## UNITED ST A TES ENVIRONMENT AL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



## MEMORANDUM

Date: 6/29/2015

SU BJECT : Methomyl: Data Evaluation Records (DERs) for EDSP Tier 

## 1 Assays

PC Code: 090301



## Decision No.: 457878, 457883

, 460540 

## DP Barcode: D396312, D396323, D398419 D399624, D403696 

## Registration No.: NA 462101

, 467223 

## Petition No.: NA 

## Risk Assessment Type: NA 

## TXR No.: 0056254 

## MRID No.: See Table 

## Regulatory Action: NA 

## Case No.: NA 

## CAS No.: 16752-77-5 

## 40CFR: NA



## FROM: Greg Akerman

, Ph.D.

# b~ Immediate Office Health Effects Division (7509P)



## THROUGH: Jess Rowland 

# de 6 1:.

# ~~ 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. 20 I 0



## 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 methomyl: Test Order# EDSP-090301-62.

Page I of2

### Page 2 of 2 

## II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for methomyl.

III. MRID Table

Chemical: Methomyl PC Code: 090301 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48701402 890.1150 Androgen Receptor Binding (Rat Prostate) 48672001 890.1200 Aromatase Assay (Human Recombinant) 48672002 890.1250 Estrogen Receptor Binding 48672003 890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) 48685901

890.1350 Fish Short-Term Reproduction 48701401 890.1400 Hershberger (Rat) 48617702 890.1450 Female Pubertal (Rat) 48701404 890.1500 Male Pubertal (Rat) 48701403 890.1550 Steroidogenesis (Human Cell Line – H295R) 48685902 890.1600 Uterotrophic (Rat) 48617701

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

EPA MRID Number 48701402

Page 1 of 39

DER Template Version: 22 September 2011 Data Requirement: EPA DP Barcode 403694

OECD Data Point 231

EPA MRID 48701402

EPA Guideline 890.1100 Amphibian Metamorphosis Assay (Frog)

Test Material: Methomyl Technical Purity (%): 99.4% Common Name Methomyl (DPX-X1179)

Chemical Name IUPAC S -methyl ( EZ )- N -(methylcarbamoyloxy)thioacetimidate
CAS Name Methyl N -[[(methylamino)carbonyl]oxy]ethanimidothioate

CAS No. 16752-77-5
Synonyms None

EPA PC Code 090301

Primary Reviewer: John Marton, Ph.D Date: 5/13/13
Environmental Scientist, CDM Smith

Secondary Reviewer: Teri S. Myers, Ph.D. Date: 5/15/13
Environmental Scientist, CDM Smith

Primary Reviewer: Tanja Crk Date: 7/24/13
Biologist, EPA/OPP/EFED

Secondary Reviewer: Elyssa Arnold Date:
Biologist, EPA/OPP/EFED

Additional Reviewer: Amy Blankinship Date:

Chemist, EPA/OPP/EFED
Date Evaluation Completed: 6/5/15 2015.06.17 07:40:04 -04'00'



### AMY BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.17 07:44:55 -04'00'

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

EPA MRID Number 48701402

Page 2 of 39

DER Template Version: 22 September 2011 CITATION: Fort, D.J. 2012. Methomyl (DPX-X1179) Technical: 21-d Amphibian Metamorphosis Assay (AMA)
with South African Clawed Frog, Xenopus laevis . Unpublished study performed by Fort Environmental
Laboratories, Inc., Stillwater, OK. Laboratory report numbe r DUPO01-00269. Study sponsored by E.I. du Pont de Nemours and Company (DuPont), Wilmington, DE. Study completed May 31, 2012.

Note: 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, andro gen, 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 determi ne 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).

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

EPA MRID Number 48701402

Page 3 of 39

DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay of methomyl on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was studied under flow-through conditions. Amphibian larvae (80 larvae per group) at Nieuwkoop-Faber (NF) stage 51 were exposed to nominal concentrations of 0 (negative control), 0.0183, 0.056, and 0.168 mg a.i./L; mean-measured concentrations were <0.0124 (<LOD; control), 0.0170, 0.0522, and 0.186 mg a.i./L. The test system was
maintained at 21.9 to 22.4°C and a pH of 6.9 to 7.2.

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control. One mortality (1.7%) was observed in the highest test concentration. All surviving tadpoles appeared normal and

healthy with no apparent clinical signs present at test termination.

There was no significant acceleration or delay of median NF developmental stage at Day 7 or Day 21 in any
treatment group. Furthermore, no asynchronous development was observed. All individuals developed beyond NF stage 60 by Day 21; the NF stage ranged from 61 to 65 in all treatment groups and in the control. The reviewer’s analysis detected significantly decreased normalized (for snout vent length) hind-limb length (HLL) at both Day
7 ( Ę 4.7%) and Day 21 ( Ę 4%) at high treatment level relative to the negative control (p<0.05). Additionally, Day 21 snout vent length (SVL) was significantly increased ( Ė 6%) at the high treatment level relative to the negative control (p<0.05). Thyroid gland histopathology revealed mild to moderate follicular cell hypertrophy in

the negative control and all treatment groups, although there was no treatment-related pattern. There were no
incidences of thyroid gland hypertrophy, atrophy, or follicular cell hyperplasia.

At Day 21, all tadpoles were great er than NF stage 60 (late stage) which may c onfound the ability to adequately
interpret the Day 21 results, however, there was no significant acceleration or delay of median NF developmental
stage at any treatment concentration throughout the test nor effects on thyroid histopathology compared to the

control.

All validity criteria were met in this study. The assay satisf ies the EDSP Tier 1 Test Order requirements for an Amphibian Metamorphosis Assay (OCSPP Guideline 890.1100).

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

EPA MRID Number 48701402

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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 Methomyl.

Treatment

(mg a.i./L)

[measured] NF Developmental
Stage Hind Limb
Length 3,4 

### Asynchronous
Development Thyroid Gross

and
Histopathology
Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21

0.0170 No No No No No No No

0.0519 No No No No No No No

0.186 No No Yes Yes No No No

Abbreviations: Diff. 

### Difference. NA 

### Not applicable.

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).

4 

### At Day 21, data are for “late stage” individuals because all individuals were at NF stage >60.



## I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guidelines outlined in U.S. EPA, Endocrine
Disruptor Screening Program Test Guidelines, OCSPP 890.1100, Amphibian

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

EPA MRID Number 48701402

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DER Template Version: 22 September 2011

Metamorphosis Assay (Frog) (October 2009). The following deviation(s) were

noted:

1. The water hardness during the definitive exposure period (134-152 mg/L as CaCO 3 

### ) exceeded the recommended range (40-48 mg/L as CaCO 3 

### ).

2. The LOQ stated in the study report is 0.0417 mg a.i./L, which is higher than the lowest test
concentration of 0.0183 mg a.i./L. However, there were no reported effects at this concentration.

These deviation(s) do not impact the interpretation of the study.

Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements
were provided. This study was conducted in compliance with U.S. EPA FIFRA (40 CFR part 160) Good Laboratory Practice Standards, which are compatible with current OECD and MAFF (Japan) Good Laboratory Practices.

A. Test Material Methomyl (DPX-X1179) Technical

Description: Powder

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. : DPX-X1179-571 (Lot No) / JUL06LP276 (Batch No)

Purity: 99.4%

Impurities: None listed

Stability of Compound: Stable. Mean-measured concentrations yielded recoveries of 93 to 111% of

nominal with coefficients of variation of 1.2 to 4.3%.

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

EPA MRID Number 48701402

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DER Template Version: 22 September 2011 Storage Conditions of

Test Chemicals: Stored under ambient conditionsData Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page7of39 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 Guidelinesfor 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 Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page8of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained asin-house stock?Yes 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):NAParental frogs were continuouslymaintained under conditions similar tothose employed in the definitive test.Details on parental feeding:Parental frogs were fed~1 tablespoon of #3frog food pellets per tanktwice a day.Food source is Zeigler Bros., Inc.Details on parental health:Parental frogs were ingood health, displayingno abnormalities orsigns of illness.Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page9of39 DER Template Version: 22 September 2011Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest single spawn?Yes EPA and OECD recommend that the best 2 – 3 individual spawns, with a minimum of 1500 larvae/spawn, be evaluated to identify the best single spawn, and that the larvae selected for testing originate from the best single spawn

(i.e., the spawns are not co-mixed)Number of spawns evaluated (ifapplicable):2Number of eggs sampled perspawn:All eggs were sampledon a given day.Embryonic viability was checked daily.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:14 days post-fertilization(dpf)Mean total length at test initiation(if reported):NARange of total length at testinitiation (if reported):NAData Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page10of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas the optional size selectionmethod used?NoDetails on larval selection:When a sufficientnumber of thepre-exposurepopulationreached NF 51, larvaewere transferred to apooling tank. Larvaerequiring >17 days toreach NF 51 were notused in the test. If >50%of the larvae in a givenclutch required >17days to reach NF 51, analternate clutch wasused. All tadpoles werefrom a single clutch.In order to guarantee at least 400 stage51 acceptable larvae at test setup, clutchsizes of ~1,500 embryos were givenpreference.Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page11of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsLoading rate (rearing density):5 larvae/L EPA recommends that rearing density (loading rate) not exceed approximately 10 larvae/L culturing system for flow - through systems or 4 tadpoles/L in static- renewal exposure systems.Type of food:Sera Micron® EPA recommends Sera Micron ® throughout

pre - exposure (after NF stage 45/46) and during the entire 21 -d definitive study. If another diet is used, the study report should provide analysis of iodide content and potential contaminants, and the diet should demonstrate equal performance to Sera

Micron ® .Source of food:Sera Micron® USA,Montgomeryville, PAIodide concentration in diet (ifknown):Not ProvidedFrequency of feeding:2 times/day EPA recommends that feeding occur at least
twice per day.Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page12of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding regime:Same as EPA guidelinesDay 0-4 : 30 mg/animal/dayDay 5-7 : 40 mg/animal/dayDay 8-10 : 50 mg/animal/dayDay 11-14 : 70 mg/animal/dayDay 15-21 : 80 mg/animal/day It is recommended that food rations during the

pre - exposure period be increased along with larval growth to approximately 30 mg/larva/day by test initiation . EPA and OECD recommend that food rations increase from 30 mg/larva/day at test initiation (Study

Day 0 - 4) to 80 mg/larva/day in the last week of the test (Study Day 15-21).Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page13of39 DER Template Version: 22 September 2011C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through

system.Type of flow-through dilutionsystem (if applicable):Continuous flow serialdiluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate (ifapplicable):25 mL/minFlow-rate provided a complete volumereplacement approximately every 2.7 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 Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page14of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems (ifapplicable):Modified BenoitMini-DiluterMixing chamber was a continuous mixchamber in which stock solution and dilutionwater were supplied. Baffle ensured completemixing since the concentration desired as ator below the limit of water solubility.Concentration prepared in mixing chamberrepresents the highest concentration, and theremaining concentrations were obtained bysequential dilution. Recommended toxicant mixing for

flow -through systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is

within 10%.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 Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page15of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water:Treated municipal tapwaterWater was prepared by passing tap waterthrough a 4 filter system: a multimedia filterto remove suspended solids in the feedwater; a 10” pre-treatment filter (5μm) toremove any additional solids;a 3.6 ft3activated virgin carbon treatment filter toremove chlorine, ammonia, and highermolecular weight organics; and a 5μmpolishing filter to remove any carbon particlesfrom the carbon treatment phase. EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with U V 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?Yes (pesticides, PAHs,PCBs, and heavy metals)Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page16of39 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 and stainless steel 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:22.5 x 14.0 x 16.5 cmFill volume:4.0 LAdditional details on exposuresystem:NoneData Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page17of39 DER Template Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsHardness (mg/L asCaCO3)134 152 142Weekly EPA recommends hardness 40 to 48 mg/L as CaCO

3.pH6.97.2 NA3x Weekly EPA recommends pH 7.5 ± 1, inter-replicate and inter-treatment differentials should not exceed 0.5.Dissolved oxygen(mg/L)3.6 6.8 4.83x Weekly EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation) . OECD recommends DO concentration

>3.5 mg/L (>40% air saturation).Temperature21.9 22.4 22.2Daily EPA recommends temperature 22±1 o C; inter-replicate and inter-treatment differentials should not exceed 0.5 o C.Iodide(μg/L) 2 5.1 3.6Semi-annually EPA recommends aquatic iodide range 0.5 – 10 μ g/L (supplemental iodide should not exceed 2 μ g/L).Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page18of39 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsAmmonia (mg/L)00.040.02Bi-monthly 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 temperature be measured continuously . OECD recommends that DO and temperature be measured at least weekly and that pH and hardness be measured at least at the beginning and end of

the test.FluorideNot ReportedPerchlorateNot ReportedChlorateNot ReportedOtherNA NA NA NAData Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page19of39 DER Template Version: 22 September 2011D. 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?Historical toxicity dataprovided in a similarspecies by the Sponsor. 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:10Test duration:4 daysData Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page20of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:A 4-day range-findingstudy was conductedunder static conditionsat nominalconcentrations of 0(negative control), 0.1,0.5, 1.0, 5.0, 10.0,25.0, and 50.0 mga.i./L. The LC50andEC50values were 2.67and 0.80 mg a.i./L,respectively.No morbidity was observed at 0.51 mga.i./L, which was the highestrange-finding test concentration withoutadverse effects. Therefore, 0.51 mg a.i./Lwas set as the highest concentration in thedefinitive test with a 0.33x dilution series.However, abnormal swimming behaviorwas observed after 24 hrs in the definitivestudy at 0.51 mg a.i./L. Since thiscompromised the collection of data for theAMA study, the study was terminated atthe direction of theSponsor andre-initiated using 0.168 mg a.i./L as thehighest test conc. The 0.33x series wasmaintained: 0, 0.0183, 0.056, 0.168 mga.i./L.Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page21of39 DER Template Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in the definitivetest:Range-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):NASeparation of test concentrations:0.33 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.Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page22of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAre nominal concentrationsadjusted for purity?NoIndicate the type of valuespresented for measuredconcentrations:Mean-measuredconcentrationsLimit of quantification (LOQ):0.0417 mg a.i./LThe lowest test concentration is 0.0183mg a.i./L, lower than the reported LOQ. EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):0.0124 mg a.i./LFrequency of measurement:7 days 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.Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page23of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in solventcontrol (ifapplicable):NA EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of larvae per treatment attest initiation:80Was a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA EPA recommends that the solvent not exceed

0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006).Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page24of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas a positive control used?NoPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12
(light:dark).Light intensity at water’s surface:0.6-2.0 Klux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:All test solutions wereclear and colorless withno precipitatethroughout the study.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 Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page25of39 DER Template Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Methomyl.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0.00<0.0124NABased onmean-measuredconcentrations. EPA and OECD recommend that test item concentrations be maintained at a coefficient of
variation (CV)≤ 20%.Treatment 10.01830.01704.3Treatment 20.0560.05192.0Treatment 30.1680.1861.2Abbreviations:CVCoefficient of variation.E. ObservationsBiological Endpoints:Day 7: NF stage, wet weight, SVL, HLL, normalized HLLDay 21: NF stage, wet weight, SVL, HLL, normalized HLL, thyroid histopathologyDaily: mortality, clinical signsWere raw (individual) data provided?YesData Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page26of39 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 Methomyl Technical to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 

## II. RESULTS AND DISCUSSION

A. Results

Throughout the entire test, a single mortality was observed, in the mean-measured 0.186 mg a.i./L treatment group (Table 9). No treatment-related patterns were noted.

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 0 0 60 0 0

0.0170 80 0 0 60 0 0

0.0519 80 0 0 60 0 0

0.186 80 0 0 60 1 2 

### 1.7

Abbreviations: NA 

### Not applicable.

1 

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

2 

### The mortality occurred in replicate C at Day 16.

By Day 7, median development stage was 54 in the control and all treatment groups (Table 10). At test termination, median development stage was 63.5 (in the control, low-, and mid-concentration treatment
groups and 63 in the mid-concentration treatment group. All individuals were at NF stage >60 at test
termination. In all treatment groups and in the control, NF stage ranged from 61 to 65. The 10 th 

### and 90 th



### percentiles of developmental stage in negative control tadpoles were 62 and 65, respectively. There was
no asynchronous development.

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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 63.5 0

0.0170 4 54 0 4 63.5 0

0.0519 4 54 0 4 63.5 0

0.186 4 54 0 4 63 0

Abbreviations: NA 

### Not applicable.

Mean Day 7 normalized HLL was 0.43 in the control and 0.0170 mg a.i./L treatment group, 0.42 in the 0.0519 mg a.i./L treatment group, and 0.41 in the 0.186 mg a.i./L treatment group (Table 11). Mean

normalized HLL on Day 21 was 1.25 in the control and mean-measured 0.0170 mg a.i./L treatment group
and 1.23 and 1.20 in the mean-measured 0.0519 and 0.186 mg a.i./L treatment groups, respectively.

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 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 1

n Mean
(mm) ±SD HLL:

SVL 2 n Mean
(mm) ±SD HLL:

SVL 2



### Negative Control 4 7.1 0.47 0.43 4 23.2 0.96 1.25

0.0170 4 7.2 0.73 0.43 4 23.3 1.26 1.25

0.0519 4 7.2 0.44 0.42 4 23.1 1.07 1.23

0.186 4 7.3 0.26 0.41 4 23.3 0.96 1.20

Abbreviations: NA 

### Not applicable. SD 

### Standard deviation.

1 

### Values are for late stage individuals because all tadpoles were at NF stage >60 at Day 21.

2 

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

Day 7 SVL averaged 16.3 mm in the negative control and 16.9, 16.5, and 17.4 mm in the mean-measured 0.0170, 0.0519, and 0.186 mg a.i./L treatment groups, respectively (Table 12). By Day 21, mean SVL
ranged from 18.9 mm in the negative control to 20.1 mm in the mean-measured 0.186 mg a.i./L treatment

group.

Day 7 wet weight averaged 0.357 g in the negative control and 0.369, 0.315, and 0.344 g in the mean-measured 0.0170, 0.0519, and 0.189 mg a.i./L treatment groups, respectively (Table 12). Day
21 wet weight averaged 0.952 g in the negative control and ranged from 0.906 to 1.04 g in the treatment

groups.

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Table 12: Larval Growth in Xenopus laevis

.

Treatment
(mg a.i./L)

[meanmeasured] Snout-Vent Length (SVL) Body Weight 1



### Day 7 Day 21 2 

### Day 7 Day 21 2

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

Negative Control 4 16.3 1.20 4 18.9 0.64 4 0.357 0.04 4 0.952 0.12

0.0170 4 16.9 1.08 4 19.0 0.64 4 0.369 0.05 4 0.906 0.08

0.0519 4 16.5 0.70 4 19.6 0.45 4 0.315 0.03 4 0.975 0.05

0.186 4 17.4 0.76 4 20.1 0.98 4 0.344 0.04 4 1.04 0.11



### Abbreviations: NA 

### Not applicable. ND 

### Not determined. SD 

### Standard deviation.

1 

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

2 

### Values are for late stage individuals because all tadpoles were at NF stage >60 at Day 21.

No thyroid gland hypertrophy, atrophy, or follicular cell hyperplasia were observed in any control or treatment group tadpoles (Table 13). Mild and moderate follicular cell hypertrophy were observed in the
negative control and mild follicular cell hypertrophy was observed in all treatment groups, though there
was no treatment-related pattern. Tadpoles selected for histopathology were stage matched to the median
development stage in the control at test termination.

### Data Evaluation Record on the Toxicity of Methomyl Technical 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)

[measured] Diagnostic Observations 1

Severity 

### Thyroid Gland
Hypertrophy Thyroid Gland
Atrophy Follicular Cell
Hypertrophy Follicular Cell
Hyperplasia

n Incidence n Incidence n Incidence n Incidence



### Negative Control 0 20 20 20 20 20 16 20 20

1 20 0 20 0 20 2 20 0

2 20 0 20 0 20 2 20 0

3 20 0 20 0 20 0 20 0

0.0170 0 20 20 20 20 20 17 20 20

1 20 0 20 0 20 3 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.0519 0 18 18 18 18 18 15 18 18

1 18 0 18 0 18 3 18 0

2 18 0 18 0 18 0 18 0

3 18 0 18 0 18 0 18 0

0.186 0 20 20 20 20 20 14 20 20

1 20 0 20 0 20 6 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

1 Thyroid gland gross pathology and histopathology are 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 Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page32of39 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 Control020--20--20--20--20--120--20--20--20--20--220--20--20--20--20--320--20--20--20--20--0.0170020--20--20--20--20--120--20--20--20--20--220--20--20--20--20--320--20--20--20--20--0.0519018--18--18--18--18--118--18--18--18--18--218--18--18--18--18--318--18--18--18--18--Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page33of39 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 CellShapenIncidencenIncidencenIncidencenIncidencenIncidence0.186020--20--20--20--20--120--20--20--20--20--220--20--20--20--20--320--20--20--20--20--1Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 forreference.No signs of clinical toxicity were observed during the study (Table 15).Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page34of39 DER Template Version: 22 September 2011Table 15: Clinical Signs in Xenopus laevis.Treatment(mg a.i./L)[measured]Clinical SignsTypenIncidenceNegative ControlNA2000.0170NA2000.0519NA2000.186NA200

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

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

The study author analyzed survival, wet weight, normalized HLL, SVL, and NF stage at days 7 and 21. Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant
effects were detected for p d 0.05 (statistical software not reported). The study author reported using statistical procedures described in the OECD (2006) guideline, which follow the flow chart and
recommendations in the OCSPP 890.1100 guideline. However, results from these statistical analyses
were not presented in the study report.

Statistical analyses presented included an ANOVA test for Day 7 SVL and wet weight. A ranked ANOVA
test was conducted for Day 21 SVL and wet weight and both Day 7 and Day 21 normalized HLL. A
Mann-Whitney U test was conducted for NF stage. Using these tests, no statistically significant differences
were identified in the study data.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: All endpoints were tested for normality using Shapiro-Wilks test ( α = 0.01) and for
homogeneity of variance using Bartlett’s test ( α = 0.01). Day 7 normalized HLL, Day 21 normalized HLL,
and Day 21 SVL exhibited monotonic trends and therefore were analyzed using the Jonckheere-Terpstra
test. All continuous endpoints met the assumptions of pa rametric statistics and therefore those without apparent monotonic trends were analyzed using Dunnett’s Multiple Comparison test. Day 7 and Day 21
developmental stage were analyzed using the Mann-Whitney U Two-Sample test using the replicate median values. All surviving tadpoles on Day 21 were considered late-stage (NF ≥ 61). These analyses
were conducted using CETIS v. 1.8.7.7. and backend database settings created by EFED on 1/30/13.

No asynchronous development was observed. Unless otherwise indicated, effects were considered
statistically significant at p < 0.05.

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Conclusions:

Normalized HLL was significantly reduced (p<0.05; Jonckheere-Terpstra) in the high-concentration
treatment group relative to the negative control on Days 7 and 21. SVL was significantly increased in the high-concentration treatment group relative to the negative control on Day 21 only (p<0.05;
Jonckheere-Terpstra). All tadpoles were at NF stage >60 at Day 21, however, there was no significant
acceleration or delay of median NF developmental stage at any treatment concentration throughout the
test nor effects on thyroid histopathology compared to the control.Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis AssayEPA MRID Number 48701402Page37of39 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Methomyl (DPX-X1179) Technical.Treatment(mg a.i./L)[mean-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 214Day 7Day 214Day 7Day 214Day 214Median pMedian p%Diff. p%Diff. p%Diff. p%Diff. pTreatment-RelatedEffects? (Yes/No)Negative Control54--63.5------------------No0.0170541.063.51.01.180.360.00.50--------No0.0519541.063.51.0-2.350.17-2.00.24--------No0.186541.0631.0-4.710.02-4.00.046--------NoStatistical TestMann-WhitneyMann-WhitneyJonckheereJonckheere------Abbreviations: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).4Values are for late stage individuals because all tadpoles were at NF stage >60 at Day 21.

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

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DER Template Version: 22 September 2011 Table 17: Growth Endpoints 1,2 

### in the AMA with Methomyl (DPX-X1179) Technical.

Treatment

(mg a.i./L)

[measured] Snout-Vent Length Body Weight
Day 7 Day 21 3 

### Day 7 Day 21 3



### % Diff. p %

Diff. p % Diff. p % Diff. p

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

0.0170 4.31 0.62 0.4 0.44 3.29 0.95 -4.78 0.83

0.0519 1.54 0.97 3.44 0.052 -11.8 0.35 2.44 0.97

0.186 7.38 0.23 6.08 0.015 -3.5 0.94 9.38 0.43

Statistical Test Dunnett’s Jonckheere Dunnett Dunnett

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 

### Values are for late stage individuals because all tadpoles were at NF stage >60 at Day 21.

E. Study Deficiencies

Although there were deviations from the guideline, as noted in the Materials and Methods section of the
DER, the study met all validity and performance criteria.

F. Reviewer’s Comments

The study author did not detect any differences in dev elopmental stage or normalized HLL in any treatment
group relative to the negative control on Day 7 or 21. However, the reviewer detected slight but statistically
significant decreases in normalized HLL in the high-concentration treatment group on both Days 7 and 21. Therefore, the reviewer’s results are reported in the Executive Summary of this DER.

An initial definitive test was conducted with nominal concentrations of 0 (negative control), 0.056, 0.168,

### Data Evaluation Record on the Toxicity of Methomyl Technical to Amphibians, Metamorphosis Assay

EPA MRID Number 48701402

Page 39 of 39

DER Template Version: 22 September 2011 and 0.510 mg a.i./L. However, this study was terminated due to erratic swimming behavior (circles) in
the highest treatment group, which was not observed in the range-finder. The test was reinitiated with
adjusted nominal concentrations reported in this DER.

Overall, the reviewer’s mean values for SVL and HLL generally agreed with those reported by the study
author, though mean values for normalized HLL slightly differed. These differences could be attributed
to rounding differences during the calculation of normalized HLL, however, these differences in rounding should not discount the consistent significant (p<0.05) findings of reduced normalized HLL on day 7 and

21, relative to control values.

The in-life portion of the definitive toxicity test was conducted from May 18 to June 15, 2011.



## III. REFERENCES

Nieuwkoop, P.D. and Faber, J. Normal Tables of Xenopus laevi (Daudin). Garland Publishing, London, 1994.

Grim, KC et al. Thyroid Histopathology Assessments for the Amphibian Metamorphosis Assay to Detect
Thyroid Active Substances. Toxicol. Pathol. 37: 415-424, 2009.

# DATA EVALUATION RECORD

METHOMYL

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

EPA Contract No. EP10H001452 Task Assignment No. 2-30-2012 (MRID 48672001)

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: 3/28/2012

Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 4/02/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/11/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/11/2012

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).METHOMYL I 090301 Androgen Receptor Binding (Rat Prostate Cytosol) (2012) I Page I of 15 OCSPP 890.1150/ OECD None



## Primary Reviewer: Karlyn Middleton 

## Health Effects Division, Office of Pesticide Programs Secondary Reviewer: Greg Akerman, Ph.D. 

## Health Effects Division, Office of Pesticide Programs Sign a tu re:**_L______,(.,C:.:::::::;:=~,-­** 

## Date:

__**_,'-'-__,,__,~-­** 

## Signature:

_**__._,.'-----"~----­** 

## Date: ------"--'----=---:.._,_..,__

__ Template version 08/2011



## DATA EVALUATION RECORD

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



## PC CODE: 090301 

## DP BARCODE: D3984 l 9



## TXR#: 0056254 

## CAS No.: 16752-77-5



## TEST MATERIAL (PURITY): Methomyl (99.4% a.i

. )



## SYNONYMS: DPX-Xl 179; methyl N-[[(methylamino)carbonyl]oxy]ethanimidothioate



## CITATION: Snajdr

, S

. (2012). Methomyl (DPX-Xl 179) technical: in vitro androgen receptor binding assay using rat prostate cytosol. DuPont Haskell Global Centers for Health & 

## Environmental Sciences, Newark, DE. Laboratory Project ID number: DuPont-32080, January 6, 2012. MRID 48672001. Unpublished.



## SPONSOR: E.l. du Pont de Nemours and Company, Wilmington

, DE



## TEST ORDER#: EDSP-090301-62



## EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48672001), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and a competitive binding experiment. The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radio-labeled reference androgen (RI 881) prior to conducting AR competitive binding experiments. The competitive binding assay was conducted to measure the binding of a single concentration of [3H]-Rl 881 (1 

## nM) in the presence of increasing concentrations (10

- 1 0 

## to 10

- 3 

## M) of methomyl (99.4% a.i., Lot # 

## J U L06LP276)

. Water was used as the solvent vehicle for methomyl, and ethanol was used as the solvent vehicle for the reference compounds at a final assay concentration of 3%. A total of three independent 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 4

. 5 fmol/l 00 μg protein and the dissociation constant (Kd) was 0

. 903 nM. The Kd was within the range reported in th e EPA validation program , but the Bm a x was below the EPA values (7-16 fmol / l 00 μg protein)

. The Scatchard plots for the three saturation assays were linear.

In the competitive binding experiment, the estimated average log ICs o was

- 9.15 M for RI 881 and

- 4.41 M for dexamethasone. Compared to Rl881

, the mean RBA for the dexamethasoneAndrogen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 2 of 15 METHOMYL / 090301 OCSPP 890.1150/ OECD None


## was 0.0019%. The performance criteria for R1881 and dexamethasone were not met across the three runs. In the first and third runs, the bottom % binding and Hill slope were outside the tolerance limits for R1881. For dexamethasone a ll of the three cr iteria were met, except for the top % binding in the third run which was hi gher than the toleran ce limits. Although, the performance criteria for both R1881 and dexameth asone were not achieved across the three runs, based on the estimated IC 50 

## s for R1881 and the weak positive c ontrol, the androgen receptor was functioning with appropriate affinity for the ligand in the competitive binding assays. It is unlikely that information from additional competitive binding runs would change the interpretation of this study.

The percent [ 3 

## H]-ligand bound to the AR was >75% at a ll concentrations of methomyl in all three runs. Therefore, a log IC 50 

## and RBA could not be calculated for methomyl.

Based on the results from the three runs, methomyl is classified as a Non-Binder in the Androgen Receptor Binding Assay.

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

COMPLIANCE: Signed and dated Data Confidentiality, GLP and Quality Assurance
statements were provided.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 3 of 15 METHOMYL / 090301 OCSPP 890.1150/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: E.I. du Pont de Nemours and Company, DuPont Haskell Global Centers for Health & Environmental Sciences
Location: Newark, DE Study Director: S. Snajdr Other Personnel: D. Styles (Staff Chemist); G. Jepson (Manager); J. O’Connor (Group Leader) Study Period: June 7, 2011 to January 6, 2012



## 2. Test substance: Methomyl
Description: Technical; solid Source: E.I. du Pont de Nemours and Company Lot/Batch #: JUL06LP276 (expiry date: May 18, 2016 Purity: 99.4% a.i. Solubility: Not reported Volatility: Not reported Stability: Formulations in water stable at room temperature for up to 8 days Storage conditions: Ambient CAS #: 16752-77-5 Molecular weight: 162.21 g/mole Structure:



## 3. Non-labeled ligand: R1881 Supplier: Perkin-Elmer (Waltham, MA) Catalog #: Lot #: NLP005005MG 590822 Purity: >97% CAS #: 965-93-5



## 4. Radioactive ligand: [ 3 

## H]-R1881 Supplier: Perkin-Elmer (Waltham, MA) Catalog #: Lot #: NET590250UC 614814 Date of production: July 1, 2010 Date of use: June 16, 2011 to July 11, 2011 Radiochemical purity: >97% Specific activity: 85.1 Ci/mmol (3.15 TBq/mmol) Concentration of stock: 1.0 mCi/mL (37 MBq/mL)



## 5. Positive control: Dexamethasone Supplier: Sigma-Aldrich (St. Louis, MO) Catalog #: Lot #: D1756 0001419230 Purity: 98.9% CAS # : 50-02-2Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 4 of 15 METHOMYL / 090301 OCSPP 890.1150/ OECD None



## 6. Solvent/vehicle control: Water (test substance) Ethanol (reference standard and positive control)
Justification for choice of solvent: Methomyl is readily water soluble

Final Concentration: 3% for ethanol; not applicable for water



## B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: Frozen prostate tissue from male Sprague Dawley rats castrated at approximately 90 days of age and euthanized 24 hours post-surgery was purchased from Charles River Laboratories, Inc. (Raleigh, NC). The prostates were excised after termination, trimmed of fat, blotted, weighed, and frozen at

− 60 to

− 80oC until shipment to DuPont Haskell. Once recei ved, the prostates were stored frozen (

− 60 to

− 80oC) until processed into cy tosol. Prostates were homogenized in cold TEDG (Tris, EDTA, DTT, glycerol, sodium molybdate) + PMSF (phenylmethylsulfonyl fluoride) buffer, and centrifuged for 30 min at 30,000 × g at 4oC. The supernatan ts were pooled, discarding the resulting pellets. Protein concentration of the cytosol was determined to be 6 mg/mL. Cytosol was divided into aliquots (a mount not specified) for storage at

− 60 to

− 80oC until

use.

