presented graphically. Analysis of the raw data indicated that specific binding reached a plateau in Run 1 and Run 3 (pp. 105, 108, and 111).

 The amount of cytosol used in the co mpetitive binding assay bound 2.20-10.73% of the radioactive estradiol, which is lower than the guideline 10-15%.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 17 of 17 METOLACHLOR/108801 OCSPP 890.1250/ OECD None



###  Tubes of unlabeled 17 β -estradiol, 19-norethindrone, and octyltriethoxysilane were prepared in duplicate at each concentration. EPA’ s protocol specifies that there should be three replicates assayed simultane ously at each concentration.

# DATA EVALUATION RECORD

METOLACHLOR

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 2-49-2012 (MRID 48617808)

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: 5/2/2012

Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 5/11/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/15/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/15/2012

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).METOLACHLOR/108801 Estrogen Rec eptor Transcriptional Activation (2012) I Page I of 13 OCSPP 890.1300 / OECD 455



### Primary Reviewer: Ayaad Assaad, D.V .M., Ph.D. 

### Health Effects Division Secondary Reviewer: Minerva Mercado Ph.D., DABT 

### Health Effects Division Signature: 

# 4**~ z~cJ**  tf**~,** 

### Date:

### ~/7,%]6

## Signature**~** Date. · 

### ./sTemplate version 08 /20 11



### DAT A EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Transcriptional Activation (Human cell Line, HeLa-9903) ; OCSPP 890.1300; OECD 455

.



### PC CODE: 108801 

### DP BARCODE: D398926



### TXR#: Not Provided 

### CAS No.: 51218-45-2



### TEST MATERIAL (PURITY): Metolachlor (97.3% a.i.)



### SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide



### CITATION: Willoughby, 

### I.A. (2012) Metolachlor: estrogen receptor transcriptional
activation (human cell line (HeLa-9903)). CeeTox, Inc

., Kalamazoo, MI. Laboratory Report No.: 9179V-100537ERTA, January 25

, 2012. MRID
48617808. Unpublished



### SPONSOR: Metolachlor Task Force, 16160 Nall, Stillwell, KS



### TEST ORDER# : CON-108801-12



### EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID 48617808), hERa-HeLa-9903 cells cultured ;n vUro 

### were exposed to metolachlor (97.3% a.i.

, Lot #2 0081115) at logarithmically increasing concentrations from 10

- 11 

### to 10

- 4 

### M in DMSO (0.1 %) for approximately 24 hours in two independent runs. The experiments were performed using 96-well plates and each metolachlor concentration was tested in 6 wells / plate in each run. Cells were exposed to the test agent for 24±2 hours at 37±2 ° C to induce reporter (luciferase)

gene products. Luciferase expression in response to activation of the estrogen receptor was measured upon using a proprietary luciferase assay

Metolachlor was tested up to a cytotoxic level, which was observed at concentrations 2:10

- 4 

### M.
The mean RPCM ax was 3.3% for the first run and 0.6% for the second

, and the associated PCMax

was 10

- 7 

### M for both.

In the main assays, the responsiveness of the cells to the very weak positive control l 7amethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the

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

very weak activity

, metolachlor responses were similar to those of the negative control
corticosterone and not comparable to the responses of l 7a-methyltestosterone

, which was able to reach a maximum of 36.5-46

. 3% PC. Metolachlor was only able to reach a maximum of 0.6-
3 .3% PC when tested up to the highest concentration possible based on cytotoxicity. BecauseEstrogen Receptor Transc riptional Activation (2012) / Page 2 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455



### the RPC Max 

### < PC 10 

### in both assay runs, metolachlor was considered 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 Compliance, Quality Assurance, and Data Confidentiality statemen ts were provided.Estrogen Receptor Transc riptional Activation (2012) / Page 3 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Metolachlor
Description: Technical, liquid, MW 283.79 g/mol Source (Cat #): WIL Research (not reported0 Lot/Batch # (Exp Date): 20081115 (Nov 15, 2013) Purity: 97.3% Solubility: Not reported Volatility: Vapor pressure: 1.3 X 10 to the minus 5 mm Hg at 20 degrees C (added by reviewer) Stability: Stable after storage at 0±1 Ԩ for 7 days, and stable after storage at 54±2 Ԩ for 14 days (added by reviewer). Storage conditions: Ambient
CAS #: 51218-45-2
Structure:

CH 3

CH 3 N
O CH 3 O Cl

CH 3



### 2. 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



### 3. Vehicle(s)

Solvent: DMSO

Solvent control (final concentration): 0.1%Estrogen Receptor Transc riptional Activation (2012) / Page 4 of 13 METOLACHLOR/108801 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 by a DNA fluorochrome assay. Cells 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 not reported), in an incubator under 5% CO 2 

### at 37 oC. Upon reaching 75-90% confluen ce, cells were subcultured prior to exposure to the test material. The cells used in this study were pa ssage 26 (range-finder), passage 27 (first run, 9/22/2011), and passage 16 (new from cryopreservation for second run, 10/20/2011).

2. Transcriptional Activation Assays: For each test, cells were plated in a 96-well plate (estrogenic activity of plastic not reported) at a density of approximately 1×10 4 

### cells/100 μL medium/well and allowed to attach for 3 hours. The growth medium was replaced with medium containing serial log dilutions of meto lachlor in DMSO. Cells were incubated for 24±2 hours at 37±2 °C. The tota l final concentration of DMSO was 0.1%. Cytotoxicity was determined by two-read propidium iodide uptak e. It was stated that transcriptional activation of the estrogen receptor was determined as described in CeeTox Standard Operating Protocol (SOP) 2041. A list of reagen ts was provided, but the assay reagent was classified as proprietary informa tion, and the SOP was not provided.

a. Preliminary Test: A preliminary test evaluating concentrations ranging from 10

− 6.5 

### to 10

− 3 

### M was conducted to determine the appropri ate concentration range and to determine concentrations resulting in insolu bility and/or cy totoxicity.

b. Proficiency Chemicals: The responsiveness of the test system was tested on March 5, April 12, and April 28, 2011 (MRID 48843501), using cells at passage 15, 25 and 28, respectively. Based on the assay dates and cell passage numbers, it is unlikely that the cells used for proficiency testing are from the same frozen stock as the cells used in the main assay. The cells were tested using the follo wing set of proficiency chemicals, and each chemical was tested in duplicate on separate days:Estrogen Receptor Transc riptional Activation (2012) / Page 5 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455

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.



### c. Reference Chemicals: To ensure the stability of the response from the cell line, eight concentrations of each of the following refere nce chemicals were included in the current assay, along with the test chemical:

3. Data analysis: To obtain the relative transcriptiona l activity to the 1 nM E2 positive control (PC), the luminescence signals from the concur rent plate were analy zed 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 expres s 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 are calculated only if a positive response is 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.

4. 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 Reference Chemical CAS No. Concentration Range Class
17 β -estradiol (E2) 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 agonistEstrogen Receptor Transc riptional Activation (2012) / Page 6 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455



### 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, a preliminary cytotoxicity and precipitation assay was conducted (Table 1). Cytotoxicity (<80% cell viability) was noted at 10

− 3 

### M, and precipitation was observed at ≥ 10

− 4 

### M metolachlor. Based on these results, logarithmically increasing concentrations from 10

− 11 

### to 10

− 4 

### M were selected for the assay.

TABLE 1. Preliminary Test for Solubility, Cytotoxi city, and Concentration-Selection for Metolachlor a Concentration (M) % Viability b Comments c

10

− 3 52 Precipitation observed, Cytotoxic
10

− 3.5 92 Precipitation observed
10

− 4 89 Precipitation observed
10

− 4.5 97
10

− 5 99
10

− 5.5 100
10

− 6 103
10

− 6.5 100 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 performed in duplicate on different days for each chemical. The reported responses are in Table 2.

TABLE 2. 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 NA = not applicable. The chemical was not tested at this time.



### 2. Reference Chemicals: The response curves for the th ree estrogenic reference chemicalsEstrogen Receptor Transc riptional Activation (2012) / Page 7 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455



### are shown in Figures 1 – 3, and the values de rived from the concentr ation response curves ( e.g ., Log PC 50 

### , Log PC 10 

### , Log EC 50 

### , Hill slope) for the concurre ntly run reference materials are presented in Table 3.

Although all four performance criteria were met for 17 β -estradiol and co rticosterone, and three of the four criteria were met for 17 α -estradiol, the available data indicate that both runs were invalid due to the lack of responsiveness of the cells to the very weak agonist, 17 α -methyltestosterone. Additionally, in R un 2, the maximum responsiveness of both 17 β -estradiol and 17 α -estradiol exceeded (122%-163%) the response of the PC.

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

− 10.4

− 10.6 X Log PC 10 <

− 11

− 11.4

− 11.4 X Log EC 50 − 11.3 to

− 10.1

− 10.3

− 10.5 X Hill Slope 0.7 to 1.5 1.2 1.5 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

− 9.6

− 9.6 X Log EC 50 − 9.6 to

− 8.4

− 8.6

− 8.8 X Hill Slope 0.9 to 2.0 1.6 2.2 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 NC X Log PC 10 − 8.0 to

− 6.2

− 5.9

− 5.9 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 pages 12 and 23 of the study report. NC = not calculableEstrogen Receptor Transcriptional Activation (2012) / Page 8 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455



### FIGURE 1. Relative Transcription Acti vation (RTA) of the Natural Ligand, 17 β

estradiol, Compared to the Positive Control, 1 nM 17 β estradiol.

Mean ± SD are presented (n=6).Estrogen Receptor Transcriptional Activation (2012) / Page 9 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455



### FIGURE 2. Relative Transcription Acti vation (RTA) of the Weak Estrogen, 17 α

estradiol, Compared to the Positive Control, 1 nM 17 β estradiol.

Mean ± SD are presented (n=6).Estrogen Receptor Transcriptional Activation (2012) / Page 10 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455



### FIGURE 3. Relative Trans cription Activation (RTA) of the Very Weak Agonist, 17 α

methyltestosterone, Compared to the Positive Control, 1 nM 17 β estradiol.

Mean ± SD are presented (n=6).



### C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls: Data for the vehicle and positive controls are included in Table 4. The overall mean TA value for the vehicle control was 18092 for the first run and 8938 for the second run; the overall mean TA value for the positive control was 516575 for the first run and 1023950 for the second run. The induction for the positive control ranged from 28.6- to 114.6-fold. The mean normalized value for the positive control was 498483 for the first run and 1015013 for the second run. The PC 50 

### (50% of the maximum response) for E2 in this assay is 249242 for the fi rst run and 507507 for the second run; the PC 10 

### (10% of the maximum response) is 49848 for the first run and 101501 for the second.Estrogen Receptor Transc riptional Activation (2012) / Page 11 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455

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 18092 3417 516575 129778 28.6 498483 129778
2 8938 2194 1023950 254690 114.6 1015013 254690 a Data were calculated by the reviewers 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 ac tivation at each concentration of the test chemical during the two assay runs is pr esented in Table 5. Th e concentration-response curves depicting fold induction of relative tran scriptional activation is presented in Figure 4 below. The mean RPC Max 

### was 3.3% for the first run a nd 0.6% for the second, and the associated PC Max 

### was 10

− 7 

### M for both. Because the RPC Max 

### < PC 10 

### in both runs, metolachlor was considered negative for estroge n receptor transcriptional activation in this test system. However, due to the failed re sponsiveness of these cells to the very weak agonist 17 α -methyltestosterone, the possibility that metalochlor is a very weak estrogen agonist could not be evaluated.

TABLE 5. Relative Transcriptional Activation (RTA) of Metolachlor a

Parameter RTA (mean ± SD); % of Positive Control (PC)
Run 1 Run 2
Conc. (M) Mean SD Mean SD

10

− 4

-- -- -- -- 10

− 5 1.0 0.5 0.2 0.3
10

− 6 1.2 0.5 0.5 0.3
10

− 7 3.3 3.2 0.6 0.2
10

− 8 1.1 1.8 0.0 0.1
10

− 9 1.3 1.6 0.2 0.3
10

− 10 1.3 1.9 0.0 0.2
10

− 11 0.9 1.9 0.1 0.3 Log EC 50 NA NA Hill Slope NA NA
RPC Max 3.3 0.6
PC Max 10

− 7 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 (2012) / Page 12 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455



### FIGURE 4. Fold Induction of Relative Tr anscription Activation (RTA) of Metolachlor Compared to the Positive Control.

3. Performance Criteria: While the Log PC 50 

### , Log PC 10 

### , Log EC 50, 

### and Hill slope values for most of the concurrent reference chemicals fe ll within or near the acceptable ranges (Table 3), the response of the cells to the reference chemical 17 α -methyltestosterone was not adequate; the log PC 10 

### for 17 α -methyltestosterone was higher than the validated range in the both runs and the log PC 50 

### for 17 α -methyltestosterone was not calcu lable in either run. For 17 α -methyltestosterone, the mean RPC Max 

### was 46.3% for the first run and 36.5% for the second run.Estrogen Receptor Transc riptional Activation (2012) / Page 13 of 13 METOLACHLOR/108801 OCSPP 890.1300/ OECD 455



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The suitable top concentration for metolachlor for use in both independent runs was 10

− 5 

### M because of excessive cytotoxicity observed at nominal concentrations of 10

− 4 

### M and higher. Precipitati on was also observed with metolachlor in the initial rangefinder at concentrations ≥ 10

− 4 

### M. In both runs, there was slight precipitation of me tolachlor observed at 10

− 4 

### M. In two independent runs of the transcriptional activation assay, metolachlor did not result in an increase in luciferase activity at any of the viable c oncentrations tested; metolachlo r is not an agonist of human estrogen receptor alpha (hER α ) in the HeLa-9903 model system.

B. AGENCY COMMENTS: Metolachlor was tested up to a cytotoxic level, which was observed at concentrations ≥ 10

− 4 

### M. The mean RPC Max 

### was 3.3% for the first run and 0.6% for the second, and the associated PC Max 

### was 10

− 7 

### M for both. Because the RPC Max 

### < PC 10 

### in both runs, metolachlor was considered ne gative for estrogen receptor transcriptional activation in this test system.

Although the conditions of this assay were not optimal to detect very weak activity, metolachlor responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 36.5-46.3% PC. Metolachlor was only able to reach a maximum of 0.6-3.3% PC when tested up to the highest concentration possible based on cytotoxicity.

C. STUDY DEFICIENCIES: The following deficiencies were noted:

 Responsiveness of the cells to th e very weak positive control 17 α -methyltestosterone was lower than the expected values, indicating a decreased sensitivity of the assay to very weak agonists.

 Certificates of Analyses were not pr ovided for the reference chemicals.

 The source of the fetal bovine serum was not provided.

### AMY
BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917
Date: 2015.06.04 12:14:32 -04'00'

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 2 of 82 CITATION: Palmer, S.J., K.H. Martin, T.Z. Kendall, and H.O. Krueger. 2012. Metolachlor: Fish Short-term
Reproduction Assay with the Fathead Minnow, ( Pimephales promelas ). Unpublished study performed by

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

Task Force, Stilwell, Kansas. Study completed January 23, 2012.

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 Metolachlor

EPA MRID Number 48617811

Page 3 of 82 EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay of metolachlor with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions. Adult fish (16 spawning groups, 2 males and 4 females in each
group; 5.5 months old) were exposed to metolachlor (97.3% purity) at nominal concentrations of 0 (negative
control), 0.050, 0.50, and 5.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.0300, negative

control), 0.046, 0.48, and 4.7 mg a.i./L. The test system was maintained at 24.1 to 25.2 o C and a pH of 7.0

to 8.2.

Overall survival was significantly reduced (p<0.05) by 66.7% for males, 73.3% for females, and 70.8% for

the combined sexes at the high treatment level compared to the negative control. Additionally, male and female

fish in the high treatment level showed signs of erratic swimming, loss of equilibrium, lethargic swimming, and

curved spine; excess feed was observed in high treatment tanks. There were no significant differences
(p>0.05) for male or female growth parameters (weight and length); however, there was a non-statistically
significant decrease (p>0.05) of 23% in male body weight at the high treatment compared to the negative

control.

Mean spawning frequency and fecundity in the negative control were at least every 4 days and 28
eggs/female/day (range 20.2-33.9), respectively; fertilization success in the negative control was 97.9%.
There was a significant reduction (p<0.05) in fecundity of 98% at the high treatment level relative to the

negative control; only one replicate in the high treatment level produced eggs, and there was 100% fertilization

success in that replicate.

Female vitellogenin (VTG) levels were significantly reduced (p<0.05) by 77% at the high treatment level
relative to the negative control. Male VTG levels were not significantly different (p>0.05) from the negative

control. There were no significant differences in female gonado-somatic index (GSI) compared to the negative
control, but there was a marginally significant increase (p=0.057; Mann-Whitney) of 29% in male GSI at the
high treatment level compared to the negative control. There was a significant reduction (p<0.05, MannWhitney) in male tubercle score at the high treatment level (median tubercle score: 5) compared to the
negative control (median tubercle score: 22). No tubercles were observed in female fish. The study authors
also reported a statistically-significant decrease (p<0.05; Jonckheere-Terpstra trend test) in mean male fatpad

scores in the mid and high treatment groups compared to the negative control. Sex steroids were not measured.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 4 of 82 Gonadal histopathologic changes in females included increased oocyte atresia related to increases in granulosa

cell hypertrophy and hyperplasia and increased granulosa cell phagocytosis as well as decreased early and

late vitellogenic oocytes and ovary size at the high treatment level. In males of the high treatment group,

germinal epithelium showed an increased presence of diploid spermatogonia with a decreased presence of

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

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

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

spermatogenesis.

All performance and validity criteria were met in this 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: 5.5 months
Mean body weight at test initiation (if measured): male 2.5 g; females 1.3 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 MetolachlorEPA MRID Number 48617811Page 5 of 82Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Metolachlor.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.046NoNoNoNoNoNoNoNoNoNoNANANANA0.48NoNoNoNoNoNoNoNoNoNoNANANANA4.7YesNoYesNoYes3NoYesYesNoYesNANANANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-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 regarding histopathology may beheavily weighted by the expert opinion of a board-certified pathologist.3The p value for this treatment was 0.057 (Mann-Whitney) suggesting a significant increase in male GSI compared to the control.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 6 of 82 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 total organic carbon, unionized ammonia, and residual chlorine in the dilution water were not

provided; EPA recommends that total organic carbon in dilution water be <2 mg/L; unionized ammonia

in the dilution water be <1 P g/L; and residual chlorine in dilution water be <10 P g/L.

2. Male and female body weight ranges for a subsample of 10 males and 10 females were ±30% of the

mean weight of each sex. EPA recommends that the individual weight of each fish selected for the

test be within ±20% of the estimated mean for each sex.

These deficiencies/deviations do not have an impact on the interpretation of the study.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality claims

statements were provided. This study was conducted in accordance with GLP
Standards as published by the U.S. EPA (40 CFR Parts 160), and OECD
Principles of GLP [ENV/MC/CHEM(98)17].

A. TEST MATERIAL: Metolachlor, CAS No. 51218-45-2

Description: The test material was a liquid; insoluble in acetone (0.05%).

OECD recommends describing water solubility, melting/boiling point stability in wa ter and light, pKa,

Pow or Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : 20081115 (Lot No.)

Purity: 97.3% (originally determined June 15, 2010).

Impurities: None reported

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 7 of 82 Stability of Compound: Analytical verification of diluter stock fortified with 4.0, 40 and 400 mg a.i./L

yielded recoveries ranging from 92.4 to 99.6% of nominal concentrations;
and pre-test (Day -1) verification of samples fortified at 0.05, 0.50 and 5.0

mg a.i./L yielded recoveries ranging from 77.6 to 89.1% of nominal

concentrations. Analytical verification of the test solutions at Days 0, 7, 14,

and 21 yielded recoveries ranging from 81.7% to 104% of nominal

concentrations. The %CV for the 0.05, 0.50 and 5.0 mg a.i./L treatment

levels were 10, 6.3 and 3.5%. These data indicate the test material was

stable under the test conditions.

Storage Conditions of

Test Chemicals: The stock solution was stored at ambient temperature in the dark. Storage

stability was determined after 7, 14, and 21 days; recoveries ranged from

81.7 to 104%.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 8 of 82B. 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 a singlelaboratory stock?YesOsage Catfisheries, Inc., Osage Beach,Missouri. EPA recommends that fish be from a single

laboratory stock.Were acclimation conditions sameas definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive test.Acclimation period:2 weeks 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 MetolachlorEPA MRID Number 48617811Page 9 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Mortalities during 7 days prior to preexposure periodwere not reported.Fish did not receive anytreatment fordisease during acclimation period.No behavioral abnormalities or clinical signswere noted. 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 during this extended acclimation period, EPA recommends that the fish not be used.Type of food:Commercial flake food(Sera Vipan) andbrineshrimp EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body condition.Source of food:Sera, North AmericaFrequency of feeding:2 times/dayDetails on feeding:Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 10 of 82Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:5-6 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at testinitiation (if determined):2.5 gBased on 10 males used to stock aquariafor pre-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):Individual weights within ±30% of theestimated mean.Mean weight of females at testinitiation (if determined):1.3 gBased on 10 females used to stock aquariafor pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):Individual weights within ±30% of theestimated mean.Mean length of males at testinitiation (if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase:14 days EPA recommends a minimum of 14 days.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 11 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsWere 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:32 tanks 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:2 males/tankNumber of females per tank:4 females/tankPre-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:>2 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:None.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 12 of 82C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through 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:Continuous flow diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:200 mL/min 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). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine DisruptorScreening 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).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 MetolachlorEPA MRID Number 48617811Page 13 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems:Solution from mixing chamber split equallyamong replicate test vessels.Treatment recoveriesprior to test initiationranged from 44.6-89.1%.The flow splitting accuracy waswithin ±10%.The test solutions were clear and colorlessduring the testwith no evidence of precipitationor surface films in any control or test solution.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 t he test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is within

10%.Aeration?No EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (<60% saturation).Source of dilution water:Well waterPrior to use, the water was sand-filtered,aerated,and UV-irradiated. 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 14 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzed forpesticides, heavy metals, andother contaminants?YesAll potential contaminants were confirmed to bebelow the detection limit.Test vessel type/materials:Glass aquaria with stainlesssteel screen drainsEPA 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:19L aquaria; solution depth10 cm.Dimensions not reported. EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Fill volume:10 L EPA recommends 10 L solution per tank.Spawning substrate material:PVC pipe cut and sectionedlengthwise and inverted toform a semicircular arch. 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:Dimensions not reported.Additional details on exposuresystem:None.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 15 of 82Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMean1Measurement IntervalGuideline RecommendationsTemperature (°C)24.1 25.2 25.0 Continuous EPA recommends temperature 25±1 oC; interreplicate and inter -treatment differentials should not exceed 1 o C.pH8.08.28.1Weekly EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)6.1 7.7 6.9 Weekly EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3)175 178 176.5 Weekly EPA recommends total alkalinity >20 mg/L

as CaCO 3.Hardness (mg/L as CaCO3)136148142WeeklyTotal organic carbon(mg/L)Not reportedNot reportedNot reportedNot reported EPA recommends that total organic carbon in dilution water be <2 mg/L.Unionized ammonia (Pg/L)Not reportedNot reportedNot reportedOnce EPA recommends that unionized ammonia in the dilution water be <1 P g/L.Residual chlorine (Pg/L)Not reportedNot reportedNot reportedOnce EPA recommends that residual chlorine in dilution water be <10 P g/L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 16 of 82ParameterMinimumMaximumMean1Measurement IntervalGuideline RecommendationsConductivity (Pmhos/cm) 306 461 182 Weekly 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 highest test concentration at least weekly. In addition, continuous temperature monitoring of at least one tank is encouraged.Abbreviations:NANot applicable.1Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels.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 MetolachlorEPA MRID Number 48617811Page 17 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsIf yes, what was the method fordetermining the highest testconcentration in the rangefinder?Not reported. 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:Fathead Minnow Pimephales promelasLife stage:Not reportedSexually mature 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-hrs EPA recommends a 96-hour exposure.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 18 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:NA 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

concentrati on-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 MetolachlorEPA MRID Number 48617811Page 19 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in the definitivetest:The acute toxicity 96-hour LC50for metolachlorwith fathead minnow is 15mg/L.Selected based on 1/3 of the 96-hour LC50(15 mg a.i./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):Not reported.Separation of test concentrations:Ten-fold EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable 1 .Number of test concentrations:4 (3 treated, and anegative control) 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 onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 20 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsLimit of quantification (LOQ):0.0300 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 reported.Frequency of measurement:0, 7, 14 and 21 days 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.Was the randomized completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks during pre - exposure. 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 21 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of male fish per replicateat test initiation:2One fish in replicate C of the 4.7 mg a.i./Lgroup was sexed incorrectly during the preexposure period (immature male considereda female). Theerror was determined duringdissection due to the presence of testis inthis fish. Therefore, this tank actually had 3males and 3 females at the start of theexposure period. EPA recommends 2 males per replicate.Number of female fish per replicateat test initiation:4 EPA recommends 4 females per replicate.Was a solvent used?NoSolvent type (if applicable):NAData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 22 of 82ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum solvent concentration (ifapplicable):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) (ifapplicable):NAPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’s surface:432 to 636 lux EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:The test solutions and stock solutionsappeared clear and colorless with noevidence of precipitate3Hutchinson 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 of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series onTesting and Assessment. No. 23. Paris, France.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 23 of 82Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Metolachlor.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0.00<LOQNA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Treatment 10.0500.04610Treatment 20.500.486.3Treatment 35.04.73.5Abbreviations:CVCoefficient of variation.LOQ=0.0300 mg a.i./LE. ObservationsBiological Endpoints:Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination (Day 21), secondary sexcharacterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal staging and histopathology,and plasma vitellogenin were evaluated.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 24 of 82Were raw (individual) data provided?Yes. It appears that one fish in the 4.7 mg a.i./L treatment group was mis-sexed (male classified as female);however, the occurrence of three males and three females in the replicate does not appear to be discussed in the study report.Data from this fishappear to be included in statistical analysis by the study author. EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinic al 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 vitellogenin,

and plasma sex steroids (testosterone and 17ȕ -estradiol, if measured). Gonado-somatic index (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 beha vior.

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Page 25 of 82 II. RESULTS AND DISCUSSION

A. Results

One male died in the 0.48 mg a.i./L treatment group and two died in the 4.7 mg a.i./L treatment

group during the study. One female died in each of the negative control, 0.046 and 0.48 mg a.i./L

treatment groups. Five females died in the 4.7 mg a.i./L treatment group. Overall mean survival

values were 95.8, 95.8, 91.7 and 70.8% in the mean-measured 0 (control), 0.046, 0.48, and 4.7

mg a.i./L treatment levels, respectively.

Table 9: Adult Fish Survival in Fathead Minnow ( Pimephales promelas ).

Treatment

(mg a.i./L)

[mean-measured] Males Females

n 1 # Surviving % Survival 2 n # Surviving % Survival 2

Negative Control (<LOQ) 8 8 100 16 15 93.8

0.046 8 8 100 16 15 93.8

0.48 8 7 87.5 16 15 93.8

4.7 9 3 6 3 66.7 15 3 11 73.3

Abbreviations: NA Not applicable.

LOQ=0.0300 mg a.i./L

1 Total number of fish at test initiation.

2 The percent survival for each sex was calculated by the reviewer based on the survival data.

3 Based on the study report, there appeared to be three females and three males present at test termination in one

replicate; therefore, the initial number of males and females in this treatment were 9 and 15, respectively.

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Page 26 of 82 Mean male body weight values were 3.12, 2.97, 3.28 and 2.39 g and female body weight values
were 1.34, 1.41, 1.39 and 1.21 g in the mean-measured 0 (control), 0.046, 0.48, and 4.7 mg a.i./L

treatment levels, respectively. Mean male body length values were 61, 60, 63, and 58 mm and female
body length values were 49, 49, 49 and 48 mm in the mean-measured 0 (control), 0.046, 0.48,

and 4.7 mg a.i./L treatment levels, respectively.