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

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

TABLE 1. Summary of Conditions for Saturation Binding Experiment a 

### Source of receptor Rat 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 approximately 23% of radioligand at 0.25 nM Temperature 4oC 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 12 and 13 of the study report.



## 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 + PMSF 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 non-specific bi nding, solutions of non-labeled R1881 were prepared in a similar manner to achieve concen trations that were 100- fold greater than eachAndrogen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 5 of 15 METHOMYL / 090301 OCSPP 890.1150/ OECD None


## respective radiolabeled concentra tion, resulting in final concen trations in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1,000 nM. In the absence of cytosol, the radioactivity found in 30 μL of 2.5, 5, 7, 10, 15, 25, 50 and 100 nM [ 3 

## H]-R1881 was measured. It was stated that the cytosol was used undiluted at a concentr ation of approximately 6 mg/mL, which has previously been shown to bind to approximately 23% of 0.25 nM [ 3 

## H]-R1881 when assayed. Each run contained three conc urrent replicates at each c oncentration, resulting in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment Run a, b 

### Total Binding Non-Specific Binding Radioligand alone Tubes 1-24 c 

### Tubes 25-48 d 

### Tubes 49-72 e 

### [ 3 

### H]-R1881 Final conc. (nM) [ 3 

### H]-R1881 Final conc. (nM) R1881 Final conc. (nM) [ 3 

### H]-R1881 Initial conc. (nM) [ 3 

### H]-R1881 (μL)
0.25 0.25 25 2.5 30 0.50 0.50 50 5 30 0.70 0.70 70 7 30 1.00 1.00 100 10 30 1.50 1.50 150 15 30 2.50 2.50 250 25 30 5.00 5.00 500 50 30 10.00 10.00 1000 100 30 a Data were obtained from pages 52 and 53 of the study report. b Each concentration was run in triplicate for a total of 72 samples. c Tubes 1-24 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 

### H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. d Tubes 25-48 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 

### H]-R1881. R1881 was added in a 100-fold mola r excess of [ 3 

### H]-R1881 in a volume of 30 μL. Samples were dried, and 300 μL of prostate cytosol were added. e Tubes 49-72 contained only 30 μL [ 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 tubes were dried, then undiluted prostate cytosol (300 μL) was added and incubated for 20 hours at 4°C. To separate bound from free R1881, hydroxya patite (HAP) slurry was added to each tube and vortexed once every 5 minutes for 20 mi nutes. The samples were then centrifuged, and the supernatant was discarded. The sample s were washed 3 times in cold 50 mM Tris buffer. Following the last wash and decanting of the Tris buffer, the pellets were then extracted by addition of 2 mL ethanol. The samples were vortexed 3 times at 5 minute intervals. Samples were centrifuged for 10 minutes at 600 × g . Each ethanol supernatant was then decanted into a scintillation vial , and the radiation was quantified by liquid scintillation counting. A total of three runs were performed.

3. Competitive Binding Experiment: The competitive binding experiment was performed according to the protocol provided in the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures the binding of a single concentration of [ 3 

## H]- R1881 (specific activity of 80.3-80.5 Ci/mmol) to the AR in the pres ence of increasing concentrations of a test substance. A summa ry of the assay conditi ons for the competitive binding experiment is included in Table 3.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 6 of 15 METHOMYL / 090301 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 approximately 11% of 1.0 nM radioligand b 

### Concentration of test substance (as serial dilutions) 10

− 10 

### to 10

− 3 

### M Incubation Temperature 4oC 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 Protease inhibitor 60 μ M a Data were obtained from pages 12 and 14 of the study report. b The study reports that the prostate cytosol was dilu ted in cold TEDG + PMSF bu ffer to 4 mg protein/mL, which was previously shown to bind approximately 11% of 1 nM [ 3 

### H]-R1881.



## Water was used as the vehicle for the test substance, and no precipitation was observed. Ethanol was used as the vehicle for the re ference standard (R1881) and positive control (dexamethasone). It was reported that the re sults from the saturati on binding experiment demonstrated that 4 mg/mL of cytosolic protein contains enough receptor to bind approximately 11% of the [ 3 

## H]-R1881.

Dilutions of the test subs tance, reference standard (R1881), weak positive control (dexamethasone), and solvent control (water and ethanol) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of three inde pendent runs on three different days. Each run incl uded triplicate sets of the blan ks (solvent controls) and 1 μ M R1881 (non-specific binding, NSB) at the beginning and end of each run, along with triplicate tubes of the reference standard, the we ak positive control, and the test chemical at each concentration, and six replicates of the hot mix resulting in a total of 87 samples per

run.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 7 of 15 METHOMYL / 090301 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, Hot Mix Water or Ethanol c 

### R1881 Dexamethasone Methomyl Tubes 1-6 and 73-78 Tubes 7-24 and 79-81 d 

### Tubes 25-48 Tubes 49-72 Tubes 82-87
1×10

− 6 

### 1×10

− 3 

### 1×10

− 3 

### 1×10

− 7 

### 1×10

− 4 

### 1×10

− 4


### 1×10

− 8 

### 1×10

− 5 

### 1×10

− 5


### 1×10

− 9 

### 1×10

− 6 

### 1×10

− 6


### 1×10

− 10 

### 1×10

− 7 

### 1×10

− 7


### 1×10

− 11 

### 1×10

− 8 

### 1×10

− 8


### -- 1×10

− 9 

### 1×10

− 9


### -- 1×10

− 10 

### 1×10

− 10 

### a Data were obtained from pages 72-82 of the study report. b Each concentration of each chemical was run in triplicat e for a total of 87 tubes per run. Tubes 1-81 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 

### H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 1-81 also contained 10 μL of the solvent control, reference standard (non-radiolabeled R1881), weak positive control, or test substance, with the exception of Tubes 7-9 and 79-81 that contained 30 μL of non-radiolabeled R1881 (used to evaluate non-specific binding). Tubes 82-87 contained only 30 μL of [ 3 

### H]-R1881. c Tubes 1-3 and 73-75 contained ethanol, and Tubes 4-6 and 76-78 contained water, for solvent controls. d Tubes 7-9 and 79-81 were used to evaluate non-specific binding 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 using Origin 8.5.1 (OriginLab Corp., Northampton, MA ). A concentration-response model was fitted for each run for each curve generated and Scatchard plots were plotted for the binding data.

5. Definitions

a. Classification of test material

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

Binder: The average curve for the test chemical across runs crosses 50% of radioligand bound.

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 8 of 15 METHOMYL / 090301 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 constant 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 mean K d 

## for [ 3 

## H]-R1881 was 0.903 nM (±0.060), and the estimated average B max 

## was 4.5 fmol/100 μ g protein (±0.08) for the si ngle batch of prostate cytosol that was prepared. The K d 

## was within the range reported in the EPA validation program, but the B max 

## was below the EPA values (7 to 16 fmol/100 μ g protein). Confidence in these numbers is high accord ing to the goodness of fit (R 2 

## = 0.821-0.872) and the small variation among runs.

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


### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean ± SE c 

### R 2 

### (unweighted) 0.865 0.872 0.821 0.821-0.865 B max 

### (nM) 0.282 0.275 0.265 0.274 ± 0.005 B max 

### (fmol/100 μ g protein) 4.7 4.6 4.4 4.5 ± 0.08
K d 

### (nM) 0.883 1.015 0.811 0.903 ± 0.060 a Data were obtained from page 22 of the study report. b The mean is reported for the c oncurrent replicates of each run. c The R 2 

### is reported as a range and the mean ± SE is reported for the other parameters. R 2 

### Goodness of fit for curve calculated for specific binding



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

## H]-R1881 to the androgen receptor. The specific binding re ached a plateau; howev er, the non-specific binding was slightly greater than 20% of total binding in 2 of the 3 runs (22% and 23%) at 10 nM [ 3 

## H]-R1881; all other NSB values were ≤ 20%. Figure 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.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 9 of 15 METHOMYL / 090301 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.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 10 of 15 METHOMYL / 090301 OCSPP 890.1150/ OECD None



## B. COMPETITIVE BINDING EXPERIMENT: Competitive binding experiment parameters are presented in Table 6 and show n graphically in Figures 3 through 6. There were no methomyl-related effects on AR binding (87-118% binding) at concentrations up to 10
− 3 

## M. An estimated log IC 50 

## and RBA for methomyl could not be determined. Since the percent [ 3 

## H]-R1881 bound to the AR was >75% at all concentrations in all three runs, methomyl is classified as a non-binder in this assay.

The estimated average log IC 50 

## was

− 9.15 M for R1881 and

− 4.41 M for dexamethasone. Compared to R1881, the mean RBA for the dexamethasone was 0.0019%. Confidence in these numbers is high due to the small variation. No precipitation of methomyl was visually observed at ≤ 10
− 3 

## M. The solvent control responses indicated no drift in the study assay.

TABLE 6. Competitive Binding Assay of Meth omyl with AR from Rat Prostate Cytosol a


### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean ± SE c


### R 2 

### R1881 d 

### 0.933 0.957 0.964 0.933-0.964 Dexamethasone e 

### 0.953 0.975 0.981 0.953-0.981 Methomyl NA NA NA NA Log IC 50 

### (M) R1881

− 9.17

− 9.19

− 9.08

− 9.15 ± 0.03 Dexamethasone

− 4.32

− 4.40

− 4.51

− 4.41 ± 0.05 Methomyl NA NA NA NA IC 50 

### (M) f 

### R1881 6.8 × 10

− 10 

### 6.5 × 10

− 10 

### 8.3 × 10

− 10 

### 7.1 × 10

− 10 

### (±0.58) Dexamethasone 4.8 × 10

− 5 

### 4.0 × 10

− 5 

### 3.1 × 10

− 5 

### 3.9 × 10

− 5 

### (±0.49) Methomyl NA NA NA NA Log RBA f 

### Dexamethasone

− 2.85

− 2.80

− 2.57

− 2.72 ± 0.09 Methomyl NA NA NA NA RBA (%) Dexamethasone 0.0014 0.0016 0.0027 0.0019 ± 0.0004 Methomyl NA NA NA NA a Data were obtained from page 24 of the study report. b The mean is reported for the concu rrent replicates within each run. c The range of r 2 

### is reported and the mean and SE are reported for the other parameters. For mean values reported in scientific notation, the SEM is reported in the same exponential. d With no weighting as bottom of the curve was below 0. e With weights equal to 1/Y. f Calculated by the reviewers. NA Not applicable. R 2 

### Goodness of fit. RBA Relative binding affinityAndrogen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 11 of 15 METHOMYL / 090301 OCSPP 890.1150/ OECD None


## FIGURE 3. Percentage R1881 Bound to the A ndrogen Receptor in the Presence of Test Compounds (Run 1).

FIGURE 4. Percentage R1881 Bound to the A ndrogen Receptor in the Presence of Test Compounds (Run 2).Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 12 of 15 METHOMYL / 090301 OCSPP 890.1150/ OECD None


## FIGURE 5. Percentage R1881 Bound to the A ndrogen Receptor in the Presence of Test Compounds (Run 3).

FIGURE 6. Mean Percentage R1881 Bound to th e Androgen Receptor in the Presence of Methomyl in Three Competitive Binding Assays.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 13 of 15 METHOMYL / 090301 OCSPP 890.1150/ OECD None



## C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay was functioning properly, each run was evaluated using the following criteria (Table 7).

TABLE 7. Criterion a 

### Tolerance Limit(s) b 

### Value Yes No Ligand depletion is minimal. The recommended ratio of total binding in the absence of competitor to total amount of [ 3 

### H]-R1881 added per assay tube. ≤ 15% 0.69 to 0.86 X

Methomyl Top (% binding) 80 to 115 102 to 118 c 

### X R1881 fitted curve parameters Top (% binding) 82 to 114 101 to 113 X Bottom (% binding)

− 2.0 to 2.0

− 3.3 to

− 0.3 d 

### X Hill Slope

− 1.2 to

− 0.8

− 0.8 to

− 0.6 e 

### X Weak positive control (dexamethasone) fitted curve parameters Top (% binding) 87 to 106 95 to 112 f 

### X Bottom (% binding)

− 12 to 12

− 5.5 to 1.8 X Hill Slope

− 1.4 to

− 0.6

− 1.2 to

− 0.8 X Saturation Binding Experiment K d 

### (nM) 0.685 to 1.57 0.903 X Non-specific binding (%) ≤ 10.0 0.05 to 0.06 g 

### X a Data were obtained from page 24 of the study report. b These values represent ranges from the validation study. c The mean highest % binding in one run wa s greater than the tolerance limits at 10

− 9 

### M methomyl; page 23 of the study report. d Bottom % binding was below the tolerance limits in 2 of the 3 runs (

− 3.3% and

− 3.3%). e Hill slope was above the tolerance limits in 2 of the 3 runs (

− 0.7 and

− 0.6); the same runs with the low bottom % binding. f The Top % binding was above the tolerance limits in one run (112%). g Calculated by the reviewers using the mean dpm of the NSB and Total activity from pages 72, 74, 76, 78, 80 and 82 of the study report.



## The performance criteria for R 1881 were not completely met acr oss the three runs. In the first and third runs, the bottom % binding and Hill slope were outside th e tolerance limits for R1881; all three parameters (Top and Bottom % binding and Hill slope) were met for the second run. However, the goodness of fit of th e curves across the three runs was tight (>0.933). For dexamethasone all of the three criteria were met, except for the Top % binding in the third run which was hi gher than the tolerance limits.

The study reported that these variances in the performance criteria did not warrant performing another run since the values were onl y slightly outside of the suggested range. All runs were within or very close to the suggested performance cr iteria and therefore the assays were considered valid by th e laboratory. Furthermore, the IC 50 

## s calculated were comparable between runs and within the e xpected ranges for R1881 and dexamethasone (a weak binder to the androgen receptor).

The non-specific binding was <1% and the curv e for the reference material showed that increasing concentrations of unlab eled R1881 generally displaced [ 3 

## H]-R1881 in a manner consistent with one-site binding, as indicated by a Hill slope of

− 0.8 in the second run; the Hill slope was slightly high (

− 0.6 and

− 0.7) in the other two runs. These variances do not appear to have affected the results for methomyl.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 14 of 15 METHOMYL / 090301 OCSPP 890.1150/ OECD None



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Radioinert R1881, the androgen receptor agonist reference standard, and dexamethasone, the weak positive control, were used to verify test system performance in the competitive binding assay (3 independent runs). As expected, radioinert R1881 showed effects consiste nt with strong competitive binding and dexamethasone showed effects consistent with weak competitive binding to the androgen receptor in all 3 runs. Under the conditions of the study, the test substance, methomyl, did not bind to the androgen receptor when tested up to a maximum concentration of 1.0 × 10

− 3 

## M. Therefore, methomyl is classified as a non-inhibitor in this androgen receptor binding
assay.

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 mean K d 

## for [ 3 

## H]-R1881 was 0.903 nM (±0.060), and the estim ated average B max 

## was 4.5 fmol/100 μ g protein (±0.08) for the single batch of prostate cytosol that was prepared. The K d 

## was within the range reported in the EPA validation program, but the B max 

## was below the EPA values (7-16 fmol/100 μ g protein). Confidence in these numbers is high according to the goodness of fit (R 2 

## = 0.821- 0.872) and the small variation among runs. The specific binding reached a plateau; however, the non-specific binding was slightly greater than 20% of total binding in 2 of the 3 runs at 10 nM [ 3 

## H]-R1881; all other NSB values were ≤ 20%.

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

## was

− 9.15 M for R1881 and

− 4.41 M for the weak positive control (dexamethasone). Compared to R1881, the mean RBA for the positive control was 0.0019% . Confidence in these numbers is high due to the small variation. No precipitation of the test compound was visually observed at ≤ 10
− 3 

## M. The solvent control responses indicated no drift in the study assay.

The performance criteria for R1881 and dexameth asone were not completely met across the three runs. In the first and third runs, the bottom % binding and Hill slope were outside the tolerance limits for R1881; all three para meters (Top and Bottom % binding and Hill slope) were met for the second run. However, the goodn ess of fit of the cu rves across the three runs was tight (>0.933). For dexamethasone all of the three criteria were met, except for the Top % binding in the third run which wa s higher than the tolerance limits.

The non-specific binding was <1% and the curv e for the reference material showed that increasing concentrations of unlab eled R1881 generally displaced [ 3 

## H]-R1881 in a manner consistent with one-site binding, as indicated by a Hill slope of

− 0.8 in the second run; the Hill slope was slightly high (

− 0.6 and

− 0.7) in the other two runs.

Although, the performance criteri a for both R1881 and dexamethasone were not achieved across the three runs, based on the estimated IC 50 

## s for R1881 and the weak positive control the androgen receptor was present in a reasonable concentration and functioning with appropriate affinity for the ligand in the comp etitive binding assays. It is unlikely that information from additional competitive binding runs would change the interpretation of this study.Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 15 of 15 METHOMYL / 090301 OCSPP 890.1150/ OECD None



## Since the percent [ 3 

## H]-R1881 bound to the AR was >75% (87-118% binding) at all concentrations of methomyl in all three runs, methomyl is cla ssified as a non-binder in this
assay.

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

 The estimated B max 

## (fmol/100 μ g protein) for the prostate cytosol in the saturation binding experiment was lower th an the EPA guideline values.

 The performance criteria (Top and Bottom pe rcent binding and/or Hill slope) for R1881 and dexamethasone were not achieved across the three runs in the competitive binding experiment.

# DATA EVALUATION RECORD

METHOMYL

Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-30-2012 (MRID 48672002)

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: Joan Harlin, M.S. Date: 4/12/2012

Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 4/13/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/17/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/17/2012

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).METHOMYL I 103301 Aromatase (Human Recombinant) Assay (2011) I Page I of 12 OCSPP 890.1200 I OECD None



## Primary Reviewer: Karlyn Midd l eton 

## Health Effects Division, Office of Pesticide Programs Secondary Reviewer: Greg Aker m an, Ph.D. 

## Health Effects Division

, Office of Pesticide Programs Sign a tu re: ---------,,..-,-,.--r-;--:;;..--- 

## D ate:**--~+---+--°----** 

## Signature:**-~~...,.,--d-.:;.L_--** 

## Da te:

___ ____,'--'-+_,_,__.._____



## DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200



## PC CODE: 090301 

## DP BARCODE: D3984 l 9



## TXR#: 0056254 

## CAS No.: 16752-77-5



## TEST MATERIAL (PURITY): Methomyl (99.4% a.i.)



## SYN ONYMS: DPX-Xl 179 ; methyl N-[[(methylamino)carbony l] oxy]et h animido thioat e



## CITAT ION: Snajdr, S. I. 

## (2012). Methomyl (DPX-Xl 179) Technica l : In vitro aromatase Inhibition using human recombinant microsomes. E.I. du Pont de Nemours and Company

, DuPont Haskell G lobal Centers for Health & 

## Environmental Sciences, P.O. Box 50

, Newark, Delaware, 19714. Laboratory Report No.: DuPont-32081. January 5

, 2012. MRID 48672002. Unp ubli s h ed.



## SP ONSOR: E.I. du Pont de Nemours and Company, Wilmington, Delaware 19898



## TEST ORDER#: EDSP-090301-62



## EXECUTIVE SUMMARY: In an 

## in vitro aromatase (CYP 19) assay (MRID 48672002), methomyl (99.4% a.i.

, Lot# JUL06LP276) was incubated with human recombinant aromatase and tritiated androstenedione**(1-~** 

## [3H(N)]-androst -4- ene

- 3

, 17-dione ; [3H]ASDN) in water at log concentrations of 10

- i o to 10

- 3 

## M for 15 minutes to assess the potential of methomyl 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). A total of four runs were conducted and each run included full enzyme activity controls, background activity controls

, a positive control series (10

- 1 0 

## to 10

- 5 

## M) using a known inhibitor

, 4-hydroxyandroste n edio n e ( 4-0H ASDN), and the methomyl series (10

- 1 0 

## to 10

- 3 

## M) with three repetitions per concentration. Because the solvent vehicle was water for methomyl and dimethyl sulfoxide (DMSO) for 4-0H ASDN, separate sets of the background activity and full enzyme activity controls were run using only water or I% DMSO (final concentration in assay). In the second run, the full act i vity controls (wate r only) had aromatase activity below the performance criteria specified in the guideline; therefore, the second run was excluded from the calculations and a fourth run was conducted.

Aromatase activity in the full activity controls for the three successful test runs averaged 0

. 381 ± 0.117 nmol·mg-protein

- 1 

## ·min

- 1 

## in water only, and 0.431 ± 0.141 nmol·mg-protein

- 1 

## ·min

- 1Aromatase (Human Recombinant) A ssay (2011) / Page 2 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None


## in 1% DMSO. Activity in the background controls averaged 0.59% of the full activity controls in water and 0.54% of full activity controls in 1% DMSO. The response of each full activity control within a run wa s between 90 to 110% of the average full control activity, except for the average initial value of 117% in 1% DMSO and the average ending value of 83% in 1% DMSO for Run 1.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.431±0.090 nmol·mg-protein

− 1 

## ·min

− 1 

## at the lowest tested concentration (10

− 10 

## M) and 0.004±0.001 nmol·mgprotein

− 1 

## ·min

− 1 

## at the highest tested concentration (10

− 5 

## M). As a percent of the control, aromatase activity for 4-OH ASDN was 1.02% at 10

− 5 

## M and 103.14% at 10

− 10 

## M.

For methomyl, aromatase activity averaged 0.358±0.096 nmol·mg-protein

− 1 

## ·min

− 1 

## at the lowest tested concentration (10

− 10 

## M) and 0.366±0.118 nmol·mg-protein

− 1 

## ·min

− 1 

## at the highest tested concentration (10

− 3 

## M). The lowest portion of the respons e curve was greater than 75% activity at concentrations from 10

− 10 

## M to 10
− 3 

## M methomyl across all runs.

Based on the data from the average response curve, methomyl is classified as a Non-inhibitor of aromatase activity 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) Assay (2011) / Page 3 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Methomyl technical (DPX-X1179) Description: Solid Source: DuPont de Nemours Lot # (expiration date): JUL06LP276 (May 18, 2016) Purity: 99.4% Volatility: Not reported Storage conditions: Ambient Stability: Stable throughout exposure phase of the study Solvent: Deionized water Solubility (in test solvent): Water soluble Highest Concentration Tested: 10

− 3 M Stock Solution Preparation: Serial dilution Molecular weight: 162.21 g/mole CAS #: 16752-77-5 Structure:



## 2. Non-Labeled Substrate: Androstenedione (ASDN)
CAS # : 63-05-8 Source: Steraloids, Newport, RI (Catalog #A6030-100) Lot # (expiration date): L1712 (not reported) Purity: 99.8%



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

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

## H]ASDN)
Source: Perkin Elmer, Waltham, MA (Catalog # NET926250UC) Batch # (expiration date): 619344 (not reported) Radiochemical Purity (Supplier): >97% Specific activity: 26.3 Ci/mmol; 0.974 TBq/mmol (vendor reported; adjusted specific activity was 24.7 Ci/mmol for all runs) Radiochemical Purity (In-lab determination): 99.972%



## 4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN)
CAS # 566-48-3 Source: Sigma Aldrich, St. Louis, MO (Catalog # F2552) Lot # (expiration date): 081K2133 (not reported) Purity: 99.6%



## 5. Solvent (Vehicle Control): DMSO (4-OH ASDN); dei onized water (Methomyl)
Source: Lot # (expiration date): DMSO: Sigma Aldrich, St. Louis, MO (Catalog # D4540) 76096AM (not reported); deionized water: not reported Justification for choice of solvent The test substance was readily water soluble Concentration (% of total volume in assays) 1% for DMSO; water – not applicableAromatase (Human Recombinant) A ssay (2011) / Page 4 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None



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

Source: Gentest TM , Woburn, MA (Catalog # 456260) Lot # (expiration date): 03897 (not reported) Protein concentration: 7.40 mg/mL Cytochrome C reductase activity: 290 nmole /mg protein/minute Aromatase activity: 6.0 pmol/pmol P450/min



## B. METHODS

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

## H]ASDN was prepared such that the final concentration of ASDN in the assay was 100 nM, and the amount of tritium ad ded to each incubation tube was 0.05 μ Ci.

Methomyl was formulated in deionized water as it is readily soluble in water and the assay is run in an aqueous solution. No indicati on of precipitation of th e test substance was observed during the assay.

A stock solution of the positive control substance, 4-OH ASDN, was formulated in DMSO. Dilutions were prepared such that the target concentrations of the pos itive control substance (10

− 10 

## to 10
− 5 

## M; Table 4) were achieved by the additi on of 20 μL of the dilution for a final assay volume of 2 mL. The final concen tration of DMSO in the assay was 1%.

Human recombinant microsomes were purchased from Gentest TM 

## , and were received on dry ice. Upon receipt, the microsomes were thawed rapidly in a water bath at approximately 37 oC, placed on ice, aliquot ed, and stored frozen at ≤− 60oC until use, within 12 months from the time of receipt.

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

TABLE 1. Assay Components and Conditions a Assay Factor Values 0.1M sodium phosphate buffer (pH 7.4) 94 mM Microsomal Protein 0.004 mg/mL NADPH 0.3 mM [ 3 H]ASDN 100 nM Propylene Glycol 5% Temperature 37°C Incubation Time 15 min a Data were obtained from page 16 of the study report.



## 2. Suitability Assessments: The protein concentration of the microsomes (Lot # 03897) was supplied by the vendor as 7.4 mg/mL. Aromat ase activity of the microsomes was also provided by the vendor as 6.0 pmol/min/ pmol P450. The minimum aromatase activities in the full activity control samples were determined to be 0.230 nmol·mg-protein

− 1 

## ·min

− 1 

## in 1% DMSO and 0.236 nmol·mg-protein

− 1 

## ·min

− 1 

## in only water, which were greater than the minimum recommended aromatase activity of 0.1 nmol·mg-protein

− 1 

## ·min

− 1



## . 3. Aromatase Assay: Because the solvent vehicle was wa ter for methomyl and DMSO for the positive control (4-OH ASDN), each assay r un contained 8 tubes for the full enzymeAromatase (Human Recombinant) A ssay (2011) / Page 5 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None


## activity (4 tubes with only water as the solven t, and 4 tubes with 1% DMSO) and 8 tubes for the background activity controls (4 with water, 4 with 1% DM SO). Two tubes of each full activity and background control we re 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 positive control, and a full concentration curve in triplicate for the test substance were established. The aromatase assay was conducte d generally according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10).

The amount of 3 

## H 2 

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

− 1 

## ·min

− 1



## . 4. Demonstration of Proficiency: It was stated that all a ssays were performed by personnel that demonstrated proficiency in conducting the assay as outl ined in the test guideline. However, the actual data from the proficiency testing were not include d in the study report.

a. Positive Control

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

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

− 1 

## ·min

− 1



## .

 Mean background control activity was ≤ 10% of the full activity control.

 The percent CV between repl icates within each sample type and concentration was <15%.

(2) Demonstration of Proficiency of Ne w Technician for Conducting Assay (when applicable): The positive control data for slope, t op and bottom percent met the criteria as listed in section (i) of OCSPP 890.1200, except that the actual upper limit for the top of the curve was outside the recommended upper li mit criteria of 110% (Table 2).

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

− 1.2

− 0.8

− 1.0

− 0.9 Top (%) 90 110 91 116 Bottom (%)

− 5 +6 0.1 0.4 Log IC 50 (M)

− 7.3

− 7.0

− 7.3

− 7.0 a Data were obtained from page 17 and Table 2 on page 24 of the study report.Aromatase (Human Recombinant) A ssay (2011) / Page 6 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None



## b. Proficiency Chemicals: Assays to classify proficienc y chemicals (econazole, fenarimol, nitrofen, and atrazine) were not performed.

TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations Econazole 24169-02-6 inhibitor No data Fenarimol 60168-88-9 inhibitor No data Nitrofen 1836-75-5 inhibitor No data Atrazine 1912-24-9 non-inhibitor No data



## 5. Determination of Aromatase Ac tivity with Test Chemical(s): The response of aromatase activity to the presence of eight concentrations of methomyl per run, in triplicate, was tested during four independent runs (T able 4). Solubility was visually assessed (presence of cloudiness or a precipitate), and no precipita tion was observed. Alt hough four independent experimental runs were conducted, the full act ivity control in only water in Run 2 had aromatase activity of approxi mately 0.06 nmol·mg-protein

− 1 

## ·min

− 1 

## , which is below the performance criteria recommended in the guideline. Therefore, this run was excluded from calculations and a fourth run was performed. The full enzymatic activity (>97% for the means of each run) was obtained at the two lowe st concentrations of methomyl, defining the top of the concentration-response curve.

TABLE 4. Test Chemical Study Design for each Test Run a
Sample Type Repetitions (Tubes) Description b Reference or Chemical (M) Full Activity Control c 4 All test components plus solvent vehicle (DMSO) N/A Bkgd Activity Control c 4 Same as above without NADPH N/A Full Activity Control d 4 All test components plus solvent vehicle (water) N/A Bkgd Activity Control d 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 3×10

− 6
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 3×10

− 7
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

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

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

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

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

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

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

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

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

− 10 a Data were obtained from page 15 of the study report. b The complete assay ("all test com ponents") contained buffer, propylene gl ycol, microsomal protein, ASDN/[3H]- ASDN substrate and NADPH. c 4-OH ASDN solvent was DMSO. d Methomyl solvent was deionized water.Aromatase (Human Recombinant) A ssay (2011) / Page 7 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None



## C. DATA ANALYSIS

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

− 1 

## ·min

− 1 

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

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

− 1 ·min

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



## 2. Statistical Methods: Statistical analyses and displays such as summary tables, graphical displays, analysis of variance, and multip le comparisons were conducted Origin 8.5.1 (OriginLab Corp., Northampton, MA). The re sponse curve was fitted by weighted least squares nonlinear regression analysis with we ights equal to 1/Y. In each run, the model parameters were estimated along with their stan dard errors. The vari ances (squared standard errors) for each parameter were compared usi ng F-tests to determine which were different. Pooled variance T-tests or Sattherthwaite T-tests were used to compare the parameter estimates (slope or log IC 50 

## ) based on whether the correspon ding F-test was significant or not. Statistical significance was at the 0.05 si gnificance level. Normality of the underlying data from each run was assessed from the residuals from the model.

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.

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.Aromatase (Human Recombinant) A ssay (2011) / Page 8 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None



## II. RESULTS

A. CONTROL ACTIVITY: Aromatase activity in the full activity controls for the three successful test runs ranged from 0.236-0.539 nmol·mg-protein

− 1 

## ·min

− 1 

## in water only with a mean and standard deviation of 0.381±0.117 nmol·mg-protein

− 1 

## ·min

− 1 

## , and 0.230-0.630 nmol·mg-protein

− 1 

## ·min

− 1 

## in 1% DMSO with a mean and standard deviation of 0.431±0.141 nmol·mg-protein

− 1 

## ·min

− 1 

## . Activity in the background controls averaged 0.59% of the full activity controls in water and 0.54% of full activity controls in 1% DMSO. The response of each full activity control within a run was between 90 to 110% of the average full activity, except in Run 1 for the average initial value in 1% DMSO of 117% and the average ending value of 83% in 1% DMSO.

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.431± 0.090 nmol·mg-protein

− 1 

## ·min

− 1 

## at the lowest tested concentration (10

− 10 

## M) and 0.004±0.001 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 3 runs is presented in Table 7, along with the overall standard deviation, standard error of the mean, and % CV. As a percent of the control, aromatase activity for 4-OH ASDN was 1.02% at 10

− 5 

## M and 103.14% at 10

− 10 

## M. The inhibition response curves for the positive cont rol are shown in Figures 1 and 2.

TABLE 7. Effect of Methomyl on Aromatase Activity (as percent of control) from Independent Runs a
Chemical Concen. (M) # Runs Overall Mean Overall SD Overall SEM Overall % CV
4-OH ASDN (positive control) 1 x 10

− 5 3 1.02 0.25 0.14 24 1 x 10

− 6 3 7.62 1.87 1.08 25 3 x 10

− 6 3 19.37 4.81 2.78 25 1 x 10

− 7 3 41.87 8.61 4.97 21 3 x 10

− 7 3 72.74 13.09 7.56 18 1 x 10

− 8 3 89.02 12.26 7.08 14 1 x 10

− 9 3 104.08 12.97 7.49 12 1 x 10

− 10 3 103.14 14.32 8.27 14 Methomyl 1 x 10

− 3 3 95.75 4.19 2.97 5 1 x 10

− 4 3 99.08 2.62 1.51 3 1 x 10

− 5 3 99.49 2.09 1.20 2 1 x 10

− 6 3 100.72 5.35 3.09 5 1 x 10

− 7 3 105.04 6.37 3.68 6 1 x 10

− 8 3 104.71 2.89 1.67 3 1 x 10

− 9 3 103.86 3.72 2.15 4 1 x 10

− 10 3 105.11 8.45 4.88 8 a Data were obtained from Table 1 on page 23 and pages 54-55, 63-64, and 68-69 of the study report. Mean, SD, SEM, and CV were calculated by the reviewers. SD Standard Deviation SEM Standard Error of the Mean CV Coefficient of Variance



## C. TEST SUBSTANCE: For methomyl, aromatase activity averaged 0.358±0.096 nmol·mg-protein

− 1 

## ·min

− 1 

## at the lowest tested concentration (10

− 10 

## M) and 0.366±0.118 nmol·mg-protein

− 1 

## ·min

− 1 

## at the highest tested concentration (10

− 3 

## M). The mean aromatase activity of methomyl (expressed as % full cont rol activity) for each concentration tested across all 3 runs is presented in Table 7 (pre sented above), along with the overall standard deviation and % CV. As a percent of cont rol, aromatase activity for methomyl wasAromatase (Human Recombinant) Assay (2011) / Page 9 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None



## ≥ 95.75% at all concentrations from 10

− 10 

## to 10
− 3 

## M. Inhibition response curves for methomyl from each run are shown in Figure 3, and the average inhibition response curve across all runs is shown in Figure 4.

FIGURE 1. Inhibition Response Curves fo r 4-OH ASDN From Each Test Run (Runs 1, 3, 4).Aromatase (Human Recombinant) A ssay (2011) / Page 10 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None


## FIGURE 2. Mean Inhibition Response Curve for 4-OH ASDN.

FIGURE 3. Inhibition Response Curves for Methomyl From Each Test Run (Runs 1, 3, 4).Aromatase (Human Recombinant) A ssay (2011) / Page 11 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None


## FIGURE 4. Mean Inhibition Response Curve for Methomyl.

The average dose-response curve indicated that the aromatase activity in the presence of methomyl at all concentrations tested was essentially equivalent to the activity observed in the full activity control (water only). These data could not be fit to the 4-parameter nonlinear regression mode l; therefore, log IC 50 

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

## averaged

− 7.2 and the Hill slope

was

− 1.0 (Table 8). The variation in the positiv e control values was acceptable (<15% CV).

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

− 7.0

− 7.2

− 7.3

− 7.2 0.05

− 0.7 Hill Slope Methomyl NR NR NR NR NR NR 4-OH ASDN

− 1.0

− 0.9

− 1.0

− 1.0 0.10 10 a Data were obtained from page 24 of the study report. b Calculated by the reviewers SD Standard Deviation CV Coefficient of variance



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: An in vitro aromatase inhibition assay was performed to evaluate the ability of methom yl to inhibit human recombinant microsomal aromatase activity, an enzyme responsible for the conversion of androgens to estrogens in vitro. A positive control chemical, 4-OH ASDN, was evaluated concurrently to verify test system performance. As expected, 4-OH ASDN showed effects consistent with aromataseAromatase (Human Recombinant) A ssay (2011) / Page 12 of 12 METHOMYL / 103301 OCSPP 890.1200 / OECD None



## inhibition in all three suc cessful independent runs. The mean estimated log IC 50 

## for across all 3 runs for 4-OH ASDN was approximately

− 7.2 M. Under the conditions of the study, the test substance, methomyl, did not inhibit aromatase activity when tested up to a maximum concentration of 10

− 3 

## M. Therefore, methomyl is classified as a non-inhibitor in this aromatase inhibition assay.

B. AGENCY COMMENTS: Aromatase activity in the full activity controls for the three successful test runs averag ed 0.381±0.017 nmol·mg-protein

− 1 

## ·min

− 1 

## in water, and 0.431±0.141 nmol·mg-protein

− 1 

## ·min

− 1 

## in 1% DMSO. The response of each full activity control within a run was betw een 90 and 110% of the average full activity, except in Run 1 for the initial value of 117% (1% DMSO) and the average ending value of 83% (1% DMSO). Activity in the backgr ound controls averaged 0.59% of the full activity controls in water and 0.54% of full activity controls in 1% DMSO.

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

## , and % CV. The estimated log IC 50 

## for 4-OH ASDN averaged

− 7.2 M and the slope was

− 1.0.

For methomyl, aromatase activity at the lowe st and the highest tested concentrations (10

− 10 

## and 10

− 3 

## M) were 105.11% and 92.93% of the cont rol, respectively. The average dose-response curve indicated that the aromatas e activity in the presen ce of methomyl at all concentrations tested was esse ntially equivalent to the activity of the full activity control; therefore, these data could not be fit to the 4-parameter nonlin ear regression model. Mean aromatase activity for methomyl, as a percent of control, was 92.9 ± 4.2% at the highest test concentration of 10

− 3 

## M. The average lowest portion of curves across runs was greater than 75% activity indicating that methomyl doe s not inhibit aromatase activity under the conditions of this assay.

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

 The required proficiency da ta were not provided.

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

# DATA EVALUATION RECORD

METHOMYL

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-30-2012 (MRID 48672003)

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: 3/29/2012
Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 4/03/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 4/11/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 4/11/2012

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).METHOMYL/090301 Estroge n R eceptor B i n di n g ( R at Uterine Cytoso l ) (201 1 ) I Page I of 14 OCSPP 890

. 1250/ OECD None



## Primary Reviewer: Karlyn Middleton 

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

## Health Effects Division Sig n a tu r e:**------Jc..._..;;...:'-b-~----==--­** 

## D a t e:**---~-=-t-L-.:.._** __ 

## S i g n a tur e:**_..,,,.'--..L-.,.+-~---­** 

## D a t e: ----""'--'-.L.:.--'---- Temp l ate version 08/2011



## D ATA EVALUAT IO N REC ORD

STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC); OCSPP 890

. 1250



## PC CODE: 090301 

## DP B A R C OD E: D398419



## TXR#: 0056254 

## CAS No.: 16752-77-5



## TEST MATERIAL (PURITY): Methomyl (99.4%)



## SYNONYMS: DPX-Xl 179 ; Methyl N-[[(methylamino)carbonyl]oxy]-ethanimidothioate



## CITATION: Snajdr

, S.I. (2012) Methomyl (DPX-Xl 179) technical: 

## in vitro estrogen receptor binding assay using rat uterine cytosol (ER-RUC). E.I. du Pont de Nemours and Company, DuPont Haskell Global Centers for Health & 

## Environmental Sciences, Newark, DE. Laboratory Project ID.: DuPont-32083

, January 5

, 2012. MRID 48672003. Unpublished



## SPONSOR: E.I. du Pont de Nemours and Company

, Wilmington

, DE



## TEST ORDER#: EDSP-090301-62



## EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48672003) for methomyl (99.4% purity

, Lot # JUL06LP276)

, uterine cytosol from Sprague Dawley rats was used as the source of ER to conduct saturation binding experiments 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 routinely conducting ER competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of**[3H]-17~-estradiol** 

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

- 1 0 

## to 10

- 3 

## M) of methomyl. Water was used as the solvent vehicle for methomyl

, and ethanol was used for the reference chemicals at a final concentration of 2%. A total of 3 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 maximum binding capacity (Bmax) was 32.5 ± 

## 2.25 fmol/l 00 μg protein and the dissociation constant (Kd) was 0.081 ± 

## 0.022 nM. The Kd for each

run was within the expected range of 0.03 to 1.5 nM. The saturation binding experiment demonstrated that the estrogen receptor was present in a reasonable concentration and functioning with appropriate affinity for the reference ligand**17~-estradiol.**Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None



## In the competitive binding experiments, the thre e reference chemicals performed as expected. The average log IC 50 

## was

− 9.16 M for 17 β -estradiol and

− 4.85 M for the weak positive control, 19-norethindrone, and the mean RBA was 0.0054% for the weak positive control. The negative control, octyltriethoxysilane, had no effect on specific binding of the [ 3 

## H]-ligand (85-105% binding).

Methomyl had no appreciable effect on specific binding (88 to 113%) of the [ 3 

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

## and RBA could not be calcu lated for methomyl. Based on the results from the three runs, methomyl is classified as Not Interactive in the Estrogen Receptor Assay.

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

COMPLIANCE: Signed and dated GLP, Data Confidentiality, and Quality Assurance statements were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: E.I. du Pont de Nemours and Company, DuPont Haskell Global Centers for Health & Environmental Sciences

Location: Newark, DE Study Director: Suzanne Snajdr Other Personnel: Dandan Wang, Research Chemist Gary Jepson, Manager John O'Connor, Group Leader Study Period: July 21, 2011 - January 5, 2012



## 2. Test substance: Methomyl Description: Technical, solid Source (Catalog #): E.I. du Pont de Nemours and Company (NR) Lot/Batch # (Exp date): JUL06LP276 (May 18, 2016) Purity: 99.4% Solubility: Readily soluble in water Volatility: Not provided Stability: 6 years Storage conditions: Ambient CAS #: 16752-77-5 Molecular weight: 162.21 g/mol Structure:



### N

S O N H O

CH 3 

### CH

3



### CH 3



## 3. Non-labeled ligand: 17 β -Estradiol Supplier: Sigma Aldrich, St Louis, MO Catalog #: E8875 Batch #: 21k1267 Purity: 99% CAS #: 50-28-2



## 4. Radioactive ligand: [ 3 

## H]-17 β -Estradiol Supplier: Perkin Elmer , Boston, MA Catalog #: NET517001MC Lot #: 650702 Radiochemical purity: >97% Specific activity: 130.2 Ci/mmol (4817 GBq/mmol) Concentration of stock: 1.0 mCi/mL (37 MBq/mL)



## 5. Positive control: 19-Norethindrone Supplier: Sigma Aldrich, St Louis, MO Catalog #: N4128 Batch #: 019K1735 Purity: 100% CAS #: 68-22-4Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None


## 6. Negative control: Octyltriethoxysilane Supplier: Spectrum Chemicals & Laboratory Products, Gardena, CA Catalog #: O1472 Batch #: XD0346 Purity: 100% CAS #: 2943-75-1



## 7. Solvent/vehicle control: Deionized Water (test substance) Ethanol (reference standard, pos itive and negative controls)
Justification for choice of solvent: Methomyl is readily soluble in water

Final Concentration: 2% for ethanol; not applicable for water



## B. METHODS

1. Preparation of Rat Uterine Cytosol (RUC): Ovariectomized adult female Crl:CD(SD) were purchased from Charles River Laboratorie s, Inc., having been ovariectomized 10 days prior to uterine collection. Animals were 95-100 days old at the time of euthanasia. The uteri were trimmed, blotted, weighed, rapidly frozen in liquid nitrogen, and stored at

− 60 to

− 80 ° C until processing. For preparation of th e cytosol, uteri were thawed on ice and homogenized at 0.1g of tissue pe r 1.0 mL ice-cold TEDG (Tri s, EDTA, DTT, glycerol) + PMSF (phenylmethylsulfonyl fluoride) buffe r. After homogenization, uteri were centrifuged for 10 min at 2500 × g at 4 oC. The resulting supe rnatants were transferred and centrifuged for 60 minutes at 105000 × g , discarding the resul ting pellets. Protein concentration of the cytosol was determined to be 3.5 mg/m L using a Biorad protein kit compatible with DTT in the TEDG buffer. It was not stated if samples were divided into aliquots for storage.

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

## H]-17 β -estradiol was performed to demonstrate that the 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.

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 27% of radioligand at 0.03 nM Temperature 4oC Incubation time 20 hours Composition of assay buffer Tris 10 mM (pH not reported) EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride 1 mM
DTT 1 mM a Data were obtained from pages 15-17 of the study report.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None


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

## H]-17 β -estradiol was 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, and 300 nM. For each batch of cytosol, the optimal protein concentration was determined by testi ng serial amounts of pr otein per tube, using 0.03 nM radiolabeled estradiol, until a concentration was reached that bound 27% of the total radioactivity added. Each assay consisted of three non-concurre nt runs. Each run included three replicates of each test substance at each concen tration, 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 serial dilutions) e



### --- 50 μL --- Non-labeled 17 β -estradiol (8 serial dilutions, 100x each respective labeled concentration) f 

### 100 μL 100 μL --- Uterine cytosol (diluted to appropriate conc.) 500 μL 500 μL 50 μL Total volume in each assay tube a Data were obtained from page 17 of the study report. b Total binding = [ 3 

### H]-17 β -estradiol bound to ER c Non-specific binding = [ 3 

### H]-17 β -estradiol and 100-fold greater non-labeled bound to ER d Total [ 3 

### H]-17 β -estradiol alone for dpm determ ination 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 gentle mixing for 20 hours at 4  C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was a dded to each tube and vortexed (3 times with 5-minute intervals). Subsequently, the 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. Ethanol was then added to the HAP pellet remaining in each tube to extract the [ 3 

## H]-17 β -estradiol, followed by vortexing, and centrifugation for 10 min at 1000 x g . An aliquot of supern atant was radioassayed by scintillation counting. The temperature was ma intained at 4oC throughout the assay prior to extraction with ethanol.

3. Competitive Binding Experiment: A summary of the experiment conditions for the competitive binding experiment is included in Table 3.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 6 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None



### 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 8.2-11% of radioligand b 

### Concentration of test substance (as serial dilutions) 10

− 10 

### to 10

− 3 

### M Temperature 4  

### C Incubation time 20 hours Composition of assay buffer Tris 10 mM (pH not reported) EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride 1 mM
DTT 1 mM a Data were obtained from pages 15, 18 and 28 of the study report. b Receptor concentration may need to be adjusted by adding volume.



## A solubility test was not perfor med as methomyl is readily sol uble in water. On the day of the assay, the specific activit y of the stock solution [ 3 

## H]-17 β -estradiol was adjusted for decay over time, and diluted in TEDG + PMSF buffer to achieve a final concentration of 1.0 nM. For each batch of cytosol, the optimal pr otein concentration was determined by testing serial amounts of protein per tube, using 1.0 nM radiolabeled estradiol, until a concentration was reached that bound 7% of the total radioac tivity added. Serial dilutions of the test substance, positive control (19-norethindrone), negative control (oct yltriethoxysilane), and reference material (non-labeled 17 β -estradiol) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of three runs, and each run contained three replicates at each concentration, including the solvent controls of ethanol and water, plus triplicate samples to determin e total radioactivity , resulting in a to tal of 114 samples.

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



### Methomyl Positive control Negative co ntrol Reference Chemical
19-norethindrone Octyltriethoxysilane Non-labeled 17 β -estradiol Tubes 100-123 c 

### Tubes 52-75 c 

### Tubes 28-51 c 

### Tubes 1-27, 124-138 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 62-64 of the study report. b Each tube contains: 10μL of either the test substance, positiv e control, negative contro l, 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), fo r a total of 500 μL. c Each concentration of each chemical was run in triplicat e, for a total of 138 tubes per run; tubes 76-99 were skipped in sequence. d Solvent is water for methomyl and ethanol for the reference chemicals (<2%)Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 7 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None


## The procedures for assay incubation, separati on of bound from free estradiol (washing with buffer and extraction with ethanol), fo llowed 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 non-specific binding data were modeled via non-linear re gression using Origin 8.5.1 (OriginLab Corp., Northampton, MA. For the competitive bi nding assay, similar methods of nonlinear regression were used to fit a curve (for 17 β -estradiol, the positive control, and the test substance) to the Hill equation formula which incorporated log IC 50 

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

## K d 

## and B max 

## ) and competitive binding experiment (log IC 50 

## and RBA), m ean and standard deviation were calculated for each run and mean and standard error were calculated for the composite three

runs.



## 1. Definitions

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

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

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

− 6 

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

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

− 6 

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

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

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

Run Classification Numerical Value 

### Interactive 2 Equivocal 1 Not interactive 0 Equivocal up to the limit of c oncentrations tested “missing”



## The values for each run were then averaged acro ss runs and the chemical classified using the following ranges:Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 8 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None



### 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 c oncentrations 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: The saturation binding experiment parameters are presented in Fi gure 1 and Table 5. The mean K d 

## for [ 3 

## H]-17 β -estradiol was 0.081 ± 0.002, and the mean estimated B max 

## was 32.5 ± 2.25 fmol/100μg protein for the prepared at uterine cytosol. The K d 

## for each run was within th e expected range of 0.03 to 1.5 nM. The data produced a linear Scatchard plots (r 2 

## = 0.88-0.91); Table 2. Confidence in these numbers is high due to the goodness of fit and the small variation among runs.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 9 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None


## FIGURE 1. Binding of [ 3 

## H]-17 β -estradiol to the Estrogen R eceptor during the Saturation Binding Experiment.

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

## H]-17 β -estradiol to the Estrogen Receptor.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 10 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None



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


### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean ± SE c 

### R 2 

### (unweighted) 0.913 0.880 0.889 0.880-0.913
B max 

### (nM) 0.020 0.024 0.024 0.023± 0.001 B max 

### (fmol/100 μ g protein) 28.0 35.1 34.5 32.5 ± 2.25
K d 

### (nM) 0.077 0.081 0.085 0.081 ± 0.002 a Data were obtained from page 26 of the study report. b The mean is reported for the c oncurrent replicat es of each run. c The range of R 2 

### is reported and the mean and SE are reported for the other parameters. R 2 

### Goodness of fit for curve calculated for specific binding



## B. COMPETITIVE BINDING EXPERIMENT: The results from the competitive binding experiment are shown graphically in Figures 3, 4 and 5 and the parameters calculated from these curves are presented Table 6.

The three reference chemicals performe d as expected. The average log IC 50 

## was

− 9.16 M for 17 β -estradiol and

− 4.85 M for the weak positive control, 19-norethindrone, and the mean RBA was 0.0054% for the weak positive control. The negative control, octyltriethoxysilane, had no effect on specific binding of the [ 3 

## H]-ligand (85-105% binding). Confidence in these numbers is high due to th e small variation. Additional runs were
unnecessary.

Methomyl had no effect on speci fic binding (88-113%) of the [ 3 

## H]-ligand indicating there was no competitive interactions w ith the ER. Therefore, a log IC 50 

## and RBA could not be calculated for the methomyl. Based on the re sults of the three valid runs, methomyl is classified as not interactive with the estroge n receptor under the condi tions of this assay (Table 7).

FIGURE 3. Percentage E2 Bound to the Estrog en Receptor in the Presence of Estradiol.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 11 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None


## FIGURE 4. Percentage E2 Bound to the Estrogen Receptor in the Presence of Octyltriethoxysilane.

FIGURE 5. Percentage E2 Bound to the Es trogen Receptor in the Presence of 19- Norethindrone.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 12 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None


## FIGURE 6. Percentage E2 Bound to the Estrog en Receptor in the Presence of Methomyl.

TABLE 6. Competitive Binding Assay of Methomyl with Estrogen Receptor from Rat Uterine Cytosol a


### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean ± SE c



### r 2 

### (unweighted) 17 β -estradiol 0.992 0.994 0.988 0.988-0.994 Positive control 0.931 0.983 0.967 0.931-0.983 Methomyl NA NA NA NA Log IC 50 

### (M) 17 β -estradiol

− 9.43

− 9.03

− 9.02

− 9.16 ± 0.13 Positive control

− 4.92

− 4.70

− 4.95

− 4.85 ± 0.08 Methomyl NA NA NA NA IC 50 

### (M) d 

### 17 β -estradiol 3.72×10

− 10 

### 9.33×10

− 10 

### 9.55×10

− 10 

### 7.53×10

− 10 

### (±1.91) Positive control 1.20×10

− 5 

### 2.00×10

− 5 

### 1.12×10

− 5 

### 1.44×10

− 5 

### (±2.79) Methomyl NA NA NA NA Log RBA (%) d 

### Positive control

− 2.51

− 2.34

− 2.09

− 2.27 ± 1.23 Methomyl NA NA NA NA RBA (%) Positive control 0.0031 0.0046 0.0085 0.0054 ± 0.0016 Methomyl NA NA NA NA a Data were obtained from page 28 of the study report.

r 2 

### Goodness of fit RBA (%) Relative binding affinity b The mean is reported for the concurrent replicates within each run. c The range is reported for r 2 

### , and the mean ± SEM is reported for the remaining parameters. For mean values reported in scientific notation, the SEM are reported in the same exponential. d Calculated by the reviewer NA Not applicable. r 2 

### is more appropriately expressed as a range, as opposed to a mean.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 13 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None



### TABLE 7. Binding Classification of Methomyl with Es trogen Receptor a


### Run 1 2 3 Mean c 

### Binding Classification d 

### Classification category value b 

### 0 0 0 0 Not interactive a Data were calculated by the reviewer. b Classification category value: Interactive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of concentrations tested (“missing”, i.e., not included in calculation of mean). c Mean of three runs expressed to the tenths place d Interactive = mean ≥ 1.5; Equivocal = 0.5 ≤ mean <1.5; Not interactive = mean <0.5



## C. PERFORMANCE CRITERIA: To ensure that the compe titive binding assay functioned properly, each run was evaluate d using the following criteria.

TABLE 8. Criterion a 

### Tolerance Limit(s) Value Yes No

17 β -estradiol 

### fitted curve parameters Log e 

### residual SD ≤ 2.35 ≤ 1.40 X Top (% binding) 94 to 111 76 to 101 X Bottom (% binding)

− 4 to 1

− 1.7 to

− 1.1 X Hill Slope (log 10 

### (M)

− 1 

### )

− 1.1 to

− 0.7

− 1.0 to

− 0.9 X Weak Positive control (19-norethindrone) 

### fitted curve parameters b 

### Log e 

### residual SD NA ≤ 2.14 Top (% binding) NA 96 to 103 Bottom (% binding) NA

− 22.1 to 3.0 Hill Slope (log 10 

### (M)

− 1 

### ) NA

− 0.8 to

− 1.0 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

a Data were obtained from page 28 of the study report. b The EPA Guideline does not define a set of tolerance limits for 19-norethi ndrone. Acceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable as they show 19-nor ethindrone to be an acceptable weak positive control. NA Not applicable



## The top % binding for 17 β -estradiol was only 76% for Run 1. However, the curve for the reference material showed that incr easing concentrations of unlabeled 17 β -estradiol displaced [ 3 

## H]-17 β -estradiol in a manner consistent with one-site binding, as indicated by a Hill Slope of

− 1.0 to

− 0.9.

Methomyl was tested over a concentration range that fully defined the top of the curve. The percent binding at this top plateau was within 25 percentage points of the value for solvent control.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Under the conditions of the study, methomyl did not bind to the estrogen receptor when tested up to a maximum of 10

− 3 

## M. Therefore, methomyl is classified as a non-inhibitor in this estrogen recep tor binding assay.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 14 METHOMYL/090301 OCSPP 890.1250/ OECD None



## B. AGENCY COMMENTS: In the saturation binding e xperiment, the maximum binding capacity (B max 

## ) was 32.5 ± 2.25 fmol/100 μg protein and the dissociation constant (K d 

## ) was 0.081 ± 0.022 nM. For the competitive binding experiment, performance criteria were generally met for 17 β -estradiol (with the exception top % binding for Run 1); the log PC 50 

## , log PC 10, 

## log EC 50, 

## and Hill Slope for the reference chemicals 17 β -estradiol and octyltriethoxysilane all performed within th e acceptable range. For 19-norethindrone, the bottom % binding for the second run was

− 22.1%. However, visual observation of the graphs indicated that that assay performed sim ilarly to the other two runs. The average log IC 50 

## was

− 9.16 M for 17 β -estradiol and

− 4.85 M for 19-norethindrone, and the mean RBA was 0.0054% for 19-norethindrone.

Methomyl had no appreciable effect on specific binding (88-113%) of the [ 3 

## H]-ligand to the ER. Therefore, a log IC 50 

## and RBA could not be calculated for methomyl. Based on the results of the three valid runs, methomyl is cl assified as not interactive with the ER as specific binding was >75% at all concentrations tested.

C. STUDY DEFICIENCIES: The following deficiency was note d that is not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The top % binding for 17 β -estradiol was only 76% for Run 1 (expected range: 94 to 111%).

# DATA EVALUATION RECORD

METHOMYL

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 2-47-2012 (MRID 48685901)

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: 5/3/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/18/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 6/08/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 6/08/2012

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).METHOMYL/090301 Estrogen Receptor Transcriptional Activation (2012) I Page I of 14 OCSPP 890.1300/ OECD 455



## Primary Reviewer: Karlyn Middleton 

## Signature:**--+-~~-.----** 

## Health Effects Division Date: 1/11/13

__**____,,,, __,_,_~**  

## Secondary Reviewer: Minerva Mercado-Feliciano PhD DABT 

## Signature y :**~c::;:~-=-** 

## Health Effects Division Date: ___.,,,,_...___..=...._,__

__ _ Template version 08/2011



## DATA EVALUATION RECORD

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



## PC CODE: 090301 

## DP BARCODE: D399624



## TXR#: Not Provided 

## CAS No.: 16752-77-5



## TEST MATERIAL (PURITY): Methomyl (99.4% a.i.)



## SYNONYMS: Methyl N-[[(methylamino)carbonyl]oxy]ethanimidothioate; DPX-Xl 179



## CITATION: Willoughby, 

## I.A (2012). Methomyl (DPX-Xl 179) Technical: Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa-9903)). CeeTox

, Inc., Kalamazoo, MI. Laboratory Report No

. : 9144V-100360ERTA, Feb 7

, 2012. MRID 48685901. Unpublished.



## SPONSOR: E.I. du Pont de Nemours and Company, Wilmington, DE



## TEST ORDER#: EDSP-090301-62



## EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID 48685901 )

, hERa-HeLa-9903 cells cultured in vitro 

## were exposed to methomyl (99.4% a.i., Lot # 

## J U L06LP276) at logarithmically increasing concentrations from 

## 10

- 108 to 

## 10

- 3 s Min water for approximately 24 hours in two independent runs. Experiments were performed using 96-well plates and each concentration was tested in 6 wells / plate in each run. Cells were exposed to the
test agent for 24 ± 2 hours at 37 ± 1 ° C to induce reporter (luciferase) gene products. Luciferase expression in response to activation of the estrogen receptor was measured using a proprietary luciferase assay

.

No cytotoxicity or precipitation were noted at the highest concentration of methomyl tested, 1

# o3

· 8 M

. 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

v ery weak activity, methomyl 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 24.3-26.6% PC. Methomyl was only able to reach a maximum of 3.1-3.7% PC when tested up to the highest concentration possible based on cytotoxicity

. Because the RPC Max < PC1 0 in two assay runs , methomyl was considered negative for estrogen receptor transcriptional activation in this test system.Estrogen Receptor Transc riptional Activation (2012) / Page 2 of 14 METHOMYL/090301 OCSPP 890.1300/ OECD 455


## This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor Transcriptional Activation assay (OCSPP 890.1300).

COMPLIANCE: Signed and dated GLP, Quality Assurance and Data Confidentiality statements were provided.Estrogen Receptor Transc riptional Activation (2012) / Page 3 of 14 METHOMYL/090301 OCSPP 890.1300/ OECD 455



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Methomyl Description: White Powder Source (cat #): DuPont (not reported) Lot/Batch # (Exp date): JUL06LP276 (May 18, 2016) Purity: 99.4% Solubility: Up to 0.16 M in water Volatility: Not reported Stability: Not reported Storage conditions: Ambient CAS #: 16752-77-5 Structure:

N

S O N H O

CH 3 CH

3

CH 3



## 2. Reference substances 17 β -estradiol (strong estrogen; positive control) Supplier: Sigma-Aldrich, St. Louis, MO Catalog and Lot #: E8875; 110M0138V Purity: 100% CAS # : 50-28-2



## 17 α -estradiol (weak estrogen) Supplier: Sigma-Aldrich, St. Louis, MO Catalog and Lot #: E8750; 041M4065V Purity: 99.72% CAS # : 57-91-0


## Corticosterone (negative compound) Supplier: Sigma-Aldrich, St. Louis, MO Catalog and Lot #: 27840; BCBC6322V Purity: 99.2% CAS # : 50-22-6



## 17 α -methyltestosterone (very weak agonist) Supplier: Sigma-Aldrich, St. Louis, MO Catalog and Lot #: M7252; 060M1543V Purity: 99% CAS # : 58-18-4



## 3. Vehicle(s)
Solvent: Ultrapure water (methomyl) a nd DMSO (reference compounds)

Solvent control (final concentration): Water – NA; DMSO – 0.1%Estrogen Receptor Transc riptional Activation (2012) / Page 4 of 14 METHOMYL/090301 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 (EMEM) without phenol re d, supplemented with kanamycin (60 mg/L) and 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS; source not reported), in an in cubator under 5% CO 2 

## at 37oC. Upon reaching 75-90% confluency, cells were subcultured at least twice pr ior to exposure to the test material. The cells used in this study were Passage 31 (range -finder and first run), and Passage 33 (second run).

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/75 μL medium/well and allowed to attach for 3 hours. The growth media was replaced with media containing serial log dilutions of methomyl in water. Cells were incubated for 24±2 hours at 37±2 °C. The final concentration of the DMSO (reference chemical vehicle) was 0.1% in the assay. Cytotoxicity was determined by two-read propidium iodide uptake.

It was stated that transcrip tional activation of th e estrogen receptor was determined as described in CeeTox Standard Operating Pr otocol (SOP) 2041. A list of reagents was provided, but the assay reagent was classified as proprietary information, and the SOP was not provided.

a. Preliminary Test: A preliminary test evaluating concentrations ranging from 10 -7.3 

## to 10 -3.8 

## M was conducted to determine the appropriat e concentration range and to determine concentrations resulting in inso lubility and/or cytotoxicity.

b. Proficiency Chemicals: The responsiveness of the test system was tested on March 5, April 12 and April 28, 2011. The cells were te sted using the following set of proficiency chemicals, and each chemical was tested in duplicate on separate days (MRID 48843501) 1 

## :

1 

### MRID 48843501 Willoughby, J.A. (2012) Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa-9903)): Proficiency Data Report. CeeTox, Inc.Estrogen Receptor Transc riptional Activation (2012) / Page 5 of 14 METHOMYL/090301 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 100 μM. 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 reference chemicals were included in each plate in the current assay, along w ith the test chemical:

Reference Chemical CAS No. Concentration Range Class 17 β -estradiol 50-28-2 10

− 15 to 10

− 8 Strong estrogen 17 α -estradiol 57-91-0 10

− 13 to 10

− 6 Weak estrogen Corticosterone 50-22-6 10

− 11 to 10

− 4 Negative compound 17 α -methyltestosterone 58-18-4 10

− 12 to 10

− 5 Very weak agonist



## 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 plateEstrogen Receptor Transc riptional Activation (2012) / Page 6 of 14 METHOMYL/090301 OCSPP 890.1300/ OECD 455



## PC 50 

## = concentration of a test chemical at which the response is 50% of the response induced by the positive control (E2 at 1 nM) in each plate

RPC Max 

## = maximum level of response induced by a test chemical, expressed as a percentage of the response induced by the positive control (1 nM E2) on the same plate

PC Max 

## = concentration of a test chemical inducing the RPC Max



## II. RESULTS

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the transcriptional activation assays, a preliminary cytotoxicity and precipitation assay was conducted. No precipitation or cytotoxicity were observed at any concentration tested (Table 1). Based on these results, logari thmically increasing concentrations from 10

− 10.8 

## to 10
− 3.8 

## M were selected for the assay.

TABLE 1. Preliminary Test for Solubility, Cytoto xicity, and Concentration-Selection for Methomyl a Concentration (M) % Viability Comments
10

− 3.8 95 No precipitation observed 10

− 4.3 97 10

− 4.8 100 10

− 5.3 100 10

− 5.8 100 10

− 6.3 101 10

− 6.8 100 10

− 7.3 99 a Data were obtained from page 20 of the study report.



## B. POSITIVE AND NEGATIVE REFERENCE CHEMICALS

1. Proficiency Chemicals: The responsiveness of the test system was tested on March 5, April 12 and April 28, 2011(report submitted separately; MRID 48843501). 2 

## The cells were tested using the following set of proficiency chemicals, and each chemical was tested in duplicate on separate days as shown in Table 2a.