Table 10: Size at Test Termination in Fathead Minnow ( Pimephales promelas ).

Treatment

(mg a.i./L)

[meanmeasured] Body Weight Length

Males Females Males Females

n Mean

(g) ±SD n Mean

(g) ±SD n Mean

(mm) ±SD n Mean

(mm) ±SD

Negative

Control

(<LOQ) 4 3.12 0.08 4 1.34 0.11 4 61 1.0 4 49 0.8

0.046 4 2.97 0.47 4 1.41 0.13 4 60 2.5 4 49 0.8

0.48 4 3.28 0.53 4 1.39 0.13 4 63 3.4 4 49 1.7

4.7 4 2.39 0.53 4 1.21 0.10 4 58 1.4 4 48 2.1

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

LOQ=0.0300 mg a.i./L

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Page 27 of 82 Mean fecundity values were 27.9, 29.9, 22.0 and 0.5 and fertilization success was 97.9, 98.6, 97.9
and 100% in the mean-measured 0 (control), 0.046, 0.48, and 4.7 mg a.i./L treatment levels,

respectively. Fertilization success ranged from 97.9 to 100%.

Table 11: Fecundity and Fertilization Success in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[mean-measured] Fecundity 1 Fertilization Success (%) 2

mean ± SD mean ± SD

Negative Control (<LOQ) 27.9 6.15 97.9 1.08

0.046 29.9 5.86 98.6 0.39

0.48 22.0 4.76 97.9 0.89

4.7 0.5 1.00 100 3 NA

Abbreviations: NA Not applicable. ND Not determined.

LOQ=0.0300 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.

3 Only one replicate produced eggs.

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Page 28 of 82 Median male tubercle scores were 22, 20, 22 and 5 in the mean-measured 0 (control), 0.046, 0.48, and
4.7 mg a.i./L treatment levels, respectively (Table 12a). None of the surviving females were found to have

tubercles.

Table 12a: Nuptial Tubercle Score in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[mean-measured] Males Females

n Median Tubercle

Score n Median Tubercle

Score

Negative Control (<LOQ) 4 22 4 0

0.046 4 20 4 0

0.48 4 22 4 0

4.7 4 5 4 0

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

LOQ=0.0300 mg a.i./L

The study author reported fatpad scores (Table 12b). Mean male fatpad scores were 3.5, 3.4, 2.8 and 1.9
in the mean-measured 0 (control), 0.046, 0.48, and 4.7 mg a.i./L treatment levels, respectively. Mean

female fatpad scores were 1.0 at all treatment levels.

Table 12b: Fatpad Score in Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[mean-measured] Males Females

n Mean Fatpad

Score n Mean Fatpad

Score

Negative Control (<LOQ) 4 3.5 4 1.0

0.046 4 3.4 4 1.0

0.48 4 2.8 4 1.0

4.7 4 1.9 4 1.0

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

LOQ=0.0300 mg a.i./L.

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Page 29 of 82 Mean male GSI was 1.45, 1.78, 1.53 and 1.87 and mean female GSI was 12.6, 12.8, 12.8 and 12.0 in
the mean-measured 0 (control), 0.046, 0.48, and 4.7 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 Mean GSI 1

(%) ±SD n Mean GSI 1

(%) ±SD

Negative Control (<LOQ) 4 1.45 0.14 4 12.6 1.1

0.046 4 1.78 0.48 4 12.8 1.4

0.48 4 1.53 0.26 4 12.8 0.15

4.7 4 1.87 0.32 4 12.0 2.6

Abbreviations: NA Not applicable.

LOQ=0.0300 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 30 of 82 Median male gonadal stage was 3, 2, 2 and 3 and median female gonadal stage was 3, 3, 3, and 2 in
the mean-measured 0 (control), 0.046, 0.48, and 4.7 mg a.i./L treatment levels, respectively (Table 14).

The study authors did indicate that testes development was clearly disrupted in one fish in the 4.7 mg

a.i./L treatment.

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) 8 3 15 3

0.046 8 2 15 3

0.48 7 2 15 3

4.7 5 3 11 2

Abbreviations: J Juvenile. NA Not applicable. ND Not determined. UTS Unable to stage.

LOQ=0.0300 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=post-ovulatory.

In males, germinal epithelium showed an increased presence of diploid spermatogonia with decreased

presence of spermatocysts containing spermatocytes and spermatids. One in six male fish showed moderate

alterations in these ratios and could not be staged under normal criteria. These changes appeared to result

from a treatment-related inability of spermatogonia in the testes of affected fish to enter into

spermatogenesis. These results indicate an inability of immature germ cells in the testes that had developed

into spermatogonia to enter into spermatogenesis in the 4.7 mg a.i./L treatment group. This directly reduces

the number of spermatocysts containing primary spermatocytes, leading to a concomitant decrease in

spermatocysts containing secondary spermatocytes and spermatids.

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Page 31 of 82 In females, an increase in atresia was related to increases in granulose cell hypertrophy and hyperplasia

and granulose cell phagocytosis, as well as decreases in late vitellogenic oocytes and ovary size. Moderate

atresia was observed in one fish in the 0.48 mg a.i./L treatment group. These results indicate mild to

marked increase in atresia that was treatment related.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 32 of 82Table 15: Gonadal Histopathology in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Diagnostic Observations1SeverityIncreased Proportion ofSpermatogoniaPresence of Testis-OvaIncreased TesticularDegenerationInterstitial CellFibrosisGerminal Epithelium,AtrophynIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control(<LOQ)088NANA87NANA88180NANA80NANA80280NANA81NANA80380NANA80NANA80480NANA80NANA800.046088NANA88NANA88180NANA80NANA80280NANA80NANA80380NANA80NANA80480NANA80NANA800.48077NANA77NANA73170NANA70NANA70270NANA70NANA74370NANA70NANA70470NANA70NANA70Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 33 of 82Treatment(mg a.i./L)[mean-measured]Diagnostic Observations1SeverityIncreased Proportion ofSpermatogoniaPresence of Testis-OvaIncreased TesticularDegenerationInterstitial CellFibrosisGerminal Epithelium,AtrophynIncidencenIncidencenIncidencenIncidencenIncidence4.7062NANA63NANA66160NANA60NANA60263NANA62NANA60361NANA61NANA60460NANA60NANA60Abbreviations:NANot applicable.LOQ=0.0300 mg a.i./L1Gonadal 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 34 of 82Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control(<LOQ)08NANANANANA888818NANANANANA808028NANANANANA808038NANANANANA808048NANANANANA80800.04608NANANANANA888818NANANANANA808028NANANANANA808038NANANANANA808048NANANANANA80800.4807NANANANANA767617NANANANANA707027NANANANANA707037NANANANANA707047NANANANANA7171Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 35 of 82Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence4.706NANANANANA666616NANANANANA626126NANANANANA636036NANANANANA616046NANANANANA6060Abbreviations:NANot applicable.LOQ=0.0300 mg a.i./L1Gonadal 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 36 of 82 Table 17: Gonadal Histopathology in Female Fathead Minnow ( Pimephales promelas)

.

Treatment

(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Increased Oocyte

Atresia Granulosa Cell

Hyperplasia/

Hypertrophy Increased Granulosa

Cell Phagocytosis Infection,

Microsporidia

n Incidence n Incidence n Incidence n Incidence

Negative

Control

(<LOQ) 0 15 15 15 15 15 15 NA NA

1 15 0 15 0 15 0 NA NA

2 15 0 15 0 15 0 NA NA

3 15 0 15 0 15 0 NA NA

4 15 0 15 0 15 0 NA NA

0.046 0 15 15 15 15 15 15 NA NA

1 15 0 15 0 15 0 NA NA

2 15 0 15 0 15 0 NA NA

3 15 0 15 0 15 0 NA NA

4 15 0 15 0 15 0 NA NA

0.48 0 15 15 15 15 15 15 NA NA

1 15 0 15 0 15 0 NA NA

2 15 0 15 0 15 0 NA NA

3 15 0 15 0 15 0 NA NA

4 15 0 15 0 15 0 NA NA

4.7 0 11 3 11 8 11 5 NA NA

1 11 0 11 0 11 0 NA NA

2 11 2 11 3 11 6 NA NA

3 11 0 11 0 11 0 NA NA

4 11 6 11 0 11 0 NA NA

Abbreviations: NA Not applicable.

LOQ=0.0300 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 37 of 82 Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( Pimephales

promelas)

.

Treatment

(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Early Vitellogenic

Oocytes Decrease Late Vitellogenic

Oocytes

Decrease Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

Negative

Control

(<LOQ) 0 15 15 15 15 15 15 NA NA

1 15 0 15 0 15 0 NA NA

2 15 0 15 0 15 0 NA NA

3 15 0 15 0 15 0 NA NA

4 15 0 15 0 15 0 NA NA

0.046 0 15 14 15 14 15 14 NA NA

1 15 0 15 0 15 0 NA NA

2 15 0 15 0 15 0 NA NA

3 15 0 15 0 15 0 NA NA

4 15 1 15 1 15 1 NA NA

0.48 0 15 15 15 15 15 15 NA NA

1 15 0 15 0 15 0 NA NA

2 15 0 15 0 15 0 NA NA

3 15 0 15 0 15 0 NA NA

4 15 0 15 0 15 0 NA NA

4.7 0 11 7 11 6 11 11 NA NA

1 11 0 11 0 11 0 NA NA

2 11 0 11 1 11 0 NA NA

3 11 2 11 0 11 0 NA NA

4 11 2 11 4 11 0 NA NA

Abbreviations: NA Not applicable.

LOQ=0.0300 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 82 Mean male VTG was 442.5, 527.5, 765.0 and 822.5 ng/mL and female VTG was 3260750, 3294750,
3371500 and 765750 ng/mL in the mean-measured 0 (control), 0.046, 0.48, and 4.7 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) 4 442.5 286.5 4 3260750 486506

0.046 4 527.5 349.0 4 3294750 597410

0.48 4 765.0 1139 4 3371500 839708

4.7 4 822.5 1028 4 765750 460589

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

LOQ=0.0300 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 39 of 82Male and female plasma testosterone and plasma 17ȕ-estradiolwere not measured.Table 20: Plasma Sex Steroids in Fathead Minnow ( Pimephales promelas). Not measured.Treatment(mg a.i./L)[mean-measured]Plasma Testosterone (T)Plasma 17ȕ-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNegative Control (<LOQ)NANANANANANANANANANANANA0.046NANANANANANANANANANANANA0.48NANANANANANANANANANANANA4.7NANANANANANANANANANANANAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.0300 mg a.i./LData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 40 of 82There appeared to be an increase in clinical signs of overt toxicity at the 4.7 mg a.i./L treatment level in both males and females that were interpretedby the study authors as treatment-related (Table 21). Also, excess feed was noted in the bottom of the test chamber at 4.7 mg a.i./L compared to thecontrol. All other fish reported as appearing normal at test termination.Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical Signs at Test TerminationMalesFemalesTypenIncidenceTypenIncidenceNegative Control (<LOQ)Redness behind operculum, dead81None1500.046Hemorrhage in or missing eye81Hemorrhage in or missing eye1510.48None70None1524.7Erratic swimming, loss of equilibrium,curved spine61Erratic swimming and lethargic swimming111Loss of equilibrium and lethargic swimming1LOQ=0.0300 mg a.i./L

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Page 41 of 82 B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, length, tubercle score, GSI, fertility (% eggs that were
fertile), fecundity (#eggs/female/reproductive day), and VTG. Data for growth, tubercle score, GSI,

and VTG were analyzed separately by sex. For all parameters, metolachlor-treated conditions were
compared to the control. The study author’s performed statistical analyses using SAS software (Version

8.2). Data from the one mis-sexed fish in the high treatment group was used in the data analysis.

Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant
effects were detected for p<0.05 unless otherwise noted (statistical program used was not reported).

Effects on survival were determined using a Fisher’s exact test. All endpoints except survival, fertility

and histopathology severity scores and stages were determined by ANOVA and a pair-wise Dunnett’s

test or Wilcoxon rank-sum test. For endpoints showing a monotonic response, a step-down
Jonckheere-Terpstra trend test was used. Prior to Dunnett’s, data were analyzed by Shapiro-Wilk’s

test and Levene’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.

Adult survival (range 70.8-95.8%) was significantly different compared to the controls (95.8%) at the
4.7 mg a.i./L treatment level (p<0.05; Fisher’s Exact test). There was also a statistically significant
decrease in fecundity in the 4.7 mg a.i./L treatment group compared to the control (p<0.05;

Jonckheere-Terpstra test and Dunnett’s test). There were no statistically differences in the growth
parameter (weight and length) in either sex compared to the control (p>0.05). There was also no

statistically significant effect on male GSI or VTG. There was a statistically significant decrease in male
tubercle score at the 4.7 mg a.i./L treatment level compared to the control (p<0.05; Dunnett’s test),
and a significant decrease in Fatpad score at the 0.48 mg a.i./L (p<0.05; Jonckheere-Terpstra test)
treatment level and the 4.7 mg a.i./L treatment levels compared to the control (p<0.05; JonckheereTerpstra trend test and Dunnett’s test). There was also a statistically significant decrease in female
VTG at the 4.7 mg a.i./L (p<0.05; Jonckheere-Terpstra and Dunnett’s test). Although not analyzed
statistically, there were generalized overt signs of toxicity in both males and females (i.e., dead, erratic

swimming, loss of equilibrium, lethargic swimming, curved spine). Histopathology results were

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Page 42 of 82 statistically analyzed and significant effects were determined in both males and females at the 4.7 mg

a.i./L treatment level. In males, there were statistically significant increase in proportion of

spermatogonia, altered proportion of spermatocytes and spermatids, and deceased gonad size, sertoli

cells, germinal epithelium thickness and milt at the 4.7 mg a.i./L treatment compared to the control
(p=0.001 to p=0.02; Jonckheere-Terpstra test). In females, there were statistically significant increase

in oocyte atresia, granulose cell hyperplasia/hypertrophy, and granulose cells phagocytosis, and

decreases in early and late vitellogenic oocytes at the 4.7 mg a.i./L treatment compared to the control
(p<0.0001 to p=0.002; Jonckheere-Terpstra test).

C. Reviewer’s Analysis and Conclusions

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via

Toxstat 3.5. Female and male weight, male length, female mean VTG, male GSI, and fertility data

were not consistent with a monotonic concentration-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 that failed parametric assumptions were analyzed using the non-parametric MannWhitney U test. Female body weight, male VTG, female GSI, male tubercle score and fecundity data

exhibited monotonic trends and were analyzed using the non-parametric Jonckheere-Terpstra test, as

recommended by the OCSPP 890.1350 guideline. There was no response for fertility; however, only

one replicate at the high concentration produced eggs, of which 100% survived. The reviewer compared

all metolachlor-treated groups to the negative control group. Female VTG exhibited a significant
reduction of 77% compared to the negative control, at the 4.7 mg a.i./L treatment levels (p<0.05;

Dunnett’s test). Fecundity was significantly reduced 98% at the 4.7 mg a.i./L treatment, compared to
the negative control (p<0.05; Jonckheere-Terpstra). Growth was unaffected in male and female fish
relative to the negative control. Survival (combined sexes) was significantly different at the 4.7 mg

a.i./L treatment level (p<0.05; Fisher’s Exact test) compared to the control.

Histopathology and secondary sex characteristics were adversely indicated at the 4.7 mg a.i./L level,

but these effects were not statistically analyzed by the reviewer. Male and female fish at the 4.7 mg

a.i./L level showed signs of erratic swimming, loss of equilibrium, lethargic swimming, and curved

spine. In males, there was an increase in the proportion of spermatogonia, altered proportion of

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

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Page 43 of 82 spermatocytes and spermatids, and deceased gonad size, sertoli cells, germinal epithelium thickness

and milt at the 4.7 mg a.i./L treatment compared to the control or lower treatment levels. One in six

male fish showed could not be staged under normal criteria at the 4.7 mg a.i./L treatment. In females,

there was an increase in atresia, granulose cell hypertrophy and hyperplasia and granulose cell

phagocytosis, as well as decreases in early and late vitellogenic, oocytes and ovary size in the 4.7

mg a.i./L treatment compared to the control and lower metolachlor-treated levels.

The reviewer did not re-analyze the fat pad score data and relied on the study author’s conclusion for

this endpoint.

It appears that the there was a mis-sexed fish in replicate C of the high treatment group, based on

visual observation of the data for the fish; however, this occurrence of three males and three females

does not appear to be discussed in the study report. Based on the raw data, the mis-sexed fish

appears to be fish #4 in that replicate, but the study report does not confirm this. In the analysis of

the data, all male endpoints for this replicate were included in the analysis; however, if fish #4 from
that replicate were removed from the analysis it doesn’t appear that it would make a difference in the

interpretation of the overall response and conclusion for that treatment group for any endpoint.

Conclusions:

Survival (combined sexes) was significantly reduced at the 4.7 mg a.i./L treatment level (p<0.05;

Fisher’s Exact test) compared to the control. Additionally, male and female fish at the 4.7 mg a.i./L

level showed signs of erratic swimming, loss of equilibrium, lethargic swimming, and curved spine.

Female VTG was reduced 77% and fecundity by 98% at the highest treatment level, compared to the

negative control. However, there was no effect on fertility due to only one replicate producing eggs

and 100% fertilization success in that replicate. There was also a significant increase in oocyte atresia

related to increases in granulose cell hypertrophy and hyperplasia and granulose cell phagocytosis, as

well as decreases in late vitellogenic oocytes and ovary size at the highest treatment level. There were

no significant differences in female GSI compared to the control; the p value for male GSI at the 4.7

mg a.i./L was 0.057, suggesting a significant increase compared to the control. A significant 76%

reduction in male tubercle score at the 4.7 mg a.i./L treatment was observed compared to the control.

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Page 44 of 82 No tubercles were observed in female fish. The study authors found a statistically significant decrease
in male Fatpad score at the 0.48 mg a.i./L (p<0.05; Jonckheere-Terpstra test) treatment level and
the 4.7 mg a.i./L treatment levels compared to the control (p<0.05; Jonckheere-Terpstra trend test

and Dunnett’s test). In males, germinal epithelium showed an increased presence of diploid

spermatogonia with decreased presence of spermatocysts containing spermatocytes and spermatids.

One in six male fish showed moderate alterations in these ratios and could not be staged under normal

criteria. These changes appeared to result from a relative inability of spermatogonia in the testes of

affected fish to enter into spermatogenesis that was treatment-related. These results indicate an inability

of immature germ cells in the testes that had developed into spermatogonia to enter into

spermatogenesis in the 4.7 mg a.i./L treatment group. This directly reduces the number of

spermatocysts containing primary spermatocytes, leading to a concomitant decrease in spermatocysts

containing secondary spermatocytes and spermatids.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 45 of 82Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with Metolachlor.Treatment(mg a.i./L)[mean-measured]Tubercle Score3GSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.p% Diff.pNegative Control(<LOQ)22 NA 0 NANA 0 NA 0 NA 0 NA0.046200.09423.140.486No19.210.282NANANANA0.48220.5005.870.486No72.8840.696NANANANA4.750.04129.190.057Yes85.8840.663NANANANAStatistical TestJonckheere-TerpstraMannWhitneyNAJonckheere-TerpstraNANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.LOQ=0.0300 mg a.i./L1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3The study authors reported a statistically significant decrease in mean male fatpad scores at the 0.48 and 4.7 mg a.i./L treatment group compared to the control (p<0.05;Jonckheere-Terpstra trend test).4These differences were due to VTG values in one fish for each group being substantially greater than the control VTG values.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 46 of 82Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with Metolachlor.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)0 NA 0 NA 0NA0NANo 0 NANANANANA0.0467.260.6140.710.3430NA1.390.718No1.040.614NANANANA0.48-21.080.094-0.08>0.990NA1.590.441No3.400.670NANANANA4.7-98.21<0.0012.1--30NA-4.370.425Yes-76.520.025NANANANAStatistical TestJonckheere-TerpstraMann Whitney UNAJonckheereTerpstraNAJonckheere-TerpstraNA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.LOQ=0.0300 mg a.i./L.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Only one replicate in this treatment produced eggs and fertilization success was 100%.

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Page 47 of 82 Table 24: Growth Endpoints 1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Metolachlor.

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

0.046 -4.96 0.927 4.66 0.801 -2.45 0.684 0.00 0.500

0.48 4.88 0.930 3.35 0.909 2.04 0.782 -0.51 0.322

4.7 -23.38 0.096 -9.87 0.311 -5.31 0.158 -2.55 0.155

Statistical Test Dunnett’s Dunnett’s Dunnett’s Jonckheere-Terpstra

Abbreviations: Diff. Difference. NA Not applicable.

LOQ=0.0300 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 deviations/deficiences from the guideline as noted in Section I. Materials and Methods of

the DER which did not impact the interpretation of the study.

F. Reviewer’s Comments

The reviewer’s and the study authors’ results were generally in agreement. The reviewer and study authors

agreed on the subjectively-determined effects on secondary sex characteristics, gonad development, and

histopathology.

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Page 48 of 82 III. REFERENCES

U.S. Environmental Protection Agency. 2009. Series 890-Endocrine Disruptor Screening Program Test

Guidelines, OCSPP Number 890.1350: Fish Short-Term Reproduction Assay.

Organization for Economic Cooperation and Development. 2009. OECD Guidelines for Testing of

Chemicals, Guideline 229. Fish Short Term Reproduction Assay. Adopted 7 September 2009.

APHA, AWWA, WEF. 1998. Standard Methods for the Examination of Water and Wastewater. 20th Edition,

American Public Health Association. American Water Works Association. Water Environment

Federation, Washington, D.C.

Organization for Economic Cooperation and Development. 2006. Current Approaches in the Statistical
Analysis of Ecotoxicity Data: A Guidance to Application. OECD ENV/JM/MONO(2008)18.

The SAS System for Windows. 1999 - 2001. Version 8.2. SAS Institute, Inc. Cary, North Carolina.

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Page 49 of 82

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Page 50 of 82Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 51 of 82APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:Reviewer’s statistical verification tableEndpointMonotonic?Parametric?OCSPP890.1350Historical EFEDCommentsFemale bodyweightNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale body weightNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale bodylengthYesNoJonckheere:n.s. p>0.05Jonckheere:n.s. p>0.05EDSP and EFED same conclusions, no effectMale body lengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale VTGNoYesDunnett’s:Dose 3p=<0.001Dunnett’s:Dose 3p=<0.001EDSP and EFED same conclusions; a significant77% reduction from control at 4.7 mga.i./L,relative to control.Male VTGYesNoJonckheere:n.s. p>0.05Jonckheere:n.s. p>0.05EDSP and EFED same conclusions, no effect forincreasing trendFemale GSINoNoJonckheere:n.s. p>0.05Jonckheere:n.s. p>0.05EDSP and EFED same conclusions, no effectMale GSINoNoMannWhitney:n.s. p=0.057MannWhitney:n.s. p=0.057EDSP and EFED same conclusions, no effect forincreasing trendData Evaluation Record onthe Fish Short-Term Reproduction Assay with MetolachlorEPA MRID Number 48617811Page 52 of 82EndpointMonotonic?Parametric?OCSPP890.1350Historical EFEDCommentsFemale tuberclescoreNANANANANo scoreMale tuberclescoreNoNoJonckheere:Dose 3 p=0.041Jonckheere:Dose 3 p=0.041EDSP and EFED same conclusions, no effectFecundityYesYesJonckheere:Dose 3p=<0.001William’s:Dose 3p=<0.001EDSP and EFED same conclusions; significantreduction at 4.7 mga.i./L (p<0.001; 98% lowerthan control)FertilityYesNoMannWhitney:n.s. p>0.05MannWhitney:n.s. p>0.05EDSP and EFED same conclusions, no effect; atthe top dose, only one replicate produced eggs ofwhich 100% survived.F testosteroneNANANANANAM testosteroneNANANANANAF estradiolNANANANANAM estradiolNANANANANA

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Page 53 of 82 Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

CONTROL 8 0 8

0.046 8 0 8

---------------------------------------------------------------------------

TOTAL 16 0 16

============================================================================

Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 8. Since b is greater than 4.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

CONTROL 8 0 8

0.48 7 1 8

---------------------------------------------------------------------------

TOTAL 15 1 16

============================================================================

Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 7. Since b is greater than 4.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

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Page 54 of 82 Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

CONTROL 8 0 8

4.7 6 2 8

---------------------------------------------------------------------------

TOTAL 14 2 16

============================================================================

Critical Fisher's value (8,8,8) (alpha=0.05) is 4.0. b value is 6. Since b is greater than 4.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

Summary of Fisher's Exact Tests

--------------------------------------------------------------------------- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 8 0
1 0.046 8 0
2 0.48 8 1
3 4.7 8 2

---------------------------------------------------------------------------

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

CONTROL 15 1 16

0.046 15 1 16

---------------------------------------------------------------------------

TOTAL 30 2 32

============================================================================

Critical Fisher's value (16,16,15) (alpha=0.05) is 10.0. b value is 15. Since b is greater than 10.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

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Page 55 of 82 Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

CONTROL 15 1 16

0.48 15 1 16

---------------------------------------------------------------------------

TOTAL 30 2 32

============================================================================

Critical Fisher's value (16,16,15) (alpha=0.05) is 10.0. b value is 15. Since b is greater than 10.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

CONTROL 15 1 16

4.7 11 5 16

---------------------------------------------------------------------------

TOTAL 26 6 32

============================================================================

Critical Fisher's value (16,16,15) (alpha=0.05) is 10.0. b value is 11. Since b is greater than 10.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

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Page 56 of 82 Summary of Fisher's Exact Tests

--------------------------------------------------------------------------- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 16 1
1 0.046 16 1
2 0.48 16 1
3 4.7 16 5

----------------------------------------------------------------------------

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

CONTROL 23 1 24

0.046 23 1 24

---------------------------------------------------------------------------

TOTAL 46 2 48

============================================================================

Critical Fisher's value (24,24,23) (alpha=0.05) is 18.0. b value is 23. Since b is greater than 18.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

CONTROL 23 1 24

0.48 22 2 24

---------------------------------------------------------------------------

TOTAL 45 3 48

============================================================================

Critical Fisher's value (24,24,23) (alpha=0.05) is 18.0. b value is 22. Since b is greater than 18.0 there is no significant difference
between CONTROL and TREATMENT at the 0.05 level.

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Page 57 of 82 Fisher's Exact Test

============================================================================ NUMBER OF

---------------------------------------

IDENTIFICATION ALIVE DEAD TOTAL ANIMALS

-------------------------- ----- ----- --------------

CONTROL 23 1 24

4.7 17 7 24

---------------------------------------------------------------------------

TOTAL 40 8 48

============================================================================

Critical Fisher's value (24,24,23) (alpha=0.05) is 18.0. b value is 17. Since b is less than or equal to 18.0 there is a significant difference
between CONTROL and TREATMENT at the 0.05 level.

Summary of Fisher's Exact Tests

--------------------------------------------------------------------------- NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED DEAD 0.05

----- ------------------------- ----------- ----------- ----- CONTROL 24 1
1 0.046 24 1
2 0.48 24 2
3 4.7 24 7 *

---------------------------------------------------------------------------

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Page 58 of 82 108801 Metolachlor 48617811 890.1350 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
0.961 0.673 0.252 0.859 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.34 0.11 0.06 8.35 1.16, 1.52 Dose1 4 1.41 0.13 0.06 8.97 1.20, 1.61 Dose2 4 1.39 0.13 0.07 9.64 1.17, 1.60 Dose3 4 1.21 0.10 0.05 8.43 1.05, 1.37

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 1.29 1.28 1.51 . .
Dose1 1.43 1.23 1.53 104.66 -4.66
Dose2 1.40 1.23 1.53 103.35 -3.35
Dose3 1.23 1.07 1.31 90.13 9.87

******************************************************************************** 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 2.19 0.142

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 1.34 . 1.38 . . . . . .
Dose1 1.41 0.801 1.38 0.748 . . . . .
Dose2 1.39 0.909 1.38 0.781 0.997 . . . .
Dose3 1.21 0.311 1.21 0.091 0.149 0.206 . . .

******************************************************************************** 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 4.63 0.201

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 1.43 0.676 0.719
Dose2 1.40 0.676 0.722
Dose3 1.23 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)

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Page 59 of 82 ******************************************************************************** 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 2.19 0.142

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 -1.34 . -1.34 . . . . . .
Dose1 -1.41 0.801 -1.34 0.614 . . . . .
Dose2 -1.39 0.909 -1.34 0.649 0.997 . . . .
Dose3 -1.21 0.311 -1.34 0.668 0.149 0.206 . . .

******************************************************************************** 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 4.63 0.201

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 -1.43 0.676 0.281
Dose2 -1.40 0.676 0.278
Dose3 -1.23 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)

108801 Metolachlor 48617811 890.1350 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
0.945 0.419 1.582 0.245 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3.12 0.08 0.04 2.61 2.99, 3.25 Dose1 4 2.97 0.47 0.24 16.00 2.21, 3.72 Dose2 4 3.28 0.53 0.27 16.33 2.42, 4.13 Dose3 4 2.39 0.53 0.27 22.16 1.55, 3.24

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 3.12 3.03 3.22 . .
Dose1 2.99 2.39 3.51 95.04 4.96
Dose2 3.28 2.62 3.93 104.88 -4.88

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 60 of 82 Dose3 2.26 1.97 3.09 76.62 23.38

******************************************************************************** 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 2.98 0.074

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 3.12 . 3.12 . . . . . .
Dose1 2.97 0.927 3.12 0.582 . . . . .
Dose2 3.28 0.930 3.12 0.616 0.767 . . . .
Dose3 2.39 0.096 2.39 0.024 0.312 0.068 . . .

******************************************************************************** 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 6.17 0.104

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 2.99 0.678 0.282
Dose2 3.28 0.346 0.768
Dose3 2.26 0.125 0.104

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 2.98 0.074

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 -3.12 . -2.94 . . . . . .
Dose1 -2.97 0.927 -2.94 0.797 . . . . .
Dose2 -3.28 0.930 -2.94 0.828 0.767 . . . .
Dose3 -2.39 0.096 -2.94 0.844 0.312 0.068 . . .

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

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 61 of 82 3 6.17 0.104

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 -2.99 0.678 0.718
Dose2 -3.28 0.346 0.232
Dose3 -2.26 0.125 0.896

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

108801 Metolachlor 48617811 890.1350 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
0.931 0.254 3.600 0.046 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 49.00 0.82 0.41 1.67 47.70, 50.30 Dose1 4 49.00 0.82 0.41 1.67 47.70, 50.30 Dose2 4 48.75 1.71 0.85 3.50 46.03, 51.47 Dose3 4 47.75 2.06 1.03 4.32 44.47, 51.03

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 49.00 48.00 50.00 . .
Dose1 49.00 48.00 50.00 100.00 0.00
Dose2 48.50 47.00 51.00 99.49 0.51
Dose3 47.50 46.00 50.00 97.45 2.55

******************************************************************************** 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 0.67 0.588

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 49.00 . 49.00 . . . . . .
Dose1 49.00 1.000 49.00 0.583 . . . . .
Dose2 48.75 0.990 48.75 0.511 0.995 . . . .
Dose3 47.75 0.503 47.75 0.163 0.631 0.768 . . .

******************************************************************************** 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 Metolachlor

EPA MRID Number 48617811

Page 62 of 82 Degrees of Freedom TestStat P-value
3 1.04 0.791

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 49.00 . .
Dose1 49.00 1.000 0.500
Dose2 48.50 0.775 0.322
Dose3 47.50 0.568 0.155

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 0.67 0.588

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 -49.00 . -48.63 . . . . . .
Dose1 -49.00 1.000 -48.63 0.726 . . . . .
Dose2 -48.75 0.990 -48.63 0.761 0.995 . . . .
Dose3 -47.75 0.503 -48.63 0.779 0.631 0.768 . . .

******************************************************************************** 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 1.04 0.791

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 -49.00 . .
Dose1 -49.00 1.000 0.500
Dose2 -48.50 0.775 0.678
Dose3 -47.50 0.568 0.845

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

108801 Metolachlor 48617811 890.1350 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. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 63 of 82 Test Stat P-value Test Stat P-value
0.963 0.716 1.579 0.246 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 61.25 0.96 0.48 1.56 59.73, 62.77 Dose1 4 59.75 2.50 1.25 4.18 55.77, 63.73 Dose2 4 62.50 3.42 1.71 5.47 57.06, 67.94 Dose3 4 58.00 1.41 0.71 2.44 55.75, 60.25

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 61.50 60.00 62.00 . .
Dose1 59.50 57.00 63.00 97.55 2.45
Dose2 62.00 59.00 67.00 102.04 -2.04
Dose3 57.50 57.00 60.00 94.69 5.31

******************************************************************************** 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 2.90 0.079

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 61.25 . 61.25 . . . . . .
Dose1 59.75 0.684 61.13 0.550 . . . . .
Dose2 62.50 0.782 61.13 0.584 0.363 . . . .
Dose3 58.00 0.158 58.00 0.042 0.705 0.068 . . .

******************************************************************************** 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 6.58 0.086

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 61.50 . .
Dose1 59.50 0.410 0.153
Dose2 62.00 0.779 0.616
Dose3 57.50 0.079 0.066

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 2.90 0.079

Dunnett - testing each trt mean signif. different than control

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 64 of 82 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 -61.25 . -60.38 . . . . . .
Dose1 -59.75 0.684 -60.38 0.786 . . . . .
Dose2 -62.50 0.782 -60.38 0.818 0.363 . . . .
Dose3 -58.00 0.158 -60.38 0.834 0.705 0.068 . . .

******************************************************************************** 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 6.58 0.086

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 -61.50 . .
Dose1 -59.50 0.410 0.847
Dose2 -62.00 0.779 0.384
Dose3 -57.50 0.079 0.934

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

108801 Metolachlor 48617811 890.1350 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
0.934 0.285 2.381 0.121 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3260750 486506.2 243253.1 14.92 2486610, 4034890 Dose1 4 3294750 597410.2 298705.1 18.13 2344137, 4245363 Dose2 4 3371500 839708.1 419854.0 24.91 2035337, 4707663 Dose3 4 765750.0 460589.7 230294.9 60.15 32848.93, 1498651

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 3122500 2875000 3923000 . .
Dose1 3531500 2423000 3693000 101.04 -1.04
Dose2 3359000 2618000 4150000 103.40 -3.40
Dose3 703000.0 318000.0 1339000 23.48 76.52

******************************************************************************** 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 17.15 <.001

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 65 of 82 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 3260750 . 3309000 . . . . . .
Dose1 3294750 1.000 3309000 0.629 . . . . .
Dose2 3371500 0.988 3309000 0.664 0.998 . . . .
Dose3765750.0 <.001 765750.0 <.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 8.54 0.036

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 3122500 . .
Dose1 3531500 0.889 0.614
Dose2 3359000 1.000 0.670
Dose3 703000.0 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 17.15 <.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 -3260750 . -2673188 . . . . . .
Dose1-3294750 1.000 -2673188 0.946 . . . . .
Dose2-3371500 0.988 -2673188 0.961 0.998 . . . .
Dose3 -765750 <.001 -2673188 0.967 <.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 8.54 0.036

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 -3122500 . .
Dose1 -3531500 0.889 0.386

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 66 of 82 Dose2 -3359000 1.000 0.330
Dose3 -703000 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)

108801 Metolachlor 48617811 890.1350 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
0.824 0.006 2.424 0.116 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 442.50 286.52 143.26 64.75 -13.41, 898.41 Dose1 4 527.50 349.03 174.52 66.17 -27.89, 1082.89 Dose2 4 765.00 1138.79 569.39 148.86 -1047.06, 2577.06 Dose3 4 822.50 1028.12 514.06 125.00 -813.46, 2458.46

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 320.00 260.00 870.00 . .
Dose1 560.00 110.00 880.00 119.21 -19.21
Dose2 245.00 100.00 2470.00 172.88 -72.88
Dose3 430.00 120.00 2310.00 185.88 -85.88

******************************************************************************** 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 0.21 0.888

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 442.50 . 639.38 . . . . . .
Dose1 527.50 0.997 639.38 0.721 . . . . .
Dose2 765.00 0.893 639.38 0.755 0.974 . . . .
Dose3 822.50 0.841 639.38 0.773 0.952 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 0.59 0.899

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 320.00 . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 67 of 82 Dose1 560.00 0.678 0.718
Dose2 245.00 0.413 0.304
Dose3 430.00 0.889 0.337

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 0.21 0.888

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 -442.50 . -442.50 . . . . . .
Dose1 -527.50 0.997 -527.50 0.519 . . . . .
Dose2 -765.00 0.893 -765.00 0.369 0.974 . . . .
Dose3 -822.50 0.841 -822.50 0.340 0.952 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 0.59 0.899

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 -320.00 . .
Dose1 -560.00 0.678 0.282
Dose2 -245.00 0.413 0.696
Dose3 -430.00 0.889 0.663

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

108801 Metolachlor 48617811 890.1350 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
0.965 0.757 12.955 <.001 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 12.58 1.06 0.53 8.45 10.88, 14.27 Dose1 4 12.75 1.41 0.71 11.06 10.51, 14.99

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 68 of 82 Dose2 4 12.78 0.15 0.08 1.17 12.54, 13.01 Dose3 4 12.03 2.59 1.29 21.50 7.91, 16.14

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 13.00 11.00 13.30 . .
Dose1 12.85 11.10 14.20 101.39 -1.39
Dose2 12.80 12.60 12.90 101.59 -1.59
Dose3 12.40 9.10 14.20 95.63 4.37

******************************************************************************** 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 0.20 0.895

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 12.58 . 12.70 . . . . . .
Dose1 12.75 0.997 12.70 0.630 . . . . .
Dose2 12.78 0.996 12.70 0.665 1.000 . . . .
Dose3 12.03 0.925 12.03 0.414 0.912 0.904 . . .

******************************************************************************** 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 0.22 0.974

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 13.00 . .
Dose1 12.85 0.678 0.718
Dose2 12.80 0.484 0.441
Dose3 12.40 1.000 0.425

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 0.20 0.895

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 -12.58 . -12.53 . . . . . .
Dose1 -12.75 0.997 -12.53 0.600 . . . . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 69 of 82 Dose2 -12.78 0.996 -12.53 0.635 1.000 . . . .
Dose3 -12.03 0.925 -12.53 0.653 0.912 0.904 . . .

******************************************************************************** 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 0.22 0.974

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 -13.00 . .
Dose1 -12.85 0.678 0.282
Dose2 -12.80 0.484 0.559
Dose3 -12.40 1.000 0.575

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

108801 Metolachlor 48617811 890.1350 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
0.973 0.883 3.718 0.042 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.45 0.14 0.07 9.76 1.22, 1.67 Dose1 4 1.78 0.48 0.24 26.93 1.02, 2.55 Dose2 4 1.53 0.26 0.13 17.22 1.11, 1.95 Dose3 4 1.87 0.32 0.16 16.93 1.37, 2.37

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 1.43 1.30 1.64 . .
Dose1 1.83 1.24 2.23 123.14 -23.14
Dose2 1.64 1.14 1.71 105.87 -5.87
Dose3 1.78 1.62 2.31 129.19 -29.19

******************************************************************************** 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 1.53 0.257

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 1.45 . 1.66 . . . . . .

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 70 of 82 Dose1 1.78 0.364 1.66 0.882 . . . . .
Dose2 1.53 0.966 1.66 0.906 0.701 . . . .
Dose3 1.87 0.207 1.66 0.917 0.980 0.482 . . .

******************************************************************************** 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 3.93 0.270

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 1.43 . .
Dose1 1.83 0.494 0.807
Dose2 1.64 0.494 0.721
Dose3 1.78 0.103 0.944

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 1.53 0.257

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 -1.45 . -1.45 . . . . . .
Dose1 -1.78 0.364 -1.66 0.226 . . . . .
Dose2 -1.53 0.966 -1.66 0.241 0.701 . . . .
Dose3 -1.87 0.207 -1.87 0.057 0.980 0.482 . . .

******************************************************************************** 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 3.93 0.270

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 -1.43 . .
Dose1 -1.83 0.494 0.193
Dose2 -1.64 0.494 0.279
Dose3 -1.78 0.103 0.056

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

108801 Metolachlor 48617811 890.1350

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 71 of 82 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
Jonckheere Dose1

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 72 of 82 ******************************************************************************** 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

108801 Metolachlor 48617811 890.1350 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
0.903 0.089 5.164 0.016 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 21.13 2.50 1.25 11.81 17.15, 25.10 Dose1 4 19.63 1.49 0.75 7.61 17.25, 22.00 Dose2 4 22.00 4.30 2.15 19.55 15.16, 28.84 Dose3 4 7.88 10.05 5.03 127.66 -8.12, 23.87

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 22.00 17.50 23.00 . .
Dose1 20.00 17.50 21.00 92.90 7.10

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 73 of 82 Dose2 22.25 16.50 27.00 104.14 -4.14
Dose3 5.25 0.00 21.00 37.28 62.72

******************************************************************************** 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 5.43 0.014

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 21.13 . 21.13 . . . . . .
Dose1 19.63 0.965 20.81 0.550 . . . . .
Dose2 22.00 0.992 20.81 0.584 0.932 . . . .
Dose3 7.88 0.016 7.88 0.004 0.053 0.019 . . .

******************************************************************************** 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 6.79 0.079

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 22.00 . .
Dose1 20.00 0.281 0.094
Dose2 22.25 1.000 0.500
Dose3 5.25 0.101 0.041

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 5.43 0.014

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 -21.13 . -17.66 . . . . . .
Dose1 -19.63 0.965 -17.66 0.871 . . . . .
Dose2 -22.00 0.992 -17.66 0.896 0.932 . . . .
Dose3 -7.88 0.016 -17.66 0.908 0.053 0.019 . . .

******************************************************************************** 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 Metolachlor

EPA MRID Number 48617811

Page 74 of 82 Degrees of Freedom TestStat P-value
3 6.79 0.079

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 -22.00 . .
Dose1 -20.00 0.281 0.906
Dose2 -22.25 1.000 0.500
Dose3 -5.25 0.101 0.959

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

108801 Metolachlor 48617811 890.1350 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
0.973 0.890 2.644 0.097 USE PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 27.88 6.15 3.07 22.06 18.09, 37.66 Dose1 4 29.90 5.86 2.93 19.59 20.58, 39.22 Dose2 4 22.00 4.76 2.38 21.63 14.43, 29.57 Dose3 4 0.50 1.00 0.50 200.00 -1.09, 2.09

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 28.70 20.20 33.90 . .
Dose1 28.70 24.20 38.00 107.26 -7.26
Dose2 22.65 16.60 26.10 78.92 21.08
Dose3 0.00 0.00 2.00 1.79 98.21

******************************************************************************** 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 30.31 <.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 27.88 . 28.89 . . . . . .
Dose1 29.90 0.886 28.89 0.700 . . . . .
Dose2 22.00 0.259 22.00 0.072 0.157 . . . .
Dose3 0.50 <.001 0.50 <.001 <.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 75 of 82 Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value
3 10.27 0.016

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 28.70 . .
Dose1 28.70 0.889 0.614
Dose2 22.65 0.346 0.094
Dose3 0.00 0.062 <.001

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 30.31 <.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 -27.88 . -20.07 . . . . . .
Dose1 -29.90 0.886 -20.07 0.992 . . . . .
Dose2 -22.00 0.259 -20.07 0.995 0.157 . . . .
Dose3 -0.50 <.001 -20.07 0.996 <.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 10.27 0.016

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 -28.70 . .
Dose1 -28.70 0.889 0.386
Dose2 -22.65 0.346 0.906
Dose3 0.00 0.062 0.999

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

108801 Metolachlor 48617811 890.1350 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.

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 76 of 82 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion
Test Stat P-value Test Stat P-value
0.587 <.001 8.722 0.002 USE NON-PARAMETRIC TESTS

********************************************************************************

BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 97.93 1.08 0.54 1.11 96.20, 99.65 Dose1 4 98.63 0.39 0.19 0.39 98.01, 99.24 Dose2 4 97.85 0.89 0.44 0.91 96.44, 99.26 Dose3 4 25.00 50.00 25.00 200.00 -54.56, 104.56

Level Median Min Max %of Control(means) %Reduction(means)
Ctrl 98.30 96.40 98.70 . .
Dose1 98.70 98.10 99.00 100.71 -0.71
Dose2 97.75 96.90 99.00 99.92 0.08
Dose3 0.00 0.00 100.00 25.53 74.47

******************************************************************************** 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 8.55 0.003

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 97.93 . 98.28 . . . . . .
Dose1 98.63 1.000 98.28 0.592 . . . . .
Dose2 97.85 1.000 97.85 0.616 1.000 . . . .
Dose3 25.00 0.004 25.00 <.001 0.006 0.007 . . .

******************************************************************************** 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 3.34 0.342

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 98.30 . .
Dose1 98.70 0.343 0.877
Dose2 97.75 1.000 0.471
Dose3 0.00 0.332 0.120

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 8.55 0.003

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811

Page 77 of 82 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 -97.93 . -79.85 . . . . . .
Dose1 -98.63 1.000 -79.85 0.901 . . . . .
Dose2 -97.85 1.000 -79.85 0.923 1.000 . . . .
Dose3 -25.00 0.004 -79.85 0.933 0.006 0.007 . . .

******************************************************************************** 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 3.34 0.342

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 -98.30 . .
Dose1 -98.70 0.343 0.123
Dose2 -97.75 1.000 0.529
Dose3 0.00 0.332 0.880

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 Metolachlor

EPA MRID Number 48617811



## 108801 48617811 890.1350 all fish Male GSI

Page 78 of 82 

### Obs **conc endpoint** 
**1** 

### 0 1.42
**2** 

### 0 1.64
**3** 

### 0 1.30
**4** 

### 0 1.43
**5** 

### 1 1.24
**6** 

### 1 1.52
**7** 

### 1 2.14
**8** 

### 1 2.23
**9** 

### 2 1.14
**10** 

### 2 1.65
**11** 

### 2 1.71
**12** 

### 2 1.63
**13** 

### 3 2.31
**14** 

### 3 1.89
**15** 

### 3 1.62
**16** 

### 3 1.66

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811



## 108801 48617811 890.1350 all fish Male GSI

The NPAR1WAY

Procedure

Page 79 of 82 

### 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 21.0 34.0 8.246211 5.250
**1** 

### 4 37.0 34.0 8.246211 9.250
**2** 

### 4 31.0 34.0 8.246211 7.750
**3** 

### 4 47.0 34.0 8.246211 11.750

**Kruskal-Wallis Test Chi-Square**  

### 3.9265

**DF** 

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

### 0.2695 **Exact Pr >= Chi-Square** 

### 0.2845

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811



## 108801 48617811 890.1350 all fish Male GSI

The NPAR1WAY

Procedure

Page 80 of 82 

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

**conc N**  **Sum of**
**Scores Expecte**  **d**
**Under H 0**  **Std Dev Under H0**  **Mean**
**Score 0**  

### 4 15.0 18.0 3.464102 3.750
**1** 

### 4 21.0 18.0 3.464102 5.250

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

### 15.0000

**Normal Approximation**

**Z** 

### -0.7217
**One-Sided Pr < Z** 

### 0.2352 **Two-Sided Pr > |Z|** 

### 0.4705

**t Approximation**

**One-Sided Pr < Z** 

### 0.2469 **Two-Sided Pr > |Z|** 

### 0.4939

**Exact Test**
**One-Sided Pr <= S** 

### 0.2429 **Two-Sided Pr >= |S - Mean|** 

### 0.4857

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

**Kruskal-Wallis Test Chi-Square**  

### 0.7500

**DF** 

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

### 0.3865

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811



## 108801 48617811 890.1350 all fish Male GSI

The NPAR1WAY

Procedure

Page 81 of 82 

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

**conc N**  **Sum of**
**Scores Expecte**  **d**
**Under H 0**  **Std Dev Under H0**  **Mean**
**Score 0**  

### 4 15.0 18.0 3.464102 3.750
**2** 

### 4 21.0 18.0 3.464102 5.250

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

### 15.0000

**Normal Approximation**

**Z** 

### -0.7217
**One-Sided Pr < Z** 

### 0.2352 **Two-Sided Pr > |Z|** 

### 0.4705

**t Approximation**
**One-Sided Pr < Z** 

### 0.2469 **Two-Sided Pr > |Z|** 

### 0.4939

**Exact Test**
**One-Sided Pr <= S** 

### 0.2429 **Two-Sided Pr >= |S - Mean|** 

### 0.4857

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

**Kruskal-Wallis Test Chi-Square**  

### 0.7500

**DF** 

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

### 0.3865

### Data Evaluation Record on the Fish Short-Term Reproduction Assay with Metolachlor

EPA MRID Number 48617811



## 108801 48617811 890.1350 all fish Male GSI

The NPAR1WAY

Procedure

Page 82 of 82 

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

**conc N**  **Sum of**
**Scores Expecte**  **d**
**Under H 0**  **Std Dev Under H0**  **Mean**
**Score 0**  

### 4 11.0 18.0 3.464102 2.750
**3** 

### 4 25.0 18.0 3.464102 6.250

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

### 11.0000

**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** 

### 0.0433

# DATA EVALUATION RECORD

METOLACHLOR

Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-49-2012 (MRID 48617802)

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: Kelly Luck, M.S. Date: 05/01/2012

Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 5/07/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/09/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/09/2012

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).METOLACHLOR/ 108801



### Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D. 

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

### Health Effects Division In vivo Hershberger Assay (2012) I Page 1 of 13
OCSPP 890.1400/ OECD 441



### S i g n a tur e: 

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___ _..cc,,__ t ...,_1....._ r _.,._. / 1

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- Temp l ate version 10/201 I


### DATA EVALUAT IO N REC ORD

STUDY TYPE: Jn Vivo 

### Hershberger Assay (Rat); OCSPP 890.1400; OECD 441



### PC CODE: 108801 

### D P BA R C OD E: D398926



### TXR#: 0052848 

### CAS#: 51218-45-2



### TEST MATERIAL (PURITY): Metolachlor (97.3% a.i.)



### SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamide



### CITATION: Sloter, E. D

. (2012)

. A Hershberger Assay of Metolachlor Administered Subcutaneously in Peripubertal Orchidoepididymectomized Rats. WIL
Research Laboratories, LLC, Ashland, OH. Laboratory project ID: WIL786010

, January 25, 2012. MRID 48617802. Unpublished.



### SPONSOR: Metolachlor Task Force, 16160 Nall, Stilwell, KS



### TEST ORDER#: CON-108801-12



### EXECUTIVE SUMMARY: In a Hershberger Assay (MRID 48617802) screening for androgenic activity, metolachlor (97.3% a.i., Lot # 20081115) in corn oil was administered daily
via subcutaneous (s.c.) injection to groups of six 54- to 56-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 500 mg / kg / day. An androgenic positive
control group consisted of six castrated rats exposed to 0.2 mg / kg / day of testosterone propionate (TP) by s

. c. injection.

To screen for potential anti-androgenic activity, metolachlor (97.3% a.i.) in com oil was administered daily via s.c. injection to groups of six 54- to 56-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 500 mg / kg / day in conjunction with a daily
dose of reference androgen TP at 0

. 2 mg / kg / day by s.c

. injection

. The anti-androgenic positive
control group consisted of six castrated rats exposed to 0

. 2 mg / kg / day TP (s.c

. injection) and
3 mg / kg / day flutamide (FT, via oral gavage). TP alone was used as the anti-androgenic negative
control.

For both components of the assay, body weights were determined daily. The animals were dosed
for I 

### 0 consecutive days and terminated approximately 24 hours after the final dose
administration. At necropsy, the five androgen-dependent tissues, adrenal glands, kidney, and
liver were collected and weighed. One animal in the 100 mg / kg / day group in the androgen agonist assay was inadvertently given FT on the last day of dose administration; therefore, this
animal was removed from the study.In vivo Hershberger Assay (201 2) / Page 2 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441



### One animal in the 300 mg/kg/day group in th e anti-androgen assay was found dead on Day 10, within one hour of dosing; no macroscopic findi ngs were noted at necropsy. All other rats survived until scheduled sacrifice. No animal s exhibited any dose-rela ted clinical signs of toxicity and there were no treatmen t-related gross pat hological findings.

In the androgen agonist assay, overall body weight gains were decreased compared to controls by 18% (not significant [NS]) and 45% (p <0.01) in the 300 and 500 mg/kg/day groups, respectively. Body weights were also decr eased on Days 6-11 in the 500 mg/kg/day group; however, the decreases were not statistically signif icant. Body weights in the 100 and 300 mg/kg/day dose groups and the positive control group were comparable to controls throughout the duration of the assay. Th ere were no treatment related e ffects in accessory sex organs, adrenal glands, kidneys, or liver for any meto lachlor treatment group. Animals in the positive control group had increased (p<0.01) accessory se x organ weights as follows: 327% in seminal vesicles; 381% in ventral prostate; 132% in levator ani-bulbocavernosus (LABC); 322% in Cowper’s gland; and 53% in glans penis. Ad renal glands, kidney, and liver weights in the positive control group were comparable to the vehicle control.

Percent CVs for the vehicle control and the 500 mg /kg/day treatment groups were compared to the performance criteria in the Guidelines. All %CV values were less than the maximum permissible values, with the ex ception of the %CV for ventral prostate for the 500 mg/kg/day group (CV = 48.7%; maximum permissible CV = 45%).

In the anti-androgen assay, overa ll body weight gains were d ecreased by 16% (NS) and 33% (p<0.01) in the 300 and 500 mg/kg/day groups, compar ed to controls. Body weights were also decreased on Days 6-11 in the 500 mg/kg/da y group; however, the decreases were not statistically significant. B ody weights in the 100 and 300 mg/kg/day dose groups and the positive control group were comparable to controls throughout the duration of the assay. At 500 mg/kg/day, the weights for ventral prostate and LABC were decreased (p<0.05) by 25% and 22%, respectively. LABC weights were also de creased (p<0.05) in the 300 mg/kg/day group ( ↓ 17%). There were no treatment related effects in seminal vesicles, Cowper’s glands, glans penis, adrenal glands, kidneys, or liver for any metolachlor group. Animals dosed with TP + FT (positive control) had decreas ed (p<0.01) accessory sex organ weights as follows: 74% in seminal vesicles; 74% in ventra l prostate; 58% in LABC; 75% in Cowper’s gland; and 33% in glans penis. Adrenal glands, kidney, and liv er weights in the positive control group were comparable to the vehicle control. The perf ormance criteria indicated that this assay was performing as expected.

The high dose of metolach lor selected for this study (500 mg /kg/day) is appropriate because it induced anti-androgenic effects but did not cause significant toxicit y. In addition, reductions in body weight grains of 16-18% (NS) at 300 mg /kg/day and 33-45% (p<0.01) at 500 mg/kg/day indicate that the doses selected were adequate.