2 

### MRID 48843501 Willoughby, J.A. (2012) Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa-9903)): Proficiency Data Report. CeeTox, Inc.Estrogen Receptor Transc riptional Activation (2012) / Page 7 of 14 METHOMYL/090301 OCSPP 890.1300/ OECD 455



### TABLE 2a. Proficiency Chemicals

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 100 μM. 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.



## In the proficiency tests, the reference chemicals 17 β -estradiol, 17 α -estradiol and 17 α

methyltestosterone were tested concurrently with each run of the assay (Table 2b). The responses do not demonstrate proficiency as the assay cannot be fully evaluated by the reviewer.

Table 2b. Performance Criteria for Ref erence Chemicals in the Proficiency test
Reference Chemical Parameter Acceptable Range Values Acceptable

Run 1 Run 2 Run 3 Yes No 17 β -estradiol Log PC 50 − 11.4 to

− 10.1

− 9.6

− 11.3

− 10.6 Run 1 Log PC 10 <

− 11

− 11.5

− 12.5

− 12.1 X Log EC 50 − 11.3 to

− 10.1

− 9.0

− 11.3

− 10.6 Run 1 Hill Slope 0.7 to 1.5 1.2 0.9 0.8 Test range (M) 10

− 14 to 10

− 8 10

− 14 to 10

− 8 10

− 14 to 10

− 8 10

− 14 to 10

− 8 X 17 α -estradiol Log PC 50 − 9.6 to

− 8.1

− 8.3

− 9.4

− 8.7 X Log PC 10 − 10.7 to

− 9.3

− 9.3

− 10.5

− 9.9 X Log EC 50 − 9.6 to

− 8.4

− 8.2

− 9.3

− 8.9 Run 1 Hill Slope 0.9 to 2.0 0.9 0.9 2.9 Run 3 Test range (M) 10

− 12 to 10

− 6 10

− 12 to 10

− 6 10

− 12 to 10

− 6 10

− 12 to 10

− 6 X 17 α -methyltestosterone Log PC 50 − 6.0 to

− 5.1

− 6.2 NC

− 5.2 Run 1, 2 Log PC 10 − 8.0 to

− 6.2

− 8.1

− 6.3

− 7.7 Run 1 Test range (M) 10

− 11 to 10

− 5 10

− 11 to 10

− 5 10

− 11 to 10

− 5 10

− 11 to 10

− 5 X



## 2. Reference Chemicals: The response curves for the th ree estrogenic reference chemicals 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.Estrogen Receptor Transc riptional Activation (2012) / Page 8 of 14 METHOMYL/090301 OCSPP 890.1300/ OECD 455


## Although all four performance criteria were generally met for 17 β -estradiol, 17 α -estradiol and corticosterone, the available data indicate that both runs we re invalid due to the lack of responsiveness of the cells to 17 α -methyltestosterone. For 17 α -methyltestosterone, the mean RPC Max 

## was 24.3% for the first run and 26.6% for the second run. Additionally, cytotoxicity was observed in cells treated with corticosterone at the hi ghest concentration in the first run and, in both runs, the ma ximum response of the cells to both 17 β -estradiol and 17 α -estradiol exceeded (149-170%) 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 -10.5 -10.7 X Log PC 10 <

− 11 -11.5 -11.8 X Log EC 50 − 11.3 to

− 10.1 -10.4 -10.7 X Hill Slope 0.7 to 1.5 1.3 1.2 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 -8.8 -8.5 X Log PC 10 − 10.7 to

− 9.3 -9.8 -9.4 X Log EC 50 − 9.6 to

− 8.4 -8.8 -8.5 X Hill Slope 0.9 to 2.0 1.0 1.5 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 NA b NA X Log PC 10 − 8.0 to

− 6.2 -5.8 -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 page 23 of the study report. b NA = not applicable; 17 α -methyltestosterone did not achieve 50% activation at up to 10

− 5 M.Estrogen Receptor Transcriptional Activation (2012) / Page 9 of 14 METHOMYL/090301 OCSPP 890.1300/ OECD 455


## FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of 17 β -Estradiol Compared to the Positive Control.Estrogen Receptor Transcriptional Activation (2012) / Page 10 of 14 METHOMYL/090301 OCSPP 890.1300/ OECD 455


## FIGURE 2. Fold Induction of Relative Transcription Activation (RTA) of 17 α -Estradiol Compared to the Positive Control.Estrogen Receptor Transcriptional Activation (2012) / Page 11 of 14 METHOMYL/090301 OCSPP 890.1300/ OECD 455


## FIGURE 3. Fold Induction of Relative Transcription Activation (RTA) of 17 α
Methyltestosterone Compared to the Positive Control.

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 32142 for the first run and 26021 for the second; the overall mean TA valu e for the positive control was 520658 for the first run and 372642 for the second. The induction for the positive control ranged from14- to 16-fold. The mean normalized value for the positive control was 488516 for the first run and 346621 for the second run. The PC 50 

## (50% of the maximum response) for E2 in this assay is 244258 for the first run a nd 173310 for the second run, and the PC 10 

## (10% of the maximum response) is 48852 for the first run and 34662 for the second run.Estrogen Receptor Transc riptional Activation (2012) / Page 12 of 14 METHOMYL/090301 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 32142 6078 520658 98029 16 488516 98029 2 26021 8191 372642 116177 14 346621 116177 a Data were calculated by the reviewer from da ta obtained from 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 3.1% for the first run a nd 3.7% for the second, and the associated PC Max 

## was 10
− 6.8 

## M for the first run and 10

− 6.8 

## M for the second run. Because the RPC Max 

## < PC 10 

## in both runs, methomyl was considered negative for estrogen receptor transcriptional activation in this test system.

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

Parameter RTA (mean ± SD); % of Positive Control (PC) Run 1 Run 2 Conc. (M) Mean SD Mean SD
10

− 3.8 0.0 1.1 0.0 1.8 10

− 4.8 2.8 1.1 1.8 2.2 10

− 5.8 2.7 0.5 1.7 2.6 10

− 6.8 3.1 1.2 3.7 2.9 10

− 7.8

− 0.2 1.6 0.0 2.7 10

− 8.8 1.6 1.9 2.4 3.2 10

− 9.8 0.2 1.0 0.8 3.2 10

− 10.8 1.0 1.7 2.3 3.7 Log EC 50 b NA NA Hill Slope b NA NA RPC Max 3.1 3.7 PC Max 10

− 6.8 10

− 6.8
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 13 of 14 METHOMYL/090301 OCSPP 890.1300/ OECD 455



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

3. Performance Criteria: While the log PC 50 

## , log PC 10 

## , log EC 50, 

## and Hill slope values for the concurrent reference chemicals fell within or near the acceptable ranges (Table 3), the full response of the cells to the reference chemicals was not satisfactory, as detailed above in Section B.2.Estrogen Receptor Transc riptional Activation (2012) / Page 14 of 14 METHOMYL/090301 OCSPP 890.1300/ OECD 455



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The suitable top concentration of methomyl for use in the transcriptional activation assays was 10 -3.8 

## M as no cytotoxicity or precipitation was observed. In two independent runs of the transcriptional activati on assay, methomyl did not result in an increase in luciferase activity (RPC Max 

## <10%) at any of the viable concentrations tested; methomyl is not an agonist of human estroge n receptor alpha (hER α ) in the HeLa-9903 model system.

B. AGENCY COMMENTS: Methomyl was tested up to 10 -3.8 

## M, with no cytotoxicity or precipitation noted. At these concen trations, the RTA of methomyl was ≤ 3.7%, indicating it is negative for estrogenic activity un der the conditions of this assay.

Although the conditions of this assay were not optimal to detect very weak activity, methomyl 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 24.3-26.6% PC. Methomyl was only able to reach a maximum of 3.1-3.7% PC when tested up to the highest concentration possible based on cytoto xicity. Because the RPC Max 

## < PC 10 

## in two assay runs, methomyl was cons idered negative for estrogen receptor transcriptional activation in this test system.

C. STUDY DEFICIENCIES: The following deficiencies were noted:

 The cells responded poorly (<50% RTA) to the very weak estrogen, 17 α -methyltestosterone.

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

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

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

EPA MRID Number 48701401

Page 1 of 51 Data Requirement: EPA DP Barcode 403694

OECD Data Point

EPA MRID 48701401
EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test material: Methomyl Purity: 99.4%

Common name

Chemical name: IUPAC:

CAS name:

CAS No.:
Synonyms: DPX-X1179; DPX-X1179-571.

EPA PC Code: 090301

Primary Reviewer: Joan Gaidos Signature:
Environmental Scientist, CDM Smith Date: 6/21/13

Secondary Reviewer: Teri S. Myers Signature:
Environmental Scientist, CDM Smith Date: 7/17/13

Primary Reviewer: Tanja Crk Date:

EPA/OPP/EFED/ERB3

Secondary Reviewer(s): Amy Blankinship Date:

EPA/OPP/EFED/ERB3

Date Evaluation Completed: 6/5/15

CITATION: S.L. Hicks. 2012. Methomyl Technical (DPX-X1179): Short-term Reproduction Assay with the Fathead

Minnow, Pimephales promelas , Determined Under Flow-Through Conditions. Performed by ABC Laboratories, Inc., 

# AMY BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.15 17:39:46 -04'00'

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

EPA MRID Number 48701401

Page 2 of 51 Columbia Missouri and Experimental Pathology Laboratories, Inc., Sterling, Virginia. Lab Study No.: 66795; Sponsor
Project No.: DuPont-32248. Submitted by E.I. du Pont de Nemours and Company, Wilmington, Delaware.

Completion date May 18, 2012



### .

Note: 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).

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

EPA MRID Number 48701401

Page 3 of 51 EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay of methomyl with fathead minnows ( Pimephales promelas) was conducted under continuous flow-through conditions. Adult fish (16 spawning groups, 2 males and 4 females

in each group; ca . 6 months old) were exposed to methomyl (99.4% purity) at nominal concentrations of 0 (control), 0.090, 0.30, and 1.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.0124, negative control), 0.0947, 0.312, and 1.03 mg a.i./L. The test system was maintained at 24.2 to 24.8 o 

### C and a pH

of 7.88 to 8.33.

Adult male and female survival was unaffected by treat ment with methomyl and was 100% for all test levels.
There were no notable observations with regards to behavior or changes in appearance, specifically
color/banding, ovipositor appearance, or size of dorsal nape pad in the control or treated groups, and no clinical signs of toxicity were observed. Male body weights were reduced (p<0.05; Jonckheere-Terpstra test)
by 9.8 and 13% at the mid and high concentrations, respectively, relative to the negative control; female
body weights were not affected. Body lengths at test termination were not reported.

Spawning occurred at least every three days, and mean fecundity in negative control was 54 eggs/female/day/replicate (replicate range was 50-62 eggs/female/day); fertilization success in the
negative control group averaged 92%. There were significant reductions in fecundity of 23 and 77% and in
fertility of 1.6 and 33% at the mid and high concentrations, respectively, compared to the negative control (p<0.05; Jonckheere-Terpstra test).

Additionally, female gonadal-somatic index (GSI) was significantly reduced by 15 and 21% at the mid and

high concentrations, respectively, compared to the negati ve control (p<0.05; Jonckheere-Terpstra test). Male
GSI was not significantly different from the control. Histopathology showed treatment-related effects at the
high concentration, consisting of increased prevalence and severity of increased spermatogonia in males and
increased prevalence and severity of increased oocyte atresia in females. There were no differences in gonadal stages between treated groups and the negative controls. Plasma vitellogenin (VTG) was not
significantly different from controls for either sex. Sex steroids were not measured in this study.

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

EPA MRID Number 48701401

Page 4 of 51 The performance criteria were met in this study except that the average fertilization success in the control
was 92%, which is slightly below the recommended value of 95%. However, this slight decrease in
fertilization rate did not impact the interpretation of the results 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: ca . 6 months Mean body weight at test initiation (if measured): Not reported. Mean length at test initiation (if measured): Not reported. Test Type (Flow-through, Static, Stat ic Renewal): Continuous flow-throughData Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 5 of 51Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Methomyl.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.0947 No No No No No No No No No No NANANANA0.312YesYesNoNoNoYesNoNoNoNoNANANANA1.03YesYesNoNoNoYesYesYesNoNoNANANANAAbbreviations: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 regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.

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

EPA MRID Number 48701401

Page 6 of 51 I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP
890.1350: “Fish Short-Term Reproductive Assay”. The following

deviations were noted:

1. The total organic carbon, unionized ammonia, residual chlorine and conductivity in the
test water were not reported. The OCSPP 890.1350 performance criteria establish

maximum levels for these values, and it is unclear if the maximum recommendations

were exceeded.
2. Individual fish weights at study initiation were not reported; however, the range was
reported as ± 20% for males. EPA recommends that the subsample of fish weighed

before the test be within ±20% of the estimated mean for each sex.
3. The average fertilization success in the control was 92% which is slightly below the

recommended value of 95%.
4. Body lengths at test termination were not measured.

These deviations do not have an impact on the acceptability of this study.

COMPLIANCE: Signed and dated No Data Confidentiality, GLP, and Quality Assurance
statements were provided. This study was conducted in compliance with all
pertinent U.S. EPA Good Laboratory Practice regulations with the following
exceptions: The most recent water characterizations performed in February
2011 were not performed in accordance to the stated Good Laboratory
Practices. This exception has no impact on the study results.

A. TEST MATERIAL: Methomyl (CAS# not reported)

Description: Solid.

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

EPA MRID Number 48701401

Page 7 of 51 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. : JUL06LP276

Purity: 99.4%

Impurities: None reported

Stability of Compound: Analytical verification of the test material from time 0, 7, 14, and 21 days
at nominal concentrations of 0.090, 0.30 and 1.0 mg a.i./L yielded mean recoveries of 105, 104
and 103%, respectively; the % CV was 1.9, 1.2 and 3.5%, respectively. Method validation of
methomyl fortified at 0.0814 and 1.22 mg a.i./L in freshwater yielded recoveries of 100 to 106% (n= 6). Analysis of exposure solutions 4 days prior to application showed recoveries of methomyl
ranging from 103 to 109% of applied. Samples collected from diluter stock had recoveries of 95 to
106% of applied. The test material appears to be stable under the test conditions.

Storage Conditions of Test Chemicals: Stored under ambient conditions.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 8 of 51B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: Fathead Minnow EPA recommends fathead minnow (Pimephales promelas).Species scientific name: Pimephales promelasSpecies strain (if stated): Not reportedWere fish obtained from asingle laboratory stock?Yes In-house stock. EPA recommends that fish be from a single laboratory stock.Were acclimation conditionssame as definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive

test.Acclimation period: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 on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 9 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:There were no mortalities during the 7days prior to pre-exposure period.Fish did not receive any treatment fordisease during acclimation period.Behavioral abnormalities or clinical signswere not reported. EPA recommends that mortality during the 7 days prior to the pre-exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA recommends that the fish be rejected. If mortality is between 5-10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur during this extended acclimation period, EPA recommends that the fish not be used.Type of food:Live and frozen brineshrimp nauplii ( Artemia

salina) EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body

condition.Source of food:Brine Shrimp Direct,Ogden, UTFrequency of feeding:3 times/dayData Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 10 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding:Fish not fed 12 hoursprior to testtermination.Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation: Ca. 6 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months

old.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 11 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsMean weight of males at testinitiation (if determined):Not reportedBased on 10 males used to stockaquaria for 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):Not reportedIndividual weights within ± 20% of theestimated mean.Mean weight of females at testinitiation (if determined):Not reportedBased on 10 females used to stockaquaria for pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):Not reportedIndividual weights within ± 10% 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 on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 12 of 51ParameterValue(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:36Additional breeding groups weremaintained to account for a potentiallack of spawning and/or mortalityduring this phase. EPA recommends that additional tanks set up at the beginning of pre-exposure will ensure that sufficient replicates with the correct sex ratio are available for the definitive test.Number of males per tank:2Number of females per tank:4Pre-exposure fecundity:Not reported 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 13 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of spawns during preexposure:> two times in 7 daysand at least 15eggs/female/day EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Details on pre-exposure:None.C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Proportional flow-throughEPA recommends the use of a flowthrough system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this assay.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).Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 14 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem:Flow-through diluters Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:10 volume additions/testvessel/day. 8.4cycles/day.Not further described Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.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 Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 15 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems:A proportional diluter was used toproduce nominal concentrations. Flowsplitting chambers were used betweendiluter cells and the 4 replicate testvessels.Treatment recoveries prior to test initiationwere 101-109% of the applied.Flow splitting accuracy was not reported;however proper operation of the diluterwas verified 2x/day.Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy
is within 10%.Aeration? No EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (< 60%

saturation).Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 16 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water:Blended moderately hardwell water and reverseosmosis demineralizedwell water.Dilution water was aerated, heated,filtered and UV sterilized prior to deliveryto dilution system. EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?YesTest vessel type/materials: Glass aquaria.EPA 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:22 cm wide x 38 cmlong x 22 cm high. EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 17 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsFill volume:12 cm depth EPA recommends 10 L solution per tank.Spawning substrate material: Stainless steel screenedtrays. 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:Not reportedAdditional details on exposuresystem:None.Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)24.2 24.824.5 Continuously EPA recommends temperature 25±1 o C; inter-replicate and inter-treatment differentials should not exceed 1 o C.pH 7.888.337.6Weekly EPA recommends pH 6.5 to 9.0.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 18 of 51ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsDissolved oxygen (mg/L) 5.17 8.246.7Weekly EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L asCaCO3)148 162 155 Weekly EPA recommends total alkalinity > 20 mg/L as CaCO 3.Hardness (mg/L as CaCO3) 136 154 145 WeeklyTotal organic carbon (mgC/L)Not reported Not reported Not reported None EPA recommends that total organic carbon in dilution water be < 2 mg/L.Unionized ammonia (Pg/L) Not reported Not reported Not reported None EPA recommends that unionized ammonia in the dilution water be < 1

P g/L.Residual chlorine (Pg/L) Not reported Not reported Not reported None EPA recommends that residual chlorine in dilution water be < 10 P g/L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 19 of 51ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsConductivity (PS/cm) Not reported Not reported Not reported None 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 20 of 51D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details orRemarksGuideline RecommendationsWas a range-finder conducted?No EPA recommends conducting a range-finder if 96-hour LC

50 data for the fathead minnow are unavailable.If yes, what was the method fordetermining the highest testconcentration in the rangefinder?NA EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or spec ies, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is

appropriate.Species:NALife stage:NA 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:NA EPA recommends a 96-hour exposure.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 21 of 51ParameterValue(s)Details orRemarksGuideline RecommendationsAdditional details:None. EPA recommends conducting a ra nge-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentration-response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the

derived 96-hr LC 50, may be selected as the highest exposure concentration in

the 21-day test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 22 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in thedefinitive test:1.0 mg a.i./L Based on existing acute methomylaquatic toxicity data for fatheadminnows provided by the sponsor. 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 reportedSeparation of testconcentrations:Ca. 3 fold EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or ten-fold) is scientifically

acceptable 1 .Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesData Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 23 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Mean measuredLimit of quantification (LOQ): 0.0124 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 24 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsWas 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 preexposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Number of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per testitem treatment level:4 EPA recommends 4 replicates.Number of male fish perreplicate at test initiation:2 EPA recommends 2 males per replicate.Number of female fish perreplicate at test initiation:4 EPA recommends 4 females per replicate.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 25 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsWas a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA EPA recommends that the solvent not exceed 0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8
(light:dark).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp. 69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testingand Assessment. No. 23. Paris, France.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 26 of 51ParameterValue(s)Details or RemarksGuideline RecommendationsLight intensity at water’ssurface:634.4-900.9 luxFluorescents EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:The test solutions were reported asclear and colorless with no visibleprecipitates, films or undissolved testsubstance.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 27 of 51Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay withMethomyl.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilutionwater only)<0.0124 <LOQ NA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Solvent control (ifapplicable)NANANATreatment 10.0900.09471.9Treatment 20.300.3121.2Treatment 31.01.033.5Diluter StockNANANAAbbreviations:CVCoefficient of variation.LOQ=0.0124 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 28 of 51E. ObservationsBiological Endpoints: Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination(Day 21),secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, gonadalsomatic index (GSI), gonadal staging and histopathology, and plasma vitellogenin were evaluated.Were raw (individual) data provided?Yes EPA recommends that observations of survival, fecundity, fertiliz ation 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 s taging and histopathology, plasma vitellogenin, and plasma sex steroids (testosterone and 17-estradiol, if measured). Gonado-somatic index (GSI) is calc ulated 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.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 29 of 51II. RESULTS AND DISCUSSIONA. ResultsThere were no treatment-related mortalities observed in the study, and no mortalities in the controls; however there were two incidentalmortalities (Table 9). On Day 12, a female in replicate D of the control and replicate D of the0.0947 mg a.i./L treatments were found deaddue to injuries sustained during tile placement. These two mortalities were excluded from the percent survival because they were nottreatment related. Also on Day 12, replicate A of the 0.0947 mga.i./L treatment was missing a male fish and an extra male fishwas foundin an adjacent chamber, replicate D of the 0.312 mg a.i./L treatment, where it remained until study termination.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 30 of 51Table 9: Adult Fish Survival in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]MalesFemalesN1# Surviving% SurvivalN1# Surviving% SurvivalControl (<LOQ)88100 15-16215 1000.0947 7-838 100 15-16215 1000.312 8-938 100 16 16 1001.03 8 8 100 16 16 100Abbreviations:NANot applicable.1Total number of fish at test initiation.2On Day 12, a female in replicate D of the control and replicate D of the 0.0947 mg a.i./L treatments were found dead due to injuries sustainedduring tile placement. These two mortalities were excluded from the percent survival because they were not treatment related.3On Day 12, replicate A of the 0.0947 mg a.i./L treatment was missing a male fish and an extra male fish was found in an adjacent chamber,replicate D of the 0.312 mg a.i./L treatment, where it remained until study termination.LOQ=0.0124 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 31 of 51Mean male body weight values were 4.374, 4.089, 3.947 and 3.795 g, and mean female body weight values were 1.867, 1.834, 1.732and1.752 g in the mean-measured 0 (control), 0.0947, 0.312 and 1.03 mg a.i./L treatment levels, respectively (Table 10). The study authordid not report mean male or female body length.Table 10: Size at Test Termination in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDControl (<LOQ) 4 4.374 0.533 41.867 0.079 4 NA NA 4 NA NA0.0947 4 4.089 0.328 4 1.834 0.12 4 NA NA 4 NA NA0.312 4 3.947 0.348 4 1.732 0.18 4 NA NA 4 NA NA1.03 4 3.795 0.167 4 1.752 0.23 4 NA NA 4 NA NAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.0124 mg a.i./L.

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

EPA MRID Number 48701401

Page 32 of 51 Mean fecundity values were 54, 47, 41 and 12 eggs/female/day, and fertilization success was 92, 94, 91 and 62% in the 0 (control), 0.0947, 0.312 and 1.03 mg a.i./L treatment levels, respectively (Table
11).

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

.

Treatment
(mg a.i./L)
[mean-measured] Fecundity 1 

### ± S.D. Fertilization
Success (%) 2 

### ± S.D.

Control (<LOQ) 54 5.6 92 6.7

0.0947 47 9.4 94 1.4

0.312 41 9.7 91 1.0

1.03 12 8.4 62 25

Abbreviations: NA 

### Not applicable. ND 

### Not determined.

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.

LOQ=0.0124 mg a.i./L.

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

EPA MRID Number 48701401

Page 33 of 51 Median male tubercle scores were 36, 38, 32 and 30 in the mean-measured the 0 (control), 0.0947, 0.312 and 1.03 mg a.i./L treatment levels, respectively (Table 12). None of the surviving females were
found to have notable tubercles; one female from the low treatment was reported to have tubercles with a

tubercle score of 6 for a treatment mean of 0.5.

Table 12: 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

Control (<LOQ) 4 36 4 0

0.0947 4 38 4 0

0.312 4 32 4 0

1.03 4 30 4 0

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.
LOQ=0.0124 mg a.i./L.

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

EPA MRID Number 48701401

Page 34 of 51 Mean male GSI was 1.31, 1.25, 1.38 and 1.33%, and mean female GSI was 13, 13, 11, and 10% in the mean-measured 0 (control), 0.0947, 0.312 and 1.03 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

Control (<LOQ) 4 1.31 0.42 4 13 1.2

0.0947 4 1.25 0.24 4 13 0

0.312 4 1.38 0.22 4 11 0.82

1.03 4 1.33 0.10 4 10 2.9

Abbreviations: NA 

### Not applicable.

1 

### Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x

100.

LOQ=0.0124 mg a.i./L.

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

EPA MRID Number 48701401

Page 35 of 51 Median male gonadal stages was 2 for all treatment levels, and female gonadal stages were 3, 3, 4 and 3 in the mean-measured 0 (control), 0.0947, 0.312 and 1.03 mg a.i./L treatment levels, respectively (Table
14).

Table 14: Gonadal Staging in Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)
[mean-measured] Males Females

n Median Stage 1 n Median Stage 2



### Control (<LOQ) 8 2 

### 15 3



### 0.0947 7 2 

### 15 3



### 0.312 9 2 

### 16 4



### 1.03 8 2 16 3



### Abbreviations: J 

### Juvenile. NA 

### Not applicable. ND 

### Not determined. UTS 

### Unable to stage.

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.
LOQ=0.0124 mg a.i./L.

In male fish, there was an increase in the prevalence and severity of increased spermatogonia (minimal to moderate) in males at the 1.03 mg a.i./L level (Tables 15-16), relative to negative control male fish.
Increased spermatogonia was characterized by an elevated proportion of spermatogonia compared to the other spermatogenic cell types with the germinal epithelium (i.e. spermatocytes and spermatids). In female fish, there was an increase in the prevalence and severity of increased oocyte atresia (minimal to moderate) at the 1.03 mg a.i./L level (Tables 17-18), relative to negative control female fish. Increased oocyte atresia
was characterized by disintegration or discontinuity of the chorion, disorganization of the ooplasm, and uptake
of yolk material by perifollicular cells.

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

EPA MRID Number 48701401

Page 36 of 51 Table 15: Gonadal Histopathology in Male Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Increased
Proportion of
Spermatogonia Presence of

Testis-Ova Increased

Testicular
Degeneration Duct

Mineralization Interstitial cell
(Leydig)
hypertrophy/
hyperplasia

n Incidence n Incidence n Incidence n Incidence n Incidence

Control
(<LOQ) 0 8 8 NA NA 8 6 8 7 8 7

1 8 0 NA NA 8 1 8 0 8 1

2 8 0 NA NA 8 1 8 1 8 0

3 8 0 NA NA 8 0 8 0 8 0

4 8 0 NA NA 8 0 8 0 8 0



### 0.0947 0 7 6 NA NA 7 7 7 7 7 6

1 7 1 NA NA 7 0 7 0 7 1

2 7 0 NA NA 7 0 7 0 7 0

3 7 0 NA NA 7 0 7 0 7 0

4 7 0 NA NA 7 0 7 0 7 0



### 0.312 0 9 6 NA NA 9 9 9 8 9 9

1 9 0 NA NA 9 0 9 1 9 0

2 9 0 NA NA 9 0 9 0 9 0

3 9 0 NA NA 9 0 9 0 9 0

4 9 0 NA NA 9 0 9 0 9 0



### 1.03 0 8 3 NA NA 8 8 8 7 8 8

1 8 3 NA NA 8 1 8 0 8 0

2 8 1 NA NA 8 0 8 1 8 0

3 8 1 NA NA 8 0 8 0 8 0

4 8 0 NA NA 8 0 8 0 8 0

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.

LOQ=0.0124 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 37 of 51Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceControl (<LOQ) 0NA NA NA NA NA NA NA NA NA NA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANANANANANANANA0.09470NA NA NA NA NA NA NA NA NA NA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANA NA NA NA NA NA NAData Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 38 of 51Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascularor InterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportionsof Spermatocytesor SpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.3120NA NA NA NANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANA NA NA NA NA NA NA1.030NA NA NA NANANANANANANA1NANANANANANANANANANA2NANANANANANANANANANA3NANANANANANANANANANA4NANANANA NA NA NA NA NA NA1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. SeeAppendix E of the test guideline for reference.LOQ=0.0124 mg a.i./L.

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

EPA MRID Number 48701401

Page 39 of 51 Table 17: Gonadal Histopathology in Female Fathead Minnow ( Pimephales promelas)

.

Treatment
(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1



### Severity Increased Oocyte

Atresia Perifollicular Cell
Hyperplasia/
Hypertrophy Decreased Yolk

Formation Aggregates of
macrophages,

multifocal

n Incidence n Incidence n Incidence n Incidence

Control (<LOQ) 0 15 12 NA NA NA NA NA NA



### 1 15 3 NA NA NA NA NA NA



### 2 15 0 NA NA NA NA NA NA



### 3 15 0 NA NA NA NA NA NA



### 4 15 0 NA NA NA NA NA NA



### 0.0947 0 15 12 NA NA NA NA NA NA



### 1 15 2 NA NA NA NA NA NA



### 2 15 1 NA NA NA NA NA NA



### 3 15 0 NA NA NA NA NA NA



### 4 15 0 NA NA NA NA NA NA



### 0.312 0 16 13 NA NA NA NA NA NA



### 1 16 0 NA NA NA NA NA NA



### 2 16 1 NA NA NA NA NA NA



### 3 16 1 NA NA NA NA NA NA



### 4 16 0 NA NA NA NA NA NA



### 1.03 0 16 7 NA NA NA NA NA NA



### 1 16 4 NA NA NA NA NA NA



### 2 16 2 NA NA NA NA NA NA



### 3 16 3 NA NA NA NA NA NA



### 4 16 0 NA NA NA NA NA NA

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



### . LOQ=0.0124 mg a.i./L.

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

EPA MRID Number 48701401

Page 40 of 51 Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( Pimephales
promelas)

.

Treatment
(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Interstitium/multifo

cal inflammation Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

Control
(<LOQ) 0 NA NA NA NA 15 10 15 9

1 NA NA NA NA 15 5 15 3

2 NA NA NA NA 15 0 15 0

3 NA NA NA NA 15 0 15 3

4 NA NA NA NA 15 0 15 0



### 0.0947 0 NA NA NA NA 15 12 15 7

1 NA NA NA NA 15 2 15 2

2 NA NA NA NA 15 1 15 1

3 NA NA NA NA 15 0 15 5

4 NA NA NA NA 15 0 15 0



### 0.312 0 NA NA NA NA 16 16 16 11

1 NA NA NA NA 16 0 16 0

2 NA NA NA NA 16 0 16 0

3 NA NA NA NA 16 0 16 5

4 NA NA NA NA 16 0 16 0



### 1.03 0 NA NA NA NA 16 15 16 9

1 NA NA NA NA 16 1 16 2

2 NA NA NA NA 16 0 16 3

3 NA NA NA NA 16 0 16 2

4 NA NA NA NA 16 0 16 0

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.
LOQ=0.0124 mg a.i./L.

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

EPA MRID Number 48701401

Page 41 of 51 Mean male VTG was 2870, 1870, 3180 and 6200 ng/mL and mean female VTG was 3583000, 4940000, 4414000 and 3309000 ng/mL in the mean-measured 0 (control), 0.0947, 0.312 and 1.03 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

Control (<LOQ) 4 2870 3290 4 3,583,000 978,000

0.0947 4 1870 1650 4 4,940,000 1,078,000

0.312 4 3180 3850 4 4,414,000 527,000

1.03 4 6200 10,200 4 3,309,000 898,000

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.
LOQ=0.0124 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 42 of 51Mean plasma testosterone and plasma 17ȕ-estradiol in male and females was not measured (Table 20).Table 20: Plasma Sex Steroids in Fathead Minnow ( Pimephales promelas). Not measured.Treatment(mg a.i./L)[mean-measured]Plasma Testosterone (T)Plasma 17ȕ-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDNMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDControl (<LOQ)NANA NA NANANANANA NA NANANA0.0947NANA NA NANANANANA NA NANANA0.312NANA NA NANANANANA NA NANANA1.03NANA NA NANANANANA NA NANANAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.0124 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 43 of 51There were no notable observations with regards to behavior orchanges in appearance, specifically color/banding, ovipositor appearance, orsize of dorsal nape pad in the control or treated groups (Table 21). In the 1.03 mg a.i./L treatment group, there was one malethatdisplayed irregular respiration (Day 17) andone female that showed a loss of equilibrium(Day 21). There were also two females in thecontrol and 0.312 mg a.i./L treatment level that showed vertical banding at test termination. One female and three males in the 1.03 mga.i./L treatment were observed to have flared operculum at test termination. All other fish in the negative controls and treatment groupsappeared normal, and no overt signs of toxicity were observed during the test period.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 44 of 51Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceTypenIncidenceControl (<LOQ)None8 0Vertical banding15 20.0947 None 70 None115 00.312 None 90 None116 01.03 Flaredoperculum 83Loss of equilibrium16 1Flared operculum16 11On Day 21, one female in the low treatment and 2 in the middle treatment were coded as “DC” in the study report, however, thedefinition of “DC”was not provided.LOQ=0.0124 mg a.i./L.