Metolachlor was negative for androgenic activity in the Hershberger assay as no statistically significant increases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist assay. Metolachlor was positive for potential anti-androgenic activity in this Hershberger assay as statistically significant decreases in organ weights were seen in two or more of the androgen sensitive tissues in the androg en antagonist assay.In vivo Hershberger Assay (201 2) / Page 3 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441



### The assay satisfies the EDSP Tier 1 Test Order require ments for a Hershberger assay (OCSPP 890.1400).

COMPLIANCE: Signed and dated GLP Compliance, Data Confidentiality, and Quality Assurance statements were provided.In vivo Hershberger Assay (201 2) / Page 4 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH Study Director: E. D. Sloter Other Personnel: E. S. Bodle (Assistant Director, Analytical Chemistry), S. A. Keets (Senior Operations Manager, Vivarium), C. A. Kopp (Manager, Gross Pathology and Developmental Toxicology Laborato ry), G. M. Maginnis (Clinical Veterinarian), T. M. Rafeld (Group Ma nager, Formulations Laboratory), M. J. Schlosser (Vice President, Chemistry Services), B. J. Varsho (Director, Operations), E. C. Vedaa (Manager, Clinical Pathology), R. A. Wally (Operations Manager, Repor ting & Technical Support Services), S. T. Coffee (Contributing Scientist, Analyses of Dosing Formulations), L. Freshwater (BioSTAT Consultants, Statistical Analysis) Study Period: January 4, 2011 - January 25, 2012



### 2. Test Substance: Metolachlor Description: Dark brown, clear liquid Source: Sipcam (Valdosta, GA) Lot #: 20081115 (expiration date 11/15/2013) Purity: 97.3% Stability: Stable in corn oil for 13 days at room temperature CAS #: 51218-45-2 Structure:



### 3. Reference Androgen: Testosterone propionate (TP) Supplier: AK Scientific, Inc. (Mountain View, CA) Lot/Batch #: 7032J (expiration date 8/9/2012) Purity: 98.3% CAS #: 57-85-2



### 4. Reference Anti-androgen: Flutamide (FT) Supplier: Spectrum Chemical Manufacturing Co rporation (New Brunswick, NJ) Lot/Batch #: YV0734 (March 1, 2011) Purity: 100% CAS #: 1311-84-7



### 5. Solvent/Vehicle Control: Corn oil Supplier: Spectrum Chemical Manufacturing Co rporation (New Brunswick, NJ) Lot/Batch #: ZT1301 (expiration date 7/1/2012) Rationale (if other than water): Metolachlor technical is insoluble in water Final concentration: Not applicable CH

3 N O

Cl CH 3

O CH

3

CH 3In vivo Hershberger Assay (201 2) / Page 5 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441



### 6. Test Animals: Species: Rat (castrated males only) Strain: Sprague Dawley Age/weight at dose initiation: Post-natal day (PND) 54-56/ 227.4 – 301.4 g Source: Charles River Laboratories, Inc. (Raleigh, NC) Housing: Rats were individually hous ed in stainless steel wi re-mesh cages suspended above cage board. Diet: Certified Rodent LabDiet® 5002, PMI International , ad libitum Phytoestrogen content was not reported
Water: Reverse-osmosis purified drinking water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 21.3-21.7 oC (mean daily temperature) 34.6-48.8% (mean daily humidity) 10/hr (minimum) 12 hrs light/12 hrs dark Acclimation period: Rats were castrated at the supplier and received at the facility approximately one week post-castration; rats were th en allowed a 6-day acclimation period at the facility



### B. STUDY DESIGN

1. In life dates: Start: January 10, 2011 End: January 21, 2011

2. Study Design: In a Hershberger Assay conducted to screen for pot ential androgenic activity, the test substance was administered daily via s.c. injection to castrated male rats. Positive androgenic activity is defined as a signif icant increase in two or more target organ weights compared to the vehicle control. To screen for the po tential anti-androgenic activity, the test substance was also administered daily via s.c. injection to castrated male rats in conjunction with a daily dose of TP (0.2 mg/kg/day) by s.c. injection. 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 organ weight

measurements.

3. Study Schedule: Male rats were castrated on PND 42 by the supplier and were received at the test facility approximately one week fo llowing castration, at PND 48-49 (rats were born over two days but were not kept separate after receipt at facil ity; therefore, exact age of each rat was not known). The animals were acclimated at the facility for at least 6 days prior to initiation of dosing. The dose administra tion period was from PND 54/55 through PND 63/64 for half the rats in each group, a nd from PND 55/56 through PND 64/65 for the remaining rats in each group. Rats were euth anized approximately 24 hours after the last dose and necropsied for organ weight measurements.

4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1. Statistical anal ysis indicated that th ere were no significant differences in group mean weight s at study initiation. Furtherm ore, the body weight of each
animal was within  20% of the group mean.In vivo Hershberger Assay (201 2) / Page 6 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Males Androgen Agonist Assay Vehicle control (negative control) 0 6 Low (Metolachlor) 100 6 Mid (Metolachlor) 300 6 High (Metolachlor) 500 6 Testosterone propionate (TP), positive control b 0.2 6 Anti-Androgen Assay Low (Metolachlor + TP) 100 + 0.2 6 Mid (Metolachlor + TP) 300 + 0.2 6 High (Metolachlor + TP) 500 + 0.2 6 Flutamide + TP, positive control 3 + 0.2 6 a Data were obtained from page 24 of the study report. b This dose group served as the positive control for the androgen agonist assay and the negative control for the anti-androgen assay.



### 5. Dose Selection Rationale: The dose levels used in this study were chosen based on the results of published toxicity da ta (Reregistration Eligibility Decision Metolachlor, U.S. EPA, 1995) and a previous study. 1 

### No discussion of the results of the previous study was provided.

6. (a) Dose Preparation: Dose formulations were prepar ed twice during the study as single formulations for each dose level, by mixing appr opriate amounts of test substance with corn oil. Formulations of TP and FT were prep ared once by dissolving the material in a small amount of 95% ethanol and dilu ting to volume with corn oil. Analyses to demonstrate homogeneity and stability were conducted with a concurrent uterotrophic study (MRID 48617801, concurrently reviewed) for dose formul ations at 20 and 200 mg/mL following up to 13 days of room temperature storage. During the study, samples of each test substance dosing formulation (middle stratum of each) prep ared during the in-life phase were analyzed for achieved concentration.

( b) Dose Analysis:

Results

Homogeneity (% CV): 1.1-2.5%

Stability (% of Day 0): 92.5% and 97.9% after 13 days of storage at room temperature

Concentration (% of nominal): 109-112%

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.

1 Sloter, E.D. A Hershberger Assay of Metolachlor Administered Subcutaneously in Peripubertal Orchidoepididymectomized Rats (Study No. WIL-786002). WIL Research Laboratories, LLC, Ashland, OH, DraftIn vivo Hershberger Assay (201 2) / Page 7 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441



### 7. Dosage administration: Test formulations were administered to the animals daily via s.c. injection (5 mL/kg bw) for 10 days. TP was gi ven via s.c. injection at 0.5 mL/kg bw, and FT was administered via oral gavage at 10 mL/kg bw. Dose volumes were adjusted daily based on the concurrent body weight measurement.

8. Statistics: Statistical analyses we re conducted for organ weight s, daily body weights, and body weight gains. Each endpoint was tested for homogeneity of variance using Levene's test. If that test was sign ificant at p=0.01, then a log transformation was applied and Levene's test conducted on the transformed data. If that test was still significant, then the square root transformation was applied to the raw data (except cumulative body weight gain) and Levene's test conducted again. If the test was still significant, then a nonparametric test, as described below, was used to analyze the data. One-sided tests were conducted for organ weight data and two-si ded tests were conducted for in-life data.

If variances were homogeneous, the one- or tw o-sided t-test (for comparing positive control data to data for negative cont rol) or an ANOVA (for comparing dose groups to the negative control) was performed on da ta; the ANOVA test was follo wed by a one- or two-sided Dunnett’s test. If the tran sformations were unsuccessful in making the variances homogeneous, the nonparametric one- or two- sided Wilcoxon rank sum test was used to compare data for the positive control to the negative control. For comparison of dose groups to the negative control, if the tran sformations were unsuccessful in making the variances homogeneous, the nonparametric Kruskal-Wallis test was used, followed by a one- or two-sided Dunn's test. Significance wa s denoted at p<0.05. Statistical analyses were performed using SAS software (version 9.2 or higher). The statis tical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortalit y and moribundity were conducted twice daily. Individu al clinical observati ons (hand-held physical examinations) were recorded daily through termination. Each rat wa s also observed for signs of toxicity at the time of dosing and approximately 1 hour following dosing.

2. Body Weight: Animals were weighed at rando mization, daily thr oughout the dosing period, and on the day of termination. Mean bo dy weight changes were calculated for each corresponding interval and also for the overall dosing period (Days 1-11).

3. Food Consumption (Optional): Food consumption was not measured.

4. Serum Hormone Measurements (Optional): At study termination, each animal was anesthetized with isoflurane, a nd blood was collected from the vena cava for potential future serum hormone analyses; however, serum hormo ne analyses were not conducted for this study.

5. Dissection and Measurement of Tissue and Organ Weights: On PND 64 to 66 (approximately 24 hours after the final administ ration of the test s ubstance), all animals were anesthetized with isoflurane, exsangui nated, and examined. The five mandatory androgen-dependent organs (ven tral prostate, seminal vesicl es with coagulating glands,In vivo Hershberger Assay (201 2) / Page 8 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441



### LABC, Cowper’s gland, and glans penis) we re excised and weighed fresh (unfixed) according to the standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400). Additionally, the liver, kidneys, and adrenals were weighed. The tissues and organs were placed in 10% ne utral buffered formalin.

II. RESULTS

A. OBSERVATIONS

1. Mortality: One animal in the 300 mg/kg/day group in the anti-androgen assay (Animal No. 92315) was found dead on Day 10, within one hour of dosing. Within 15 minutes of dosing on that day, the rat was prostrate, gasping, convulsing, and had labored respiration. No macroscopic findings were noted at necropsy. All other rats surv ived until scheduled sacrifice. The death of this animal does not a ffect the validity of this study as a screening assay because no other rats were found dead or exhibited similar clinical findings.

One animal in the 100 mg/kg/day group in the androgen agonist assay (Animal No. 92317) was inadvertently given FT on the last day of dos e administration; therefore, this animal was removed from the study resulting in only 5 males in that dose group.

2. Clinical signs of toxicity: No treatment-related clinical si gns of toxicity were observed in animals for any dose groups. Any clinical findings noted in th e dose groups occurred infrequently and in a manner that was not dose related.

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data for the androgen agonist assay are presen ted in Table 2. Overall body weight gains (Days 1-11) were decreased by 20% (NS), 18% (NS) and 45% (p<0.01) in the 100, 300 and 500 mg/kg/day groups, respectively, compared to controls. Body weights in the 500 mg/kg/day group were also decreased by 6% and 10% on Days 6 and 11; however, the decreases were not statistically significant. Body weights in the 100 and 300 mg/kg/day dose groups and the positive control group were comparable to controls throughout the duration of the assay.In vivo Hershberger Assay (201 2) / Page 9 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441

TABLE 2. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Androgen Agonist Assay a

Study Day # Dose (mg/kg/day)

Vehicle Control Positive Control Vehicle + TP (0.2) Metolachlor (100) Metolachlor (300) Metolachlor (500)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 6 263.4 22.4 6 263.8 26.5 5 267. 7 23.8 6 261.9 26.4 6 262.7 27.4
4 6 283.2 24.4 6 282.5 23.2 5 288. 6 25.2 6 279.1 25.1 6 275.2 28.0
7 6 310.1 22.4 6 309.7 21.0 5 309. 4 24.0 6 300.9 24.1 6 290.1 28.2
11 6 336.9 23.5 6 339.0 18.2 5 326. 8 37.2 6 322.2 22.9 6 302.8 33.4

B ody Weight Gain (Days 1-11) 6 73.5 12.8 6 75.1 11.0 5 59.1 ( ↓ 20) 29.8 6 60.4 ( ↓ 18) 14.6 6 40.1** ( ↓ 45) 9.4

a Data were obtained from Tables S3, S4, and S6 on pages 49 -51, 54, and 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 ** Significantly different from controls at p<0.01



### Selected body weight and body weight gain data for the anti-androgen assay are presented in Table 3. Overall body weight gains (Days 1- 11) were decreased by 16% (NS) and 33% (p<0.01) in the 300 and 500 mg/kg/day groups, re spectively, compared to controls. Body weights were also decreased by on Da ys 6-11 in the 500 mg/kg/day group ( ↓ 5% on Day 6 to ↓ 7% on Day 11); however, the decreases were no t statistically signifi cant. Body weights and body weight gains in the 100 and 300 mg/kg/day dose groups and the positive control group were comparable to controls throughout the duration of the assay.

TABLE 3. Selected Group Mean Body Weights an d Cumulative Body Weight Gains (g) in the Anti-Androgen Assay a

Study Day # Dose (mg/kg/day)
Vehicle Control + TP (0.2) Positive Control TP + FT (0.2 + 3) Metolachlor + TP (100 + 0.2) Metolachlor + TP (300 + 0.2) Metolachlor + TP (500 + 0.2)
N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 6 263.8 26.5 6 258.6 24.6 6 262. 5 26.4 6 260.7 24.1 6 262.5 23.4
4 6 282.5 23.2 6 273.8 26.7 6 284. 4 26.1 6 277.4 24.1 6 272.8 22.4
7 6 309.7 21.0 6 297.0 24.3 6 308. 3 24.7 6 300.3 22.7 6 294.0 20.9
11 6 339.0 18.2 6 323.7 26.2 6 339. 2 25.3 5 328.6 25.1 6 312.9 20.2

B ody Weight Gain (Days 1-11) 6 75.1 11.0 6 65.1 5.2 6 76.7 ( ↑ 2) 6.6 5 63.2 ( ↓ 16) 7.4 6 50.4** ( ↓ 33) 14.8

a Data were obtained from Tables S3, S4, and S8 on pages 49 -51, 54, and 66 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<0.01



### C. FOOD CONSUMPTION (Optional): Food consumption was not measured.

D. SERUM HORMONE CONCE NTRATIONS (Optional): Serum hormone concentrations were not measured.

E. ORGAN WEIGHTS: Accessory sex organ, adrenal gl ands, kidney, and liver weights for the androgen agonist assay are presented in Ta ble 4. There were no tr eatment related effects in accessory sex organs, adrenal glands, kidne ys, or liver for any metolachlor treatmentIn vivo Hershberger Assay (201 2) / Page 10 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441



### group. Animals in the TP group had increased (p<0.01) accessory sex organ weights as follows: 327% in seminal vesicles; 381% in ventral prostate; 132% in LABC; 322% in Cowper’s gland; and 53% in glans penis. Ad renal glands, kidney, and liver weights in the positive control group were comparable to the vehicle control.

Percent CVs for the vehicle control and the 500 mg/kg/day treatment groups were compared to the performance criteria in the Guidelines. All %CV values were less than the maximum permissible values, with th e exception of the %CV for ventral prostate for the 500 mg/kg/day group (CV = 48.7%; maximu m permissible CV = 45%).

There were no treatment-relate d gross pathological findings.

TABLE 4. Accessory Sex Organ, Adrena l Glands, Kidney, and Liver Weights from Androgen Agonist Assay in Sprague Dawley Rats a

Organ Dose (mg/kg/day)

Vehicle Control Positive Control Vehicle + TP (0.2) Metolachlor (100) Metolachlor (300) Metolachlor (500)

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)
Seminal vesicles (mg) 6 100.4 11.1 11.0 6 429.0** ( ↑ 327) 42.0 9.8 5 100.0 12.3 12.3 6 98. 0 15.4 15.7 6 89.6 9.8 11.0

Ventral prostate (mg) 6 23.6 4.7 19.8 6 113.4** ( ↑ 381) 7.0 6.2 5 19.2 8.6 44.6 6 21. 5 5.1 23.5 6 19.1 9.3 48.7

LABC (mg) 6 195.7 33.4 17.1 6 455.0** ( ↑ 132) 48.9 10.8 5 167.8 23.0 13.7 6 152. 8 22.6 14.8 6 151.2 24.8 16.4

Cowper’s glands (mg) 6 7.8 1.6 20.3 6 32.9** ( ↑ 322) 4.8 14.7 5 8.1 0.7 9.2 6 6.2 1.1 17.8 6 6.8 0.9 13.0

Glans penis (mg) 6 94.1 9.1 9.7 6 144.1** ( ↑ 53) 16.5 11.5 5 102.2 24.1 23.5 6 88. 9 10.1 11.4 6 82.6 30.0 36.4

Adrenal glands (mg) 6 66.8 13.3 19.9 6 59.8 5.3 8.8 5 57.6 10.1 17.4 6 57.9 7.3 12.6 6 54.8 9.6 17.5

Kidneys (g) 6 2.062 0.259 12.6 6 2.058 0.133 6.5 5 2.002 0.115 5.7 6 2.17 0.259 11.9 6 2.101 0.302 14.4

Liver (g) 6 12.9 1.19 9.2 6 13.3 0.92 6.9 5 12.9 1.67 13.0 6 14.2 0.94 6.7 6 13.2 2.54 19.2
a Data were obtained from Tables S15 and S18 on pages 81, 82, and 89-91 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<0.01In vivo Hershberger Assay (201 2) / Page 11 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441



### Accessory sex organ, adrenal glands, kidney, an d liver weights for the anti-androgen assay are presented in Table 5. At 500 mg/kg/da y, the weights for ventral prostate and LABC were decreased (p<0.05) by 25% and 22%, respectively. LABC weights were also decreased (p<0.05) in the 300 mg/kg/day group ( ↓ 17%). Adrenal glands weights were decreased (p<0.05) in the 300 mg/kg/day group ( ↓ 19%); however, because significant decreases were not observed in the 500 mg /kg/day group, these differences were not considered to be dose related. There were no treatment related effects in seminal vesicles, Cowper’s glands, glans penis, kidneys, or liver for any metolachlor treatment group. Animals dosed with TP + FT (positive contro l) had decreased (p<0.01) accessory sex organ weights as follows: 74% in seminal vesicles ; 74% in ventral prostate; 58% in LABC; 75% in Cowper’s gland; and 33% in glans penis. Adrenal glands, kidney, and liver weights in the positive control group were comparable to the vehicle control.

Percent CVs for the vehicle control and the 500 mg/kg/day treatment groups were compared to the performance criteria in the Guidelines. All %CV values were less than the maximum permissible values.

There were no treatment-relate d gross pathological findings.

TABLE 5. Accessory Sex Organ, Adre nal Glands, Kidney, and Liver Weig hts from Anti-Androgen Assay in Sprague Dawley Rats a

Organ Dose (mg/kg/day)
Vehicle Control + TP (0.2) Positive Control TP + FT (0.2 + 3) Metolachlor + TP (100 + 0.2) Metolachlor + TP (300 + 0.2) Metolachlor + TP (500 + 0.2)
N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Seminal vesicles (mg) 6 429.0 42.0 9.8 6 113.0** ( ↓ 74) 14.6 12.9 6 392.2 49.5 12.6 5 376.4 95.9 25.5 6 355.8 51.5 14.5

Ventral prostate (mg) 6 113.4 7.0 6.2 6 29.4** ( ↓ 74) 9.5 32.3 6 106.3 17.2 16.1 5 90.6 17.6 19.5 6 84.8* ( ↓ 25) 24.9 29.4

LABC (mg) 6 455.0 48.9 10.8 6 189.0** ( ↓ 58) 34.8 18.4 6 410.9 65.7 16.0 5 376.0* ( ↓ 17) 59.1 15.7 6 354.3** ( ↓ 22) 45.3 12.8

Cowper’s glands (mg) 6 32.9 4.8 14.7 6 8.1** ( ↓ 75) 1.7 20.7 6 29.1 8.2 28.3 5 27.4 6.0 22.0 6 26.6 6.8 25.5

Glans penis (mg) 6 144.1 16.5 11.5 6 96.1** ( ↓ 33) 7.5 7.8 6 141.9 12.1 8.5 5 144. 1 8.5 5.9 6 134.3 7.9 5.9

Adrenal glands (mg) 6 59.8 5.3 8.8 6 56.2 10.8 19.2 6 59.2 7.7 13.0 5 48.5* ( ↓ 19) 9.0 18.5 6 51.2 6.2 12.2

Kidneys (g) 6 2.058 0.133 6.5 6 1.974 0.128 6.5 6 2 .130 0.080 3.8 5 2.057 0.100 4.8 6 2.143 0.074 3.5 Liver (g) 6 13.3 0.92 6.9 6 13.7 1.94 14.2 6 13. 5 0.99 7.4 5 13.6 1.32 9.8 6 13.8 0.94 6.8 a Data were obtained from Tables S15, S 17, and S19 on pages 81, 82, 86-88, and 92-94 of the study report. Percent differences from controls, calculated by the review ers, 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<0.01In vivo Hershberger Assay (201 2) / Page 12 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the results of this study, statistically significant lower weights of ≥ 2 androgen-dependent organs in males given 500 mg/kg/day with TP suggested an anti-androgenic res ponse when administered by subcutaneous injection to peripubertal, castrat ed male Crl:CD(SD) rats. No anti-androgenic effects were noted at 100 and 300 mg/kg/day. No androgeni c activity was demonstrated at metolachlor dosage levels up to 500 mg/kg/day, the highest dosage level of metolachlor tested, as evidenced by the lack of increase in the wei ghts of the androgen-dependent organs relative to the vehicle control group. The anti-androgenic positive control substance, flutamide, generally elicited the expected reduction in androgen-dependent organ weights. Theandrogenic positive control substance, TP, elicited the expected increase in androgendependent organ weights.

B. AGENCY COMMENTS: One animal in the 300 mg/ kg/day group in the anti-androgen assay was found dead on Day 10, within one hour of dosing; no macros copic findings were noted at necropsy. All other rats survived unt il scheduled sacrifice. The death of this animal does not affect the valid ity of this study as a screeni ng assay because no other rats were found dead or exhibited similar clinical findings. One animal in the 100 mg/kg/day group in the androgen agonist as say was inadvertently given FT on the last day of dose administration; therefore, this animal was removed from the study. No animals exhibited any dose-related clinical signs of toxicology and there were no treatment-related gross pathological findings.

In the androgen agonist assay, overall body weig ht gains were decreased by 18% (NS) and 45% (p<0.01) in the 300 and 500 mg/kg/day gr oups, respectively, compared to controls. Body weights were also decreased on Days 6-11 in the 500 mg/kg/day group ( ↓ 6% on Day 6

to ↓ 10% on Day 11); however, the decreases we re not statistically significant. Body weights in the 100 and 300 mg/kg/day dose gr oups and the positive control group were comparable to controls throughout the durati on of the assay. Th ere were no treatment related effects in accessory sex organs, adrena l glands, kidneys, or liver for any metolachlor treatment group. Animals in the positive contro l group had the expected increases accessory sex organ weights. Adrenal glands, kidney, a nd liver weights in the positive control group were comparable to the vehicle control.

Percent CVs for the vehicle control and the 500 mg/kg/day treatment groups were compared to the performance criteria in the Guidelines. All %CV values were less than the maximum permissible values, with th e exception of the %CV for ventral prostate for the 500 mg/kg/day group (CV = 48.7%; maxi mum permissible CV = 45%).

In the anti-androgen assay, overa ll body weight gains were d ecreased by 16% (NS) and 33% (p<0.01) in the 300 and 500 mg/kg/day groups, re spectively, compared to controls. Body weights were also decreased by on Da ys 6-11 in the 500 mg/kg/day group ( ↓ 5% on Day 6 to ↓ 7% on Day 11); however, the decreases were not statistically si gnificant. Body weights in the 100 and 300 mg/kg/day dose groups and the positive control group were comparable to controls throughout the duration of the assay. At 500 mg/kg/day, the weights for ventral prostate and LABC were decr eased (p<0.05) by 25% and 22%, respectively. LABC weights were also decreased (p<0.05) in the 300 mg/kg/day group ( ↓ 17%). There were no treatmentIn vivo Hershberger Assay (201 2) / Page 13 of 13 METOLACHLOR/ 108801 OCSPP 890.1400/ OECD 441



### related effects in seminal vesicles, Cowper’s gl ands, glans penis, adrena l glands, kidneys, or liver for any metolachlor group. Animals dosed with TP + FT (positive control) had the expected decreases in accessory sex organ we ights. Adrenal glands, kidney, and liver weights in the positive control group were comparable to the vehicle control. The performance criteria indicated that this assay was performing as expected.

The high dose level tested in the study (500 mg /kg/day) is appropriate because it induced anti-androgenic effects but did not cause signi ficant toxicity. A dditional support for the high dose selected includes reductions in body weight grains of 16-18% (NS) at 300 mg/kg/day and 33-45% (p< 0.01) at 500 mg/kg/day.

No statistically significant increases in organ weights were seen in two or more of the five androgen responsive tissues in the androgen a gonist assay. Metolachlor was negative for androgenic activity. Statistically significant de creases in tissue weights were seen in LABC and ventral prostate in the androgen antagonist assay. Metolachlor was positive for potential anti-androgenic activity in this Hershberger assay

C. STUDY DEFICIENCIES: None

# DATA EVALUATION RECORD

METOLACHLOR

Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-49-2012 (MRID 48617804)

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: Joseph Rieth, Ph.D., D.A.B.T. Date: 5/9/2012

Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 5/11/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/16/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/16/2012

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).METOLACHLOR/ 108801 Fema l e Puberta l Assay (2012)/ Page I of 15
OCSPP 890.1450/ OECD None



### Primary Reviewer: Ayaad Assaad, D.V

. M, Ph.D 

### Health Effects Division Secondary Reviewer: Elizabeth Mendez, Ph.D.


### Health Effects Division

DAT A EVALUAT IO N REC OR D

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None.



### PC CODE: 108801 

### DP BA R C OD E: D398926



### TXR#: 0052848 CAS No: 51218-45-2



### TEST MATERIAL (PURITY): Metolachlor Technical (97.3% a.i.)



### SYN 0 NYMS: 2-chloro-N-(2-ethyl-6-methyl pheny l)-N-(2-methoxy- l -methylethy l)acetamide



### CITATION: Sloter, E

. D. (2012) Pubertal Development and Thyroid Function in Intact
Juvenile / Peripubertal Female Rats Following Oral Administration of
Metolachlor

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

, January 25

, 2012. MRID 48617804. Unpublished.



### SPONSOR: Metolachlor Task Force

, 16160 Nall, Stilwell, KS



### TEST ORDER#: CON 108801-112



### EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48617804)

, 15 Crl:CD(SD) Sprague-Dawley rats / dose group were treated daily via oral gavage (5 mL/kg) with metolachlor
technical (97.3% a.i., Lot # 20081115) in com oil at doses ofO, 300 or 600 mg/kg / day from postnatal day (PND) 22 to 42

. Animals were examined for vaginal opening daily beginning on PND 22, and weight at day of attainment was recorded. Following sacrifice on PND 42, total thyroxine (T4) and thyroid stimulating hormone (TSH) levels were analyzed using an electrochemiluminescent immunoassay (T4) and a magnetic 

## [1 2 5 

### I]rTSH gamma counter immunoassay (TSH). Kidneys, liver, adrenal glands, thyroid

, pituitary, and ovary weights were recorded

, and microscopic examination of the thyroid

, kidneys, ovary and uterus were performed.

One animal in the control group was sacrificed 

### in extremis on PND 25 due to impairment of the
left forelimb. All other rats survived until scheduled sacrifice

. No treatment-related clinical findings were noted during the daily examinations at any dose level. There were no significant
differences in body weight at vaginal opening or final body weights in the treated groups relative

to controls. Adjusted age at VO was significantly earlier at 300 mg / kg / day (p < 0.05) but not at 600 mg / kg / day. One female in the control group failed to attain vaginal opening. There were no statistically significant differences in mean age at first vaginal estrous or mean cycle length. The

c y cle status at necropsy was similar among groups. Serum TSH was not affected by treatment.Female Pubertal Assay (2012)/ Page 2 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None



### Unadjusted, adjusted and relative liver wei ghts were increased at 600 mg/kg/day (p<0.05, ↑ 20- 22%); only relative liver we ight was significantly increa sed at 300 mg/kg/day (p<0.05, ↑ 9%). Unadjusted, adjusted and relative kidney we ights were increased at 600 mg/kg/day (p<0.05, ↑ 8- 10%). No other effects on organ weights were reported. The percent of females cycling (93.3%) and regularly cycling (77.8%) were lower at 600 mg/kg/day (p<0.05) than the controls (100% for both). Percent regularly cyc ling was also reduced at 300 mg /kg/day (p<0.05, 53.8%). There were dose-related increases in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, respectively; the increa se was significant (p<0.05) at 600 mg/kg/day only. Mean globulin levels were significantly higher in the 300 mg/kg/day (p<0.05, ↑ 7%) and 600 mg/kg/day (p<0.05, ↑ 7%) groups; there were corre sponding reductions in A/ G ratios of 8% and 12%, respectively, with only the high dos e being significant (p <0.05). GGT was significantly elevated at 600 mg/kg/day (p<0.05, ↑ 700%).

There was an increased incidence of Grade 2 follicular cell height at 600 (11/14) mg/kg/day relative to control (4/14); also, one animal at the high dose was observed with Grade 3. There was an increased incidence of Grade 4 colloid ar ea at 600 mg/kg/day (2/14, 4/15 and 11/14 in the control, 300 and 600 mg/kg/day respectively). Th e differences in follicular cell height and colloid area were significant at 600 mg/kg/day (p=0.005). Th e ovaries appeared normal histopathologically. There were no microscopic findings in the uter us related to treatment with the test material. Renal tubular cysts were in creased in the 300 and 600 mg/kg/day groups (1/14, 2/15 and 4/15 in the 300 and 600 mg/ kg/day groups, respectively).

No effects were observed on body weights or body weight gain in the study. Increased liver weights accompanied by elevated GGT levels an d increased kidney weights demonstrate organ toxicity as a result of trea tment. However, histopathology was not conducted on the liver and microscopic findings in the kidney were minimal, so the degree of toxicity in these organs is unclear. Age at vaginal openi ng was not affected by treatment. There was a dose-related increase in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, respectively; the increase was significant (p<0.05) at 600 mg/kg/day only. Serum TSH was unaffected by treatment. The dosing levels tested in this assay are adequate based on systemic toxicity (increased liver and kidney weights) at the high dose.

The assay satisfies the EDSP Tier 1 Test Order require ments for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.Female Pubertal Assay (2012)/ Page 3 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH Study Director: E. D. Sloter
Other Personnel: J. B. Nold, Pathologist L. Freshwater, Statistics Study Period: July 29, 2010 – January 25, 2012



### 2. Test Substance: Metolachlor
Description: Dark brown, clear liquid, MW 283.79
Source: Sipcam (Valdosta, GA)
Lot/Batch #: 20081115 (exp. November 15, 2013) Purity: 97.3% Stability: Stable in corn oil for 13 days at room temperature
CAS #: 51218-45-2
Structure:

CH 3

CH 3 N
O CH 3 O Cl

CH 3



### 3. Vehicle: Corn oil

4. Test Animals:
Species: Rat
Strain: Crl:CD(SD) Sprague-Dawley Age/Weight at Study Initiation: PND [22]/47.0 – 61.6 g females only
Source: Charles River Laboratories; Raleigh, NC Housing: Two-three/cage in plastic cages with heat treat ed laboratory-grade pine shavings for bedding.
Diet: Harlan Laboratories 2016CM Teklad Global 16% protein rodent diet, ad libitum Phytoestrogen content average 10 ppm tota l isoflavones (genistein+daidzein+glycitein)
Water: Municipal water, reverse-os mosis filtered (on site), ad libitum
Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 22 ± 3 °C
50 ± 20%
10/hr 14 hrs light/ 10 hrs dark



### B. STUDY DESIGN

1. In-Life Dates: Start: August 28, 2010 End: September 18, 2010

2. Mating: Time-mated pregnant dams were receive d from the supplier on gestation days (GD) 11 or 12. The day an animal was found to be sperm positive was designated GD 0. Litters with more than 8 pups were standardi zed by culling on PND 4 to 8 pups per litter.Female Pubertal Assay (2012)/ Page 4 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None



### 3. Animal Assignment: Following weaning on PND 21, animals were assigned to the test groups noted in Table 1 using a randomiza tion procedure designed to ensure equal stratification of body weights. Littermates were not assigned to th e same treatment group.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Females
Control 0 15
Low 300 15 High 600 15 a Data were obtained from page 26 of the study report.



### 4. Dose Selection Rationale: It was stated that the dose le vels were selected based on the results of published toxicity studies ( no further details were provided).

5. Dose Preparation and Analysis: Studies on the homogeneity and stability of the test substance in formulations were conducte d in a previous study (MRID 48617801, reviewed concurrently) 1 

### and the results are reported below. Concentration of the test material as administered to the study animals was assess ed pre-study and once dur ing the in-life phase of the study.

Results of Dose Analysis

Homogeneity (% RSD): 1.8-2.5%

Stability (% of initial): 92.5-97.9% following storage for 13 days at room temperature.

Concentration: The formulated concentration of th e test material ranged from 101% to 103% of nominal.

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: All doses were administered once daily by oral gavage, from PND 22 through PND 42, in a volume of 5 mL /kg of body weight. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: Statistics were conducted at WIL Research and at BioSTAT Consultants, Inc. At WIL, Cycling status (cyc ling vs. non-cycling) and per cent of animals cycling were assessed using Chi-square analysis. Estrous cycle length and day of first estrus were analyzed using a one-way analysis of vari ance (ANOVA) followed by Dunnett’s test when intergroup differences were indicated by ANOVA. Dichotomous pathology data were analyzed with pairwise Fisher’s exac t test. Tests were conducted at the p ≤ 0.05 significance level.

1 Sloter, E.D. (2012) A Uterotrophic Assay of Metolachlor Administered Subcutaneously in Ovariectomized Rats Study No. WIL-786001. WIL Research Laboratories, LLC, Ashland, OH.Female Pubertal Assay (2012)/ Page 5 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None



### At BioSTAT, the data that were statistical ly analyzed included organ weights (liver, kidneys, pituitary and adrenals), organ wei ght to final body weight ratios, daily body weights, weight gains, serum chemistries, hormones and vaginal opening data. Each endpoint was tested for homogeneity of variance using Levene’s test. If this was positive (p ≤ 0.01) then log or square root transformations were applied to the data; if these were positive then a non-parametric test was used. Organ weights, age at vaginal opening and weight at vaginal opening data, when para metric, were analyzed using analysis of covariance (ANCOVA) followed by Dunnett’s test using PND 21 body weights as the covariate. Other parameters were anal yzed with ANOVA followed by Dunnett’s test. Linear trend tests were used in conj unction with both ANOV A and ANCOVA. For nonparametric data the Kruskal-Wallis test was used followed by Dunn’s test. Pathology data presented as a graded response were analy zed with a pairwise Mann-Whitney U test. Dichotomous histopathology data were an alyzed using a Fish er’s exact test.

C. METHODS

1. Mortality and Clin ical Examinations: All animals were examined twice daily for mortality and moribundity. Clinical exam inations were conducted on the day of randomization and daily thr ough the day of euthanasia.

2. Body Weight: Animals were weighed on the day of randomization, daily prior to dosing and the day of euthanasia.

3. Vaginal Opening: Beginning on PND 22, all animals were examined daily for onset of vaginal opening. The appearance of a small “pin hole”, a vaginal thread, and complete vaginal opening were recorded for all days th ey were observed. Age and weight on the day of completion of vagina l opening were recorded.

4. Estrous Cyclicity: Beginning on the day of vaginal ope ning, up to and including the day of necropsy, daily vaginal smears were obtained and evaluated to determine the stage of estrus for each female. Cycle length was determined for a complete estrous cycle by counting the number of days from one diestr us to the next diestrus. The overall pattern for each female was characterized as regular, irregular, non-cycling or insuffici ent data. The definitions for these terms are as follows:

 Regular cycling (RC): the animal has at leas t 6 days of data collected and displays 1 complete cycle with no cycles greater than 5 days of duration, no cycles with 3 or more days of proestrus (P) and/or estrus (E), and no cycles less than 4 days in duration.

 Irregular cycling (IC): 1) the animal does not display 1 complete cycle but has at least 1 E and/or P and a partial cycle greater than 5 days, 2) the animal has at least 1 E and either at least 1 cycle (complete or partia l) greater than 5 days in durat ion or at least 1 cycle with 3 or more consecutive days of P and/or E, 3) th e animal has at least 1 cycle less than 4 days in duration, or 4) the animal has 1 irregular cycle and 1 regular cycle.

 Non-cycling (NC): no E or P present on any da ys of estrous cycle determination and at least 5 days of data collected.

 Insufficient data (ID): 1) the animal does not display at least 1 complete cycle but has at least 1 E and/or P and a partial cycle of 5 days or fewer, 2) no E is present on any days ofFemale Pubertal Assay (2012)/ Page 6 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None



### estrous cycle determination and 4 or fewer days of data collected, or 3) at least 1 E or P present and only 1-4 days of data collected.

Percent cycling and percent regularly cycling were calculated as follows:

Percent cycling: [(RC + IC + ID ) / (RC + IC + ID + NC)] X 100

Percent cycling regularly: [RC / (RC + IC)] X 100

Note that, 1) as long as there are no animals cl assified as NC, the % cycling will be 100, and 2) when no animals are classified as IC, th e % cycling regularly will be 100. Also, the equation presented on page 26 of the repor t results in a rati o, not a percentage.

5. Sacrifice and Pathology: On the day before euthanasia, rats were moved to a holding room separate from where the necropsies were to be performed. Dosing was performed in the holding room on the day of termination and th e animals were moved to the necropsy room one at a time for terminal procedures. A pproximately 2 hours following dosing, animals were sacrificed by decapitation without anesthesia and all sacrifices were completed by 1300 hours to minimize hormonal variability due to normal diurnal fluctuation. Immediately following decapitation, trunk blood samples were taken for thyroxine (T4), thyroid stimulating hormone (TSH) and serum chemistry analyses. The sample for TSH analysis was stored at ≤ 20° C and the sample for T4 was analyzed fresh.

a. Hormone Analysis: Total thyroxine (T4) was analyzed using an electrochemiluminescent assay and thyroid stimulating hormone (T SH) levels were analyzed using an [ 125 

### I] radioimmunoassay. For both assays, multiple qu ality control samples were run prior to analysis.

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 Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Albumin globulin ratio (A/G Rati o, by calculation) X Alanine aminotransferase (A LT/also SGPT) X Bile acids X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in fe male rats based on guideline 890.1450.Female Pubertal Assay (2012)/ Page 7 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None



### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological examination.

UROGENITAL OTHER XX Ovaries (paired, without oviducts)*+ XX Thyroid*+
XX Uterus*+ X Liver* XX Kidneys (paired)*+ X Adrenals (paired)* X Pituitary* * Weights required base d on guideline 890.1450 + Histopathological examination re quired based on guideline 890.1450



### All organs except the thyroid/ trachea were weighed prior to fixation. Paired organs (kidneys, adrenals, and ovaries) were weighed together. The uterus was separated from the vagina and weighed. The uterus was weighe d “wet” and then again following removal of the fluid in the lumen (blotted weight).

The ovaries (left or right not specified), uterus and kidneys were fixed in 10% buffered formalin, rinsed and stored in 70% ethanol pr ior to embedding. The thyroid was fixed in 10% buffered formalin for at least 24 hrs. Following fixation, the thyroid was dissected from the trachea. All collected tissues we re routinely processed into paraffin blocks, sectioned, stained with hematoxylin a nd eosin, and examined microscopically.

Thyroid sections were subjectively evaluated for follicular cell height and colloid area, using a five point grading scale (1 = shortes t/smallest; 5 = tallest/largest), and any abnormalities/lesions were noted. At least two se ctions of the thyroid were examined. Five random sections of an ovary were evaluated for follicular development and any abnormalities/lesions (such as atrophy). Uter ine histology documented cases of uterine hyper- or hypotrophy as characteri zed by changes in uterine horn diameter and myometrial, stromal, or endometrial gland de velopment. The stage of the es trous cycle of the female at the time of necropsy was evaluated.

II. RESULTS

A. Mortality: One animal in the control group (A nimal No. 79829-10) was sacrificed in
extremis on PND 25 due to impairment of the left forelimb; this animal lost 5.9 g body weight during PND 24-25. All other rats survived until scheduled sacrifice.

B. Clinical Signs of Toxicity: No treatment-related clinical findings were noted during the daily examinations at any dose le vel. Clinical signs infrequently noted in the study included clear material around the mouth, salivation, vocalizati on, and red material on the eye, nose or mouth.

C. General Growth and Vaginal Opening: Body weights, body weight gains, age of attainment of vaginal opening and weight at day of attainment are presented in Table 2. There were no significant di fferences in body weight at vaginal opening or final body weights in the treated groups relative to cont rols. Adjusted age at VO was significantlyFemale Pubertal Assay (2012)/ Page 8 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None



### earlier (p<0.05) at 300 mg/kg/ day but not at 600 mg/kg/day. One female in the control group failed to attain vaginal opening.

The control CV for the mean age at vagi nal opening (9.39%) exceeded the performance criteria maximum (6.52%).

TABLE 2. General Growth and Vaginal Opening (VO) a

Parameter Evaluated Vehicle Control Metolachlor 300 mg/kg/day Metolachlor 600 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 22; g) U 15 54.6 3.97 7.27 15 54.6 3.88 7.10 15 54.9 3.67 6.69

Body weight at vaginal opening (g) U 14 121.0 14.78 12.22 15 109.8 15. 29 13.92 15 116.6 17.36 14.89
A 14 122.0 14.78 12.22 15 110.4 15. 29 13.92 15 117.0 17.36 14.89 Final body weight(g) U 14 153.8 10.38 6.75 15 156.0 9.80 6.28 15 157.4 8.99 5.71 Final body weight (% of control) U 15 101 15 102

Body weight gain (final – initial; g) U 14 98.7 9.7 9.83 15 101.4 8.55 8.43 15 102.5 7.91 7.72

Age at vaginal opening (PND) U 14 35.4 3.32 9.39 15 33.4 2. 29 6.86 15 34.5 2.53 7.35
A 14 35.6 3.32 9.39 15 33.4* 2. 29 6.86 15 34.6 2.53 7.35 Age at vaginal opening (incomplete) (PND) 14 34.7 2.49 7.18 15 33.4 2. 29 6.86 15 34.5 2.53 7.35
14 34.9 2.49 7.18 15 33.4 2. 29 6.86 15 34.6 2.53 7.35 Proportion unopened (#/N) 1/14 0/15 0/15 a Data were obtained from page 63 of the study report U Unadjusted for body weight on PND 22 A Adjusted for body weight on PND 22 N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p<0.05



### D. Organ Weights: Organ weights at necrops y are presented in Table 3. Unadjusted, adjusted and relative liver weights were increased at 600 mg/kg/day (p<0.05, ↑ 20-22%); only relative liver weight was significantly increased at 300 mg/kg/day (p<0.05, ↑ 9%). Unadjusted, adjusted and relative kidney weights were increased at 600 mg/kg/day (p<0.05, ↑ 8-10%). No other effects on organ weights were reported.

The control group CV for liver weight (13.360%) was higher than the performance criteria maximum (13.13%).Female Pubertal Assay (2012)/ Page 9 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None

TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control Metolachlor 300 mg/kg/day Metolachlor 600 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 14 6.91 0.923 13.360 15 7.62 0.670 8.786 15 8.45* ( ↑ 22) 0.962 11.377

A 14 6.91 0.923 13.360 15 7.64 0.670 8.786 15 8.44* ( ↑ 22) 0.962 11.377

R 14 4.48 0.349 7.777 15 4.89* ( ↑ 9) 0.265 5.416 15 5.37* ( ↑ 20) 0.436 8.125

Kidneys (g) U 14 1.19 0.104 8.807 15 1.25 0.100 8.001 15 1.31* ( ↑ 10) 0.093 7.129

A 14 1.18 0.104 8.807 15 1.25 0.100 8.001 15 1.3* ( ↑ 10) 0.093 7.129

R 14 0.77 0.061 7.896 15 0.80 0.064 7.927 15 0.83* ( ↑ 8) 0.059 7.074

Pituitary (mg) U 14 8.1 1.26 15.46 15 7.6 0. 820 10.74 15 8.2 0.82 10.07
A 14 8.1 1.26 15.46 15 7.6 0. 820 10.74 15 8.1 0.82 10.07
R 14 5.3 0.83 15.60 15 4.9 0. 390 8.03 15 5.2 0.49 9.52

Adrenals (mg) U 14 32.5 4.44 13.65 15 31.8 3. 74 11.73 15 33.0 4.76 14.4
A 14 32.5 4.44 13.65 15 32.0 3. 74 11.73 15 32.9 4.76 14.4
R 14 21.2 3.13 14.73 15 20.5 2. 91 14.18 15 21.0 2.91 13.86
Ovaries (mg) U 14 72.5 11.96 16.50 15 70.0 6. 25 8.93 15 70.7 8.30 11.75
A 14 72.4 11.96 16.50 15 70.1 6. 25 8.93 15 70.5 8.30 11.75 Uterus, wet (mg) U 14 296.3 108.24 36.54 15 309.4 178. 10 57.56 15 337.4 195.36 57.91
A 14 297.6 108.24 36.54 15 309.3 178. 10 57.56 15 338.7 195.36 57.91 Uterus, blotted (mg) U 14 246.5 46.17 18.73 15 238.5 98. 69 41.37 15 260.5 71.18 27.32
A 14 245.4 46.17 18.73 15 238.2 98. 69 41.37 15 259.9 71.18 27.32 Thyroid, fixed (mg) U 14 9.59 2.154 22.468 15 9.67 2. 093 21.64 15 10.15 1.763 17.367
A 14 9.67 2.154 22.468 15 9.72 2. 093 21.64 15 10.18 1.763 17.367 a Data were obtained from page 64 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. U Unadjusted for body weight on PND 22 A Adjusted for 100 g body weight on PND 22 N Number of animals examined SD Standard Deviation CV Coefficient of Variation R Organ-to-body weight ratio (relative to body weight) * Significantly different fro m controls at p<0.05.



### E. Estrous Cyclicity: Estrous cycle data are provided in Table 4. There were no statistically significant differences in mean age at first va ginal estrous or mean cycle length. The cycle status at necropsy was similar among groups. The percent of females cycling (93.3%) and regularly cycling (77.8%) was lower at 600 mg/ kg/day (p<0.05) than th e controls (100% for both). Percent regularly cy cling was also reduced at 300 mg/kg/day (p<0.05, 53.8%).Female Pubertal Assay (2012)/ Page 10 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None

TABLE 4. Estrous Cyclicity a

Treatment

Groups N Mean Age at First Vaginal Estrus (PND) Mean Cycle Length (days) Cycling (%) Regularly Cycling

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not Cycling

Vehicle 14 35.8 4.7 100 100 12 0 1 0
Metolachlor 300 mg/kg/day 15 36.1 4.6 100 53.8* 12 0 2 0

Metolachlor 600 mg/kg/day 15 37.2 4.6 93.3* 77.8* 13 0 2 1

a Data were obtained from pages 64 & 65 of the study report. N Number of animals * Significantly different from controls at p<0.05.



### F. Clinical Chemistry and Hormone Levels: Mean clinical chemistry and hormone levels are presented in Table 5. In addition, the study repor t provided historical control ranges onpages 421-424 for the clinical chemistry, but not hormone measures. There was a doserelated increase in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, respectively; the increase was significant at 600 mg/kg/day (p<0.05) only. Serum TSH was not affected by treatment.

Mean globulin levels were significantly higher in the 300 and 600 mg/kg/day groups (p<0.05, ↑ 7%); there were corres ponding reductions in A/G ratios of 8% and 12%, respectively, with only the hi gh dose being signifi cant (p<0.05). GGT was significantly elevated at 600 mg/kg/day (p<0.05, ↑ 700%).

It is noted that 12/20 of the control clinical chemistry means are outside the historical control range for the performing laboratory.Female Pubertal Assay (2012)/ Page 11 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control Metolachlor 300 mg/kg/day Metolachlor 600 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Hormones

Serum T 4 , Total (μg/dL) 14 4.42 1.143 25.851 15 5.05 ( ↑ 14) 1.147 22.727 15 5.51* ( ↑ 25) 1.399 25.41

Serum TSH (ng/mL) 13 8.76 5.686 64.901 15 9.02 3. 712 41.153 15 10.41 5.893 56.594

Clinical Chemistry
A/G ratio 13 2.97 0.305 10.289 15 2.73 ( ↓ 8) 0.219 8.030 15 2.61* ( ↓ 12) 0.103 3.930

Albumin (g/dL) 13 4.1 0.22 5.36 15 4.1 0.22 5.38 15 4 0.21 5.34
Alkaline phosphatase (U/L) 13 329 73.0 22.2 15 289 42.7 14.8 15 300 83.8 27.9

Alanine amino transferase (U/L) 13 96 19.0 19.8 15 92 13.7 14.9 15 96 13.4 14.0

Aspartate aminotransferase (U/L) 13 341 105.8 31.0 15 324 77.2 23.8 15 311 75.9 24.4

Bile acids (μmol/L) 13 23.8 10.27 43.13 15 29.3 19.49 66.52 15 32.2 13.66 42.40 Calcium (mg/dL) 13 11.9 0.77 6.48 15 12.1 0.46 3.77 15 12.0 0.23 1.93 Chloride (mEq/L) 13 101 1.5 1. 5 15 101 1.7 1.6 15 101 1.3 1.3
Cholesterol (mg/dL) 13 92 14.4 15.7 15 101 10.9 10.7 15 97 11.5 11.8

Creatinine (mg/dL) 13 0.2 0.03 14.42 15 0.2 0.05 22.15 15 0.2 0.05 24.21

Gamma glutamyl transferase (U/L) 13 0.2 0.30 121.44 15 0.3 0.39 115.95 15 1.6* ( ↑ 700) 1.44 89.11

Globulin (g/dL) 13 1.4 0.13 9.25 15 1.5* ( ↑ 7) 0.15 9.95 15 1.5* ( ↑ 7) 0.08 5.39

Glucose (mg/dL) 13 145 12.7 8. 8 15 142 8.0 5.6 15 142 7.5 5.3 Phosphorus (mg/dL) 13 10 0.78 7.82 15 10.3 0.75 7.32 15 10.2 0.85 8.38

Potassium (mEq/L) 13 7.04 0.825 11.722 15 7.01 0.603 8.603 15 6.91 0.682 9.875 Sodium (mEq/L) 13 139 2.4 1. 7 15 139 1.9 1.3 15 140 1.4 1.0
Total bilirubin (mg/dL) 13 0.15 0.039 26.105 15 0.14 0. 029 21.670 15 0.16 0.025 15.510

Total protein (g/dL) 13 5.5 0.25 4.61 15 5.6 0.31 5.63 15 5.5 0.29 5.14 Triglyceride (mg/dL) 13 132 71.7 54.20 15 142 60.2 42.5 15 117 25.7 22.0

Serum urea nitrogen (mg/dL) 13 12.7 1.81 14.20 15 14.1 2. 26 16.05 15 13 3.12 24.04

a Data were obtained from pages 66 & 67 of the study report. Pe rcent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different fro m controls at p<0.05.



### G. Histopathology: The incidences of histopathological findings of the thyroid gland are presented in Table 6. There was an increased incidence of Grade 2 follicular cell height at 300 (8/15) and 600 (11/14) mg/kg/day relative to control (4/14); also, one animal at the high dose was observed with a Grade 3. There was an increased incidence of Grade 4 colloid area at 600 mg/kg/day (2/14, 4/15 and 11/14 in the control, 300 and 600 mg/kg/day respectively. The differences in follicular cel l height and colloid area were significant atFemale Pubertal Assay (2012)/ Page 12 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None



### 600 mg/kg/day (p=0.005) and, with the excepti on of the single female with Grade 3 follicular cell height, all animals had values in the historical range. The only other significant difference in the thyroid gland was an increase in ultimobranchial cyst at 300 mg/kg/day that was not dose related.

TABLE 6. Incidence of Histopathologi cal Lesions of the Thyroid Gland a

Treatment

Groups Parameter Evaluated
Colloid Area Follicular Cell Height

Grade b Incidence Grade b Incidence
O E O E

Vehicle Control 1 0 14 1 10 14
2 0 14 2 4 14
3 0 14 3 0 14
4 2 14 4 0 14
5 12 14 5 0 14

Metolachlor 300 mg/kg/day 1 0 15 1 7 15
2 0 15 2 8 15
3 0 15 3 0 15
4 4 15 4 0 15
5 11 15 5 0 15

Metolachlor 600 mg/kg/day 1 0 14 1 2 14

2 0 14 2 11 14

3 0 14 3 1 14

4 11 14 4 0 14

5 3 14 5 0 14 a Data were obtained from pages 45 & 113 of the study report. b Thyroid histopathology is graded on a 1 – 5 scale: Follicular cell height,1 = lowest, 5 = highest; and Colloid area, 1 = most colloid, 5 = least colloid. O Number Observed E Number Examined



### The incidence of histopathological findings of the ovaries, uterus and kidneys are presented in Table 7. The ovaries appeared normal hi stopathologically. Ther e were no microscopic findings in the uterus related to treatment with the test materi al. Renal tubular cysts were increased in the 300 and 600 mg/kg/day gr oups (1/14, 2/15 and 4/15 in the 300 and 600 mg/kg/day groups, respectively). Other observations were consid ered to be incidental and included renal tubular degeneration, mine ralization, pelvis dilatation and acute inflammation.Female Pubertal Assay (2012)/ Page 13 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None

TABLE 7. Incidence of Histopathological Le sions of the Ovaries, Uterus and Kidney a

Findings Dose Level (mg/kg/day)

Vehicle Control Metolachlor 300 mg/kg/day Metolachlor 600 mg/kg/day

# Observed # Examined # Observed # Examined # Observed # Examined

Ovaries
Unremarkable 14 14 15 15 15 15

Uterus Apoptosis minimal 1 14 3 15 1 15 Apoptosis mild 4 14 2 15 1 15 Glans, developing minimal 5 14 3 15 3 15

Glans, developing mild 7 14 9 15 11 15 Infiltrate, eosinophil minimal 2 14 5 15 6 15

Infiltrate, eosinophil mild 11 14 10 15 9 15

Infiltrate, eosinophil moderate 1 14 0 15 0 15

Kidney
Unremarkable 0 14 0 15 1 15 Cyst, tubular 1 14 2 15 4 15 Dilitation, pelvis minimal 0 14 2 15 1 15

Dilitation, pelvis mild 1 14 0 15 0 15 Inflammation,, acute mild 1 14 0 15 0 15

Mineralization, minimal 3 14 2 15 2 15 Mineralization, mild 0 14 1 15 0 15 Regeneration, renal tubular minimal 13 14 15 15 14 15

Regeneration, renal tubular mild 1 14 0 15 0 15

a Data were obtained from pages 110 - 114 of the study report.



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: No evidence of altered female pubertal development was noted at any dose level, based on the lack of effects on the mean age and body weight at attainment of vaginal opening, ag e at the first occurren ce of estrus, estrous cycle length and regularity, uter ine weights (wet and blotted) , ovary weights, or test substance-related macroscopic and microscopic findings in the uterus and ovaries. This indicates that metolachlor technical does not demonstrate or mimi c biological activities consistent with agonism of natural estrogens wh en administered orally to peripubertal female rats at dosage levels up to 600 mg/kg/day.

At 600 mg/kg/day, increases in fol licular cell height and colloid depletion in the thyroid were noted; however, the changes were generally mini mal and some control animals also exhibited the same findings. These slight changes in the thyroid of the 300 and 600 mg/kg/day females are consistent with an adaptive response to test substance metabolism and most likely secondary to chemical metabolizing enzyme induction and increased hepatic clearance of thyroxine. Liver weights were significantly higher and this is consistent with hepatocellular hypertrophy; however, liver histopathology was not performed in this study. Serum gammaFemale Pubertal Assay (2012)/ Page 14 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None



### glutamyl transferase levels we re also higher in the 600 mg/kg/ day group, which may indicate an effect on hepatobiliary function. Mean T4 concentration was slight ly, yet significantly higher; however, there were no corresponding effects on TSH levels or thyroid gland weights. Serum globulin levels were increas ed and thus A/G ratios were reduced. Test substance-related higher mean kidney weight s were noted in the 600 mg/kg/day group.

At 300 mg/kg/day, increases in fol licular cell height and colloid depletion in the thyroid were noted; liver weights were signifi cantly higher. Serum globulin levels were increased and thus A/G ratios were lower.