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

EPA MRID Number 48701401

Page 45 of 51 B. Study Author’s Analysis and Conclusions

The study author analyzed survival, wet weight, tubercle score, gonadal development, GSI, fertility,
fecundity, VTG, and incidence and severity of gonad abnormalities. Data were gender specific and
analyzed in comparison to the negative control.

Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant
effects were detected for p d 0.05 (SAS, version not reported). Survival data were analyzed using
Fisher’s Exact Test with Hochberg adjustment. Reproductive endpoints were analyzed by either
one-tailed or two-tailed Dunnett’s test to determine which treatment groups differed statistically from
the control. Prior to Dunnett’s, data were analyzed by Shapiro-Wilk’s test and Levene’s to test for normality and homogeneity of variances, respectively ( D = 0.01). If normality and homogeneitytests passed (p>0.01), a parametric analysis was performed using the transformed data. If nonnormality or unequal variance were indicated (p<0.01), a non-parametric analysis was performed.

These methods appear to be consistent with the methods recommended in the guideline. Length
was not reported or statistically analyzed.

There were significant differences in fertility and fecundity at the 1.03 mg a.i./L concentration compared to the negative control (p<0.05; Dunnett’s test). There were no other statistically significant differences in treated groups compared to negative control for any endpoint (p>0.05).
Histopathology showed there appeared to be treatment-related effects in males due to increased
prevalence and severity of increased spermatogonia at the 1.03 mg a.i./L treatment level; and in
females due to increased prevalence and severity of increased oocyte atresia at the 1.03 mg a.i./L
treatment level. There were no statistically significant differences between gonadal stages in treated
groups compared to the negative controls.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: The reviewer assessed survival (mortality) data based on visual observation.
Endpoints consistent with a decreasing monotonic concentration-response included fecundity, female

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

EPA MRID Number 48701401

Page 46 of 51 GSI, and male body weight; the median values for fertility exhibited a concentration-dependent
reduction and, while male tubercle scores were not linearly decreasing, the pattern of reduction was
suggestively concentration-dependent. These endpoints were analyzed using the JonckheereTerpstra test. Fertility and male VTG were the only endpoints which did not satisfy the parametric assumptions of normality using Shapiro-Wilks test and homogeneity of variances using Levene’s
test. Male VTG was analyzed using the nonparametric Mann-Whitney U test, while remaining endpoints satisfying parametric assumptions were analyzed using Dunnett’s test. Unless otherwise
indicated, effects were considered statistically si gnificant at p<0.05. These analyses were conducted
using CETIS version 1.8.7.7 and backend settings approved for use by EFED on 5/29/13. Treated
groups were compared to the negative control. Mean-measured concentrations were used to
discuss effects in this study.

Conclusions:

Adult male and female survival were unaffected by treatment with methomyl and was 100% for all

test levels.

There was a significant decrease in fecundity, female GSI, and fertility at the 0.312 and 1.03 mg a.i./L concentrations compared to the negative control (p<0.05; Jonckheere-Terpstra test). There
was also a significant reduction in male body weights at the 0.312 and 1.03 mg a.i./L concentrations compared to the negative control (p<0.05; Jonckheere-Terpstra test). There were
no other statistically significant differences in tr eated groups compared to negative control for any statistically analyzed endpoint (p>0.05). Histopathology showed treatment-related effects in males
due to increased prevalence and severity of increased spermatogonia at the 1.03 mg a.i./L
treatment level; and in females due to increased prevalence and severity of increased oocyte atresia
at the 1.03 mg a.i./L treatment level. There were no differences between gonadal stages in treated
groups compared to the negative controls. There were no notable observations with regards to
behavior or changes in appearance, specifically color/banding, ovipositor appearance, or size of
dorsal nape pad in the cont rol or treated groups. There were no treatment-related clinical signs of
toxicity.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 47 of 51Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with Methomyl.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTG3Plasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.p% Diff.pNegative Control(<LOQ)36 NA0 NANo0 NANA NA NA NA0.0947 380.56-4.57 0.98 No -34.8 0.89 NA NA NA NA0.312 320.284.95 0.97 No 10.9 0.89 NA NA NA NA1.03 300.091.71 1.0Yes116.030.89 NA NA NA NAStatistical Test Jonckheere-Terpstra Dunnett’sNA Mann-WhitneyNANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.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<0.05.3This percent difference is due to one fish in replicate A with a substantially greater VTG value than the control fish, this percent difference does not reflect anincrease in male VTG from the population of fish at this treatment compared to the control.LOQ=0.0124 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with MethomylEPA MRID Number 48701401Page 48 of 51Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with Methomyl.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercleScoreGSIGonadalStagingand Histo.Plasma VTGPlasma TPlasma E2%Diff.p%Diff.PMedianp%Diff.pEffect?(Yes/No)%Diff.p%Diff.p%Diff.pControl(<LOQ)0 NA 0 NA 0 NA 0 NA No 0 NA NA NA NA NA0.0947-12.6 0.15 2.17 0.32 0 NA 0.00 0.56 No 37.9 0.15 NA NA NA NA0.312-23.3 0.02 -1.63 0.0210 NA-15.4 0.011No 23.2 0.53 NA NA NA NA1.03-77.2 0.0002 -33.2 0.00020 NA-21.0 0.0065 Yes-7.64 0.95 NA NA NA NAStatistical TestJonckheereTerpstraJonckheere-TerpstraNAJonckheere-TerpstraNA Dunnett’s NA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.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.LOQ=0.0124 mg a.i./L.

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

EPA MRID Number 48701401

Page 49 of 51 Table 24: Growth Endpoints 1,2 

### in the Fish Short-Term Reproduction Assay (FSTRA) with Methomyl.

Treatment
(mg a.i./L)
[mean-measured] Body Weight Length

Males Females Males Females

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

Diff. p

Control (<LOQ) 0 NA 0 NA 

### NA NA NA NA

0.0947 -6.52 0.17 -1.74 0.98 

### NA NA NA NA

0.312 -9.76 0.046 -7.22 0.52 

### NA NA NA NA

1.03 -13.2 0.022 -6.17 0.63 

### NA NA NA NA

Statistical Test JonckheereTerpstra Dunnett’s NA NA

Abbreviations: Diff. Difference. NA Not applicable.

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



### . LOQ=0.0124 mg a.i./L.



### E. Study Deficiencies

1. The total organic carbon, unionized ammonia and residual chlorine in the test water
were not reported. The OCSPP 890.1350 performance criteria establish maximum

levels for these values, and it is unclear if the maximum recommendations were

exceeded.

2. Individual fish weights at study initiation were not reported; however the range was
reported as ± 20% for males. EPA recommends that the subsample of fish weighed

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

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

EPA MRID Number 48701401

Page 50 of 51 3. The body lengths of the fish were not measured at test termination as recommended in
the guideline.
4. The average fertilization success in the controls was 92%, whereas the recommended

rate is 95%.

The remaining validity and performance criteria for OCSPP 890.1350 were met.

F. Reviewer’s Comments

The reviewer’s and the study authors’ results differed. While both the study author’s and reviewer’s
analysis detected statistically significant reductions in fecundity and fertility, the reviewer relied on the
results of the monotonic trend tests because these endpoints exhibited a decreasing trend; the reviewer
additionally relied on monotonic trend test results for female GSI and male body weight, while the study
author used non-monotonic trend tests for these endpoints. The monotonic trend tests ultimately
revealed more sensitive findings than the non-mono tonic tests for these endpoints. The reviewer’s
conclusions based on the OCSPP 890.1350 flowchart are presented in the Executive Summary and

Conclusions sections of this DER.

On Day 12, replicate A of the 0.0947 mg a.i./L treatment was missing a male fish and an extra male
fish was found in an adjacent chamber, replicate D of the 0.312 mg a.i./L treatment, where it remained
until study termination. All test chambers were covered with a screen afterwards to prevent future

escapes.

Also on Day 12, a female in replicate D of the control and replicate D of the 0.0947 mg a.i./L
treatments were found dead due to injuries sustained during tile placement. These two mortalities were
excluded from the percent survival because they were not treatment related.

For male VTG, one result in the middle concentration was not reported, as the study report indicated
that the result was greater than the highest standard and was therefore excluded from further analyses.

In regards to the histopathology, the pathologist’s report indicated that minimal to mild granulomatous
inflammation in the ovary was observed in several contro l and treated fish (5 fish in the control; 3 fish in

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

EPA MRID Number 48701401

Page 51 of 51 the low concentration and one fish in the high concentration). In two instances, the inflammation was associated spatially with the presence of microsporidian fungi (minimal). However, the report indicates
that the presence of the inflammation or microsporidia did not appear to have any effects on the study

results.

III. REFERENCES

U.S. Environmental Protection Agency. 2009. Endocrine Disruptor Screening Program Test
Guidelines, OCSPP 890.1350, Fish Short-Term Reproduction Assay, EPA 740-C-09-007, 93 pp.
Mount DI, Brungs WA. 1967. A Simplified Dosing Apparatus for Fish Toxicological Studies. Water

Res 1: 21-29.

American Public Health Association. 1998. Methods 2320 and 2340 In: Standard Methods for the

Examination for Water and Waste-water. 20th ed . Washington, D.C. 2-27 to 2-29, 2-36 to 2-39.
U.S. Environmental Protection Agency. 1989. Pesticide Programs; Good Laboratory Practice Standards; Final Rule (40 CFR, Part 160). Federal Register, 54(158):34067-34074.ABC.Report Date: 17 Jul-13 12:50 (p 1 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jun-13 12:31 Endpoint: Fecundity CETIS Version: CETISv1.8.7 Analysis ID: 00-8527-2873 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control Group Dunnett Multiple Comparison Test

DF P-Type
1.13 2.68 16 0.5525 Non-Significant Effect Negative Control 0.0947 6 CDF
2.1 2.68 16 0.1373 Non-Significant Effect 0.312 6 CDF
6.96 2.68 16 <0.0001 Significant Effect 1.03* 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
0.312 1.03 29.7% 0.5669 C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 4005.688 1335.229 3 18.8 <0.0001 Significant Effect
Error 852.25 71.02084 12 4857.938 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
0.883 11.3 0.8294 Equal Variances Bartlett Equality of Variance Variances
0.928 0.841 0.2252 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL Fecundity Summary

53.8 50 62 51.5 0 4 Negative Control 2.78 10.3% 0.0% 44.9 62.6
47 34 56 49 0.0947 4 4.71 20.0% 12.6% 32 62
41.3 31 50 42 0.312 4 4.84 23.5% 23.3% 25.9 56.6
12.3 3 22 12 1.03 4 4.21 68.7% 77.2% -1.15 25.7CenteredUntransformed

Rankits -2

-4

-6

-8

-10

-12

-14 0 2 4 6 8 10

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0Fecundity

Group Reject Null

Reject Null

0 10 20 30 40 50 60 70

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 2 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jul-13 11:45 Endpoint: Fecundity CETIS Version: CETISv1.8.7 Analysis ID: 20-4554-5955 Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control Group Jonckheere-Terpstra Step-Down Test

DF P-Type
1.02 1.64 1 0.1536 Non-Significant Effect Negative Control 0.0947 -2 Asymp
2.06 1.64 2 0.0197 Significant Effect 0.312* -2 Asymp
3.56 1.64 2 0.0002 Significant Effect 1.03* -2 Asymp NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed
0.0947 0.312 0.1719 C > T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 4005.688 1335.229 3 18.8 <0.0001 Significant Effect
Error 852.25 71.02084 12 4857.938 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
0.883 11.3 0.8294 Equal Variances Bartlett Equality of Variance Variances
0.928 0.841 0.2252 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL Fecundity Summary

53.8 50 62 51.5 0 4 Negative Control 2.78 10.3% 0.0% 44.9 62.6
47 34 56 49 0.0947 4 4.71 20.0% 12.6% 32 62
41.3 31 50 42 0.312 4 4.84 23.5% 23.3% 25.9 56.6
12.3 3 22 12 1.03 4 4.21 68.7% 77.2% -1.15 25.7CenteredUntransformed

Rankits -2

-4

-6

-8

-10

-12

-14 0 2 4 6 8 10

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0Fecundity

Group 0 10 20 30 40 50 60 70

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 3 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jun-13 12:31 Endpoint: FemaleBodyWt CETIS Version: CETISv1.8.7 Analysis ID: 06-8901-7753 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control Group Dunnett Multiple Comparison Test

DF P-Type
0.284 2.68 0.307 0.9837 Non-Significant Effect Negative Control 0.0947 6 CDF
1.18 2.68 0.307 0.5236 Non-Significant Effect 0.312 6 CDF
1.01 2.68 0.307 0.6324 Non-Significant Effect 1.03 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
1.03 >1.03 16.4% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 0.05017923 0.01672641 3 0.64 0.6036 Non-Significant Effect
Error 0.3135445 0.02612871 12 0.3637237 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
2.93 11.3 0.4019 Equal Variances Bartlett Equality of Variance Variances
0.978 0.841 0.9496 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL FemaleBodyWt Summary

1.87 1.75 1.94 1.89 0 4 Negative Control 0.0396 4.25% 0.0% 1.74 1.99
1.83 1.68 1.94 1.86 0.0947 4 0.0607 6.62% 1.74% 1.64 2.03
1.73 1.5 1.9 1.77 0.312 4 0.0898 10.4% 7.22% 1.45 2.02
1.75 1.49 2.04 1.74 1.03 4 0.113 12.9% 6.17% 1.39 2.11CenteredUntransformed

Rankits -0.1

-0.2

-0.3 0.0 0.1 0.2 0.3

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleBodyWt

Group Reject Null

Reject Null

0.0 0.5 1.0 1.5 2.0 2.5

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 4 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jun-13 12:31 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 17-4347-5156 Analysis: Parametric-Multiple Comparison Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control Group Dunnett T3 Multiple Comparison Test

DF P-Type
0 4.43 2.56 >0.05 Non-Significant Effect Negative Control 0.0947 3 CDF
2.83 3.31 2.34 0.0869 Non-Significant Effect 0.312 5 CDF
1.76 3.77 5.86 0.3459 Non-Significant Effect 1.03 4 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
1.03 >1.03 45.0% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 23.37687 7.792291 3 3.03 0.0713 Non-Significant Effect
Error 30.9075 2.575625 12 54.28437 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
6.24 5.95 0.0085 Unequal Variances Mod Levene Equality of Variance Variances
7.16 5.95 0.0052 Unequal Variances Levene Equality of Variance Variances
0.912 0.841 0.1235 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL FemaleGSI Summary

13 12 14 13 0 4 Negative Control 0.577 8.88% 0.0% 11.2 14.8
13 13 13 13 0.0947 4 0 0.0% 0.0% 13 13
11 10 12 11 0.312 4 0.408 7.42% 15.4% 9.7 12.3
10.3 7.5 14 9.8 1.03 4 1.44 28.0% 21.0% 5.69 14.9CenteredUntransformed

Rankits -0.5

-1.0

-1.5

-2.0

-2.5

-3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleGSI

Group Reject Null

Reject Null

0 5 10 15 20

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 5 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jul-13 11:45 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 19-7827-7497 Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control Group Jonckheere-Terpstra Step-Down Test

DF P-Type
8 NA 0.5571 Non-Significant Effect Negative Control 0.0947 -2 Exact
2.28 1.64 3 0.0114 Significant Effect 0.312* -2 Asymp
2.48 1.64 4 0.0065 Significant Effect 1.03* -2 Asymp NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed
0.0947 0.312 0.1719 C > T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 23.37687 7.792291 3 3.03 0.0713 Non-Significant Effect
Error 30.9075 2.575625 12 54.28437 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
6.24 5.95 0.0085 Unequal Variances Mod Levene Equality of Variance Variances
7.16 5.95 0.0052 Unequal Variances Levene Equality of Variance Variances
0.912 0.841 0.1235 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL FemaleGSI Summary

13 12 14 13 0 4 Negative Control 0.577 8.88% 0.0% 11.2 14.8
13 13 13 13 0.0947 4 0 0.0% 0.0% 13 13
11 10 12 11 0.312 4 0.408 7.42% 15.4% 9.7 12.3
10.3 7.5 14 9.8 1.03 4 1.44 28.0% 21.0% 5.69 14.9CenteredUntransformed

Rankits -0.5

-1.0

-1.5

-2.0

-2.5

-3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleGSI

Group 0 2 4 6 8 10 12 14

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 6 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jun-13 12:31 Endpoint: FemaleMedianTubercleScore CETIS Version: CETISv1.8.7 Analysis ID: 16-2853-4104 Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control Group Mann-Whitney U Two-Sample Test

DF P-Type
8 NA 1 1.0000 Non-Significant Effect Negative Control 0.0947 6 Exact
8 NA 1 1.0000 Non-Significant Effect 0.312 6 Exact
8 NA 1 1.0000 Non-Significant Effect 1.03 6 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed
1.03 >1.03 NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 0 0 3 65500 <0.0001 Significant Effect
Error 0 0 12 0 15 Total

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL FemaleMedianTubercleScore Summary

0 0 0 0 0 4 Negative Control 0 0 0
0 0 0 0 0.0947 4 0 0 0
0 0 0 0 0.312 4 0 0 0
0 0 0 0 1.03 4 0 0 0CenteredUntransformed

Rankits 0.0E+00 2.5E-01 5.0E-01 7.5E-01 1.0E+00

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleMedianTubercleScore

Group 0.0E+00 2.5E-01 5.0E-01 7.5E-01 1.0E+00

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 7 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jun-13 12:31 Endpoint: FemaleVTG CETIS Version: CETISv1.8.7 Analysis ID: 11-3914-0061 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control Group Dunnett Multiple Comparison Test

DF P-Type
2.08 2.72 2E+06 0.1462 Non-Significant Effect Negative Control 0.0947 6 CDF
1.18 2.72 2E+06 0.5289 Non-Significant Effect 0.312 5 CDF
0.42 2.72 2E+06 0.9529 Non-Significant Effect 1.03 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
1.03 >1.03 49.5% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 6.631514E+12 2.210505E+12 3 2.61 0.1044 Non-Significant Effect
Error 9.329782E+12 8.481619E+11 11 1.59613E+13 14 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
0.934 11.3 0.8172 Equal Variances Bartlett Equality of Variance Variances
0.929 0.833 0.2630 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL FemaleVTG Summary

3.58E+6 2.71E+6 4.46E+6 3580000 0 4 Negative Control 4.89E+5 27.3% 0.0% 2.03E+6 5.14E+6 4.94E+6 3.61E+6 6.13E+6 5010000 0.0947 4 5.39E+5 21.8% -37.9% 3.22E+6 6.66E+6 4.41E+6 3.89E+6 4.94E+6 4410000 0.312 3 3.04E+5 11.9% -23.2% 3.10E+6 5.72E+6 3.31E+6 2.44E+6 4.31E+6 3240000 1.03 4 4.49E+5 27.1% 7.64% 1.88E+6 4.74E+6CenteredUntransformed

Rankits -2.0E+05

-4.0E+05

-6.0E+05

-8.0E+05

-1.0E+06

-1.2E+06

-1.4E+06 0.0E+00 2.0E+05 4.0E+05 6.0E+05 8.0E+05 1.0E+06 1.2E+06

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0FemaleVTG

Group Reject Null

Reject Null

0.0E+00 1.0E+06 2.0E+06 3.0E+06 4.0E+06 5.0E+06 6.0E+06 7.0E+06

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 8 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jun-13 12:30 Endpoint: Fertility CETIS Version: CETISv1.8.7 Analysis ID: 15-1782-6111 Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control Group Mann-Whitney U Two-Sample Test

DF P-Type
9.5 NA 3 0.7429 Non-Significant Effect Negative Control 0.0947 6 Exact
12 NA 0 0.2286 Non-Significant Effect 0.312 6 Exact
16 NA 0 0.0286 Significant Effect 1.03* 6 Exact NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
0.312 1.03 24.3% 0.5669 C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 2846 948.6667 3 5.66 0.0118 Significant Effect
Error 2010 167.5 12 4856 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
25.1 11.3 <0.0001 Unequal Variances Bartlett Equality of Variance Variances
0.8 0.841 0.0027 Non-normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL Fertility Summary

92 82 96 95 0 4 Negative Control 3.37 7.32% 0.0% 81.3 103
94 93 96 93.5 0.0947 4 0.707 1.5% -2.17% 91.7 96.3
90.5 90 92 90 0.312 4 0.5 1.1% 1.63% 88.9 92.1
61.5 28 81 68.5 1.03 4 12.5 40.5% 33.2% 21.8 101CenteredUntransformed

Rankits -5

-10

-15

-20

-25

-30

-35 0 5 10 15 20

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0Fertility

Group 0 20 40 60 80 100

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 9 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jul-13 11:45 Endpoint: Fertility CETIS Version: CETISv1.8.7 Analysis ID: 17-5007-2084 Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control Group Jonckheere-Terpstra Step-Down Test

DF P-Type
0.461 1.64 3 0.3224 Non-Significant Effect Negative Control 0.0947 -2 Asymp
2.03 1.64 4 0.0213 Significant Effect 0.312* -2 Asymp
3.54 1.64 4 0.0002 Significant Effect 1.03* -2 Asymp NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed
0.0947 0.312 0.1719 C > T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 2846 948.6667 3 5.66 0.0118 Significant Effect
Error 2010 167.5 12 4856 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
25.1 11.3 <0.0001 Unequal Variances Bartlett Equality of Variance Variances
0.8 0.841 0.0027 Non-normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL Fertility Summary

92 82 96 95 0 4 Negative Control 3.37 7.32% 0.0% 81.3 103
94 93 96 93.5 0.0947 4 0.707 1.5% -2.17% 91.7 96.3
90.5 90 92 90 0.312 4 0.5 1.1% 1.63% 88.9 92.1
61.5 28 81 68.5 1.03 4 12.5 40.5% 33.2% 21.8 101CenteredUntransformed

Rankits -5

-10

-15

-20

-25

-30

-35 0 5 10 15 20

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0Fertility

Group 0 20 40 60 80 100

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 10 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jun-13 12:30 Endpoint: MaleBodyWt CETIS Version: CETISv1.8.7 Analysis ID: 19-9910-1693 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control Group Dunnett Multiple Comparison Test

DF P-Type
1.1 2.68 0.697 0.5743 Non-Significant Effect Negative Control 0.0947 6 CDF
1.64 2.68 0.697 0.2811 Non-Significant Effect 0.312 6 CDF
2.23 2.68 0.697 0.1098 Non-Significant Effect 1.03 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
1.03 >1.03 15.9% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 0.7292412 0.2430804 3 1.8 0.2005 Non-Significant Effect
Error 1.619025 0.1349187 12 2.348266 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
3.08 11.3 0.3790 Equal Variances Bartlett Equality of Variance Variances
0.889 0.841 0.0539 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL MaleBodyWt Summary

4.37 3.59 4.77 4.57 0 4 Negative Control 0.266 12.2% 0.0% 3.53 5.22
4.09 3.61 4.33 4.21 0.0947 4 0.164 8.02% 6.52% 3.57 4.61
3.95 3.49 4.32 3.99 0.312 4 0.174 8.81% 9.76% 3.39 4.5
3.79 3.63 3.97 3.79 1.03 4 0.0835 4.4% 13.2% 3.53 4.06CenteredUntransformed

Rankits -0.1

-0.2

-0.3

-0.4

-0.5

-0.6

-0.7

-0.8 0.0 0.1 0.2 0.3 0.4

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleBodyWt

Group Reject Null

Reject Null

0 1 2 3 4 5 6

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 11 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jul-13 11:46 Endpoint: MaleBodyWt CETIS Version: CETISv1.8.7 Analysis ID: 07-6773-6472 Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control Group Jonckheere-Terpstra Step-Down Test

DF P-Type
12 NA 0.1714 Non-Significant Effect Negative Control 0.0947 -2 Exact
36 NA 0.0463 Significant Effect 0.312* -2 Exact
70 NA 0.0215 Significant Effect 1.03* -2 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed
0.0947 0.312 0.1719 C > T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 0.7292412 0.2430804 3 1.8 0.2005 Non-Significant Effect
Error 1.619025 0.1349187 12 2.348266 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
3.08 11.3 0.3790 Equal Variances Bartlett Equality of Variance Variances
0.889 0.841 0.0539 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL MaleBodyWt Summary

4.37 3.59 4.77 4.57 0 4 Negative Control 0.266 12.2% 0.0% 3.53 5.22
4.09 3.61 4.33 4.21 0.0947 4 0.164 8.02% 6.52% 3.57 4.61
3.95 3.49 4.32 3.99 0.312 4 0.174 8.81% 9.76% 3.39 4.5
3.79 3.63 3.97 3.79 1.03 4 0.0835 4.4% 13.2% 3.53 4.06CenteredUntransformed

Rankits -0.1

-0.2

-0.3

-0.4

-0.5

-0.6

-0.7

-0.8 0.0 0.1 0.2 0.3 0.4

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleBodyWt

Group 0 1 2 3 4 5

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 12 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jun-13 12:30 Endpoint: MaleGSI CETIS Version: CETISv1.8.7 Analysis ID: 13-6902-2141 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control Group Dunnett Multiple Comparison Test

DF P-Type
0.307 2.68 0.524 0.9798 Non-Significant Effect Negative Control 0.0947 6 CDF
0.333 2.68 0.524 0.9747 Non-Significant Effect 0.312 6 CDF
0.115 2.68 0.524 0.9989 Non-Significant Effect 1.03 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
1.03 >1.03 40.0% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 0.03256874 0.01085625 3 0.142 0.9328 Non-Significant Effect
Error 0.917125 0.07642709 12 0.9496938 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
4.48 11.3 0.2137 Equal Variances Bartlett Equality of Variance Variances
0.926 0.841 0.2132 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL MaleGSI Summary

1.31 1.03 1.95 1.13 0 4 Negative Control 0.214 32.6% 0.0% 0.631 1.99
1.25 1.01 1.53 1.24 0.0947 4 0.122 19.5% 4.57% 0.864 1.64
1.38 1.05 1.56 1.45 0.312 4 0.114 16.6% -4.95% 1.01 1.74
1.34 1.2 1.44 1.35 1.03 4 0.0524 7.84% -1.71% 1.17 1.5CenteredUntransformed

Rankits -0.1

-0.2

-0.3

-0.4 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleGSI

Group Reject Null

Reject Null

0.0 0.5 1.0 1.5 2.0

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 13 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jun-13 12:30 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7 Analysis ID: 14-4243-4245 Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) MSD vs Control Group Dunnett Multiple Comparison Test

DF P-Type
0.483 2.68 11.1 0.9301 Non-Significant Effect Negative Control 0.0947 6 CDF
0.785 2.68 11.1 0.7757 Non-Significant Effect 0.312 6 CDF
1.33 2.68 11.1 0.4356 Non-Significant Effect 1.03 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
1.03 >1.03 31.8% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 133.6875 44.5625 3 1.3 0.3199 Non-Significant Effect
Error 411.75 34.3125 12 545.4375 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
2.72 11.3 0.4362 Equal Variances Bartlett Equality of Variance Variances
0.909 0.841 0.1122 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL MaleMedianTubercleScore Summary

35 31 37 36 0 4 Negative Control 1.35 7.74% 0.0% 30.7 39.3
37 29 44 37.5 0.0947 4 4.06 22.0% -5.71% 24.1 49.9
31.8 24 37 33 0.312 4 2.75 17.3% 9.29% 23 40.5
29.5 24 35 29.5 1.03 4 2.9 19.7% 15.7% 20.3 38.7CenteredUntransformed

Rankits -2

-4

-6

-8 0 2 4 6 8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleMedianTubercleScore

Group Reject Null

Reject Null

0 10 20 30 40 50

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 14 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jul-13 11:46 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7 Analysis ID: 13-4172-9067 Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control Group Jonckheere-Terpstra Step-Down Test

DF P-Type
-0.147 1.64 2 0.5586 Non-Significant Effect Negative Control 0.0947 -2 Asymp
0.592 1.64 4 0.2770 Non-Significant Effect 0.312 -2 Asymp
1.36 1.64 5 0.0867 Non-Significant Effect 1.03 -2 Asymp NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed
1.03 >1.03 NA C > T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 133.6875 44.5625 3 1.3 0.3199 Non-Significant Effect
Error 411.75 34.3125 12 545.4375 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
2.72 11.3 0.4362 Equal Variances Bartlett Equality of Variance Variances
0.909 0.841 0.1122 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL MaleMedianTubercleScore Summary

35 31 37 36 0 4 Negative Control 1.35 7.74% 0.0% 30.7 39.3
37 29 44 37.5 0.0947 4 4.06 22.0% -5.71% 24.1 49.9
31.8 24 37 33 0.312 4 2.75 17.3% 9.29% 23 40.5
29.5 24 35 29.5 1.03 4 2.9 19.7% 15.7% 20.3 38.7CenteredUntransformed

Rankits -2

-4

-6

-8 0 2 4 6 8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleMedianTubercleScore

Group 0 5 10 15 20 25 30 35 40 45

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________Report Date: 17 Jul-13 12:50 (p 15 of 15)
Test Code: 090301 48701401 | 19-3528-4756 

### CETIS Analytical Report

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) ABC Labs

Analyzed: 17 Jun-13 12:30 Endpoint: MaleVTG CETIS Version: CETISv1.8.7 Analysis ID: 12-7263-9993 Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 00-0868-7414
Start Date: 06 May-11 Ending Date: Test Type: EDSP FSTRA Tier 1

Duration: NA Protocol: OCSPP 890.1350 Tier I FSTRA Diluent:
Brine: Species: Pimephales promelas
Source: Lab In-House Culture Analyst:

Age:

Test Stat Critical P-Value Decision( α :5%) Ties vs Control Group Mann-Whitney U Two-Sample Test

DF P-Type
9 NA 0 0.8857 Non-Significant Effect Negative Control 0.0947 6 Exact
9 NA 0 0.8857 Non-Significant Effect 0.312 6 Exact
9 NA 0 0.8857 Non-Significant Effect 1.03 6 Exact NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed
1.03 >1.03 347.0% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)
Between 41623550 13874520 3 0.419 0.7427 Non-Significant Effect
Error 397395000 33116250 12 439018600 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)
8.63 11.3 0.0347 Equal Variances Bartlett Equality of Variance Variances
0.821 0.841 0.0052 Non-normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err Group CV% %Effect 95% LCL 95% UCL MaleVTG Summary

2.87E+3 1.07E+3 7.80E+3 1310 0 4 Negative Control 1.64E+3 115.0% 0.0% -2.36E+3 8.10E+3 1.87E+3 1.00E+2 4.09E+3 1650 0.0947 4 8.26E+2 88.2% 34.8% -7.56E+2 4.50E+3 3.18E+3 1.30E+2 8.62E+3 1990 0.312 4 1.92E+3 121.0% -10.9% -2.94E+3 9.30E+3 6.20E+3 5.60E+2 2.15E+4 1370 1.03 4 5.10E+3 165.0% -116.0% -1.00E+4 2.24E+4CenteredUntransformed

Rankits -2000

-4000

-6000

0 2000 4000 6000 8000 10000 12000 14000 16000

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.0MaleVTG

Group 0 5000 10000 15000 20000 25000

0 N 0.0947 0.312 1.03 Graphics

CETISTM v1.8.7.7 000-516-187-1 QA:________ Analyst:________

# DATA EVALUATION RECORD

METHOMYL

Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-09-2012 (MRID 48617702)

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.Sc. Date: 01/12/2012

Secondary Reviewer Signature: David A. McEwen, B.S. Date: 01/16/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 01/19/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 01/19/2012

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).METHOMYL/090301



## Primary Reviewer: Lisa Austin, Ph.D. Registration Division Secondary Reviewer: Greg Akerman, Ph.D. Health Effects Division In vivo Hershberger Assay (2011) I Page 1of12 OCSPP 890.1400/ OECD 441



## Sig n at u re:

# ~~ Da t e:**cp Q.Jf** **~L---­** 

## Signat u re:

## ___.**...,.. __...._** **:_ =+-** **~--­** Date: 

# "'2(i/J£1t? Template version I0/2011 

## DATA EVALUATI O N REC O RD

STUDY TYPE: In Vivo 

## Hershberger Assay (Rat); OCSPP 890.1400; OECD 441



## PC CODE: 103801 

## DP B A R C OD E: D396261



## TXR#: 0056254 

## CAS#: 16752-77-5



## TEST MATERIAL (PURITY): Methomyl (99.4% purity)



## SYNONYMS: Methyl N-[[(methylamino )carbonyl]oxy ]ethanimidothioate; DPX-Xl 179



## CITATION: Shajdr

, S

. (2011). Methomyl (DPX-X 11 79) Technical: 10-Day Hershberger Bioassay for Detecting Androgenic Activity

. DuPont Haskell Global Centers for Health & 

## Environmental Sciences

, Newark

, DE. Laboratory Study ID : DuPont32085

, November 8

, 2011

. MRID 48617702

.