In conclusion, a separate amphibian assay c onducted as part of this same package of endocrine studies using metolachlor showed no e ffects on the thyroid at dose levels up to 8.4 mg/L (Palmer et al ., 2012, Wildlife Interna tional, Ltd. Project Number 568A-103). In addition, metolachlor has been shown to i nduce liver cytochrome P450 enzymes (Dalton, et al., 2003). Therefore, despite the larger proporti on of females in the 300 and 600 mg/kg/day groups with minimal microscopic changes in th e thyroid relative to the control group, the weight of evidence suggests that metolachlor is not a clear disrupter of thyroid homeostasis at oral dose levels up to 600 mg/kg/ day in peripubertal female rats.

B. AGENCY COMMENTS: One animal in the control group was sacrificed in extremis on PND 25 due to impairment of the left forelimb. All other rats survived until scheduled sacrifice. No treatment-related clinical findi ngs were noted during the daily examinations at any dose level. There were no significant differences in body weight at vaginal opening or final body weights in the treated groups rela tive to controls. Adjusted age at VO was significantly earlier at 300 mg/kg/day (p<0.05) but not at 600 mg/kg/day. One female in the control group failed to attain vaginal openi ng. There were no stat istically significant differences in mean age at first vaginal estrous or mean cycle length. The cycle status at necropsy was similar among groups. Serum TSH was not affected by treatment.

Unadjusted, adjusted and relative liver wei ghts were increased at 600 mg/kg/day (p<0.05, ↑ 20-22%); only relative liver weight was significantly incr eased at 300 mg/kg/day (p<0.05, ↑ 9%). Unadjusted, adjusted and relative ki dney weights were increased at 600 mg/kg/day (p<0.05, ↑ 8-10%). No other effects on organ we ights were reported. The percent of females cycling (93.3%) and regularly cyc ling (77.8%) were lowe r at 600 mg/kg/day (p<0.05) than the controls (100% for both). Pe rcent regularly cycling was also reduced at 300 mg/kg/day (p<0.05, 53.8%). There were dose-re lated increases in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, respectively; the increase was significant (p<0.05) at 600 mg/ kg/day only. Mean globulin leve ls were significantly higher in the 300 mg/kg/day (p<0.05, ↑ 7%) and 600 mg/kg/day (p<0.05, ↑ 7%) groups; there were corresponding reductions in A/G ratios of 8% and 12%, respectively, with only the high dose being significant (p<0.05). GGT was signi ficantly elevated at 600 mg/kg/day (p<0.05, ↑ 700%).

There was an increased incidence of Grade 2 follicular cell height at 600 (11/14) mg/kg/day relative to control (4/14); also, one animal at the high dose was observed with Grade 3. There was an increased incidence of Grade 4 co lloid area at 600 mg/kg/day (2/14, 4/15 and 11/14 in the control, 300 and 600 mg/kg/day respectively). The differences in follicular cell height and colloid area were significant at 600 mg/kg/day (p= 0.005). The ovaries appearedFemale Pubertal Assay (2012)/ Page 15 of 15 METOLACHLOR/ 108801 OCSPP 890.1450/ OECD None



### normal histopathologically. There were no micr oscopic findings in the uterus related to treatment with the test material. Renal t ubular cysts were incr eased in the 300 and 600 mg/kg/day groups (1/14, 2/15 and 4/15 in the 300 and 600 mg/kg/day grou ps, respectively).

No effects were observed on body weights or body weight gain in the study. Increased liver weights accompanied by elevated GGT levels and increased kidney weights demonstrate organ toxicity as a result of treatment. Ho wever, histopathology was not conducted on the liver and microscopic findings in the kidney were minimal, so the degree of toxicity in these organs is unclear. Age at vaginal opening wa s not affected by treatment. There was a doserelated increase in serum T4 levels of 14% and 25% in the 300 and 600 mg/kg/day groups, respectively; the increase was significant (p <0.05) at 600 mg/kg/day only. Serum TSH was unaffected by treatment.

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

 The control CV for the mean age at vagi nal opening (9.39%) exceeded the performance criteria maximum (6.52%).

 The control CV for the mean TSH level (64.901%) exceeded the performance criteria maximum (58.29%).

 The control CV for liver weight (13.360%) was higher than the performance criteria maximum (13.13%).

# DATA EVALUATION RECORD

METOLACHLOR

Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-49-2012 (MRID 48617803)

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: Joseph Rieth, Ph.D., DABT Date: 5/9/2012

Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 5/16/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/17/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/17/2012

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).Male Pubertal Assay (2012) I Page I of 16
METOLACHLOR/108801 OCSPP 890.1500 I OECD None



### Primary Reviewer: Ayaad Assaad,D.V.M., Ph.D. 

### Health Effects Division Secondary Reviewer: John Liccione Ph.D. 

### Health Effects Division

DATA EVALUATION RECORD

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500



### PC CODE: 108801 

### DP BARCODE: D398926



### TXR#: 0052848 

### CAS No.: 51218-45-2



### TEST MATERIAL (PURITY): Metolachlor Technical (97.3% purity)



### SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamide



### CITATION: Sloter

, E.D. (2012) Pubertal Development and Thyroid Function in Intact
Juvenile / Peri pubertal Male Rats Following Oral Administration of Metolachlor.
WIL Research Laboratories, LLC, Ashland Ohio. WIL- 786004, January 25,
2012. MRID 48617803. Unpub li shed.



### SPONSOR: Metolachlor Task Force, 16160 Nall, Stilwell, KS



### TEST ORDER#: CON-108801-112



### EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48617803), 15 Crl:CD(SD) Sprague-Dawley rats / dose group were treated daily via oral gavage with metolachlor technical (97.3% a.i., Lot #20081115) in corn oil at doses of 0, 400 or 800 mg / kg / day from post-natal day (PND) 23 to 53

. Animals were examined for preputial separation (PPS) daily beginning on PND
30 and weight at day of attainment was recorded

. Following sacrifice on PND 53, blood was taken for total thyroxine (T4)

, testosterone

, thyroid stimulating hormone (TSH) and clinical chemistry analysis. Although the hormones were reportedly analyzed by radioimmunoassay ( RIA) or chemiluminescence, the specific details of the methods were not provided. weights

were recorded for kidneys

, liver

, adrenal glands, thyroid

, pituitary, epididymides, ventral prostate

, dorsal prostate, seminal vesicle

, levator ani plus bulbocavernosus (LABC) muscles and

testes

, and microscopic examinations were performed on the thyroid, kidneys

, testis and epididymides.

One male in the 800 mg / kg / day group (Animal No. 79812-07) was found dead on PND 40 ; no significant clinical or macroscopic findings were observed in this animal. One male in the
control group (Animal No. 79823-03) was sacrificed 

### in extremis on PND 4 7 following clinical findings of a swollen right forelimb resulting in impaired use; gross examination of this animal at

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

. Clinical findings included sa livation prior to dosing and clear materialMale Pubertal Assay (2012) / Page 2 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



### around the mouth approximately 1 hour post-dosing in the 400 and 800 mg/kg/day dose groups. At 800 mg/kg/day, clear material was also observed around the ventral neck.

No treatment-related changes in body weights, body weight gains, or age and weight at PPS occurred in either treatment group. Liver weights were in creased (p<0.05) by 15-20% and kidney weights were increased (p<0.05) by 11% each at 400 and 800 mg/kg/day, respectively. A significant trend for increased thyroid weights was observed, although pairwise comparisons were not significant; the increasing trend in th yroid weights did not correlate with higher TSH levels or microscopic findings. Although a sign ificant trend for decreas ing right epididymis weights was observed, it was not si gnificant by pairwise comparis on and was not observed in the left epididymis.

TSH levels were increased (p<0.05) by 54 a nd 134% at 400 and 800 mg/kg/day, respectively. There was no corresponding effect on T 4 

### levels, and the increas ed TSH levels did not consistently correlate with thyr oid gland weight or histopathol ogical findings on an individual animal basis.

Mean testosterone levels were d ecreased in a dose-related manner ( ↓ 45%, NS and ↓ 69%, p<0.05) in the 400 and 800 mg/kg/day group s, respectively. The control group had three animals with unusually high testosterone levels (>8.00 ng/mL), and the control mean was above the performance criteria maximum (3.96 ng/mL) when th ese animals are included in the analysis. When these animals were excluded from the analysis, the mean value for the control group was 2.27 ng/mL, which is similar to the values fo r the treated groups (2.29 and 1.35 ng/mL) although a statistical analysis was not c onducted using the adjusted mean.

At the scheduled euthanasia, no treatment-related findings were noted. A s light treatment-related effect on thyroid morphology is considered possi ble based the larger number of animals with follicular height Grade 1 and colloid area Grade 5 in the control group relative to the 400 and 800 mg/kg/day animals; this is consistent with a higher number of treated animals with follicular height and colloid area scores of 3. These findings are consistent with higher mean thyroid weights and elevated TSH levels in the treated animals; how ever, these endpoints were not correlated when evaluated on an individual anim al basis. Additionally, increased follicular height was not necessarily correlated with decreased colloid area in individual animals. There were no treatment-related findings in the testes, epididymides or kidneys.

The high dose tested was considered to be adeq uate based on the results of a study in which a metolachlor dose of 1000 mg/kg/day (route of ad ministration not reporte d) resulted in overt toxicity (all of the males found d ead, euthanized in extremis or terminated early). In addition, organ toxicity was observed in the current study with both liver and kidney weights significantly increased at 400 and 800 mg/kg/day.

The assay satisfies the EDSP Tier 1 Test Order requirement s for a Male Pubertal Assay (OCSPP 890.1500).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.Male Pubertal Assay (2012) / Page 3 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH 44805 Study Director: E. D. Sloter
Other Personnel: G. B. Marit, Pathologist L. Freshwater, Statistics Study Period: July 27, 2010 – January 25, 2010



### 2. Test Substance: Metolachlor
Description: Dark brown, clear liquid, MW 283.79
Source: Sipcam (Valdosta, GA)
Lot/Batch #: 20081115, exp. 15 November 2013 Purity: 97.3% Stability: Stable in corn oil for 13 days at room temperature
CAS #: 51218-45-2
Structure:

CH 3

CH 3 N
O CH 3 O Cl

CH 3



### 3. Vehicle: Corn oil

4. Test Animals
Species: Rat
Strain: Crl:CD(SD) Sprague-Dawley Age/Weight at Study Initiation: PND 23 /42.2 – 69.1 g males only
Source: Charles River Laboratories; Raleigh, NC Housing: Two-three/cage in plastic cages with heat treat ed laboratory-grade pine shavings for bedding
Diet: Harlan Laboratories 2016CM Teklad Global 16% protein rodent diet, ad libitum Phytoestrogen content average 10 ppm tota l isoflavones (genistein+daidzein+glycitein)
Water: Municipal water, reverse-os mosis filtered (on site), ad libitum
Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 22 ± 3 °C
50 ± 20%
10/hr 14 hrs light/ 10 hrs dark



### B. STUDY DESIGN

1. In-Life Dates: Start: 08/29/2012 End: 09/29/2010

2. Mating: Time-mated pregnant dams were received from the supplier between gestation days (GD) 11 or 12. The day an animal was found to be sperm positive was designated GD 0. Litters with more than 8 pups were standardi zed by culling on PND 4 to 8 pups per litter.Male Pubertal Assay (2012) / Page 4 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



### 3. Animal Assignment: Animals were assigned to the test groups noted in Table 1 using a randomization procedure designed to ensure equal stratification of body weights. Littermates were not assigned to the same treatment group.

TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Males
Control 0 15
Low 400 15 High 800 15 a Data were obtained from page 28 of the study report.



### 4. Dose Selection Rationale: Dose levels were selected ba sed on the results of published toxicity data and the results of a previously conducted Hers hberger assay (WIL-786002). In Phase 1 of the Hershberger assay, a dose leve l of 1000 mg/kg/day (rout e of administration not reported) resulted in all of the males found dead, euthanized in extremis or terminated early. In a subsequent Phase, oral gavage dose levels of 400 and 800 mg/kg/day for 5 days were tolerated by the animals and these dose levels were selected for the current study.

5. Dose Preparation and Analysis: Dose formulations were prepared every 10 days by mixing appropriate amounts of test substance w ith corn oil. Homogeneity and stability of the test substance in formulations were as sessed in a previous ut erotrophic study (MRID 48617801, reviewed concurrently) and data from the uterotrophic study are reported below. During the current study, samples from all dos e formulations prepar ed during the in-life phase were analyzed for achieved concentration.

Results of Dose Analysis

Homogeneity (% RSD): 1.8-2.5%

Stability (% of initial): 92.5-97.9% following storage for 13 days at room temperature.

Concentration: Mean concentrations ranged from 101% to 106% of nominal.

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: All doses were administered once daily by gavage from PND 23 through PND 53, in a volume of 5mL/kg of body weight. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: Statistics were conducte d by BioSTAT Consultants, Inc using SAS® software. Data statistically anal yzed included the organ weights, organ weight to body weight ratio (liver, kidneys, pituitary, and adrenal gla nds), daily body weights, cumulative body weight gain, serum chemistries, hormones, and weight and age at PPS. When an animal did not attain PPS, PND 54 (last Study Day + 1) and term inal body weight were used as the age and body weight at PPS, respectively. Gamma glut amyltransferase values under range were assigned a value of 0.1 (half the LLQ) fo r statistical anal ysis and reporting.Male Pubertal Assay (2012) / Page 5 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



### Endpoints were tested for homogeneity of variance using Levene's test. If that test was significant (p=0.01), then a log or square root transformation was applied to the raw data; if the test was still significant th en a nonparametric test was used to analyze the data. If the variances were homogeneous then an analys is of variance (ANOVA) was conducted. The statistical model contained a factor for trea tment group and a blocking factor based on the time of study start. A two-sided Dunnett's test was conducted, regardless of the outcome of the ANOVA, looking for significant differences in the test ar ticle groups when compared with the vehicle control. If the variances were not homogeneous then the nonparametric Kruskal-Wallis test was used, ignoring the blocking factor, followed by Dunn's test, to compare each of the test article groups with the vehicle control. Since these were preplanned pairwise comparisons, Dunn's test was conducted regardless of the outcome of the Kruskal-Wallis test. The tests were two-side d (p=0.05), looking for significant differences from the vehicle control. In addition, organ weights, age and we ight at PPS were subject to the following analyses if they met the homogene ity of variance criteria: 1) Analysis of covariance (ANCOVA) with Dunnett's test, us ing body weight at PND 21 as the covariate; 2) linear trend test using the ANOVA model; and 3) linear trend test using the ANCOVA model.

Histopathology findings presented as a graded response were analyzed with pairwise MannWhitney U tests. The tests were one-sided at p=0.05 for increased severity. The tests were two sided for graded responses presented on a Grade 1-5 scale at p=0.05 for increased or decreased severity.

C. METHODS

1. Mortality and Clin ical Examinations: All animals were examined twice daily for mortality and moribundity. Clinical exam inations were conducted on the day of randomization and daily thr ough the day of euthanasia.

2. Body Weight: Animals were weighed on the day of randomization, daily prior to dosing and the day of euthanasia. Mean body weight changes were calculated for each corresponding interval and for the overall treatment period (PND 23-53).

3. Preputial Separation (PPS): Beginning on PND 30, all animals were examined daily for onset of PPS. The appearance of a partial or a persistent thread of tissue between the glans and prepuce was recorded. Age and weight at on the day of completion of PPS were recorded.

4. Sacrifice and Pathology: On the day before euthanasia, rats were moved to a holding room separate from where the necropsies were to be performed. Dosing was performed in the holding room on the day of termination and the rats were moved to the necropsy room one at a time for terminal procedures. Approxi mately 2 hours following dosing, animals were sacrificed by decapitation without anesthesia and all sacrifices were completed by 1300 hours to minimize hormonal variability due to normal diurnal fluctuation. Immediately following decapitation, trunk blood samples were taken for T 4 

### , TSH, testosterone, andMale Pubertal Assay (2012) / Page 6 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



### serum chemistry analyses. The hormone samples were stored at ≤− 20° C for subsequent analyses.

a. Hormone Analysis: The study authors report ed that hormones were analyzed using either RIA or chemiluminescence; however, specifi c details and supporting data (i.e. method recovery and specificity data) were not provided.

b . Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER

X Calcium X Albumin

X Chloride Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol
X Potassium X Globulins (by calculation)

X Sodium X Glucose

ENZYMES X Total bilirubin
X Alkaline phosphatase (ALK) X Total protein Cholinesterase (ChE) X Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X A/G ratio (b y calculation) X Alanine aminotransferase (ALT/also SGPT) X Creatinine X Aspartate aminotransferase (AST/also SGOT) X Bile acids Sorbitol dehydrogenase X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended based on guideline 890.1500.



### c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological examination.

UROGENITAL OTHER XX Testes (left and right separately)* + XX Thyroid* + XX Epididymides (left and right separately)* + X Liver* X Seminal vesicle plus coagulating glands (with fluid)* X Adrenals (paired)* X Ventral prostate* X Pituitary* X Dorsolateral prostate* X Levator ani-bulbocavernosus (LABC) muscle complex* XX Kidneys (paired)* + * Weights required base d on guideline 890.1500 + Histopathological examination re quired based on guideline 890.1500



### The right testis, right epidi dymis and kidneys were weighe d prior to fixation. Following weighing, the testis and epididymis were fixed in Bouin’s solution (dur ation of fixation not specified). The thyroid (with parathyroid) was collected with the trachea and fixed in 10% buffered formalin for at least 24 hrs. Followi ng fixation, the thyroid wa s dissected free of the trachea and weighed. All collected tissues we re routinely processed into paraffin blocks, sectioned, stained with hematoxylin and eosi n, and examined microscopically. ThyroidMale Pubertal Assay (2012) / Page 7 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



### sections were subjectively evaluated for follicul ar cell height and colloid area using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest), and any abnormalities/lesions noted. Two sections from the thyroid were examined in order to obtain representative samples of the thyroid tissue.

II. RESULTS

A. Mortality: One male in the 800 mg/kg/day grou p (Animal No. 79812-07) was found dead on PND 40; no significant clinical or macroscopic findings were observed in this animal. One male in the control group (Animal No. 79823-03) was sacrificed in extremis on PND 47 following clinical findings of a swollen right forelimb resulting in impaired use; gross examination of this animal at necropsy reve aled a punctured esophagus probably due to a dosing error. All other rats survived until scheduled sacrifice.

B. Clinical Signs of Toxicity: Clinical findings included sali vation prior to dosing and clear material around the mouth approximately 1 hour post-dosing in th e 400 and 800 mg/kg/day dose groups (Table 2). At 800 mg/kg/day, clear material was also observed around the ventral neck.

TABLE 2. Incidence of Clinical Observations a

Observation Dose Level (mg/kg/day)

Vehicle Control Metolachlor 400 mg/kg/day Metolachlor 800 mg/kg/day

# Observed # Examined # Observed # Examined # Observed # Examined
No clinical observations 472 b 15 480 1 15 450 b 15
Found dead 0 15 0 15 1 15
Sacrificed in extremis 1 15 0 15 0 15 Salivation prior to dosing 0 15 8 15 10 15
Wet clear material ventral neck-1 hr post-dose 0 15 2 15 6 15

Wet clear material, mouth1 hr post-dose 3 15 10 15 15 15

a Data were extracted from pages 68-70 of the study report b Total number of observations. All other observations refer to total number of anim als with that observation at least once.



### C. General Growth and Preputial Separation: Body weights, body weight gains, age of attainment of PPS and weight at day of PPS are presented in Table 3. No treatment-related changes in body weights or body weight gain s occurred at 400 or 800 mg/kg/day. The control group CV values for body weight at PND 21 (10.94%) and PND 53 (11.27%) were above the performance crit eria acceptable ranges of 10.25% and 7.47%, respectively.

The mean ages of PPS were 43.4, 42.4 and 43.4 da ys in the control, 400 and 800 mg/kg/day groups, respectively; there was no effect of treatment on this developmental landmark. The CV value for the age at attainment of PPS (6.08%) was above the acceptable performance criteria range (5.67%). The elevated CV was attributed to one control animal (# 79813-01) that did not achieve se paration until PND 52.Male Pubertal Assay (2012) / Page 8 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None

TABLE 3. General Growth and Preputial Separation (PPS) a

Parameter Evaluated Vehicle Control Metolachlor 400 mg/kg/day Metolachlor 800 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 23; g) U 15 60.7 6.69 11.02 15 61.2 4. 73 7.73 14 61.8 5.4 8.75 Body weight at PPS (g) U 15 198.2 13.8 6.96 15 194.4 13 6.69 14 196.2 15.63 7.97
A 15 198.1 13.8 6.96 15 194.1 13 6.69 14 196.2 15.63 7.97 Final body weight (g) U 15 276.7 31.18 11.27 15 279.9 27. 57 9.85 14 272.4 22.81 8.37 Body weight gain (final – initial; g) U 15 215.8 25.48 11.81 15 218.8 25. 52 11.67 14 211 22.01 10.43 Final body weight percent of control NA NA NA NA 15 101 NA NA 15 98 NA NA

Age at PPS (PND) U 15 43.4 2.64 6.08 15 42.4 1. 45 3.43 14 43.4 1.7 3.91
A 15 43.4 2.64 6.08 15 42.5 1. 45 3.43 14 43.5 1.7 3.91 Proportion unseparated (#/N) 15 0 15 0 14 0

a Data were obtained from page 63 of the study report. U Unadjusted for body weight on PND 23 A Adjusted for body weight on PND 23 N Number of animals examined SD Standard Deviation CV Coefficient of Variation NA Not applicable



### D. Organ Weights: Organ weights at necr opsy are presented in Tabl e 4. Liver weights were significantly increased by 15-20% at 400 a nd 800 mg/kg/day (p<0.05) . Kidney weights were significantly increased by 11% each at 400 and 800 mg/kg/day (p<0.05). A significant trend for increased thyroid weights was obser ved, although pairwise comparisons were not significant. The increasing tr end in thyroid weights did no t correlate with the observed higher TSH levels or with microscopic findi ngs when evaluated on an individual animal basis. A significant trend for decreasing right epididymis weights was observed, but this was not significant by pairwise comparison a nd was not observed in the left epididymis.

The mean control absolute thyroid (11.62 mg) and kidney weights (1.84 g) were below the performance criteria minimum acceptable valu e (14 mg and 2.242 g, respectively) but were within the laboratory’s historical control ranges fo r rats of this age. The mean CV values for ventral prostate (24.18%), adrenal gland ( 24.51%) and pituitary gland (20.39%) weights were above the performance criteri a maximums of 22.32%, 22.77% and 15.98%, respectively.Male Pubertal Assay (2012) / Page 9 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None

TABLE 4. Organ Weights at Necropsy a

Organ Vehicle Control Metolachlor 400 mg/kg/day Metolachlor 800 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 14 13.13 1.891 14.404 15 15.11* ( ↑ 15) 1.323 8.757 14 15.77* ( ↑ 20) 1.499 9.508

A 14 13.11 1.891 14.404 15 15.06* ( ↑ 15) 1.323 8.757 14 15.79* ( ↑ 20) 1.499 9.508

R 14 4.73 0.246 5.207 15 5.41* ( ↑ 14) 0.23 4.25 14 5.79* ( ↑ 22) 0.32 5.518

Kidneys (g) U 14 1.84 0.23 12.47 15 2.05* ( ↑ 11) 0.16 7.806 14 2.05* ( ↑ 11) 0.151 7.392

A 14 1.84 0.23 12.47 15 2.04* ( ↑ 11) 0.16 7.806 14 2.04* ( ↑ 11) 0.151 7.392

R 14 0.67 0.031 4.724 15 0.74* ( ↑ 10) 0.043 5.868 14 0.75* ( ↑ 12) 0.05 6.638

Pituitary (mg) U 14 8.9 1.82 20.39 15 9 2. 04 22.6 14 8 1.34 16.81
A 14 9 1.82 20.39 15 9 2.04 22.6 14 8.1 1.34 16.81
R 14 3.1 0.36 11.55 15 3.3 0.8 24.45 14 3.1 0.47 15.45
Adrenals (mg) U 14 36.1 8.84 24.51 15 34.6 6. 47 18.72 14 33.7 7.88 23.41
A 14 36.2 8.84 24.51 15 34.6 6. 47 18.72 14 34.1 7.88 23.41
R 14 12.9 2.31 17.89 15 12.4 2. 15 17.34 14 12.4 3.01 24.2
Seminal vesicle + coagulating gland, with fluid (mg) U 14 493.9 84.24 17.06 15 488.1 84.22 17.26 14 509.4 94.4 18.53

A 14 493.8 84.24 17.06 15 487.2 84. 22 17.26 14 510.4 94.4 18.53

Seminal vesicle + coagulating gland, without fluid (mg) U NR NR NR NR NR NR NR NR NR NR NR NR

A NR NR NR NR NR NR NR NR NR NR NR NR

Ventral prostate (mg) U 14 210.9 50.99 24.18 15 208.8 41. 58 19.91 14 198.7 53.39 26.87
A 14 210.5 50.99 24.18 15 207.9 41. 58 19.91 14 198.6 53.39 26.87 Dorsolateral prostate (mg) U 14 103.6 25.66 24.78 15 105.3 20.2 19.18 14 103 21.34 20.71
A 14 104.2 25.66 24.78 15 105.9 20.2 19.18 14 104.9 21.34 20.71
LABC (mg) U 14 478.8 78.63 16.42 15 485.8 50.2 10.33 14 450.8 76.26 16.92
A 14 477.5 78.63 16.42 15 483.1 50.2 10.33 14 449.4 76.26 16.92 Epididymis, left (mg) U 14 189.9 32.56 17.15 15 196.4 22. 24 11.32 14 176.6 31.05 17.58
A 14 190 32.56 17.15 15 196.1 22. 24 11.32 14 177.5 31.05 17.58 Epididymis, right (mg) U 14 193.4 31.18 16.13 15 192 12.09 6.29 14 176.7 17.74 10.04
A 14 193.1 31.18 16.13 15 191.3 12. 09 6.29 14 176.7 17.74 10.04 Testis, left (mg) U 14 1360.7 165.05 12.13 15 1349.6 126.42 9.37 14 1339 155.25 11.59
A 14 1361.9 165.05 12.13 15 1348.4 126. 42 9.37 14 1346.2 155.25 11.59 Testis, right (mg) U 14 1352.4 159.52 11.8 15 1372.5 132.73 9.67 14 1278.4 97.06 7.59
A 14 1354.1 159.52 11.8 15 1372 132.73 9.67 14 1286.5 97.06 7.59 Thyroid, fixed (mg) U 14 11.62 1.796 15.457 15 13.38 2.471 18.473 14 13.85 3.479 25.108
A 14 11.56 1.796 15.457 15 13.28 2.471 18.473 14 13.76 3.479 25.108 a Data were obtained from page 66 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. U Unadjusted for body weight on PND 23 A Adjusted for body weight on PND 23 R Organ-to-body weight ratio (relative to body weight) N Number of animals examined SD Standard Deviation CV Coefficient of Variation NR Not recorded * Significantly different fro m controls at p<0.05.Male Pubertal Assay (2012) / Page 10 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



### E. Clinical Chemistry and Hormone Levels: Mean hormone levels are presented in Table 5. TSH levels were increased (p<0.05) by 54% and 134% at 400 and 800 mg/kg/day, respectively. However, there was no corresponding effect on T 4 

### levels and the TSH levels did not consistently correlate with thyroid gl and weight or histopath ological findings when evaluated on an indivi dual animal basis.

Mean testosterone levels were decreased in a dose related manner; the levels were 4.2, 2.3 ( ↓ 45%) and 1.3 ( ↓ 69%, p<0.05) ng/mL in the contro l, 400 and 800 mg/kg/day groups, respectively. The control group had three animals (Nos. 79826-02, 79828-02 & 79829-04) with unusually high testosterone levels (> 8.00 ng/mL); the control mean was above the performance criteria maximum level (3.96 ng/mL) when these animals were included in the analysis. However, when these animals were excluded from the analysis, the mean value for testosterone in the control group was 2.273 ng/mL. Statistical analysis using the adjusted mean was not presented but the values between treated and cont rol groups appear similar. The following study deficiencies in the testostero ne data were noted: 1) the details for the analytical method used testoste rone analysis were not presen ted in the report, 2) no method validation or concurrent quality control data were presented, and 3) no historical control data from the performing laboratory were pres ented. Because of these limitations, it is not possible to determine if the control animals w ith high testosterone levels should or should not be excluded from the analysis. Thus, it is not possible to definitively determine if there is an effect of the test material on testoste rone levels and reliabl e interpretation of the testosterone results.

Globulin was increased (p<0.05) by 13-25% at 400 and 800 mg/kg/day, resulting in the A/G ratio being decreased (p<0.05) at both dose levels. Higher me an total protein was observed at 800 mg/kg/day ( ↑ 7%, p<0.05). GGT was increased ( ↑ 600%, p<0.05) at 800 mg/kg/day. Lower mean ALP levels were reported at 400 and 800 mg/kg/day but the control mean was well above the laboratory’s hi storical range, and the treat ed groups were within the historical range, so the toxico logical significance is doubtful. No other clinical chemistry findings were noted.Male Pubertal Assay (2012) / Page 11 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control Metolachlor 400 mg/kg/day Metolachlor 800 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Hormones

Serum T 4 , Total (μg/dL) 14 6.82 1.192 17.48 15 7.78 1.514 19.458 14 7.27 1.196 16.445

Serum TSH (ng/mL) 14 9.76 3.994 40.938 15 15.01* ( ↑ 54) 6.912 46.057 14 22.87* ( ↑ 134) 12.143 53.092

Testosterone (ng/mL) 11 4.2 3.52 84 15 2.3 1.58 69.18 14 1.3* ( ↓ 69) 0.76 56.56

Clinical Chemistry
A/G ratio 14 2.39 0.267 11.142 15 2.15* ( ↓ 10) 0.164 7.602 14 2.03* ( ↓ 15) 0.145 7.132

Albumin (g/dL) 14 3.9 0.19 4.79 15 3.9 0.21 5.4 14 4 0.22 5.55 Alkaline phosphatase (U/L) 14 405 75.6 18.7 15 361 76.7 21.3 14 291* ( ↓ 28) 59.9 20.6

Alanine amino transferase (U/L) 14 69 11.8 17.1 15 70 11.2 16 14 71 9 12.8

Aspartate aminotransferase (U/L) 14 157 42.1 26.9 15 151 52.6 34.8 14 148 31.3 21.2

Bile acids (μmol/L) 14 40.4 27.15 67. 28 15 31 12.9 41.55 14 29.9 10.3 34.44 Calcium (mg/dL) 14 11.9 0.43 3.67 15 12 0.37 3.06 14 12 0.4 3.34 Chloride (mEq/L) 14 101 1.1 1. 1 15 101 1.2 1.2 14 100 1.4 1.4 Cholesterol (mg/dL) 14 94 13.2 14.1 15 100 14.8 14.7 14 94 8.2 8.7
Creatinine (mg/dL) 14 0.2 0.05 34.59 15 0.1 0.05 36.22 14 0.2 0.04 19.55

Gamma glutamyl transferase (U/L) 14 0.1 0.11 83.15 15 0.2 0.26 127.32 14 0.7* ( ↑ 600) 0.87 128.76

Globulin (g/dL) 14 1.6 0.15 9.16 15 1.8* ( ↑ 13) 0.18 9.81 14 2* ( ↑ 25) 0.14 7.37

Glucose (mg/dL) 14 138 7.7 5. 6 15 137 7.5 5.5 14 139 9.5 6.9 Phosphorus (mg/dL) 14 9.6 0.68 7.09 15 9.6 0.85 8.87 14 9.7 1.13 11.67 Potassium (mEq/L) 14 6.29 0.419 6.671 15 6.29 0.356 5.669 14 6.16 0.755 12.268 Sodium (mEq/L) 14 141 1.5 1.1 15 141 2 1.4 14 142 1.5 1 Total bilirubin (mg/dL) 14 0.15 0.03 20. 331 15 0.16 0.045 28.224 14 0.16 0.02 12.377 Total protein (g/dL) 14 5.6 0.17 3.04 15 5.7 0.33 5.81 14 6* ( ↑ 7) 0.3 4.96

Triglyceride (mg/dL) 14 471 183.9 39 15 581 164 28.2 14 477 193 40.5 Serum urea nitrogen (mg/dL) 14 15.1 2.06 13.64 15 14.8 1.74 11.83 14 14.5 2.38 16.44

a Data were obtained from pages 64 and 65 of the study repor t. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different fro m controls at p<0.05.



### F. Macro- and Histopathology: At the scheduled euthanasia, no treatment-related macroscopic findings were noted. Dilated re nal pelvis was reported in 1 male at 800 mg/kg/day. The incidences of histopathological findings of th e thyroid gland are presented below in Table 6. A slight treatment-rela ted effect on thyroid morphology was notedMale Pubertal Assay (2012) / Page 12 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



### possible based the larger number of animals with follicular height Grade 1 and colloid area Grade 5 in the control group relative to the 400 and 800 mg/ kg/day animals; this is consistent with a higher number of treated an imals with follicular height and colloid area scores of 3. These findings are consistent wi th higher mean thyroid weights and elevated TSH levels in the treated animals; however , these endpoints were not correlated when evaluated on an individual animal basis. Further complicating the evaluation is that the animals with abnormal follicular heights were not necessarily the same animals observed with abnormal colloid area. There were no treatment-related findings in the testes, epididymides or kidneys.

TABLE 6. Incidence of Histopathologi cal Lesions of the Thyroid Gland a

Treatment Groups Parameter Evaluated

Colloid Area Follicular Cell Height

Grade b Incidence Grade b Incidence

O E O E

Vehicle Control 1 0 14 0 4 14

2 0 14 2 10 14

3 1 14 3 0 14

4 7 14 4 0 14

5 6 14 5 0 14

Metolachlor 400 mg/kg/day 1 0 15 1 1 15
2 1 15 2 11 15
3 2 15 3 3 15
4 10 15 4 0 15
5 2 15 5 0 15

Metolachlor 800 mg/kg/day 1 0 14 1 1 14

2 1 14 2 8 14

3 5 14 3 5 14

4 7 14 4 0 14

5 1 14 5 0 14 a Data were obtained from page 397 of the study report. b Thyroid histopathology is graded on a 1 – 5 scale: Follicular cell height,1 = lowest, 5 = highest; and Colloid area, 1 = most colloid, 5 = least colloid. O Number Observed E Number Examined



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: There was no evidence of altered pubertal development at any dose level of metolachlo r tested (400 or 800 mg /kg/day) based on the absence of effects on the attainment of PPS a nd male reproductive organs. Higher globulin levels, and consequently lower A/G ratios, a nd higher kidney weights were also noted at 400 and 800 mg/kg/day, and increased total protein levels were no ted at 800 mg/kg/day. Mean testosterone levels were lower in the 400 a nd 800 mg/kg/day groups due to extraordinarily high testosterone values in 3 control males, resu lting in an atypically high mean testosterone concentration in the control group. However, there were no perturbations in male pubertal development or effects on the male reproductive organs in this study at oral dose levels up to 800 mg/kg/day, the highest dose level of metolachlor tested.Male Pubertal Assay (2012) / Page 13 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



### Although mean TSH levels were increased at dosage levels of 400 and 800 mg/kg/day; there was no corresponding effect on T 4 

### levels and the higher TSH va lues did not consistently correlate on an individual animal basis with thyroid gland wei ghts or the subtle microscopic changes observed in the thyroid (minimally in creased follicular cell height or decreased colloid area). The thyroid changes observed in this study were consistent with an adaptive response to test substance metabolism, and mo st likely secondary to chemical metabolizing enzyme induction and increased hepatic cleara nce of thyroxine (Cu rran and DeGroot, 1991). Metolachlor has been shown to induce liv er cytochrome P450 enzymes (Dalton, et. al.

, 2003). This is consistent with the higher liver weights noted in the 400 and 800 mg/kg/day group animals and apparent hepatocellular hypertrophy; however, li ver histopathology was not performed in this study. Serum GGT le vels were also higher for the 800 mg/kg/day group animals, which may indicate an eff ect on hepatobiliary function. A separate amphibian metamorphosis assay conducted using metolachlor concentra tions up to 8.4 mg/L for 21 days found no changes in the thyroi d glands of treated tadpoles (Palmer, et al. , 2012, Wildlife International, Ltd. Project Number 568A-103). Therefore, based on the overall weight of evidence from this and other studies , metolachlor does not appear to produce an endocrine effect on the thyroid gland or disr upt thyroid homeostasis in peripubertal male rats, especially at an oral dose level of 400 mg/kg/day.

B. AGENCY COMMENTS: One male in the 800 mg/ kg/day group (Animal No. 79812-07) was found dead on PND 40; no significant clinic al or macroscopic findings were observed in this animal. One male in the control group (Animal No. 79823- 03) was sacrificed in
extremis on PND 47 following clinical findings of a swollen right forelimb resulting in impaired use; gross examination of this anim al at necropsy revealed a punctured esophagus probably due to a dosing error. All other rats survived until scheduled sacrifice. Clinical findings included salivation prior to do sing and clear material around the mouth approximately 1 hour post-dosing in the 400 and 800 mg/kg/day dose groups. At 800 mg/kg/day, clear material was also observed around the ventral neck.

No treatment-related changes in body weights or body weight gains occurred at 400 or 800 mg/kg/day, and there was no effect on the age or weight at PPS. Liver weights were increased (p<0.05) by 15 and 20% at 400 and 80 0 mg/kg/day, respectively. Kidney weights were increased (p<0.05) by 11% each at 400 and 800 mg/kg/day. A significant trend for increased thyroid weights was observed, a lthough pairwise comparisons were not significant; the increasing trend in thyroid weights did not corr elate with higher TSH levels or microscopic findings. A si gnificant trend for decreasing right epididymis weights was observed, but this was not significant by pairwi se comparison and was not observed in the left epididymis.

TSH levels were increased (p<0.05) by 54- 134% at 400 and 800 mg/kg/day. However, there was no corresponding effect on T 4 

### levels and the TSH leve ls did not consistently correlate with thyroid gland weight or hist opathological findings when evaluated on an individual animal basis.

Mean testosterone levels were decrease d in a dose related manner, by 45% and 69% (p<0.05) at 400 and 800 mg/kg/da y groups, respectively. Howeve r, the control group hadMale Pubertal Assay (2012) / Page 14 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



### three animals with unusually high testosterone levels (>8.00 ng/mL); the control mean for testosterone (4.2 ng/mL) was above the perf ormance criteria maximum level (3.96 ng/mL) when these animals were included in the analysis. When these animals were excluded from the analysis, the mean value for the contro l group was 2.273 ng/mL; statistical analysis using the adjusted mean was not presented but the values between treated and control groups appear similar.

Globulin was increased (p<0.05) by 13-25% at 400 and 800 mg/kg/day; this resulted in the A/G ratio being decreased (p<0.05) at both dos e levels. Higher m ean total protein was observed at 800 mg/kg/day ( ↑ 7%, p<0.05). GGT was increased ( ↑ 600%, p<0.05) at 800 mg/kg/day. Lower mean ALP levels were reported at 400 and 800 mg/kg/day but the control mean was well above the laboratory’s historical range, and th e treated groups were within the historical ra nge, so the toxicological significan ce is doubtful. No other clinical chemistry findings were noted.

At the scheduled euthanasia, no treatment-relate d findings were noted. Dilated renal pelvis was reported in 1 male at 800 mg/kg/day. A slight treatment-relate d effect on thyroid morphology is considered possible based the larger number of animals with follicular height Grade 1 and colloid area Grade 5 in the cont rol group relative to th e 400 and 800 mg/kg/day animals; this is consistent with a higher numbe r of treated animals with follicular height and colloid area scores of 3. These findings seem consistent with higher mean thyroid weights and elevated TSH levels in the treated animals, howev er, these endpoints were not correlated when evaluated on an individual animal basis. Further complicating the evaluation is that the animals with abnormal fo llicular heights were not necessarily the same animals observed with abnormal colloid area. There were no treatment-related findings in the testes, epididymides or kidneys.

The highest dose tested was cons idered to be adequate based on the results of a Phase 1 of the Hershberger assay in whic h a dose level of 1000 mg/kg/day (route of administration not reported) resulted in all of the males found dead, euthanized in extremis or terminated early. Also, in this pubertal study, organ toxicity was observed; both liver and kidney weights were significantly increase d at 400 and 800 mg/kg/day.

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 control group CV values for body weights at PND 21 (10.94%) and PND 53 (11.27%) were above the perf ormance criteria acceptable ranges of 10.25% and 7.47%, respectively.

 The CV value for the age at attainment of PPS (6.08%) was above the acceptable performance criteria range (5.67%). The el evated CV was attributed to one control animal (# 79813-01) that did not achieve separation until PND 52.

 The mean control absolute thyroid (11.62 mg ) and kidney weights (1.84 g) were below the performance criteria acceptable ranges ( 14 mg and 2.242 g, respectively) but were within the laboratory’s historical co ntrol ranges for rats of this age.Male Pubertal Assay (2012) / Page 15 of 15 METOLACHLOR/108801 OCSPP 890.1500 / OECD None



###  The mean CV values for ventral pros tate (24.18%), adrenal gland (24.51%) and pituitary gland (20.39%) weights were above the performance criteria maximums of 22.32%, 22.77% and 15.98%, respectively.

 The testis and epididymis were fixed in Bouin’s solution (dura tion of fixation not specified).

 8/20 of the clinical chemistr y control values were out of historical range at the performing laboratory.

 Seminal vesicle + coagulating gland, without fluid (mg) were not weighed as per the guideline.

# DATA EVALUATION RECORD

METOLACHLOR

Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-49-2012 (MRID 48617809)

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: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/3/2012

Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 5/7/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/8/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/8/2012

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).METOLACHLOR I 108801



### Primary Reviewer: Ayaad Assaad, D.V.M., Ph.D.
Health Effects Division Secondary Reviewer: John Liccione, Ph.D.
Health Effects Division Steroidogenesis Assay (2012) I Page 1 of 15
OCSPP 890.1550/ OECD None



### S igna tu re:**--1+--'Y---'+-..,f-iZ->--~** 

### Date:**~r+-"7L~....::m----1£,;;--** 

### Sign a tu**r~ ~~~::!;g::;:=:::;;;~--­**  

### Date: ---'"""""'-r-+-..!......-,'---



### DATA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550

PC CODE: 108801 DP BARCODE: D398926

TXR#: 0052848 CAS No.: 51218-45-2

TEST MATERIAL (PURITY): Metolachlor (97.3%)

SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamide

CITATION: Wagner, H. (2012) Metolachlor: H295R steroidogenesis assay. CeeTox

, Inc., Kalamazoo, MI. Laboratory Study No.: 9179V-100537STER, January 26,
2012. MRID 48617809. Unpublished.

SPONSOR: Metolachlor Task Force, I 6 I 60 Nall, Stilwell, KS.

TEST ORDER# : CON-108801-12

EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48617809), H295R cells
cultured 

### in vitro in 24-well plates were incubated with metolachlor, (97.3% a.i., Batch#
2008 I 115) at log concentrations from 0.000 I to 100 μM in triplicate for 48 hours. Dimethyl
sulfoxide (DMSO) was used as a vehicle, at a final concentration was 0.05%.

Testosterone and estradiol concentrations were measured by HPLC / MS-MS with positive ion multiple reaction monitoring. Three independent experiments were performed. A Quality
Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration
levels ; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of
estradiol and testosterone production. The highest suitable concentration of metolachlor in all
three runs was 100 μM.

Guideline recommendations were met, including adequate production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls. Cytotoxicity greater than 20% was not observed.

Statistically significant changes in testosterone production after incubation with metolachlor

were not observed

, although the mean fold-difference was decreased 17% for 100 μM metolachlor. Statistically significant increases in estradiol production were observed in two of
the three runs at I 0 μM, and in all three runs at the 100 μM concentration. The mean foldSteroidogenesis Assay (2012) / Page 2 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### differences were approximatel y 1.2- and 1.3-fold, respectively, and showed a concentrationdependent response.

Based on the hormone responses in each of the three independent runs, metolachlor treatment resulted in statistically signifi cant and reproducible in creases in estradiol production, but did not result in a statistically signi ficant and reproducible alteration s in testosterone 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 (2012) / Page 3 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc.

Location: Kalamazoo, MI Study Director: H. Wagner Other Personnel: C. Toole, Director of Project Management K. Rutherford, Director of Laboratory Operations D. Blakeman, Senior Scientist B. Wallace, Lead Cell Culture Scientist F. Wong, Scientist L. Blakeman, Associate Scientist A. Cutter, Scientist Study Period: August 31, 2011 to January 26, 2012



### 2. Test Substance: Metolachlor Description: Liquid Batch #: (Expiration Date) 20081115 (November 15, 2013) Purity: 97.3% Solubility (in Solvent): Soluble in DMSO up to 200 mM Volatility: Not reported Stability: Stability in test preparations was not conducted. Storage conditions: Room temperature CAS #: 51218-45-2 Molecular weight: 283.8 Structure:

CH 3

CH 3 N
O CH 3 O Cl

CH 3



### 3. Positive Control (Inducer): Forskolin Description: White powder, MW 410.50 Source: Sigma Aldrich, St. Louis, MO (catalog # not reported) Lot #: (Expiration Date): 109K50571V (July and August, 2016) Purity: 98% Solubility (in Solvent): Soluble in DMSO up to 100 mM Storage conditions: Room temperature CAS #: 66575-29-9



### 4. Positive Control (Inhibitor): Prochloraz Description: White powder, MW 376.67 Source: Sigma Aldrich, St. Louis, MO (catalog # not reported) Lot #: (Expiration Date) SZE6220X (August 8, 2012) Purity: 99.1% Solubility (in Solvent): Soluble in DMSO up to 100 mM Storage conditions: Room temperature CAS #: 67747-09-5Steroidogenesis Assay (2012) / Page 4 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### 5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) Description: Not reported Source: Not reported Lot #: (Expiration Date) Not reported Purity: Not reported Storage conditions: Not reported CAS #: Not reported Justification for choice of solvent: Not reported (Metolachlor, forskolin, and prochloraz soluble at test concentrations) Final concentration: (% volume in assay) 0.05% (v/v)



### 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 (DMEM/Ham-F12) Description: Included 15 mM HEPES Source: Not reported Lot / Batch #: (Expiration Date) Not reported Sodium bicarbonate: Not reported Nu-Serum: Becton Dickinson, Catalog #355500, Lot #81515; tested for background hormone concentrations. ITS+ premix: Not reported



### 8. Test Cells: H295R human adrenocortical carcinom a cells (ATCC, Manassas, VA, CLR2128; Lot #7635054). Cells were maintained in DMEM/Ham-F12 with 15 mM HEPES, sodium bicarbonate, ITS+Premix, and 2.5% Nu-Serum in a 5% CO 2 

### incubator at 37±2oC. The background hormone concentrations pres ent in the Nu-Serum were 3754 pg/mL for testosterone and 3846 pg/mL for estradiol. Cell s were initially grown for five passages, and then frozen in liquid nitrogen. After thawing, cells were cultured addi tionally for seven or eight passages prior to use in the steroi dogenesis assay. 22R-Hydroxycholesterol (SigmaAldrich, St. Louis, MO, Lot #060M4098, 99.0% pur e, 10 μM) was added to the culture medium at plating, dosing, and harvesting. Cell s were plated into we lls of 24-well culture plates at a density of approximately 3 × 10 5 

### cells/mL. Plates were incubated in a 5% CO 2 

### atmosphere at 37±2oC for approximately 24 hours prior to exposure to the test chemical or the positive controls.

The following performance criteria were met (indicated by an “x”): x Cell passage identifier. Cell Passage #: ≥ 7.5 x Cells frozen down at passage 5 x Frozen cells cultured for 4 additional passages x Total number of passages does not exceed 10 (not reported)Steroidogenesis Assay (2012) / Page 5 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### B. METHODS

1. Pre-Test Information

a. Hormone Assay Interference Test: Hormone cross-reactivity or interference tests were not conducted because testosterone and estr adiol concentrations were determined by HPLC/MS-MS analysis; no interference was expected.

b. Hormone Extraction: Testosterone and estradiol were extracted from H295Rsupplemented medium by liquid/liquid extraction with methyl tert-butyl ether spiked with [ 2 

### H 5 

### ]-testosterone and [ 2 

### H 5 

### ]-estradiol as internal standards. The extracts were analyzed using HPLC/MS-MS with positive ion multiple reaction monitoring. The method MDL for testosterone and estradiol were 100 and 10 pg/mL, respectiv ely, for a 300-μL portion of medium.

c. Laboratory Proficiency Test: Laboratory proficiency testin g was not conducted for the current study.

2. Test Solutions: A 200-mM stock of metolachlor wa s prepared in DMSO, followed by serial dilutions (1:10) in DMSO. A 40-m M stock of 22R-hydroxycholesterol was dissolved in ethanol and further diluted in supplemented medium to achieve a final concentration of 10 μM. Mastermix solutions with metolach lor were prepared by 1:2000 dilutions in supplemented medium containing 10 μM 22R-hydroxycholesterol. Forskolin and prochloraz solutions were prepared in a sim ilar manner by preparing 100 mM solutions in DMSO, followed by serial dilutions in DM SO and 1:2000 dilutions in supplemented medium containing 10 μM 22R-hydroxycholesterol. Final concentrations after addition to the 24-well culture plates were 1 and 10 μM for forskolin, 0.1 and 1 μM for prochloraz, and 0.0001 to 100 μM for metolachlor. Final con centrations of DMSO and ethanol in the medium were 0.05% and 0.025% (v/v), respectively.

3. Cell Plating and Preincubation: H295R cells (ATCC CLR-2128, Manassas, VA, Lot #7635054) were initially grown for five passages, and then frozen in liquid nitrogen. After thawing, cells were cultured a dditionally for seven or eight passages prior to use. 22RHydroxycholesterol (10 μM) was added to the culture medium at plating, dosing, and harvesting. Cells (1 mL) were seeded into well s of 24-well culture plat es at a density of approximately 3 × 10 5 

### cells/mL. Plates were incubated in a 5% CO 2 

### atmosphere at 37±2oC for approximately 24 hours prior to exposure to the test chemical or the positive controls. The percent confluency after 24-hour incubation was not report ed, but the protocol stated that 50-60% confluence was e xpected after plating 3 × 10 5 

### cells/mL followed by a 24-hour incubation period.

4. Exposure: The cells were checked microscopi cally for good attachment and proper morphology, and the medium was removed and replaced with 1 mL of supplemented medium containing 10 μM 22R-hydroxycholestero l. The cells were then exposed to identical volumes of either each serial dilu tion of the test compound or DMSO (solvent control; SC) in triplicate according to the schematic presented in Table 1.Steroidogenesis Assay (2012) / Page 6 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

TABLE 1. Dosing Schematic for the Exposure of H295 R Cells to Metolachlor (Final Concentrations 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 13 of the study report. Dosing was calculated based on a to tal volume of 1 mL per well.



### A concurrent quality control (QC) plate was incl uded with each of the four independent runs of the test chemical plates to demonstrate th e assay’s response to forskolin (an inducer of testosterone and estradiol pr oduction) and prochloraz (an inhi bitor of testosterone and estradiol production). The QC plate was prepar ed and dosed in the same manner with either forskolin or prochloraz according to the schematic presented in Table 2.

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 14 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±2 hours under the conditions described previously. After the inc ubation period, each well was examined microscopically for cytotoxicity and cell attachment. Precipitati on, if present, was noted. The medium from each well was removed, split into two equal volumes, and frozen at

− 80±10°C until hormone measurements were conducted. Cell vi ability was determined after media removal was completed.

5. Cell Viability/Cytotoxicity Assay: Cell viability was determined with the MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test described by Mosman (1983) 1 

### immediately after removal of the culture me dium after the 48-hour incubation. The QC plate wells designated to receive methanol (MeOH) were rinsed twice with phosphate buffered saline (PBS), followed by an inc ubation with MeOH for 30 minutes at room temperature, and three additional rinses with PBS. Next, 0.5-mL portions of a 0.5 mg/mL MTT solution in supplemented medium c ontaining 10 μM 22R-hydroxycholesterol were added to each well on the test chemical and QC plates. MTT-treated plates were incubated in a 5% CO 2 

### atmosphere at 37±2oC for 3 hours, followed by removal of the MTT solutions and addition of 0.5 mL of isopr opanol to each well. Plates were incubated for a further 20 minutes at room temperature with shaking. After the 20-minute in cubation, the absorbance values at 570 and 650 nm were determined with a Packard Fusion plate reader, and the

1 Mosman T. (1983). Rapid colorimetric assay for gr owth and survival: application to proliferation and cytotoxicity. J. Immunol. Methods . 100:45-50.Steroidogenesis Assay (2012) / Page 7 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### absorbance at 650 nm was subtracted from th e absorbance at 570 nm to calculate the MTT value. A reduction of ≥ 20% in cell viability was considered evidence of cytotoxicity.

6. Hormone Measurement System: Concentrations of testoste rone and estradiol in the supplemented medium were determined by HPLC-M S/MS at a separate analytical facility (OpAns, LLC, Durham, NC). The extracti on method (with spiked, labeled internal standards) was described previously (B.1.b.). All back-calculated calibration standard concentrations and QC concentrations were accep table. The percent recovery across the test runs for the QC samples ranged from 87.1- 111.9% for testosterone and 86.4-114.7% for estradiol.

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 for two concentrations of testosterone and estrad iol (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 (SD) for testosterone and estradiol concentrations were calculated for each concentration of the reference chemicals and SC, as well as the blank and b ackground wells, on the QC plates and for each concentration of metolachlor a nd SC on the test chemical plate. Relative changes in hormone production were calculated with the following equation:

Relative change = (hormone concentrati on in each well) ÷ (mean SC hormone concentration)

For forskolin induction of testosterone, th e background hormone production was subtracted from the forskolin-treated wells, and blank and SC wells, prior to calculation of the relative change. Background hormone production was dete rmined from three wells on the QC plate that received H295R cells with no 22R-hydroxycholesterol.

Concentrations that exhibited ≥ 20% cytotoxicity, or where pr ecipitation was observed, were omitted from further analysis.

Normality of the data was evaluated with the Shapiro-Wilk’s test. Homogeneity of the variances between the treatment groups was evaluated by using Levene’s test. Statistical significance between each treatment group and control was ev aluated with Dunnett’s test if p>0.05 in both tests. If p ≤ 0.05 in the normality or homogeneit y tests, a log transformation was performed on the data to attempt to approx imate a normal distribut ion. Dunnett’s test was conducted to evaluate statistical significan ce between the treatm ent and control groups if p>0.05 in both the normality and homoge neity tests after tr ansformation. If p ≤ 0.05 in either the normality or homogeneity tests after transformation, th e non-transformed data were analyzed with the nonparametric Krus kal-Wallis test, followed by Dunn’s test, to evaluate statistical significance between the treatment and control groups.

Data analysis was conducted with Microsoft Exce l, and statistics were calculated with the Unistat 6.0 Light program for Excel. These anal yses are considered to be appropriate.Steroidogenesis Assay (2012) / Page 8 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### II. RESULTS

A. TEST COMPOUND: Three of the four assay runs met the guideline criteria for further evaluation. The hormone concentrations afte r exposure to metolach lor, and the folddifference change relative to SC and mean ± SD for the three suitable runs (2 through 4), are presented in Table 3. The fold-changes in hormone concentration for metolachlor in the three runs are shown graphically in Figures 1 and 2 for testosterone and estradiol, respectively. The highest suit able concentration of metolachlor in all three runs was 100 μM.

The %CVs for absolute hormone concentrations fo r the SC replicates within the test plates were 3.41 to 8.82% for testosterone, and 1.66 to 4.68% for estradiol. The between-plate %CV for the absolute concentrations in the SC (calculated by the reviewer) was 12.4% for testosterone and 3.6% for estrad iol. These data met the r ecommended performance criteria of ≤ 30%.