## SPONSOR: E.I. du Pont de Nemours and Company

, Wilmington

, DE



## TEST ORDER #: EDSP-090301-62



## EXECUTIVE SUMMARY: In a Hershberger assay (MRID 48617702) testing for androgenic acti v it y, methomyl (99.4 % a.i

., lot # JUL06LP276) in deionized water was administered daily

v ia oral g a v age to six 56-day old (approximate age)

, castrated male Sprague Dawley rats at dose l ev els o f 0 (vehicle)

, 0.25

, 0

. 5

, or 1.0 mg / kg / day

. An androgenic positive control group

c onsi s ted of 6 castrated rats exposed to 0.4 mg / kg / day testosterone propionate (TP) in corn oil wi th 1 % 

## ethanol by subcutaneous (s.c.) injection.

To s creen for potential anti-androgenic activity

, methomyl (99.4% a

. i

., lot # JUL06LP276) in d e ioni z ed water was administered daily via oral gavage to six 56-day old, castrated male Sprague Dawle y rats at dose le v els of 0 (vehicle)

, 0

. 25

, 0

. 5

, or 1.0 mg / kg / day in conjunction with a daily do s e of TP at 0.4 mg/kg / da y in corn oil with 1 % ethanol by s

. c. injection. The anti

- androgenic positi v e control group consisted of 6 castrated rats dosed with 0.4 mg / kg / day TP in corn oil with 1 % e thanol b y s.c. injection and flutamide (FT) in 0

. 1 % Tween ® 80 in 0

. 5% methylcellulose at 3

m g/kg/ da y by oral gavage

. TP alone was used as the anti

- androgenic negative control.

F or both components of the assay , the animals were dosed for 10 consecutive days and

ne c ropsied approximately 24 hours after the final dose administration to determine weights of li v er and the five androgen-dependent tissues

. Body weights

, body weight gain and food

c o n s umption were measured throughout the dosing period

.In vivo Hershberger Assay (201 1) / Page 2 of 12 METHOMYL/090301 OCSPP 890.1400/ OECD 441



## All animals survived until schedul ed termination. There were no c linical signs of toxicity and no treatment-related gross pathological findings.

In the androgen agonist assay, there were no e ffects on body weights, body weight gains, or food efficiency in the methomyl treated groups. Ov erall food consumption was increased (p<0.05) in the 0.5 mg/kg/day group (9%), but the effect wa s not dose dependent. Rats dosed with TP (positive control) had incr eases (p<0.05) in overall body we ight gains (23%) and food consumption (18%), but there was no effect on food efficiency. There were no significant increases in the weights of the five accessory sex organs in any of the methomyl-treated groups. Liver weights were comparable to the vehicle control in all dose groups. Rats in the positive control group responded appropriately with signif icant increases in all five accessory sex organ weights (p<0.05). The performance criteria indicat ed that the assay was performing as expected.

In the anti-androgen assay, there were no e ffects on body weights, body weight gains, food consumption, or food efficiency in the methom yl groups or positive control (TP + FT) group when compared to the TP control. There wa s a 31% decrease (p<0.05) in Cowper’s glands weight in the 0.5 mg/kg/day methomyl group; however, no significant decreases in organ weights were seen in the remaining four target accessory sex tissues. The liver weights in the methomyl-treated groups were comparable to the TP control. Rats in the positive (TP + FT) control group responded with significant decreases in all five accessory sex organ weights. The performance criteria indicated that the assay was performing as expected.

The dose levels tested in this study are considered to be adequate based on data from earlier studies showing toxicologically-a dverse clinical signs of cholinesterase inhibition at doses ≥ 2.0 mg/kg/day.

Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. Methomyl was negative for androgenicity and anti-androgenicity in the Hershberger assay

The assay satisfies the EDSP Tier 1 Test Order requireme nts for a Hershberger assay (OCSPP 890.1400).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.In vivo Hershberger Assay (201 1) / Page 3 of 12 METHOMYL/090301 OCSPP 890.1400/ OECD 441



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: DuPont Haskell Global Centers for Health & Environmental Sciences
Location: Newark, DE Study Director: S. Snajdr Other Personnel: Wang, D (Research Chemist); Singh, B (Senior Research Pathologist); Jepson, G. (Manager); and Malley, L (Senior Research Toxicologist) Study Period: May 2 to November 8, 2011



## 2. Test Substance: Methomyl Description: Solid Source: E.I. du Pont de Nemours and Company Lot #: JUL06LP276, expires May 18, 2016 Purity: Solubility: 99.4 % a.i. 58 g/L in water Stability: Stable in deionized water for up to 8 days CAS #: 16752-77-5 Structure:



## 3. Reference Androgen: Testosterone propionate (TP) Supplier: Steraloids (Newport, RI) Lot #: B0562 (no expiration date provided) Purity: 96.5% CAS # : 57-85-2



## 4. Reference Anti-androgen: Flutamide (FT) Supplier: Sigma-Aldrich (St. Louis, MO) Lot #: 099K1112 (no expiration date provided) Purity: >99% CAS # : 13311-84-7



## 5. Solvent/Vehicle Control (methomyl): Deionized water Supplier: Not applicable Lot/Batch #: Not applicable Rationale (if other than water): Not applicable Final concentration: Not applicable



## Solvent/Vehicle Control (TP): Corn oil with 1% ethanol

Supplier: Commercial supplier (not specified) Lot/Batch #: Not provided Rationale (if other than water): Not provided Final concentration: Not applicable



## Solvent/Vehicle Control (FT): 0.1% Tween ®



## 80 in 0.5% methylcellulose Supplier: Commercial supplier (not specified) Lot/Batch #: Not provided Rationale (if other than water): Not provided Final concentration: Not applicable N

S O N H O

CH 3 CH

3

CH 3In vivo Hershberger Assay (201 1) / Page 4 of 12 METHOMYL/090301 OCSPP 890.1400/ OECD 441



## 6. Test Animals: Species: Rat Strain: Sprague Dawley [Crl:CD(SD)] Age/weight at dose initiation: Post-natal day (PND) 56 (approximate); 248.4 – 292.7 g Source: Charles River Laboratories, Inc. (Kingston, NY) Housing: Rats were housed 2/cage, in solid bottom caging with alpha cellulose bedding (Shepherd’sTM ALPHA-dri® bedding) Diet: PMI® Nutrition International LLC Certified Rodent LabDiet® 5002, ad libitum ; phytoestrogen concentration not reported
Water: Filtered tap water, ad libitum
Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 18-26 oC 30-70% Not reported 12 hrs light/12 hrs dark Acclimation period: Rats were castrated by supplier at appr oximately 49 days of age, at least 2 days prior to shipment to study facility and at least 7 days prior to initiation of dosing. Castrated rats were quarantine d for at least 3 days at facility prior to initiation of dosing



## B. STUDY DESIGN

1. In-life dates: Start: May 3, 2011 End: May 26, 2011

2. Study Design: To screen for potential androgen ic activity, the test substance was administered daily via oral gavage to castrated male rats. Positive androgenic activity is defined as a significant increase in weight of two or more androgen-dependent organs compared to the vehicle control. Additiona lly, to screen for potential anti-androgenic activity, the test substance was administered daily via oral gavage to castrated male rats in conjunction with a daily dose of TP (0.4 mg/ kg/day, s.c.). Anti-androgenic activity is indicated by a statistically signi ficant decrease in two or more target organ weights of the treated groups (test substan ce + TP) compared to the TP- only control group. For both assays, the animals were dosed for 10 consecu tive days and necropsied approximately 24 hours after the final dose administrati on for organ weight measurements.

3. Study Schedule: Sexually mature male rats were castrated on PND 45 (approximate) by the supplier (Charles River Laboratories) and allowed at least 7 days for recovery and regression of accessory sex organ weights prior to initiation of dosing. The dose administration period was from PND 56 th rough PND 65 (approximate). Rats were euthanized approximately 24 hours after the last dose and necropsied for organ weight
measurements.

4. Animal Assignment: Animals were distributed by co mputerized randomization, stratified by body weight, to the test groups noted in Table 1. Statistical analysis indicated that there were no significant differences in gro up body weight means at study initiation. Furthermore, the body weight of each animal was within ±20% of the overall mean.In vivo Hershberger Assay (201 1) / Page 5 of 12 METHOMYL/090301 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 0.25 6 Mid 0.5 6 High 1.0 6 Testosterone propionate (TP) (s.c.), positive control 0.4 6 Anti-Androgen Assay Vehicle control (+TP b 

### ) 0 6 Low (+TP b 

### ) 0.25 6 Mid (+TP b 

### ) 0.5 6 High (+TP b 

### ) 1.0 6 Flutamide (oral gavage) + TP, b 

### positive control 3 6 a Data were obtained from pages 14 and 15 of the study report. b TP administered once daily by s.c. on all test days at 0.4 mg/kg/day.



## 5. Dose Selection Rationale: The dose levels were chosen with the objective of having midand high-dose levels in which adverse reduc tions (>10%) in cholinesterase had been established to occur, but not high enough to induc e clinical signs of toxicity. In acute oral neurotoxicity (HL-1998-01080; MRID number not provided) and relative sensitivity (MRID 46646401) studies, toxicologically adverse clinic al signs of cholinesterase inhibition, accompanied by marked decreases in brain a nd red blood cell cholines terase activity (46- 57%), were observed at dose levels of ≥ 2.0 mg/kg/day. At dose levels ranging 0.5-0.75 mg/kg/day, adverse reductions of brain and red blood cell ch olinesterase activity were observed in male (up to 25% and 43%, resp ectively) and female (up to 30% and 38%, respectively) rats (MRID 46646401, and revers ibility study HL-1997-00641; MRID number not provided). There were no clinical signs of toxicity or ad verse effects on red blood cell, plasma, or brain cholinesterase activity in male rats at a dose level of 0.25 mg/kg/day (MRID 46646401).

6. (a) Dose Preparation: Dose formulations for the test substance were prepared daily (method of preparation not reported). Dose form ulations for TP were prepared once prior to study initiation by dissolving TP in ethanol and d iluting in corn oil; the final concentration of ethanol in corn oil was 1%. TP solution wa s aliquoted and stored at room temperature for the duration of the study. Dose formulations for FT were prepared twice during the study, once prior to study initiation and once on study da y 7; each dose solution was aliquoted and stored refrigerated. Dose solutions for FT were prepared in 0.1% Tween® 80 in 0.5% methyl cellulose, in deionized water. Doses were adjusted daily based on individual body weight measurements. Stability of methomyl dosing solutions was tested at all dose levels following storage at room temperature for 3 a nd 8 days. Concentrati on verifications were conducted near the beginning of the study in the 0.25, 0.5, and 1.0 mg/kg dose formulations.In vivo Hershberger Assay (201 1) / Page 6 of 12 METHOMYL/090301 OCSPP 890.1400/ OECD 441


## ( b) Dose Analysis:

Results

Homogeneity: not applicable; methomyl is soluble in water at the concentrations used for dosing.

Stability (% of nominal): 102-103% after 3 days at room temperature; 101-102% after 8 days at room temperature

Concentration (% of nominal): 102.0-104.0%

The analytical data indicated that the varia tion between nominal and actual dosage to the animals was acceptable. The dose formulation was found to be stable for up to 8 days at room temperature. Although the tested storag e interval is shorter than the duration of dosing (10 days), the dose formulations were reportedly prepared daily. Because the test substance dose formulation was not a susp ension, homogeneity measurements are not required.

7. Dosage administration: Test formulations were administ ered to the animals daily via oral gavage (5 mL/kg bw) for 10 days. TP was gi ven via s.c. injection at 0.5 mL/kg bw, and flutamide was administered via oral gavage at 5 mL/kg bw. Dose vol umes were adjusted daily based on the concurrent body weight measurement.

8. Statistics: Body weights, body weight gains, food consumption, food efficiency, and organ weights were analyzed using Levene’s test for homogeneity and Shapiro-Wilk test for normality. Significance was noted at p < 0.05. For parameters for which the homogeneity and normality tests were not found to be signi ficant, analyses were conducted using oneway ANOVA followed by Dunnett’s test; otherwise, the data were transformed (using log, square-root, or rank order) to achieve normality and/or variance homogeneity. The statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortality, moribundity, and clin ical signs of toxicity were conducted twice da ily. Health examinations were performed prior to initiation of dosing and at termination.

2. Body Weight: Animals were weighed at randomization, study initiation, and daily throughout the dosing period.

3. Food Consumption and Efficiency: Food consumption was measured for each cage by subtracting the amount of food remaining in the cage from the amount supplied, accounting for spillage (only spillage >25 g was recorded). Values were reported as group mean daily food consumption (g/animal/day). Mean dail y food efficiency was determined using the food consumption and body weight data.In vivo Hershberger Assay (201 1) / Page 7 of 12 METHOMYL/090301 OCSPP 890.1400/ OECD 441


## 4. Serum Hormone Measurements (Optional): Serum hormone measurements were not conducted.

5. Dissection and Measurement of Tissue and Organ Weights: On Day 10, approximately 24 hours after the final administration of the test substance, all rats were euthanized by exsanguination under isoflurane anesthesia and subjected to a gross necropsy. The five mandatory androgen-dependent organs (ventral prostate, seminal vesi cles, LABC, Cowper’s gland, and glans penis) were excised, trimme d free of adhering tissue, and weighed fresh (unfixed) according to the sta ndard operating procedures deta iled in the U.S. EPA Guideline (OCSPP 890.1400). Additionall y, the liver was weighed.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived unt il scheduled termination.

2. Clinical signs of toxicity: No clinical signs of toxicity were observed in animals for any dose groups.

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data for the androgen agonist assay are pr esented in Table 2. Body weights in all methomyl-treated groups were comparable to c ontrols throughout the du ration of the assay. There was a statistically significant incr ease (p<0.05) in mean body weight gain ( ↑ 23%) over the duration of the study in rats dos ed with TP compared to controls.

TABLE 2. Selected Group Mean Body Weights and Cu mulative Body Weight Gains (g) in the Androgen Agonist Assay a



### Study Day Dose (mg/kg/day) Vehicle Control TP (0.4) 0.25 0.5 1.0
N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

### 1 6 267.2 9.2 6 270.2 9.6 6 271.9 10.1 6 273.0 11.0 6 269.7 10.7 5 6 297.4 11.1 6 307.1 9.7 6 301.5 10.2 6 304.7 13.5 6 299.8 14.2 8 6 319.1 13.1 6 334.8 13.4 6 323.1 13.3 6 326.1 16.7 6 320.6 16.4 11 6 347.5 14.4 6 369.1 16.5 6 348.7 13.8 6 351.7 20.2 6 346.9 19.3
B ody Weight Gain (Days 1-11) 6 80.3 9.6 6 98.9* ( ↑ 23) 7.0 6 76.8 5.9 6 78.7 10.8 6 77.3 11.0

a Data were obtained from Tables 1 and 3 on pages 29, 30 and 33 of the study report. Percent differences from controls are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05



## Selected body weight and body weight gain data for the anti-androgen assay are presented in Table 3. Body weights and overall body weight gain in all methomyl-treated groups, as well as the positive control group, were comparable to controls throughout the duration of the
assay.In vivo Hershberger Assay (201 1) / Page 8 of 12 METHOMYL/090301 OCSPP 890.1400/ OECD 441



### TABLE 3. Selected Group Mean Body Weights and Cumu lative Body Weight Gains (g) in the Anti-Androgen Assay a



### Study Day Dose (mg/kg/day) Vehicle Control + TP (0.4) FT (3) + TP (0.4) Positive Control 0.25 + TP (0.4) 0.5 + TP (0.4) 1.0 + TP (0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

### 1 6 267.6 11.4 6 266.4 8.8 6 267.7 11.7 6 265.4 10.9 6 268.8 8.2 5 6 303.4 10.3 6 302.1 11.1 6 306.2 14.3 6 306.3 14.1 6 304.7 11.9 8 6 331.9 12.3 6 326.2 15.3 6 334.2 16.6 6 335.0 17.6 6 330.2 12.6 11 6 361.9 13.8 6 351.8 16.3 6 364.7 19.0 6 368.0 18.7 6 358.9 15.2
B ody Weight Gain (Days 1-11) 6 94.3 10.5 6 85.4 8.9 6 96.9 10.5 6 102.6 10.1 6 90.1 9.5

a Data were obtained from Tables 2 and 4 on pages 31, 32 and 34 of the study report. N Number of animals in the group SD Standard Deviation



## C. FOOD CONSUMPTION AND EFFICIENCY: Overall food consumption and food efficiency data for the androgen agonist assa y and anti-androgen assay are presented in Tables 4 and 5, respectively. For the androge n agonist assay, overall food consumption was increased (p<0.05) by 9% in the 0.5 mg/kg/day group compared to the control. Because there was no effect in the high-dose group, the increase in food consumption was not related to test substance treatment. Overall food consumption was increased (p<0.05) by 18% in the positive control group compar ed to the vehicle control; however, mean food efficiency was comparable to the vehicle control. Averag e daily food efficiency was decreased in rats from the 0.5 and 1 mg/kg/day groups ( ↓ 10%) compared to the control; however, the decreases were not statistically significant.

In the anti-androgen assay, overall food consum ption in all methomyl-treated groups, as well as the positive control, was comparable to the negative control. Average daily food efficiency was decreased ( ↓ 12%) in the positive control comp ared to the negative control (not statistically significant).

TABLE 4. Food Consumption and Effici ency in the Androgen Agonist Assay a

Study Days Dose (mg/kg/day) Vehicle Control TP (0.4) 0.25 0.5 1.0 N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD Food Consumption (g/animal/day) Days 1-11 6 23.9 0.7 6 28.0* ( ↑ 18) 0.4 6 24.1 0.7 6 26.1* ( ↑ 9) 2.2 6 25.5 1.3

Food Efficiency (g body weight gain/g food consumption per day) Days 1-11 6 0.337 0.038 6 0. 353 0.027 6 0.319 0.032 6 0.302 0.034 6 0.302 0.034

a Data were obtained from Tables 5 and 7 on pages 35 and 37 of the study report. Percent differences from controls are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05In vivo Hershberger Assay (201 1) / Page 9 of 12 METHOMYL/090301 OCSPP 890.1400/ OECD 441



### TABLE 5. Food Consumption and Effi ciency in the An ti-Androgen Assay a

Study Days Dose (mg/kg/day) Vehicle Control + TP (0.4) FT (3) + TP (0.4) Positive Control 0.25 + TP (0.4) 0.5 + TP (0.4) 1.0 + TP (0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD Food Consumption (g/animal/day) Days 1-11 6 25.6 1.0 6 26.3 1.0 6 26.7 2.3 6 26.5 0.5 6 25.5 0.9 Food Efficiency (g body weight gain/g food consumption per day) Days 1-11 6 0.368 0.041 6 0.325 0.034 6 0.364 0.042 6 0.388 0.038 6 0.324 0.035 a Data were obtained from Tables 6 and 8 on pages 36 and 38 of the study report. N Number of animals in the group SD Standard Deviation



## D. SERUM HORMONE CONCE NTRATIONS (OPTIONAL): Serum hormone concentrations were not determined.

E. ORGAN WEIGHTS: Accessory sex organ weights a nd liver weights for the androgen agonist assay are presented in Table 6. No significant increases in the accessory sex organ weights were observed in the methomyl-treat ed groups. Liver weights in the treatment groups were comparable to the vehicle contro l. One rat from the 0.25 mg/kg/day group had a discolored liver; however, as no other rats in any dose group had similar findings, it was concluded that this finding was unrelated to treatment. Rats in the positive control (TP) group had accessory sex organ weight increas es (p<0.05) as follows: 1140% in seminal vesicles; 1340% in ventral prostate; 212% in LABC; 594% in Cowper’s glands; and 75% in glans penis. Liver weights in the positive control group were comparable to the vehicle control. The coefficients of variance (%CVs) for the accessory sex organs in the control and 1.0 mg/kg/day group were compared to the perf ormance criteria in th e Guideline; all %CV values were less than the maximum recommended values for each organ.In vivo Hershberger Assay (201 1) / Page 10 of 12 METHOMYL/090301 OCSPP 890.1400/ OECD 441



### TABLE 6. Accessory Sex Organ Weights (mg) and Liver Weights (g) from Androgen Agonist Assay in Sprague Dawley Rats a



### Organ Dose (mg/kg/day) Vehicle control Methomyl 0.25 Methomyl 0.5 Methomyl 1.0 TP (0.4)
N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Seminal vesicles 6 73.8 15.9 22 6 72.8 20.8 29 6 78. 8 25.1 32 6 74.2 25.9 35 6 914.1* ( ↑ 1140) 116.8 13

Ventral prostate 6 12.8 3.3 26 6 16.5 2.4 15 6 10. 3 3.3 32 6 13.0 5.2 40 6 184.2* (1340) 38.9 21

LABC 6 207.8 31.5 15 6 196.4 21.2 11 6 187.0 28.3 15 6 191.1 27.0 14 6 647.8* ( ↑ 212) 66.2 10

Cowper’s glands 6 6.3 1.2 19 6 6.6 1.9 29 6 6. 2 1.8 29 6 5.6 1.2 21 6 43.7** ( ↑ 594) 14.5 33

Glans penis 6 53.4 7.8 15 6 55.6 4.8 9 6 48. 2 2.7 9 6 50.3 4.5 9 6 93.5* ( ↑ 75) 7.2 8

Liver b 6 13.85 0.97 7 6 14.22 1.21 9 6 14.83 1.70 11 6 13.85 0.97 7 6 15.04 1.18 8 a Data were obtained from Tables 11 and 16 on pages 41-43 and 50 of the study report. Percent differences from controls were calculated by the reviewer and included in parentheses. B %CV for liver calculated by study reviewer. 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<0.001



## Accessory sex organ weights and liver wei ghts for the anti-androge n agonist assay are presented in Table 7. At 0.5 mg/kg/day, there was a 31% decrease (p<0.05) in Cowper’s glands weight. The other four target sex acce ssory organ weights in the methomyl-treated groups were comparable to vehicle controls. Rats in the positive control (FT + TP) group had accessory sex organ weight decreases (p<0.0 5) as follows: 69% in seminal vesicles; 73% in ventral prostate; 49% in LABC; 66% in Cowper’s glands; and 26% in glans penis. The Cowper’s glands in rats from the positive control group showed mild atrophy in five rats and moderate atrophy in one rat. Liver weights in the positive control group and methomyl treatment groups were comparable to the vehicle control. The %CVs for the accessory sex organs in the control and high-dos e groups were compared to the performance criteria in the Guideline; all %CV values were less than the maximum recommended values for each organ.In vivo Hershberger Assay (201 1) / Page 11 of 12 METHOMYL/090301 OCSPP 890.1400/ OECD 441



### TABLE 7. Accessory Sex Organ Weights (mg) and Liver We ights (g) from Anti-Androgen Agonist Assay in Sprague Dawley Rats a



### Organ Dose (mg/kg/day) Vehicle Control + TP (0.4) Methomyl + TP (0.25 + 0.4) Methomyl 0.5 + TP (0.5 + 0.4) Methomyl + TP (1.0 + 0.4) FT (3) + TP (0.4) Positive Control
N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Seminal vesicles 6 936.5 185.9 20 6 804.0 161.5 20 6 726. 4 176.1 24 6 854.0 94.8 11 6 293.0* ( ↓ 69) 53.3 18

Ventral prostate 6 204.9 22.8 11 6 181.1 55.0 30 6 174. 4 24.7 14 6 181.9 27.3 15 3 55.6* ( ↓ 73) 15.1 27

LABC 6 659.3 92.3 14 6 646.8 118.9 18 6 581. 8 106.0 18 6 640.7 77.4 12 6 335.9* ( ↓ 49) 23.1 7

Cowper’ s glands 6 46.1 7.7 17 6 35.2 8.9 25 6 31.7* ( ↓ 31) 6.7 21 6 44.3 10.4 23 6 15.9** ( ↓ 66) 3.5 22

Glans penis 6 100.0 9.5 10 6 85.8 9.6 11 6 94. 5 5.5 6 6 90.6 14.3 16 6 74.2** ( ↓ 26) 11.3 15

Liver b 6 14.91 1.56 10 6 14.23 0.94 7 6 15.33 1.51 10 6 14.38 1.66 12 6 15.13 1.96 13 a Data were obtained from Tables 12 and 17 on pages 44-46 and 51 of the study report. Percent differences from controls were calculated by the reviewer s and included in parentheses. b %CV for liver calculated by study reviewer. 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<0.001



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Under the conditions of this study, methomyl did not affect androgen sensitive organ weights consistent with potential androgenic or antiandrogenic activity in the Hershberger bioa ssay when administered to castrated male rats at up to 1.0 mg/kg/day for 10 days. As expected, the positive control chemicals showed effects consistent with their mode-of-action in the Hershberger bioassay. Rats administered TP showed increased organ weights for all andr ogen-dependent tissues, consistent with an androgen receptor agonist. Rats administered FT showed effects consistent with an androgen receptor antagonist. When administer ed to rats receiving a TP as a reference agonist, FT administration decr eased organ weights for all a ndrogen-dependent tissues. All performance criteria were met for a valid study.

B. AGENCY COMMENTS: All animals survived until scheduled termination. There were no clinical signs of toxicity and no trea tment-related gross pathological findings.

In the androgen agonist assay, there were no treatment-related e ffects on body weights, body weight gains, or food efficien cy in the methomyl treated groups. Overall food consumption was increased (p<0.05) by 9% in the 0.5 mg/kg/day group compared to the control; however, there was no correspond ing effect in the 1 mg/kg/day group. Compared to the vehicle control, rats in the positive control group (TP) had in creases (p<0.05) in mean body weight gains ( ↑ 23%) and overall food consumption ( ↑ 18%), but there was no effect on food efficiency. There were no signi ficant increases in the five target accessory sex organ weights in the methomyl-treated groups. Liver weights in the treatment groups were comparable to vehicle controls. Rats in th e positive control group had accessory sex organIn vivo Hershberger Assay (201 1) / Page 12 of 12 METHOMYL/090301 OCSPP 890.1400/ OECD 441


## weight increases (p<0.05) as follows: 1140% in seminal vesicles; 1340% in ventral prostate; 212% in LABC; 594% in Cowper’s gla nds; and 75% in glans penis. Liver weights in the positive control group were comparable to the vehicle control. The performance criteria indicated that the assay was performing as expected.

In the anti-androgen assay, there were no e ffects on body weights, body weight gains, food consumption, or food efficiency in the methomyl + TP dose groups or in the positive control group (TP + FT) when compared to the TP control group. In the 0.5 mg/kg/day dosing group, there was a 31% decrease (p <0.05) in Cowper’s glands we ight. However, there were no significant organ weight increases in the re maining four target accessory sex organs at any dose. Rats in the positive control gr oup (TP + FT) had accessory sex organ weight decreases (p<0.05) as follows: 69% in semina l vesicles; 73% in ventral prostate; 49% in LABC; 66% in Cowper’s glands; and 26% in glans penis. The performance criteria indicated that the assay wa s performing as expected.

The %CV values for the androgenic and anti-and rogenic portions of the study were less than the maximum CV values indicated in the test guideline. Based on a lack of statistically significant changes in weight of two or more of the target accessory sex organs, methomyl was negative for androgenicity and anti-andr ogenicity under conditions of this assay.

C. STUDY DEFICIENCIES: None

# DATA EVALUATION RECORD

METHOMYL

Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-13-2012 (MRID 48701404)

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: Rebecca L. Bryan, B.S. Date: 8/21/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 8/22/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 8/29/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 8/29/2012

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).METHOMYL/ 090301



## Primary Reviewer: Karlyn Middleton 

## Health Effects Division Secondary Reviewer: Elizabeth Mendez, Ph.D. 

## Health Effects Division Female Pubertal Assay (2012)/ Page 1 of 15 OCSPP 890.1450/ OECD None



## Signature: ---+-----.,--=- 

## Date:**--=~~~~~ S-b,,L.....** **Signature·~~~~~eJ/,~~ ~~**  

## Date: -------''-Pl'--Y-L-"-L

## DATA EVALUATION RECORD

STUDY TYPE: Female Pubertal Assay; OCSPP 890

. 1450; OECD None.



## PC CODE: 090301 

## DP BARCODE: D403696



## TXR#: 0056254 

## CASNo: 16752-77-5



## TEST MATERIAL (PURITY): Methomyl Technical (99.4%)



## SYNONYMS: Methyl N-[[(methylamino)carbonyl]oxy]ethanimidothioate; DPX-Xl 179



## CITATION: Toot

, J.D. (2012) Pubertal Development and Thyroid Function in Intact Juvenile / Peripubertal Female Rats Following Oral Administration of Methomyl (DPX-Xl 179) Technical. WIL Research Laboratories, LLC, Ashland, OH

. Laboratory Project ID: WIL-189227, June 5, 2012

. MRID 48701404. Unpublished.



## SPONSOR: E.I. du Pont deNemours and Company, Wilmington

, DE 19898



## TEST ORDER#: EDSP-090301-62



## EXECUTIVE SUMMARY: 

## In a Female Pubertal Assay (MRID 48701404), 15 Crl:CD(SD) Sprague-Dawley rats / dose group were treated daily by oral gavage with methomyl technical ( 99.4 %; Lot # JUL06LP276) in deionized water at doses of 0, 0.5 or 1.0 mg / kg / day from postnatal day (PND) 22 to PND 42. Animals were examined for vaginal opening (VO) daily beginning on PND 22

, and the age and weight at day of attainment was recorded. Following sacrifice on PND 42

, blood was collected for clinical chemistry analyses, including total thyroxine (T4) and thyroid stimulating hormone (TSH)

, which were analyzed with electrochemiluminescent immunoassays or radioimmunoassays

, respectively. Kidney, liver, adrenal gland, thyroid, pituitary

, uterus
, and ovary weights were recorded, and microscopic examinations were conducted on the thyroid

, kidney

, ovary , and uterus.

All animals survived until scheduled termination and no treatment-related clinical findings were observed. No effects were observed on body weights or body weight gain in the study. No significant differences were observed in mean age at VO, body weight at VO

, or final body weights in the treated groups relative to control. There were no dose-related effects on mean age at first vaginal estrus, mean cycle length, percentage of females cycling

, percentage of females regularly cycling, or cycle status at necropsy

. Serum T4 and TSH were not affected by treatment.Female Pubertal Assay (2012)/ Page 2 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None


## There were no treatment-related microscopic findings in the thyroid, ovaries, uterus, or kidneys. No effects on organ weights were observed durin g the study. All clinical chemistry parameters in the treated groups were similar to control.

The dose levels tested in this assay are consider ed to be adequate based on the cholinesterase inhibition seen at doses ≥ 2.0 mg/kg/day in the toxicology database for this chemical.

The assay satisfies the EDSP Tier 1 Test Order require ments for a Female Pubertal Assay (OCSPP 890.1450).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Female Pubertal Assay (2012)/ Page 3 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC
Location: Ashland, OH Study Director: J.D. Toot Other Personnel: J.B. Nold, Pathologist L. Freshwater, Statistics Study Period: August 15, 2011 through June 5, 2012



## 2. Test Substance: Methomyl Description: White coarse powder, MW 162.21 Source: E.I. du Pont de Nemours and Company (Newark, DE) Lot #: JUL06LP276 (exp. May 18, 2016) Purity: 99.4% Stability: Not reported CAS #: 16752-77-5 Structure:

N

S O N H O

CH 3 CH

3

CH 3 

## 3. Vehicle: Deionized water
Source: On-site Lot #: Not applicable



## 4. Test Animals: Species: Rat Strain: Crl:CD(SD) Sprague-Dawley Age/Weight at Study Initiation: PND 22/47.4-58.2 g females only Source: Charles River Laboratorie s, Inc., Portage, MI Housing: Two/cage in in plastic cages with non-heat-treated Aspen bedding. Diet: Harlan Laboratories 2016CM Teklad Global Cert ified 16% protein rodent diet, ad libitum Phytoestrogen content average 11.0 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: 20.8-22.1 °C 43.4-56.0% ≥ 10 fresh air changes/hour 14 hrs light/ 10 hrs dark



## B. STUDY DESIGN

1. In-Life Dates: Start: 9/21/2011 End: 10/12/2011

2. Mating: Time-mated pregnant dams were received from the supplier on gestation day (GD) 7 and were allowed to deliver naturally and rear their pups to PND 21. Litter size was standardized to 10 pups (5 males/5 females) on PND 4, using a computer randomization procedure.