Statistically significant changes in testosterone production after incubation with metolachlor were not observed, although the mean fold -difference was decreased 17% for 100 μM metolachlor. Statistically significant increa ses in estradiol produc tion were observed in Runs 2 and 3 at 10 μM, and in all three runs at the 100 μM concentration. The mean folddifferences were approximatel y 1.2- and 1.3-fold, respectively, and showed a concentrationdependent response.Steroidogenesis Assay (2012) / Page 9 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None

TABLE 3. Mean (±SD) Hormone Concentrations Follo wing Treatment with Metolachlor for 48 Hours. a
Nominal Concentration (μM) Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b ± SD b Statistical Significance

Testosterone (pg/mL) Fold Difference DMSO (SC) 1832 1706 2164 — — — — — —
0.0001 1819 1710 2150 0.99 1.00 0.99 0.99 0.006 —
0.001 1760 1743 2123 0.96 1.02 0.98 0.99 0.031 —
0.01 1886 1768 2163 1.03 1.04 1.00 1.02 0.021 —
0.1 1808 1810 2233 0.99 1.06 1.03 1.03 0.035 —
1 1821 1705 2099 0.99 1.00 0.97 0.99 0.015 —
10 1906 1803 2095 1.04 1.06 0.97 1.02 0.047 —
100 1505 1336 1903 0.82 0.78 0.88 0.83 0.050 — Estradiol (pg/mL) Fold Difference DMSO (SC) 99 93 99 — — — — — —
0.0001 95 97 105 0.96 1.04 1.06 1.02 0.053 —
0.001 100 98 103 1.01 1.05 1.04 1.03 0.021 —
0.01 96 97 104 0.97 1.04 1.05 1.02 0.044 —
0.1 98 99 101 0.99 1.06 1.02 1.02 0.035 —
1 95 94 99 0.96 1.01 1.00 0.99 0.026 —
10 108 121 122 1.09* 1.30* 1.23 1.21 0.107 Run 2 and 3
100 114 116 145 1.15* 1.24* 1.46* 1.28 0.159 Run 2, 3, and 4 a Data were obtained from page 26-27 and 34-36 of the study report. b Calculated by the reviewers SC Solvent control * Statistically significant (p ≤ 0.05)Steroidogenesis Assay (2012) / Page 10 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### FIGURE 1. Change in Testosterone Production Relative to Metolachlor Concentration in Test Runs #2 to #4.Steroidogenesis Assay (2012) / Page 11 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### FIGURE 2. Change in Estradiol Production Relative to Metolachlor Concentration in Test Runs #2 to #4.Steroidogenesis Assay (2012) / Page 12 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### B. CYTOTOXICITY: No evidence of cytotoxicity was present from the MTT cell viability assay conducted on the QC and metolachlor test plates (Tab le 4 and Figure 3), although viability at the 100 μM concentration was decr eased approximately 10%. The % viability values ranged from 97.5-106.8% on the QC plat es and 87.1-104.6% on the test chemical plates; methanol exposure redu ced cell viability to 3.6-4.7%.

TABLE 4. Mean (±SD) MTT Cell Vi ability Results after Treatment with Forskolin, Prochloraz, or Metolachlor 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 100.4 2.85 98.3 1.14 101.6 4.49 Background NA 100.8 1.16 99.6 0.8 101.1 3.65
SC + Methanol NA 4.70 1. 06 4.40 1.22 3.60 1.61
Forskolin b 1 106.2 1.63 105 0.6 106.8 1.05
Forskolin 10 105.1 1.63 104.4 3.65 106.4 2.52
Prochloraz 0.1 100.3 1.52 97.5 2.87 101.9 2.55
Prochloraz 1 102.5 5.39 99.3 2.61 101.8 5.07
Metolachlor 0.0001 94.2 0.48 96.7 1.56 101.0 4.38
Metolachlor 0.001 95.2 3.69 99.6 1.97 100.5 3.90
Metolachlor 0.01 96.9 2.18 102.3 5.83 103.9 5.34
Metolachlor 0.1 96.1 1.47 104.6 4.21 98.1 3.06
Metolachlor 1 93.7 5.33 101.6 5.29 93.3 3.06
Metolachlor 10 92.8 2.18 98.5 3.04 96.6 2.05
Metolachlor 100 87.1 6.68 92.8 0.8 89.9 1.85 a Data were obtained from page 20-21 of the study report. SC Solvent controlSteroidogenesis Assay (2012) / Page 13 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### FIGURE 3. MTT cell cytotoxicity results for Test Runs 2 and 3.

C. QC PLATE: The hormone concentrations after expos ure to the reference chemical, SC, blank, and background samples, as well as th e fold-difference change relative to SC (individual and mean ± SD) for the three suitab le assay runs, are presented in Table 5. Minimum basal hormone production was met in all blank and SC wells (500 pg/mL for testosterone, 40 pg/mL for estradiol), and ba sal hormone production met the fold-increase criteria in SC of ≥ 5-fold for testosterone and ≥ 2.5-fold for estrogen above the MDL of the assay. Exposure to 10 μM forskolin induced te stosterone production 1.4- to 1.7-fold, which is below the Guideline criteria of ≥ 2-fold; however, when the background is subtracted, 10 μM forskolin induced testosterone an average of 2.3-fold. Estradiol production was inducedSteroidogenesis Assay (2012) / Page 14 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### with 10 μ M forskolin by 12.5- to 17.6-fold. Cells incubated with 1 μ M prochloraz inhibited testosterone and es tradiol production by ≤ 0.5-fold. Thus, the guideline requirements were met indicating that the assay was sensitive to induction and inhibition of testosterone and estradiol.

The %CVs based on absolute hormone concentr ation in SC well repl icates were 2.49-7.65% for testosterone, and 2.22-8.11% for estradiol. The between-plate variability (calculated by the reviewer) for the QC plates based on the ab solute hormone concentrations in the SC was 8.8% for testosterone and 8.3% for estradiol. These data met the performance criteria recommended by the Guideline ( ≤ 30%).

TABLE 5. Mean (±SD) Hormone Concentrations Followi ng Treatment with Forskolin or Prochloraz for 48 Hours. a

Concentration (μM) Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b ± SD b Testosterone (pg/mL) Fold Difference (Relative to DMSO) Background 1213 1030 1081 — — — — —
Blank 1736 1892 2102 — — — — —
DMSO 1857 1844 2148 — — — — — 1 μM Forskolin 2518 2539 3050 1.36 c 1.38 c 1.42 c 1.39 c 0.03 10 μM Forskolin 2585 3011 3649 1.39 d 1.63 d 1.70 d 1.57 d 0.16 0.1 μM Prochloraz 1534 1503 1697 0.83 0.82 0.79 0.81 0.02 1 μM Prochloraz 735 700 803 0.40 0.38 0.37 0.38 0.02 Estradiol (pg/mL) Fold Difference (Relative to DMSO) Background 28 26 43 — — — — —
Blank 95 104 92 — — — — —
DMSO 97 95 83 — — — — — 1 μM Forskolin 662 867 775 6.80 9.14 9.33 8.42 1.41 10 μM Forskolin 1213 1534 1463 12.47 16.17 17.62 15.4 2.66 0.1 μM Prochloraz 90 89 78 0.93 0.93 0.94 0.93 0.01 1 μM Prochloraz 52 33 35 0.53 0.34 0.42 0.43 0.10 a Data were obtained from page 22-23 of the study report. b Calculated by the reviewers. c When the background is subtracted, the valu es are 2.03-, 1.85-, and 1.84-fold difference for Runs 2, 3, and 4, respectively, with a mean of 1.91-fold change. d When the background is subtracted, the valu es are 2.13-, 2.43-, and 2.41-fold difference for Runs 2, 3, and 4, respectively, with a mean of 2.32-fold change.Steroidogenesis Assay (2012) / Page 15 of 15 METOLACHLOR / 108801 OCSPP 890.1550/ OECD None



### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Metolachlor exposure did not result in increases or decreases in testosterone at any of the concentrations te sted in any of the assays. Statistically significant increases in estradiol re lative to the solvent control were observed in all three runs of the assay at nominal concentrations of 10 μ M and/or 100 μ M metolachlor. The increases were observed at the two highest metolachlor concentratio ns tested in Runs 2 and 3 and at the highest metolachlo r concentration tested in Run 4.

B. AGENCY COMMENTS: Laboratory proficiency testi ng results were not conducted as detailed in a protocol amendment for the cu rrent study because laboratory proficiency data had previously been determined for this batc h of cells. The details of this previous proficiency determination, including estimates of EC 50 

### s for prochloraz and forskolin were not reported in the current study report. No ev idence of cytotoxicity was present from the MTT cell viability assay conducted on the QC a nd metolachlor test plates, although viability at the 100 μM concentration was decreased approximately 10%. The highest suitable concentration of metolachlor in all three runs was 100 μM.

Minimum basal hormone production was met in all blank and SC wells, and basal hormone production met the fold-incr ease criteria in SC of ≥ 5-fold for testosterone and ≥ 2.5-fold for estrogen above the MDL of the assay. Exposur e to 10 μM forskolin induced testosterone production 1.4- to 1.7-fold, which is below the Guideline criteria of ≥ 2-fold; however, when the background is subtracted, 10 μM forskolin in duced testosterone an average of 2.3-fold. Estradiol production was induced with 10 μ M forskolin by 12.5- to 17.6-fold. Cells incubated with 1 μ M prochloraz inhibited testoste rone and estradiol production by ≤ 0.5- fold. These data generally met the performa nce criteria recommended by the Guideline.

The %CVs based on the absolute hormone c oncentration in the SC met the Guideline performance criteria of ≤ 30% for testosterone and estradio l in the test and QC plates..

Statistically significant changes in testosterone production after incubation with metolachlor were not observed, although the mean fold -difference was decreased 17% for 100 μM metolachlor. Statistically significant increa ses in estradiol produc tion were observed in Runs 2 and 3 at 10 μM, and in all three runs at the 100 μM concentration. The mean folddifferences were approximatel y 1.2- and 1.3-fold, respectively, and showed a concentrationdependent response. Based on the hormone respons es in each of the three independent runs, metolachlor treatment resulted in a statistically significant and reproducible alteration in estradiol production, but not in testoste rone production.

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:

 Laboratory proficiency testi ng prior to the initiation of the current study was not conducted, and the details of a previous pr oficiency determinati on with the current batch of cells, including estimates of EC 50 

### s for prochloraz and forskolin, were not reported in the study report.

# DATA EVALUATION RECORD

METOLACHLOR

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-49-2012 (MRID 48617801)

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: 5/1/2012

Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 5/7/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/8/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/8/2012

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).METOLACHLOR / 108801



### Primary Reviewer: Ayaad Assad, D.V.M., Ph

. D. 

### Health Effects Division Secondary Reviewer: _J"""""'e=s-=-s -=-R=o--'-w=la=n=d::.._____

_____ _ 

### Health Effects Division /11 vivo Vterotrophic Assay (2012) I Page I of9
OCSPP 890.1600/ OECD 440



### S i g n ature:

# t:Jb~d**Date:~~_!5** Sig n a tu re:**------1~'-'~--'-'----==-::--=---** 

### Date: 

## '= r'>(r 2 Temp l ate version 09/2011



### DATA EVALUATION RECORD

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440



### PC CODE: 108801 

### DP BARCODE: D398926



### TXR#: 0052848 

### CAS#: 51218-45-2



### TEST MATERIAL (PURITY): Metolachlor (97.3% a.i.)



### SYNONYMS: 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamide



### CITATION: Sloter

, E.D

. (2012) A Uterotrophic Assay ofMetolachlor Administered Subcutaneously in Ovariectomized Rats. WIL Research Laboratories, LLC, Ashland, OH. Laboratory Study No.: WIL-786001, January 3, 2012

. MRID
48617801

. Unpublished

.



### SPONSOR: Metolachlor Task Force, 16160 Nall

, Stilwell

, KS



### TEST ORDER#: CON-108801-12



### EXECUTIVE SUMMARY: In an uterotrophic assay (MRID 48617801) conducted to screen for potential estrogenic activity

, metolachlor (97

. 3% a.i., lot# 20081115) in com oil was administered daily via subcutaneous (s.c.) injection to groups of six ovariectomized Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300

, or 1000 mg / kg / day on post-natal days (PND)
57-59. A positive control group was treated with a daily dose of 17a-ethynyl estradiol (EE) at 0.5 μg / kg / day by s.c. injection. Body weights were determined daily. All animals were terminated and necropsied on PND 60 approximately 24 hours after the final dose administration

to determine wet and blotted uterine weights.

All animals survived until scheduled termination. No test substance-related clinical findings

were noted in any of the animals during the study. Body weights were comparable to the
controls throughout the duration of the assay , but overall (Days 1-4) body weight gains were decreased (p < 0.01) in the 300 mg / kg / day (142%) and 1000 mg / kg / day (153%) metolachlor

groups compared to the controls. Body weight gains in the positive control (EE) group were also
decreased (p < 0.01) by 57% compared to the controls.

Uterine weights in the metolachlor treated groups were comparable to the controls. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p < 0.05) as expected by 288 % and 175%

, respectively.In vivo Uterotrophic Assay (2012) / Page 2 of 9 METOLACHLOR / 108801 OCSPP 890.1600/ OECD 440



### Based on the observed decreases (42% and 53%) in body weight gain at 300 and 1000 mg/kg/day, adequate dose levels were achieved in this study.

No statistically significant changes were seen in uterine weight in this assay. Metolachlor is negative in the uterotrophic assay.

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 (2012) / Page 3 of 9 METOLACHLOR / 108801 OCSPP 890.1600/ OECD 440



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC

Location: Ashland, OH Study Director: E.D. Sloter
Other Personnel: S.T. Coffee (dose formulation analyses); L. Freshwater (BioSTAT Consultants, statistical analyses); E.S. Bodle (Assistant Director, Analytical Chemistry); C.A. Kopp (Manager, Gross Pathology and Developmental Toxicology Laboratory); T.M. Rafeld (Manager, Fo rmulations Laboratory); Study Period: July 28, 2010 to January 3, 2012



### 2. Test Substance: Metolachlor
Description: Dark brown, clear liquid
Source: Sipcam (Valdosta, GA)
Lot #: 20081115 (expiry date: November 15, 2013) Purity: 97.3% a.i. Stability: Stable at room temperature (ambient)
CAS #: 51218-45-2
Structure:



### 3. Reference Estrogen: 17 α -ethynyl estradiol (EE)
Supplier: Sigma Aldrich, Inc. (St. Louis, MO)
Lot #: 028K1411 (expiry date: August 4, 2011) Purity: 99.0%
CAS #: 57-63-6



### 4. Solvent/Vehicle Control: Corn oil
Supplier: Spectrum Chemical Manufacturing Co rporation (New Brunswick, NJ)
Lot #: ZR0032 and ZQ0791 (expiry dates: March 30, 2012 and May 3, 2012, respectively) Rationale (if other than water): Corn oil was selected as the vehicle due to the insolubility of metolachlor in water
Final concentration: Not applicable



### 5. Test Animals:
Species: Rat (ovariectomized)
Strain: Sprague Dawley, Crl:CD(SD) Age/weight at dose initiation: 57 days old/ 195.2 – 267.1 g females
Source: Charles River Laborat ories (Portage, MI) Housing: Individually caged in stainless steel wire -mesh cages suspended above cage-boards
Diet: Harlan Laboratories 2016CM Teklad Global 16% Protein Rodent Diet , ad libitum Average of 10.0 ppm total isoflavones (g enistein + daidzein + glycitein)
Water: Reverse-osmosis filtered tap water , ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 20.6-21.6 oC
37.5-45%
>10/hr 12 hrs light/ 12 hrs dark Acclimation period: 10 days CH

3 N O

Cl CH 3

O CH

3

CH 3In vivo Uterotrophic Assay (2012) / Page 4 of 9 METOLACHLOR / 108801 OCSPP 890.1600/ OECD 440



### B. STUDY DESIGN

1. In Life Dates: Start: August 27, 2010 End: August 30, 2010

2. Study Design: Sexually mature ovariectomized rats (PND 47) were received from Charles River Laboratories (ovariectomized on PND 42) . Animals were acclimated for 10 days prior to initiation of dosing. Vaginal lavage wa s conducted daily during th e last five days of the acclimation period to verify complete remova l of the ovaries as indicated by a lack of estrous cycling. The dose administration pe riod was from PND 57 through 59. Rats were euthanized approximately 24 hours later on PND 60, and necropsied for uterine weight

measurements.

3. Animal Assignment: Animals were assigned to the test groups noted in Table 1, using a computer program which randomized the animal s based on stratification of body weights in a block design. Statistical anal ysis indicated that there we re no significant differences between group mean weights at study initiation. Furthermore, the body weight of each
animal was within  20% of the overall mean.

TABLE 1. Study Design a Test Group Dose (mg/kg/day) # of Females Estrogen Agonist Assay Vehicle Control (Corn oil) 0 6

Metolachlor Low 100 6
Metolachlor Mid 300 6 Metolachlor High 1000 6
17 α -ethynyl estradiol (EE), Reference Estrogen 0.0005 6 a Data were obtained from page 22 of the study report.



### 4. Dose Selection Rationale: The dose levels were selected based on published toxicity data in which a pilot range -finding study (U.S. EPA 1 

### ) showed no effects on body weights at 800 mg/kg/day metolachlor. Therefore, the lim it dose of 1000 mg/kg/day was selected for the high-dosage level for the uterotr ophic study. The dosage level for 17 α -ethynylestradiol was based on the validation study (WIL-99579 2 

### ).

5. Dose Preparation and Analysis: The metolachlor formulations were prepared once as single formulations for each dosage level, divide d into sterile aliquots for daily dispensation, and stored at room temperature. The positiv e control substance (EE) was dissolved in a minimal amount of 95% v/v aqueous ethanol solu tion prior to addition of corn oil for a 0.05 mg/mL stock solution. The stock solution was prepared once and stored at room

1 United States Environmenta l Protection Agency. (1995) Registration Eligibility Decision (RED): Metolachlor

. (EPA 738-R-95-006), Washington, DC: Government Printing Office.

2 Kuhl, A.J. (2011) A Baseline Pos itive Control Uterotrophic Assay of 17 α -Ethynylestradio l Administered Subcutaneously in Ovariectomized Sp rague Dawley Rats (WIL-99579). WIL Research Laboratories, LLC, Ashland, OH.In vivo Uterotrophic Assay (2012) / Page 5 of 9 METOLACHLOR / 108801 OCSPP 890.1600/ OECD 440



### temperature, protected from light. EE dosing formulations were prepared daily by dilution of the stock solution with corn oil. The test substance and positive control substance formulations were stirre d continuously throughout the preparation, sampling, and administration.

Prior to the initiation of dose administration, quadruplicate sa mples for homogeneity determination were collected from the top, middle, and bottom strata of the 20 and 200 mg/mL dosing formulations (100 and 1000 mg /kg/day, respectively). For stability determinations, quadruplicate samples for resu spension homogeneity determinations were collected from the top and bottom strata of these same dosing suspensions following room temperature storage for 13 days. In add ition, quadruplicate samp les for concentration analysis were collected from the middle stra tum of each dosing formulation. All analyses were conducted by the Analytical Chem istry Department at WIL Research.

Results of Dose Analysis

Homogeneity (% RSD): 1.8-2.5%

Stability (% of initial): 92.5-97.9% following 13 days at room temperature.

Concentration (% of nominal): 98.8-106%

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: Animals were administered the metolachlor formulations, EE, or vehicle (corn oil) daily via s.c. injection for three consecutiv e days in a dose volume of 5 mL/kg body weight. Each daily dosage of th e test substance was divided between two injection sites (two distinct regions on the scapular region of the rat). The dosage volume for all groups was 5 mL/kg and individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/ day dose. All animals were dosed at approximately the same time each day.

7. Statistics: Data statistically an alyzed by WIL Laboratories included body weights and body weight gains. Analyses were two-tailed for significance levels of 5% and 1%. Two subset analyses were conducted. Body weights and body weight gains were analyzed using a twosample t-test to compare the positive cont rol group to the vehicle control group. Body weights and body weight gains were analy zed using Dunnett’s test to compare the metolachlor-treated groups to the vehicle control group.

Data statistically analyzed by BioSTAT Consultants, Inc. in cluded wet uterine weight and blotted uterine weight. Two s ubset analyses were conducted. All analyses were conducted using SAS version 9.1 (SAS Institute, Inc., 2002-2003), or higher, software.

Levene’s test was applied for uterine weight, at the 0.01 sign ificance level, to test for homogeneity of variances. If that test was si gnificant, then a log tr ansform was applied toIn vivo Uterotrophic Assay (2012) / Page 6 of 9 METOLACHLOR / 108801 OCSPP 890.1600/ OECD 440



### the data and Levene’s test repe ated. If that test was still si gnificant, then the nonparametric Wilcoxon rank sum test (EE compared to vehi cle control) or the nonparametric KruskalWallis test followed by Dunn’s test (test substan ce compared to vehicle control) was used. The tests were one-sided, at the 0.05 significan ce level, looking for significant increases from the vehicle control. If Le vene’s test was not significant, then an analysis of covariance (ANCOVA) was conducted, on the raw or log-transformed data , as appropriate. The necropsy body weight was used as the covariate in the analysis of wet and blotted uterine weight. For the metolachlor groups, one-sided Dunnett’s test was conducted regardless of the outcome of the ANCOVA for significant increas es (p<0.05) compared with the vehicle control.

C. METHODS

1. Clinical Examinations: All rats were observed (cage-si de) twice daily for moribundity and mortality. Individual clinical observations (hand-he ld physical examination) were recorded daily (prior to test substance administration during the treatment peri od) through the day of euthanasia. Animals were also observed for si gns of toxicity at th e time of dosing (within 15 minutes) and approximately 1 hour following dose administration.

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing period and at termination. Mean body weight ga ins were calculated for each corresponding interval and for the overa ll (Days 1-4) study period.

3. Food Consumption (Optional): Food consumption was not measured.

4. Necropsy and Measurement of Uterine Weight: On PND 60 (approximately 24 hours after final administration of the test substance), all anim als were euthanized by carbon dioxide inhalation and subjected to a gross necropsy. Animals were examined for residual ovarian tissue. Dissection of the uterus was performed according to the U.S. EPA Guideline. Briefly, the vagina was removed ju st below the cervix in order to retain the luminal fluid in the uterus. The “wet” uter us (i.e., containing th e luminal fluid) was weighed. Subsequently, the uterine horns were cut longitudinally and blotted with filter paper to remove the luminal fluid while prev enting desiccation. The blotted uterus was weighed.

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: No test substance-related clin ical findings were noted at the daily examinations, at the time of dose administration, or 1 hour following dose administration at any dosage level. Findings noted in the metolachlor-treated groups (300In vivo Uterotrophic Assay (2012) / Page 7 of 9 METOLACHLOR / 108801 OCSPP 890.1600/ OECD 440



### and 1000 mg/kg/day), including red or yellow material on various body surfaces occurred infrequently, on the day following administration of the last dose, and/or in a manner that was not considered treatment-related.

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data are presented in Table 2. Body weights were comparable to the vehicle controls throughout the duration of the assay, but ove rall (Days 1-4) body weight gains were decreased (p<0.01) in the 300 mg/kg/day ( ↓ 42%) and 1000 mg/kg/day ( ↓ 53%) metolachlor groups compared to the vehicle controls. B ody weight gains in the positive control (EE) group were also decreased (p<0.01) by 57% compared to 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 Metolachlor (100) Metolachlor (300) Metolachlor (1000) Reference Estrogen EE (0.0005)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD
1 6 227.3 16.9 6 226.1 18.6 6 226.4 16.1 6 232.4 23.0 6 225.6 17.6

2 6 233.2 18.0 6 227.8 17.4 6 228.1 17.5 6 235.3 23.5 6 230.6 17.8
3 6 239.3 17.6 6 234.0 17.9 6 233.8 16.8 6 238.9 22.0 6 231.4 16.6 4 (termination) 6 245.2 18.8 6 239.8 18.1 6 236.7 15.9 6 240.8 19.6 6 233.3 16.0 Body Weight Gain (Days 1-4) 6 17.9 3.0 6 13.7 4.3 6 10.3** ( ↓ 42) 3.0 6 8.4** ( ↓ 53) 4.0 6 7.7** ( ↓ 57) 4.1

a Data were obtained from Tables S3 and S4 on pages 42 and 43 of the study report; note that the study numbered days started with 0 (dose initiation), but the re viewer numbered the study days starting with 1 (dose initiation). BWG percent differences (calculated by the reviewers) are included in parentheses. N Number of animals in the group SD Standard Deviation ** Significantly different from controls at p<0.01



### C. FOOD CONSUMPTION (Optional): Food consumption was not measured.

D. PATHOLOGY

1. Uterine Weights: No residual ovarian tissue was noted for any of the animals at necropsy. Selected uterine weight data are presented in Table 3. Uterine weights in the metolachlor treated groups were comparable to the vehicl e controls. Absolute wet and blotted uterus weights for the positive control (EE) group we re increased (p<0.05) as expected by 288% and 175%, respectively.

The mean blotted uterine weight for the ve hicle control group was 0.03% of terminal body weight, which was within the performance criteria (<0.09%) of th e test guideline.In vivo Uterotrophic Assay (2012) / Page 8 of 9 METOLACHLOR / 108801 OCSPP 890.1600/ OECD 440

TABLE 3. Uterine Weights from Estr ogen Agonist Assay in SD Rats a

Parameters Dose (mg/kg/day)

Vehicle Control Metolachlor (100) Metolachlor (300) Metolachlor (1000) Reference Estrogen EE (0.0005)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

Terminal BW (g) 6 245.2 18.8 6 239.8 18.1 6 236.7 15.9 6 240.8 19.6 6 233.3 16.0

Wet, absolute (g) 6 0.0817 0.0111 6 0.0764 0.0096 6 0.0733 0.0136 6 0.0754 0.0067 6 0.3171* ( ↑ 288) 0.0465

Wet, relative (%) 6 0.030 0.004 6 0.032 0.005 6 0.031 0.006 6 0.031 0.002 6 0.136 0.019

Blotted, absolute (g) 6 0.0729 0.0088 6 0.0683 0.0089 6 0.0663 0.0135 6 0.0674 0.0064 6 0.2006* ( ↑ 175) 0.0325

Blotted, relative (%) 6 0.030 0.004 6 0.029 0.005 6 0.028 0.006 6 0.028 0.002 6 0.086 0.0120

a Data were obtained from Tables S6, S7, and S8 on pages 45-47 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. BW Body weight N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05



### 2. Microscopic Examination (Optional): No microscopic examinations were conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the lack of higher mean uterine weights (wet and blotted), metolachlor technical did no t demonstrate or mimic biological activities consistent with agonism of natural estrogens when administered by subcutaneous injection to ovariectomized female rats at dosag e levels of 100, 300, and 1000 mg/kg/day. The positive control substance (17 α -ethynylestradiol) elicited the expected increases in uterine (wet and blotted) weights.

B. AGENCY COMMENTS: All animals survived until scheduled termination. No test substance-related clinical findings were not ed in any of the animals during the study. However, minor clinical findi ngs (red/yellow material on va rious body surfaces) were noted for the 300 and 1000 mg/kg/day groups; these occurrences were infrequent and not considered to be treatment related.

Body weights were comparable to the vehicle controls throughout the duration of the assay, but overall (Days 1-4) body we ight gains were decreased (p<0.01) in the 300 mg/kg/day ( ↓ 42%) and 1000 mg/kg/day ( ↓ 53%) metolachlor groups compared to the vehicle controls. Body weight gains in the positive control (EE) group were al so decreased (p<0.01) by 57% compared to the vehicle controls.In vivo Uterotrophic Assay (2012) / Page 9 of 9 METOLACHLOR / 108801 OCSPP 890.1600/ OECD 440



### No abnormal gross observations were seen at necropsy. Uterine weights in the metolachlor treated groups were comparable to the vehicl e controls. Absolute wet and blotted uterus weights for the positive control (EE) group we re increased (p<0.05) as expected by 288% and 175%, respectively.

Based on the observed decreases (42% and 53%) in body weight gain at 300 and 1000 mg/kg/day, adequate dose levels were achieved in this study.

No statistically significant changes were s een in the uterine wei ght in this assay. Metolachlor was negative in the uterotrophic assay.

C. STUDY DEFICIENCIES: None.