3. Animal Assignment: Following weaning on PND 21, female rats from the extreme ends of the weight range were eliminated. Animals were assigned to the test groups noted in TableFemale Pubertal Assay (2012)/ Page 4 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None


## 1 using a randomization procedur e designed to ensure equal st ratification of body weights. Littermates were not assigned to the same treatment group.

TABLE 1. Study Design a 

### Test group Dose (mg/kg/day) # of Females Control 0 15 Low 0.5 15 High 1.0 15 a Data were obtained from page 25 of the study report.



## 4. Dose Selection Rationale: The doses of methomyl in the present study, 0.5 and 1.0 mg/kg/day, were selected based on the re sults of previously-conducted studies with methomyl that have documented the doses required to achieve decreased acetylcholinesterase activity and associated clin ical toxicity. These previous studies include an acute neurotoxicity study (MRID 44487501, Du Pont Haskell Laboratories, 1998; HL1998-01080) 1 

## , a reversibility study with carbama te insecticides (DuPont Haskell Laboratories, 1997; HL-1997-00641) 2 

## , and a sensitivity study comparing cholinesterase activity in adult versus preweanling rats (MRID 46646401, DuPont Haskell Laboratories, 2005; DuPont-15433) 3



## . Doses of 0.5 and 0.75 mg/kg/day resulted in adverse reductions of brain and red blood cell cholinesterase activity in male rats (up to 25% and 43%, respectively) and female rats (up to 30% and 38%, respectively) , and toxicologically-adverse clinical signs of cholinesterase inhibition accompanied by marked decreases in brain and red blood cell cholinesterase activity (46%-57%) were observed at dose concentrations of ≥ 2.0 mg/kg/day. It was stated that the study objective for dose selection was to set doses that would achieve reductions in cholinesterase of >10%, but not high enough to elicit clinical signs of toxicity.



## 5. Dose Preparation and Analysis: Dose formulations were prepared weekly by mixing the appropriate amounts of test substance in deioni zed water. The test substance formulations were divided into aliquots for daily dispensati on and stored refrigerate d. Dose formulations were stirred continuously duri ng preparation and dosing, and we re visually inspected for homogeneity at first dosing. Concentration analyses were performed on all dose levels.

During a previous study 4 

## , homogeneity and stability anal yses were performed; however, results were not reported in this study. Stability and ho mogeneity analyses were not conducted in the current study. Samples for con centration analysis we re collected from the middle stratum of each dosing formulation (inc luding the control group) prepared during the in-life phase of the study.

1 DuPont Haskell Laboratories. Acute Oral Neurotoxicity Study in Ra ts (HL-1998-01080). Unpublished Report, 1998.

2 DuPont Haskell Laboratories. Reversib ility Study with Carbamat e Insecticides in Rats (HLR-1997-00641). Unpublished Report, 1997.

3 DuPont Haskell Laboratories. Methomyl (DPX-X1179) Technical: Comp arison of Cholinesterase Activity in Adult and Preweanling Rats (DuPont- 15433). Unpublished Report, 2005.

4 Freer, S.D. (2011) An Analytical Me thod Validation and Stability Study of Me thomyl (DPX-X1179) Te chnical in Aqueous Formulations (Study No. WIL-189240). WIL Research Laboratories, LLC, Ashland, OH.Female Pubertal Assay (2012)/ Page 5 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None


## Results of Dose Analysis

Homogeneity (% RSD): Not provided

Stability (% of initial): Provided in Study No. WIL-189240

Concentration (% of nominal): 96.6-102%.

The analytical data indicated that the varia tion 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/k g of body weight. Individual dosages were based on the most recently recorded body weight s to provide the correct mg/kg/day dose. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: Cycling status (cycling vs. non-cycling) and percent of an imals 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.

The data that were statistically analyzed incl uded organ weights (liv er, kidneys, pituitary and adrenals), organ weight to final body wei ght ratios, daily body wei ghts, weight gains, serum chemistries, hormones and vaginal openi ng 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 nonparametric test was used. Organ weights, ag e at vaginal opening a nd weight at vaginal opening data, when parametric, were analyzed using analysis of covariance (ANCOVA) followed by Dunnett’s test using PND 21 body weight s as the covariate. Other parameters were analyzed with ANOVA followed by Dunnett’s test. Linear trend tests were used in conjunction with both ANOVA and ANCOVA. For non-parametric data the Kruskal-Wallis test was used followed by Dunn’s test. Pathology data presented as a graded response were analyzed with a pairwise Mann-Whitney U te st. Dichotomous hist opathology data were analyzed using a Fisher’s exact test.

Mean values were presented with standard deviations and coeffi cient of variation. Analyses were conducted using SAS ® 

## (SAS Institute, Inc., Cary, NC) version 9.2 (or higher) software.Female Pubertal Assay (2012)/ Page 6 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None

Classification Procedure Cycling status Percent of animals cycling regularly Chi-square analysis was performed.

Estrous cycle length Day of first estrus One-way ANOVA was conducted, follow ed by Dunnett’s test when ANOVA demonstrated significant intergroup variance. Pathology (dichotomous response) One-sided Fisher’s exact test was performed.

Organ weights Organ weight to terminal body weight ratio Body weight Cumulative body weight gain Clinical chemistry Hormone levels Vaginal opening data Each endpoint was tested for hom ogeneity of variance using Levene ’s test. If that test was significant at p=0.01, then a log transform wa s 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 and Levene’s test conduct ed again. If the test was still significant, then a nonparametric test, as described be low, was used to analyze the data.

If the variances were homoge neous, then an an alysis of variance (ANOVA) was conducted, on the raw or transformed data, as appropriate. The statis tical model contained a factor for treatment group and a blocking fa ctor based on necropsy date. A 2-sided Dunnett’s test was conducted, regardless of the outcome of the ANOVA, looking for significant differences in the test substa nce groups when compared with the vehicle control. If the transformations were unsuc cessful in making th e variances homogeneous, then the nonparametric Kruskal-Wallis test was used, ignoring the blocking factor, followed by Dunn’s test, to compare each of the test substance gr oups with the vehicle control. Since these are pre-planned pair wise comparisons, Dunn’s test was conducted regardless of the outcome of the Kruskal-Wallis test. The tests were 2-sided, at the 0.05 significance level, looking for significant differences from the vehicle control.

In addition, organ weights and vaginal opening data were subj ect to the following analyses if they met the homogeneity of variance criteria: 1) Anal ysis of covariance (ANCOVA) with Dunnett’s test. The model was as de scribed above for ANOVA with the exception that PND 21 body weight was included as a co variate; 2) linear trend test using the ANOVA model; and 3) linear trend test using the ANCOVA model.

When an animal did not atta in vaginal opening, PND 43 or 44 (last study day + 1) and body weight at necropsy were used as the age and body weight at vaginal opening, respectively. Graded histopathology findings The pairwise Mann-Whitney U test was used to compare each treated group with the control. The tests were 1-side d at the 0.05 significa nce level testing for increased severity. The tests were 2-sided for graded responses presented on a Grade 1-5 scale at the 0.05 significance level testing for increased or decreased severity. Exact p-values were calculated for the Mann-Whitney U test.



## The statistical analyses were consid ered appropriate by the reviewers.

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 through the day of euthanasia. Animals were also observed approximately 45 minutes after dosing.

2. Body Weight: Animals were weighed on the day of randomization, daily prior to dosing and the day of euthanasia. Mean body we ight changes were calculated for each corresponding interval and also for PND 22-42.Female Pubertal Assay (2012)/ Page 7 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None


## 3. Vaginal Opening (VO): Beginning on PND 22, all animals were examined daily for onset of VO. The appearance of a small “pin hol e”, a vaginal thread, and complete VO were recorded. The age and body weight on the day at which the vaginal lumen was first observed to open was recorded for each animal . However, if any animal within any group showed incomplete opening (persistent pin holes and/or threads) for greater than 3 days, a separate second analysis was conducted using the age at which incomplete opening was first observed. Examination of the females was continued daily until VO was present or until euthanasia.

4. Estrous Cyclicity: Beginning on the day vaginal openi ng was observed, vaginal lavages were performed daily, and the slides were eval uated microscopically to determine the stage of estrus of each female until necropsy. The ag e of first vaginal estrus after vaginal opening was recorded. The average cycle length was calculated and reported for complete estrous cycles. At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.

5. Sacrifice and Pathology: On the day prior to sacrifi ce, all animals were moved to a holding room separate from where the necropsies were performed so as not to induce stressrelated responses in other animals which ma y affect hormone measurements. On PND 42, all females were euthanized randomly across all groups by decapitation without anesthesia, and the time of euthanasia was recorded. Animals were euthanized beginning at least 2 hours following dose administration that day, an d all sacrifices we re completed by 1300 hours. Immediately following decapitati on, trunk blood samples were taken for T 4 

## , TSH, and serum chemistry analyses. The samples were stored at ≤− 20° C. Necropsies were performed on all females found dead or that surv ived to the scheduled sa crifice. Any tissues with gross lesions were saved in 10% neut ral-buffered formalin for possible future histopathologic examination.

a. Hormone Analysis: Total thyroxine (T 4 

## ) was analyzed using an electrochemiluminescent assay. Thyroid stimulating hormone (TSH ) levels were analyzed using the [ 125 

## I]rTSH kit obtained from Izotop, Institute of Isotopes Ltd. (Budapest, Hungary); the assay system relies on magnetic separation for qua ntitative measurement of TSH using a GAMMA-C12 gamma counter. For both assays, multiple quality control samples were included during analysis.

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.Female Pubertal Assay (2012)/ Page 8 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None

ELECTROLYTES OTHER

Calcium Albumin
Chloride X Creatinine* Magnesium X Urea nitrogen* Phosphorus Total cholesterol
Potassium Globulins
Sodium Glucose

ENZYMES Total bilirubin Alkaline phosphatase (ALP) Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) Albumin globulin ratio (A/G Rati o, by calculation) Alanine aminotransferase (A LT/also SGPT) Bile acids Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in fe male rats based on guideline 890.1450.



## c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. 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 weighed “wet” and then again following removal of the fl uid 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 hours. The thyroid gland was dissected from the trachea, blotted, weighed, and placed in 70% et hanol until histopathological processing. The liver, adrenals, and pituitary were placed in 10% neutral-buffered formalin for potential future histopathologic examination.

After fixation, protocol-specified tissues were shipped to WIL Research (Hillsborough, NC) for processing. Tissue samples from the thyr oid, kidney, ovary, and ut erus were routinely processed into paraffin blocks, sectioned, stained with hemat oxylin and eosin, and examined microscopically.Female Pubertal Assay (2012)/ Page 9 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None


## Thyroid sections were subjectively evaluated fo r follicular epithelial cell height and colloid area using a five point grading scale (1 = s hortest/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). The ova ry sections were evaluated on a five point grading scale (1=markedly reduced number or absent; 3=normal; 5=markedly increased number) for primordial, primary, secondary, ter tiary, and atretic follic les and growth phase, mature, and regressive corpora lutea. Uter ine histology documented cases of uterine hyperor hypotrophy as characterized by changes in uterine horn diameter and myometrial, stromal, or endometrial gland development. The stage of th e estrous cycle at the time of necropsy was taken into account in the final hi stological assessment, as ovarian and uterine cellular changes are depende nt upon endocrine status.

II. RESULTS

A. MORTALITY: All animals survived until scheduled termination.

B. CLINICAL SIGNS OF TOXICITY: No clinical findings were observed.

C. GENERAL GROWTH AND VAGINAL OPENING: Body weights, body weight gains, age at attainment of VO, and weight at day of attainment are presented in Table 2. There were no treatment-related effects on final body we ights, overall body weight gain, or age or body weight at VO.

The mean CV value for age at VO (10.54%) in the controls exceeded the performance criteria maximum (6.52%).Female Pubertal Assay (2012)/ Page 10 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None



### TABLE 2. General Growth and Vaginal Opening (VO) a

Parameter Evaluated Vehicle Control Methomyl 0.5 mg/kg/day Methomyl 1.0 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 22; g) U 15 52.9 2.49 4.72 15 53.3 2. 40 4.51 15 52.9 2.18 4.12

Body weight at VO (g) U 15 123.1 15.22 12.36 15 125.0 13.55 10.84 14 133.0 14.69 11.05 A 15 123.5 15.22 12.36 15 124.9 13.55 10.84 14 132.7 14.69 11.05 Final body weight(g) U 15 156.5 7.99 5.11 15 156.4 8.41 5.38 15 155.7 8.57 5.50 Final body weight (% of control) U 15 15 99.9 15 99.5
Body weight gain (final – initial; g) U 15 103.6 7.61 7.34 15 103.1 7. 12 6.91 15 102.8 9.38 9.13

Age at VO (PND) U 15 35.5 3.74 10.54 15 35.5 2. 80 7.88 15 36.5 3.14 8.60 A 15 35.6 3.74 10.54 15 35.4 2. 80 7.88 15 36.5 3.14 8.60 Age at VO, Incomplete (PND) U 15 34.2 2.48 7.26 15 35.0 2. 17 6.20 15 35.1 2.10 5.98 A 15 34.3 2.48 7.26 15 34.9 2. 17 6.20 15 35.2 2.10 5.98 Proportion unopened (#/N) 1/15 0/15 1/15 a Data were obtained from page 45 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



## D. ORGAN WEIGHTS: Organ weights at necropsy are pr esented in Table 3. No effects on organ weights were observed during the study.

Guideline criteria were met for organ weight s in the controls, except the mean adrenal weight (33.6 mg) was slightly outside the acceptable range of 38.34-48.84 mg.Female Pubertal Assay (2012)/ Page 11 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None



### TABLE 3. Organ Weights at Necropsy a

Organ Vehicle Control Methomyl 0.5 mg/kg/day Methomyl 1.0 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 15 6.94 0.456 6.57 15 7.01 0. 616 8.79 15 7.05 0.585 8.29 A 15 6.92 0.456 6.57 15 7.02 0. 616 8.79 15 7.04 0.585 8.29 R 15 4.43 0.163 3.67 15 4.48 0. 208 4.65 15 4.53 0.270 5.97
Kidneys (g) U 15 1.24 0.089 7.17 15 1.25 0. 089 7.11 15 1.26 0.064 5.04 A 15 1.24 0.089 7.17 15 1.25 0. 089 7.11 15 1.26 0.064 5.04 R 15 0.80 0.045 5.66 15 0.80 0. 041 5.11 15 0.81 0.033 4.02
Pituitary (mg) U 15 9.8 1.95 19.94 15 10.2 2. 35 23.04 15 10.3 1.27 12.26 A 15 9.8 1.95 19.94 15 10.2 2. 35 23.04 15 10.4 1.27 12.26 R 15 6.3 1.26 20.16 15 6.5 1. 34 20.59 15 6.6 0.77 11.64

Adrenals (mg) U 15 33.6 4.70 13.97 15 32.8 4. 64 14.14 15 33.1 4.07 12.30 A 15 33.4 4.70 13.97 15 33.0 4. 64 14.14 15 33.0 4.07 12.30 R 15 21.5 2.67 12.43 15 21.0 3. 02 14.39 15 21.3 2.76 12.96 Ovaries (mg) U 15 73.7 10.58 14.35 15 70.2 9. 50 13.52 15 70.6 11.97 16.94 A 15 73.6 10.58 14.35 15 70.3 9. 50 13.52 15 70.6 11.97 16.94 Uterus, wet (mg) U 15 326.9 127.0 38.84 15 389.4 102. 7 26.38 15 294.0 102.3 34.80 A 15 325.5 127.0 38.84 15 390.5 102. 7 26.38 15 295.5 102.3 34.80 Uterus, blotted (mg) U 15 275.5 70.69 25.66 15 316.0 52. 97 16.76 15 257.1 60.87 23.67 A 15 274.4 70.69 25.66 15 317.0 52. 97 16.76 15 257.4 60.87 23.67 Thyroid, fixed (mg) U 15 9.87 2.42 24.51 15 8.21 1. 84 22.38 15 8.65 2.35 27.18 A 15 9.87 2.42 24.51 15 8.21 1. 84 22.38 15 8.67 2.35 27.18 a Data were obtained from page 46 of the study report. 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)



## E. ESTROUS CYCLICITY: Estrous cycle data are provi ded in Table 4. There were no dose-related effects on mean age at first vagina l estrus, mean cycle length, percentage of females cycling, percentage of females regularly cycling, or cycle stat us at necropsy. The cycling status was determined for only 6, 4, and 6 females in the control, 0.5, and 1.0 mg/kg/day groups, respectively, due to insufficient data.

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 15 36.9 4.6 100 75.0 10 0 4 1 b Methomyl 0.5 mg/kg/day 15 37.4 4.4 100 66.7 10 3 2 0

Methomyl 1.0 mg/kg/day 15 37.4 5.0 100 100.0 8 1 5 2 c
a Data were obtained from pages 46 and 47 of the study report. b One female was in diestrus, but insuffi cient data to determine cycling status. c Two females were in diestrus, but insufficient data to determine cycling status. N Number of animalsFemale Pubertal Assay (2012)/ Page 12 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None



## F. HORMONE LEVELS AND CLINICAL CHEMISTRY: Mean hormone levels and clinical chemistry values are presented in Table 5. Serum T 4 

## and TSH levels were not affected by treatment. The creatinine and urea n itrogen clinical chemistry parameters of the treated groups were similar to the control group. In addi tion, the study report provided normal ranges for the clinical chemistry meas ures, and these ranges are provided in the Appendix. It is noted that the urea nitrogen control value was outside the historical range of the performing laboratory.

The Guideline criterion was met for serum T 4 

## level in the controls.

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control Methomyl 0.5 mg/kg/day Methomyl 1.0 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)
Hormones
Serum T 4 , Total (μg/dL) 15 4.38 0.779 17.8 15 4.46 0.666 14.92 15 4.47 0.951 21.28

Serum TSH (ng/mL) 15 5.62 2.19 39.01 15 5.75 2. 23 38.77 15 7.29 4.55 62.36

Clinical Chemistry Creatinine (mg/dL) 15 0.1 0.04 34.50 15 0.1 0.05 35.21 15 0.1 0.04 31.05

Urea nitrogen (mg/dL) 15 12.6 2.59 20.50 15 13.0 1.58 12.12 15 14.1 1.80 12.72

a Data were obtained from pages 48 and 49 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



## G. GROSS PATHOLOGY: No treatment-related gross findings were observed.

H. HISTOPATHOLOGY: The incidences of histopathological findings of the thyroid gland are presented in Table 6. There were no trea tment-related findings in the thyroid gland.Female Pubertal Assay (2012)/ Page 13 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None



### 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 15 1 15 15 2 0 15 2 0 15 3 0 15 3 0 15 4 15 15 4 0 15 5 0 15 5 0 15

Methomyl 0.5 mg/kg/day 1 0 15 1 15 15 2 0 15 2 0 15 3 1 15 3 0 15 4 14 15 4 0 15 5 0 15 5 0 15

Methomyl 1.0 mg/kg/day 1 0 15 1 14 15
2 0 15 2 1 15
3 2 15 3 0 15
4 13 15 4 0 15
5 0 15 5 0 15 a Data were obtained from page 85 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. There were no treatment-related fi ndings in the ovaries, uterus, or kidneys.Female Pubertal Assay (2012)/ Page 14 of 15 METHOMYL/ 090301 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 Methomyl 0.5 mg/kg/day Methomyl 1.0 mg/kg/day
# Observed # Examined # Observed # Examined # Observed # Examined Ovaries Unremarkable 15 15 14 15 15 15 Edema, interstitial mild 0 15 1 15 0 15
Uterus Unremarkable 15 15 15 15 15 15 Kidney Unremarkable 1 15 0 15 1 15 Basophilic tubules, minimal 14 15 13 15 14 15

Cast, hyaline, minimal 0 15 1 15 0 15 Cyst 0 15 3 15 1 15 Dilatation, pelvis, minimal 0 15 2 15 0 15 Dilatation, pelvis, mild 0 15 1 15 0 15 Dilatation, tubular, minimal 2 15 0 15 0 15
Fibrosis, interstitial, minimal 0 15 1 15 0 15
Infiltrate, mononuclear, minimal 2 15 0 15 2 15
a Data were obtained from pages 82 and 86 of the study report.



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The investigators concluded methomyl did not alter pubertal development when administered or ally to peripubertal female rats at dosage levels of 0.5 or 1.0 mg/kg/day. Based on the lack of effects on the mean age and body weight at attainment of vaginal patency, estrous cyclicity, wet and blotted uterus weights, ovary weights, and histopathology of the female reproductive organs, or test substance-relatedmacroscopic and microscopic findings, methomyl did not alter the hypothalamic-pituitarygonadal axis function when administered orally to peripubertal female rats at dose levels of 0.5 and 1.0 mg/kg/day.

Based on the absence of effects on serum hormone levels, thyroid weights, and microscopicalterations in the thyroid, methomyl did not display the potential to disrupt the hypothalamicpituitary-thyroid axis.



## B. AGENCY COMMENTS: All animals survived until scheduled termination and no treatment-related clinical findings were observed. No effects were observed on body weights or body weight gain in the study.

There were no significant diffe rences in mean age at vaginal opening, body weight at vaginal opening, or final body weights in the trea ted groups relative to c ontrols. There were no dose-related effects on mean age at first vagi nal estrus, mean cycle length, percentage of females cycling, percentage of females regularly cycling, or cycle stat us at necropsy. Serum T 4 

## and TSH were not affected by treatment.Female Pubertal Assay (2012)/ Page 15 of 15 METHOMYL/ 090301 OCSPP 890.1450/ OECD None


## There were no treatment-related microscopic fi ndings in the thyroid, ovaries, uterus, or kidneys. No effects on organ weights were observed during the study. All clinical chemistry parameters in the treated groups were similar to control.

Although the high dose of 1.0 mg/kg/ day was selected from previous studies based on the presence of decreased brain and red blood cell c holinesterase activity at methomyl doses of ≥ 0.5 mg/kg/day, cholinesterase activity was not determined in the present study. In this study, there was no evidence to support an effect of methomyl 

## on pubertal development or for the disruption of thyroid function.

C. 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 mean CV value for age at vaginal ope ning (10.54%) in the cont rols exceeded the performance criteria maximum (6.52%).

 The control group mean for adrenal gla nd weight (33.6 mg) was less than the performance criteria minimum (38.34 mg).

# DATA EVALUATION RECORD

METHOMYL

Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-13-2012 (MRID 48701403)

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: 8/20/2012

Secondary Reviewer: Signature: Rebecca L. Bryan, B.S. Date: 8/22/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 8/29/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 8/29/2012

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).METHOMYL/ 090301



## Primary Reviewer: Karlyn Middleton Health Effects Division Seco ndary Reviewer: John Liccione

, Ph

. D. Health Effects Division Male Pubertal Assay (2012)/ Page I of 14
OCSPP 

# ')!!°"

I OECD Noo•



## DATA EVALUATION RECORD

STUDY TYPE: Male Pub e rtal Assay; OCSPP 890.1500



## PC CODE: 090301 

## DP BARCODE: D403696



## TXR#: 0056254 

## CASNo: 16752-77-5



## TEST MATERIAL (PURITY): Methom y l Technical (99.4%)



## SYNONYMS: Methyl N-[[(methylamino)carbonyl]oxy]ethanimidothioate; DPX-Xl 179



## CITATION: Toot, J .D. (2012) Pubertal Development and Thyroid Function in Intact Juvenile / Peripubertal Male Rats Following Oral Administration of Methomyl (DPX-Xl 179) Technical. WIL Research Laboratories

, LLC

, Ashland, OH

, Laboratory Project ID : WIL-189230

, June 5, 2012. MRID 48701403. Unpublished.



## SPONSOR: E.I. du Pont de Nemours and Company, Wilmington, DE



## TEST ORDER#: EDSP-090301-62



## EXECUTIVE SUMMARY: In a Male Pubertal Assay (MRID 48701403)

, 15 Crl:CD(SD) Sprague-Dawley rats / dose group were treated daily via oral gavage (5.0 mL/kg) with methomyl technical (99.4%; Lot# JUL06LP276) in deionized water at doses of 0

, 0.5 or 1.0 mg / kg/day from post-natal day (PND) 23 to PND 53

. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and their age and weight at day of attainment was recorded. Fo llowing sacrifice on PND 53, blood was collected for clinical chemistry analyses, including total thyroxine (T4), testosterone
, and thyroid stimulating hormone (TSH). Testosterone and T4

were analyzed with an electrochemiluminescent assay and TSH levels were analyzed by using radioimmunoassay. Liver, adrenals, pituitar y, and urogenital organ weights were recorded

, and microscopic examinations were performed on the thyroid

, kidney

, testis and epididymis.

Red material around the mouth or ear was observed infrequently during the daily examinations. No treatment-related effects were observed on mortality

, body weights, body weight gain, organ weights

, or gross pathology. Age and body weight at PPS in the treated groups were similar to controls. Thyroid hormone levels

, colloid area, and follicular cell height scores for the thyroid

were simila r in the treated groups to the control group. Serum testosterone levels were similar in the treated groups to the control group.Male Pubertal Assay (2012)/ Page 2 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None


## The dose levels tested in this study are considered to be adequate based on data from earlier studies showing toxicologically-a dverse clinical signs of cholinesterase inhibition at doses ≥ 2.0 mg/kg/day.

The assay satisfies the EDSP Tier 1 Test Or der requirements for a Male Pubertal Assay (OCSPP 890.1500).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Male Pubertal Assay (2012)/ Page 3 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: WIL Research Laboratories, LLC
Location: Ashland, OH 44805 Study Director: J.D. Toot Other Personnel: J. B. Nold, Pathologist L. Freshwater, Statistics Study Period: August 15, 2011- June 5, 2012



## 2. Test Substance: Methomyl Description: White, coarse powder, MW 162.21 Source: E.I. du Pont de Nemours and Company (Newark, DE) Lot #: JUL06LP276/ DPX-X1179-571 (exp. May 18, 2016) Purity: 99.4% Stability: Not reported CAS #: 16752-77-5 Structure:

N H CH

3 O

O N S

CH 3 CH 3



## 3. Vehicle: Deionized Water
Source: Prepared on site Lot #: Not applicable



## 4. Test Animals Species: Rat Strain: Crl:CD(SD) Sprague-Dawley Age/Weight at Study Initiation: PND 23/53.7-64.7 g males only Source: Charles River Laboratorie s, Inc., Portage, MI Housing: Two/cage in plastic cages with non-heat-treated Aspen bedding. Diet: Harlan Laboratories 2016CM Teklad Global Cert ified 16% protein rodent diet, ad libitum Phytoestrogen content average 11.0 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 : 20.9-22.0°C 41.1-56.0% ≥ 10 fresh air changes/hour 14 hrs light/10 hrs dark



## B. STUDY DESIGN

1. In-Life Dates: Start: September 22, 2011 End: October 23, 2011

2. Mating: Time-mated pregnant dams were received from the supplier at gestation day (GD) 7, and were allowed to deliver naturally and rear their pups to PND 21. Litter size was standardized to 10 pups (5 males/5 females) on PND 4, using a computer randomization procedure. 3. Animal Assignment: Following weaning on PND 21, male rats from the extreme ends of the weight range were eliminated. Animals were assigned to the test groups noted in TableMale Pubertal Assay (2012)/ Page 4 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None


## 1 using a randomization procedur e designed to ensure equal st ratification 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 0.5 15 High 1.0 15 a Data were obtained from page 25 of the study report.



## 4. Dose Selection Rationale: The doses of methomyl in the present study, 0.5 and 1.0 mg/kg/day, were selected based on the re sults of previously-conducted studies with methomyl that have documented the doses required to achieve decreased acetylcholinesterase activity and associated clin ical toxicity. These previous studies include an acute neurotoxicity study (Dupont Hask ell Laboratories, 1998; HL-1998-01080, MRID 44487501) 1 

## , a reversibility study with carbamate in secticides (Dupont Haskell Laboratories, 1997; HL-1997-00641) 2 

## , and a sensitivity study comparing cholinesterase ac tivity in adult versus preweanling rats (Dupont Haske ll Laboratories, 2005; DuPont-15433, MRID 46646401) 3



## . Doses of 0.5 and 0.75 mg/kg/day resulted in adverse reductions of brain and red blood cell cholinesterase activity in male rats (up to 25% and 43%, respectively) and female rats (up to 30% and 38%, respectively), and toxicologically-adverse clinical signs of cholinesterase inhibition accompanied by marked decreases in brain and red blood cell cholinesterase activity (46%-57%) were observed at dose concentrations of ≥ 2.0 mg/kg/day. It was stated that the study objective for dose selection was to set doses that would achieve reductions in cholinesterase of >10%, but not high enough to elicit clinical signs of toxicity.



## 5. Dose Preparation and Analysis: Dose formulations were pr epared approximately weekly by mixing the appropriate amounts of test substan ce in deionized water. The test substance formulations were divided into aliquots fo r daily dispensation, stor ed refrigerated, and stirred continuously. Concentration analys es were performed on all dose levels.

Stability of the test substance formulations was established during a previous study (WIL Research Laboratories Study No. WIL- 189240). Additionally, because the dose formulations were solutions, homogeneity and st ability analyses were not conducted in the present study.

Results of Dose Analysis

Homogeneity (% RSD): Not conducted Stability (% of initial): Not conducted

Concentration (% of nominal): 99.2-105%.

1 DuPont Haskell Laboratories. Acut e Oral Neurotoxicity Study in Rats (HL-1998-01080). Unpublished Report, 1998.

2 DuPont Haskell Laboratories. Reversib ility Study with Carbamat e Insecticides in Rats (HLR-1997-00641). Unpublished Report, 1997.

3 DuPont Haskell Laboratories. Methomyl (DPX-X1179) Technical: Comp arison of Cholinesterase Activity in Adult and Preweanling Rats (DuPont-15433) . Unpublished Report, 2005.Male Pubertal Assay (2012)/ Page 5 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None



## The variation between nominal and actual dosages to the animals was acceptable. Data to assess stability and homogeneity were not reported. These data (from Study No. WIL189240) 4 

## should be submitted for independent verification.

6. Dosage Administration: All doses were administered once daily by oral gavage, from PND 23 through PND 53, in a volume of 5 mL/k g of body weight. Individual dosages were based on the most recently recorded body weight s to provide the correct mg/kg/day dose. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: Data statistically anal yzed included the organ we ights, organ weight to body weight ratio (liver, kidneys, pituitary, and adrenal glands), daily body weights, cumulative body weight gain, serum chemistries, hormone concentrations, and weight and age at balanopreputial separation (PPS).

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 varian ces were not homogeneous 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.

Mean values were presented with standard deviations and coeffi cient of variation. Analyses were conducted using SAS ® 

## (SAS Institute, Inc., Cary, NC) version 9.2 (or higher) software.

4 Freer, S.D. (2011) An Analytical Me thod Validation and Stability Study of Me thomyl (DPX-X1179) Te chnical in Aqueous

Formulations (Study No. WIL-189240). WIL Research Laboratories, LLC, Ashland, OH.Male Pubertal Assay (2012)/ Page 6 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None

Classification Procedure Pathology (dichotomous response) One-sided Fisher’s exact test was performed.

Organ weights Organ weight to terminal body weight ratio Body weight Cumulative body weight gain Hormone assay Clinical chemistry Preputial separation data Each endpoint was tested for homogeneity of va riance using Levene’s test. If that test was significant at p=0.01, then a log transf orm was applied and Leve ne’s test conducted on the transformed data. If that test was still significant, then the square root transformation was applied to the raw data and Levene’s test co nducted again. If the test was still significant, then a nonparametric test , as described below, was used to analyze the data.

If the variances were homoge neous, then an an alysis of variance (ANOVA) was conducted, on the raw or transformed data, as appropriate. Th e statistic al model contained a factor for treatment group and a blocking factor based on necropsy date. A 2-sided Dunnett’s test was conducted, rega rdless of the outcome of the ANOVA, looking for significant differences in the test substance groups when compared with the vehicle control. If the transformations were unsuc cessful in making th e variances homogeneous, then the nonparametric Kruskal-Wallis test was used, ignoring the blocking factor, followed by Dunn’s test, to compare each of the test substance gr oups with the vehicle control. Since these are pre-planned pair wise comparisons, Dunn’s test was conducted regardless of the outcome of the Kruskal-Wallis test. The tests were 2-sided, at the 0.05 significance level, looking for significant differences from the vehicle control.

In addition, organ weights a nd preputial separation data we re subject to the following analyses if they met the homogeneity of variance criteria: 1) Analysis of covariance (ANCOVA) with Dunnett’s test. The mode l was as described above for ANOVA with the exception that PND 21 body weight was included as a covariate; 2) linear trend test using the ANOVA model; and 3) linear trend test using the ANCOVA model. Graded histopathology findings The pairwise Mann-Whitney U test was used to compare each treated group with the control. The tests were 1- sided at the 0.05 significanc e level testing for increased severity. The tests were 2-sided for graded responses presented on a Grade 1-5 scale at the 0.05 significance level testing for increased or decreased severity. Exact p-values were calculated for the Mann-Whitney U test.



## The statistical analyses were consid ered appropriate by the reviewers.

C. METHODS

1. Mortality and Clin ical Examinations: All males were observed twice daily, once in the morning and once in the afternoon, for moribund ity and mortality. Individual clinical observations were recorded daily (prior to test substance ad ministration during the treatment period) through the day of euthanasia. Each an imal was observed for signs of toxicity at approximately 45 minutes after dose administra tion. The absence or presence of findings was recorded for individual animals.

2. Body Weight: Animals were weighed on the day of randomization, daily prior to dosing and the day of euthanasia. Mean body we ight 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. In addition, if any animal with in a group displayed incomplete separation (persistent threads) for >3 days, a separate second analysis was conduc ted with the age at which incomplete separation was observed.Male Pubertal Assay (2012)/ Page 7 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None



## 4. Sacrifice and Pathology: On the day before euthanasia (PND 52), rats were moved to a holding room separate from where the necr opsies were to be performed. Dosing was performed in the holding room on the day of termination (PND 53) and the rats were moved to the necropsy room one at a time for te rminal procedures. Beginning 2 hours following dosing, animals were euthanized randomly across all groups by decapitation without anesthesia, and all sacrifi ces were completed by 1300 hour s. Immediately following decapitation, trunk blood samples were taken for T 4 

## , TSH, testosterone, and serum chemistry analyses. The hormone samples were stored at ≤− 20° C for subsequent analyses. Necropsies were performed on all males at the scheduled necropsy. Any tissues with gross lesions were saved in 10% neutral-buffe red formalin for potential histopathologic examination.

a. Hormone Analysis: Serum concentrations of T 4 

## and testosterone were analyzed with an electrochemiluminescent assay on the Cobas e411® . Serum TSH levels were analyzed with the [ 125 

## I]rTSH kit obtained from Izotop, Institute of Isotopes Ltd. (Buda pest, Hungary); the assay system relies on magnetic separation for quantitative measurement of TSH by using a GAMMA-C12 gamma counter. For both assays, multiple quality control samples were included during analyses.

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER

Calcium Albumin
Chloride X Creatinine* Magnesium X Urea nitrogen* Phosphorus Total cholesterol
Potassium Globulins
Sodium Glucose

ENZYMES Total bilirubin Alkaline phosphatase (ALP) Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) Albumin globulin ratio (A/G Rati o, by calculation) Alanine aminotransferase (A LT/also SGPT) Bile acids Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase 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.Male Pubertal Assay (2012)/ Page 8 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None

UROGENITAL OTHER XX Testes (left and right separately)* + XX Thyroid* + XX Epididymides (left and right separately)* + X Liver* X Seminal vesicle plus coagulating glan d (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 fixe d in Bouin’s solution for no more than 24 hours. The thyroid (with parathyroid) was collected with the tr achea and fixed in 10% neutral-buffered formalin for at least 24 hrs. Following fixation, the thyroid was dissected free of the trachea and weighed. Kidneys were fixed for at least 24 hours in 10% neutralbuffered formalin, and then placed in 70% etha nol until histological processing. All other tissues listed above were placed in 10% ne utral-buffered formalin for possible future histopathologic examination.

After fixation, protocol-specified tissues were shipped to WIL Research (formerly Biotechnics, LLC, Hillsborough, NC) for processing. Tissue samples from the thyroid, kidney, testis, and epididymis were routinel y processed into para ffin blocks, sectioned, stained with hematoxylin and eosin, and examin ed microscopically. PAS staining was also used for the right testis and epididymis.

Thyroid sections (minimum of two) were subjectively evaluated for follicular epithelial cell height and colloid area using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest), and any abnormalities/lesions noted.

II. RESULTS

A. Mortality: All animals survived until scheduled te rmination, with two exceptions. Male No. 24161-01 (vehicle control group) was found dead on PND 45 and male No. 24158-02 (1.0 mg/kg/day group) was found dead on PND 42. The death of the high-dose animal was determined to be caused by an intubation error due to finding a perforat ion in the esophagus and yellow contents in the thoracic cavity. Th e cause of death for the vehicle control group animal was not determined, but a dark red disc oloration of the lungs was noted at necropsy.

B. Clinical Signs of Toxicity: Daily clinical observations ar e summarized in Table 2. Red material around the mouth or ear was observed infrequently during the daily examinations.Male Pubertal Assay (2012)/ Page 9 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None



### TABLE 2. Incidence of Daily Clinical Observations a

Observation Dose Level (mg/kg/day)

Vehicle Control Methomyl 0.5 mg/kg/day Methomyl 1.0 mg/kg/day
# Observed b # Examined # Observed b # Examined # Observed b # Examined No significant clinical observations 457 15 465 15 444 15

Dried red material around right ear 0 0 0 0 9 3

Found dead 1 1 0 0 1 1 No significant clinical observations, post-dose 456 15 465 15 450 15

Dried red material around nose, post-dose 1 1 0 0 3 3
Animal expelled dose, post-dose 6 4 8 5 7 6
a Data were extracted from pages 50 and 51 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 we ight gains, age at attainment of PPS and weight at day of PPS are presented in Table 3. There were no treatment-related effects on fi nal body weights, overall body we ight gain, or age or body weight at PPS.

The mean % CV for weight at age of attain ment of PPS in the control group (9.55%) was above the performance criteria maximum of 7.57%, but the mean we ight was within the acceptable range.Male Pubertal Assay (2012)/ Page 10 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None



### TABLE 3. General Growth and Preputial Separation (PPS) a

Parameter Evaluated Vehicle Control Methomyl 0.5 mg/kg/day Methomyl 1.0 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 23; g) U 15 59.3 2.99 5.04 15 59.5 2. 89 4.86 15 58.7 2.64 4.50
Body weight at PPS (g) U 14 217.4 20.76 9.55 15 213.8 14. 17 6.63 14 210.5 12.92 6.14 A 14 217.7 20.76 9.55 15 213.5 14. 17 6.63 14 210.7 12.92 6.14 Final body weight (g) U 14 286.9 19.19 6.69 15 286.6 16. 73 5.84 14 277.7 18.54 6.67

Body weight gain (final – initial; g) U 14 227.5 18.16 7.98 15 227.1 15. 67 6.9 14 219.4 17.69 8.06
Final body weight percent of control U NA NA NA NA NA -0.10 NA NA NA -3.21 NA NA
Age at PPS (PND) U 14 44.1 2.27 5.15 15 43.8 1. 74 3.97 14 44.1 2.32 5.25 A 14 44.2 2.27 5.15 15 43.8 1. 74 3.97 14 44.1 2.32 5.25 Age at PPS, Incomplete (PND) U 14 42.0 1.92 4.58 15 41.8 1.82 4.36 14 43.6 2.14 4.91 A 14 42.1 1.92 4.58 15 41.8 1. 82 4.36 14 43.6 2.14 4.91 Proportion unseparated (#/N) 14 0 15 0 14 1
a Data were obtained from page 45 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 n ecropsy are presented in Table 4. There were no significant effects observed for organ weight s, with one exception. The LABC muscle weight in the 1.0 mg/kg/day gr oup was significantly decreased (p ≤ 0.0374) compared to control, but this decrease was c onsidered related to a decreased ( ↓ 3.21%) mean body weight, and was not biologically significan t or dose related. The LABC muscle weight reported in Table 4 is the Dunnett p -value (0.0678), which is not significantly decreased.

Guideline criteria were met for organ weights in the controls with one exception. The mean control absolute kidney weight (2.02 g) was below the perf ormance criteria minimum value (2.242 g), but the % CV value for the kidneys was within the acceptable range in the performance criteria.Male Pubertal Assay (2012)/ Page 11 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None



### TABLE 4. Organ Weights at Necropsy a

Organ Vehicle Control Methomyl 0.5 mg/kg/day Methomyl 1.0 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Liver (g) U 14 13.13 1.309 9.966 15 13.38 1.177 8.797 14 12.41 1.821 14.675 A 14 13.09 1.309 9.966 15 13.40 1.177 8.797 14 12.42 1.821 14.675 R 14 4.57 0.284 6.201 15 4.67 0. 303 6.485 14 4.45 0.451 10.12 Kidneys (g) U 14 2.02 0.195 9.638 15 2.03 0. 159 7.860 14 1.98 0.143 7.222 A 14 2.02 0.195 9.638 15 2.03 0. 159 7.860 14 1.98 0.143 7.222 R 14 0.70 0.041 5.834 15 0.71 0. 042 6.010 14 0.71 0.048 6.726 Pituitary (mg) U 14 12.0 1.20 10.00 15 11.0 1. 26 11.43 14 10.9 1.89 17.38 A 14 12.0 1.20 10.00 15 11.0 1. 26 11.43 14 10.9 1.89 17.38 R 14 4.2 0.47 11.21 15 3.8 0. 40 10.29 14 3.9 0.50 12.85 Adrenals (mg) U 14 47.4 6.98 14.72 15 49.7 8. 03 16.14 14 44.2 6.20 14.03 A 14 47.1 6.98 14.72 15 49.8 8. 03 16.14 14 44.2 6.20 14.03 R 14 16.6 2.79 16.76 15 17.3 2. 26 13.08 14 15.9 1.79 11.24 Seminal vesicle + coagulating gland, with fluid (mg) U 13 660.4 87.64 13.27 14 653.0 147. 21 22.54 14 581.0 130.28 22.42

A 13 653.3 87.64 13.27 14 652.3 147.21 22.54 14 582.3 130.28 22.42

Seminal vesicle + coagulating gland, without fluid (mg) U 14 388.4 37.57 9.67 15 386.5 61. 69 15.96 14 363.7 60.16 16.54

A 14 388.3 37.57 9.67 15 386.3 61. 69 15.96 14 364.1 60.16 16.54
Ventral prostate (mg) U 14 266.7 45.42 17.03 15 263.7 40. 48 15.35 14 251.3 57.21 22.77 A 14 265.0 45.42 17.03 15 264.3 40. 48 15.35 14 251.6 57.21 22.77 Dorsolateral prostate (mg) U 14 107.4 25.53 23.76 15 104.3 24. 41 23.41 14 95.3 18.37 19.27 A 14 107.1 25.53 23.76 15 104.2 24. 41 23.41 14 95.6 18.37 19.27 LABC muscle (mg) U 14 555.5 52.22 9.40 15 530.1 68. 59 12.94 14 505.6 67.56 13.36 A 14 557.6 52.22 9.40 15 528.8 68. 59 12.94 14 506.0 67.56 13.36 Epididymis, left (mg) U 14 211.2 24.02 11.37 15 210.3 33. 24 15.80 14 197.1 37.68 19.12 A 14 210.2 24.02 11.37 15 210.8 33. 24 15.80 14 197.1 37.68 19.12 Epididymis, right (mg) U 14 216.0 23.32 10.79 15 212.6 26. 86 12.63 14 198.9 27.47 13.81 A 14 215.4 23.32 10.79 15 212.9 26. 86 12.63 14 198.9 27.47 13.81 Testis, left (mg) U 14 1430.8 42.50 2.97 15 1454.9 87. 61 6.02 14 1421.5 203.71 14.33 A 14 1435.7 42.50 2.97 15 1451.9 87. 61 6.02 14 1422.6 203.71 14.33 Testis, right (mg) U 14 1448.0 59.83 4.13 15 1458.8 92. 09 6.31 14 1424.4 228.43 16.04 A 14 1456.4 59.83 4.13 15 1453.9 92. 09 6.31 14 1426.0 228.43 16.04 Thyroid, fixed (mg) U 14 16.19 2.598 16.052 15 14.81 2.173 14.680 14 15.38 1.771 11.516 A 14 16.14 2.598 16.052 15 14.83 2.173 14.680 14 15.37 1.771 11.516 a Data were obtained from page 46 of the study report. 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



## E. Clinical Chemistry and Hormone Levels: Serum T 4 

## , TSH, and testosterone were not affected by treatment, and the two serum chemis try parameters evaluated were not affected by treatment (Table 5).Male Pubertal Assay (2012)/ Page 12 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None



## Guideline criteria for serum T 4 

## , TSH, and testosterone levels were met in the controls, except for the % CV for the TSH level ( 61.742%) which was greater than the maximum acceptable value in the performance criteria ( 58.29%). The mean value for TSH was within the performance criteria acceptance range.

TABLE 5. Hormone Levels and Clinical Chemistry a

Parameter Evaluated Vehicle Control Methomyl 0.5 mg/kg/day Methomyl 1.0 mg/kg/day N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)
Hormones
Serum T 4 , Total (μg/dL) 14 5.49 0.560 10.210 15 5.44 0.553 10.181 14 5.12 0.826 16.126 Serum TSH (ng/mL) 14 8.91 5.499 61.742 15 5.98 2.817 47.104 14 7.69 5.141 66.828 Testosterone (ng/mL) 14 2.7 1.46 53.47 15 2.5 1.48 59.25 14 2.2 1.70 77.67 Clinical Chemistry Creatinine (mg/dL) 14 0.1 0.05 51.11 15 0.1 0.04 51.75 14 0.1 0.03 28.78 Serum urea nitrogen (mg/dL) 14 13.4 1.67 12. 48 15 13.7 1.18 8.58 14 13.7 1.37 9.99 a Data were obtained from pages 47 and 48 of th e study report. N Number of animals examined SD Standard Deviation CV Coefficient of Variation



## F. Macro- and Histopathology: At the scheduled euthanasia, no treatment-related macroscopic findings were noted.

The incidences of histopathological findings of the thyroid gland are presented below in Table 6. There were no treatmen t-related findings in the thyr oid gland or kidneys. Mild seminiferous tubular degenerati on was noted in the right testis of one animal in the 1.0 mg/kg/day group (No. 24151-03), characterized as random mild degeneration of spermatocytes and round spermatid s within the seminiferous tubules. The right epididymis in this animal exhibited mild hypospermia and moderate cellular debris . The right testes and epididymides from all remaining animals in this dose group were normal microscopically.Male Pubertal Assay (2012)/ Page 13 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None



### 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 4 14 3 0 14 4 10 14 4 0 14 5 0 14 5 0 14

Methomyl 0.5 mg/kg/day 1 0 15 1 14 15 2 0 15 2 1 15 3 1 15 3 0 15 4 14 15 4 0 15 5 0 15 5 0 15

Methomyl 1.0 mg/kg/day 1 0 14 1 12 14
2 0 14 2 2 14
3 4 14 3 0 14
4 10 14 4 0 14
5 0 14 5 0 14 a Data were obtained from page 98 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: Based on the lack of effects on the mean age and body weight at the attainment of PPS, male reproductive organ weights, serum testosterone levels, or test substance-related macroscopic and microscopic findings, methomyl did not alter pubertal development when administered orally to peripubertal male rats at dosage levels of 0.5 and 1.0 mg/kg/day. Based on the absence of effects on serum thyroid hormone levels, thyroid weights, and microscopic alterations in the thyroid, methomyl did not display the potential to disrupt hypothalamic-pituitary-thyroid axis.



## B. AGENCY COMMENTS: All but two animals survived until scheduled termination. One vehicle control group male was found dead on PND 45 and one male in the 1.0 mg/kg/day group was found dead on PND 42. The death of th e high-dose animal was determined to be caused by an intubation error; the cause of the death for th e control group animal was unknown. Red material around the mouth or ear was observed infrequently during the daily examinations.

There were no treatment-related effects on fina l body weights, overall body weight gain, or the age or body weight at PPS. There were no significant e ffects observed for organ weights.

Serum T 4 

## , TSH, and testosterone were not affected by treatment. No clinical chemistry findings were noted. At the scheduled eu thanasia, no treatment-related macroscopic findings were noted in the thyr oid gland or kidneys. Mild se miniferous tubular degeneration was noted in the right testis of one animal in the 1.0 mg/kg/day group, characterized asMale Pubertal Assay (2012)/ Page 14 of 14 METHOMYL/ 090301 OCSPP 890.1500 / OECD None


## random mild degeneration of spermatocytes an d round spermatids within the seminiferous tubules. The right epididymis in the same an imal exhibited mild hypospermia and moderate cellular debris. The right testes and epididymides from all other animals were normal microscopically.

The dose levels tested in this study are considered to be adequate based on data from earlier studies showing toxicologically-a dverse clinical signs of cholinesterase inhibition at doses ≥ 2.0 mg/kg/day.

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

 The mean % CV for weight at age of attainment of PPS in the control group (9.55%) was above the performance criteria maxi mum of 7.57%, but the mean weight was within the acceptable range.

 The mean control absolute kidney weight ( 2.02 g) was below the performance criteria minimum value (2.242 g), but the % CV va lue for the kidneys was within the acceptable range in the performance criteria.

 The % CV for the TSH level (61.742%) was greater than the maximum acceptable value in the performance criteria (58.29 %), although the mean value for TSH was within the performance cr iteria acceptance range.

# DATA EVALUATION RECORD

METHOMYL

Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-47-2012 (MRID 48685902)

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: 5/29/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 6/8/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 6/11/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 6/11/2012

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).METHOMYL I 090301



## Primary Reviewer: Karlyn Middleton 

## Health Effects Division Secondary Reviewer: John Liccione Ph.D. 

## Health Effects Division Steroidogenesis Assay (2012) I Page I of 11 OCSPP 890.1550/ OECD None



## Signature: --+--_..,___

___ _ 

## Date:**~-~f-=--,LJ.'...-"'--** 

## Signa ture:**~;::::::::it:;=:~===--** 

## Date:**~**



## DATA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550



## PC CODE: 09030 

## l DP BARCODE: D399624



## TXR#: 0056254 

## CAS No.: 16752-77-5



## TEST MATERIAL (PURITY): Methomyl (99.4%)



## SYNONYMS: Methyl N-[[(methylamino)carbonyl]oxy]ethanimidothioate; DPX-Xl 179



## CITATION: Nabb

, D.L (2012). Methomyl (DPX-Xl 179) Technical: H295R Steroidogenesis Assay. E.I. du Pont de Nemours and Company, Newark, DE. Laboratory Report No.: DuPont-32086, February 8, 2012. MRID 48685902. Unpublished

.



## SPONSOR: E.I. du Pont de Nemours and Company

, Wilmington

, DE



## TEST ORDER#: EDSP-090301-62



## EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48685902)

, H295R cells cultured 

## in vitro in 24-well plates were incubated with methomyl (99.4% a.i., Lot # JUL06LP276) at log concentrations from 0.000 l to l 00 μM for 48 hours in triplicate in three independent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicle for the positive controls and water was used as the vehicle for methomyl.

Testosterone and estradiol levels were measured using HPLC / MS / MS method with internal standards. 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 minimum basal hormone production levels for estradiol were below the recommended concentration of 40 pg / mL

, except in the blank and DMSO cells in Run 1. Although the basal estradiol production was generally less than 40 pg / mL

, all values were > 12.5 pg / mL (2.5-fold greater than the minimum detection limit (MDL) as per the Guideline recommendation). Estradiol production in the presence of 10 μM forskolin met induction requirements ( 2: 7.5-fold). However

, induction of testosterone production at 10 μM forskolin was 1.76- to 1.80-fold, which is slightly less than the**~2-fold** 

### requirement

, and induction production in the presence of l μM forskolin was slightly higher than expected ( 1.59- to 1.63-fold). Testosterone and estradiol inhibition were both greater than the required 50% in the presence of 1 μM prochloraz. BecauseSteroidogenesis Assay (2012) / Page 2 of 11 METHOMYL / 090301 OCSPP 890.1550/ OECD None


## all other guideline requirements for basal con centrations and hormone induction and inhibition were met for testosterone and estradiol, these results were deemed acceptable.

The %CVs within each plate fo r the SC on the test chemical plates analyzed for hormone production ranged from 3.12 to 12.37% for testoste rone and 0.78 to 12.45% for estradiol. The between-plate %CVs for the test plates were 6.93% and 7.0%, resp ectively, for testosterone and estradiol. These values are within the Guideline limit of ≤ 30%.

Precipitation was not observed at any tested me thomyl concentration, and methomyl was not found to be cytotoxic to the H295R cells at concentrations up to 100 μ M. No effect on estradiol production was observed at any tested methomyl concentration. Across the three runs, there were significant (p<0.05) decreases in mean testosterone levels at methomyl concentrations of 0.01 μ M (0.89-fold) and 100 μM (0.83-fold) methomyl. The effect was consistent across all three runs, but it was not dose-dependent, as it occurred only at two non-sequential concentrations.

Based on hormone responses in three independent runs, methomyl treatm ent did not result in statistically significant and repr oducible alterations in testoste rone or estradiol production.

The assay satisfies the EDSP Tier 1 Test Order requi rements for a Steroidogenesis assay (OCSPP 890.1550).

COMPLIANCE: Signed and dated Data Confiden tiality, GLP Compliance, and Quality Assurance statements were provided.Steroidogenesis Assay (2012) / Page 3 of 11 METHOMYL / 090301 OCSPP 890.1550/ OECD None



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: E.I. du Pont de Nemours and Company

Location: Newark, DE Study Director: Nabb, D.L Other Personnel: Styles, D. A., Staff Chemist Jepson, G.W. , manager O'Connor, J.C., Group Leader Study Period: November 14, 2011 – February 8, 2012



## 2. Test Substance: Methomyl Description: Technical, White Solid Lot / Batch #: (Expiration Date) JUL06LP276 Purity: 99.4% Solubility (in Solvent): Not reported Volatility: Not reported Stability: 6 year shelf life Storage conditions: Ambient CAS #: 16752-77-5 Molecular weight: 162.21 Structure:

N

S O N H O

CH 3 CH

3

CH 3



## 3. Positive Control (Inducer): Forskolin Description: White powder Source: Sigma-Aldrich, Cat. # F3917 Lot #: (Expiration Date): 109K50571V (Exp date not reported) Purity: 98% Solubility (in Solvent): 50 mg/mL in EtOH Storage conditions: Not reported CAS #: 66575-29-9



## 4. Positive Control (Inhibitor): Prochloraz Description: Not reported Source: Sigma-Aldrich, Cat. # 45631 Batch #: (Expiration Date) SZE6220X (August 8, 2012) Purity: 99.1% Solubility (in Solvent): Not reported Storage conditions: Room temperature CAS #: 67747-09-5



## 5. Solvent/Vehicle Control: Deionized Water (for methomyl) Description: NA Source: Mediatech, Manassas, VA (Cat. # 25-055-CM) Lot #: (Expiration Date) 25055393 (not applicable) Purity: Not applicable Storage conditions: Not reported CAS #: Not applicable Justification for choice of solvent: Methomyl was stable in water during the dosing period Final concentration: Not applicableSteroidogenesis Assay (2012) / Page 4 of 11 METHOMYL / 090301 OCSPP 890.1550/ OECD None

(% volume in assay)



## 6. Solvent/Vehicle Control: DMSO (for positive controls) Description: Not reported Source (Cat. #): Sigma-Aldrich, St. Louis, MO. (Cat. # D2438) Lot #: (Expiration Date) RNBB9459 (not provided) Purity: Not reported Storage conditions: Not reported CAS #: 67-68-5 Justification for choice of solvent: One of the solvents recommended in the Guideline. Final concentration: (% volume in assay) 0.05%



## 7. Stock Medium: Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture
Source: Invitrogen, Grand Island, NY (Cat # 11039-021) Lot #: (Expiration Date) 988654 ( not reported) Sodium bicarbonate: Not Reported Nu-Serum: BD Bioscience, Bedford, MA, Cat # 355100, Lot # 11268; cells were maintained with media containing unstripped Nu-S erum. Stock dosing media contained 90% charcoal-stripped media and 10% uns tripped media, to adjust the basal concentration of testosterone. Adjust ed background hormone concentrations were not reported by the performing laboratory ITS+ premix: BD Bioscience, Cat # 35452, Lot # 14793



## 8. Test Cells: H295R human adrenocortical carcin oma cells (ATCC CLR-2128; Lot # 58333130)

. Cells were stored frozen in liquid nitr ogen for no longer than 24 months. From an original ATCC batch, H295R cells were grow n for 5 passages and subsequently frozen in liquid nitrogen. Cells started from frozen ba tches were cultured for at least 4 additional passages prior to their use in an assay. The maximum passage number used in any assay was passage 10 after freeze down. The H295R cells were maintained in supplemented media containing DMEM:F12 media base with ITS + Premix and Nu-Serum. Cells were incubated at 37 ° C in an atmosphere of 5% CO 2 

## . The passage number used in each test was β /8.5/13/B, β /9.5/14/B and β /9.5/14/C

The following performance criteria were met (indicated by an “x”): X Cell passage identifier. Cell Passage #: 8.5-9.5 X Cells frozen down at passage 5 X Frozen cells cultured for 4 additional passages X Total number of passages does not exceed 10



## B. METHODS

1. Pre-Test Information

a. Hormone Assay Interference Test: Analysis of the production of testosterone and 17 β -estradiol by H295R cells was conducted by HP LC/MS/MS using internal standards to measure extraction and quantitati on efficiency. No interference would be anticipated with this analytical methodology.

b. Hormone Extraction: Portions of each sample were processed by different methods to extract estradiol and testosterone prior to HP LC/MS/MS analyses. For estradiol analysis, samples were extracted with methylene chloride after addition of the internal standardSteroidogenesis Assay (2012) / Page 5 of 11 METHOMYL / 090301 OCSPP 890.1550/ OECD None


## (D4-estradiol, Cambridge Isotope Laboratorie s, Andover, MA). The methylene chloride was evaporated to dryness, followed by estr adiol solubilization with 100 mM sodium bicarbonate buffer (pH 10.5), and derivatization with dansyl ch loride. For testosterone analysis, samples were aliquote d onto a 96-well protein-precip itation plate with acetonitrile, and the internal standard (D2-testosterone, Cambridge Isotope Laboratories, Andover, MA) was added. After shaking and then allowing th e plate to stand for 10 minutes, the eluent was filtered off, concentrated to dryness, and resuspended in 50% acetonitrile/water for LC/MS/MS analysis.

c. Laboratory Proficiency Test: It was stated that all assays were performed by personnel that demonstrated proficiency performing the assay as outlined in the test guideline. Proficiency records are referenced in the study records, but were not included in the study report.

2. Test Solutions: A 200-mM stock solution was prepar ed by dissolving an appropriate amount of methomyl in water. Six dilutions serial 1:10 dilutions were then prepared by adding 200 μL of the test solution to 1800 μL wate r, yielding a range of 100 nM to 100 mM. Forskolin and prochloraz solutions were prepar ed in a similar manner with seven and eight serial dilutions, respectively.

3. Cell Plating and Preincubation: H295R cells (ATCC CLR-2128) were grown for five passages, frozen in liquid nitrogen, then thaw ed and cultured for at least four additional passages. The cells were then seeded in 24-well plates using 1 mL of cells at a concentration of 300,000 cells/mL, yielding approximately 50-60% confluency at 24 hours. The seeded plates were incubate d for 24 hours at 37°C in a 5% CO 2 

## atmosphere.

4. Exposure: The cells were checked microscopi cally for good attachment and proper morphology, and the medium was removed and re placed with 1 mL of a mastermix of each concentration of methomyl, posit ive control, or solvent in me dium with charcoal stripped Nu-Serum. Cells were exposed in triplicate acco rding to the schematic presented in Table 1.

TABLE 1. Dosing Schematic for the Exposure of H295R Cells to Methomyl (Final Concentrations in μM) a
1 2 3 4 5 6
A Water Water Water 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 15 of the study report. Dosing was calculated based on a to tal volume of 1 mL per well.



## A concurrent QC plate was included with each of the three independent runs of the test chemical plates to demonstrate the assay’s res ponse to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testoste rone and estradiol production). The QC plate was prepared and dosed in the same manner w ith either forskolin or prochloraz according to the schematic presented in Table 2.Steroidogenesis Assay (2012) / Page 6 of 11 METHOMYL / 090301 OCSPP 890.1550/ OECD None



### TABLE 2. Dosing Schematic for the QC Plate for Po sitive Controls (Final Concentrations in μM) a
1 2 3 4 5 6
A Blank b Blank Blank Blank + MeOH c Blank + MeOH Blank + MeOH
B DMSO DMSO DMSO DMSO + MeOH DMSO + MeOH DMSO + MeOH C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochlor az 0.1μM Prochloraz 0.1μM Prochloraz 0.1μM
D Forskolin 10μM Forskolin 10μM Forskolin 10μM Proc hloraz 1μM Prochloraz 1μM Prochloraz 1μM a Data were obtained from page 16 of the study report. Dosing was calculated based on a to tal volume of 1 mL per well. b Blank wells received medium only. c MeOH = 70% methanol was added to these wells for 30 mi nutes at room temperature following medium removal.



## Following dosing, the plates were incubated for 48 hours under the conditions previously described. The medium from each well was rem oved, split into two aliquots, and frozen at

− 80°C until hormone measurements.

5. Cell Viability/Cytotoxicity Assay: Cell viability was determined using the LIVE/DEAD ® 

## Viability/Cytotoxicity kit (Molecular Probes, E ugene, OR) immediately after removal of the culture medium. The plate wells were rins ed twice with 300 μL of phosphate buffered saline (PBS) (with Ca 2+ 

## and Mg 2+ 

## ). Next, 0.5 mL viability reagent containing the polyanionic dye calcein AM and Ethidium homodimer l (EthD-l) was added to each well. The plates were incubated at room temperature for 1 hour, and scanned with a fluorometer (Molecular Devices, Sunnyvale , CA). Calcein AM is retained within live cells and produces an intense uniform green fluorescence (Ex/Em -495/-515 nm) when it is enzymatically converted by intracellular esterase to calcein. EthD-1 enters damaged cells and undergoes a 40-fold enhancement of fluorescence after bind ing to nucleic acids, producing a bright red fluorescence in dead cells (Ex/Em ~495/~635 nm). EthD-1 is excluded by the intact plasma membrane of live cells. The measurements of calcein AM and EthD-1 for triplicate wells at each test concentration were averaged, and th e average calcein AM fluorescence for each sample was divided by its EthD-1 fluor escence to obtain a live/dead ratio.

6. Hormone Measurement System: The concentration of each hormone (testosterone and estradiol) was evaluated in triplicate in the culture medium. Hormones were extracted from the samples with either methylene chloride (estradiol) or by pr otein precipitation and filtration (testosterone). The estradiol was also derivatized with dansyl chloride for analysis, and both hormones were quantified by LC/MS/MS using deuterated internal standards. The lower limit of quantification (LLQ, or MDL fo r this system was 100 pg/mL for testosterone and 5 pg/mL 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 Samples were corrected for recovery losses by using internal standards. NA Hormone cross-reactivity (antibody-based assays only; ≤ 30% of basal production of the respective hormone) NA 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: To evaluate the relative increase/decrease in chemically altered hormone production, the results within each assay were normalized to the mean solvent control (SC) value. The results were expressed as changes relative to the solvent control in each exposure plate. Relative changes were calculated using the following equation:Steroidogenesis Assay (2012) / Page 7 of 11 METHOMYL / 090301 OCSPP 890.1550/ OECD None


## Relative change = (hormone concentrati on in each well) ÷ (mean SC hormone concentration).

Data were expressed as mean ± standard deviat ion. All doses exhibiti ng cytotoxicity greater than 20% were omitted from further evaluation. Prior to conducting statistical analyses, the assumptions of normality and variance homogeneity were evaluated. Normality was evaluated with standard proba bility plots or any other appr opriate statistical method (e.g., Shapiro-Wilk's test). If the data were not nor mally distributed, the data were transformed to approximate a normal distribution. If the data were normally distributed or approximately normally distributed, differences between chemi cal treatments and the SC were analyzed using a parametric test (e.g., Dunnett's Test). If data were not normally distributed, an appropriate non-parametric test was used (e.g., Kruskal Wallis, Steel's Many-one rank test). Differences were consider ed significant at p<0.05.

II. RESULTS

A. TEST COMPOUND: The hormone concentrations afte r exposure to methomyl, and the fold-difference change relative to the SC, and mean ± SD, for the three assay runs are presented in Table 3 and shown graphically in Figures 1 and 2. Precipitation was not observed at any tested me thomyl concentration.

The %CVs within each plate fo r the SC on the test chemical plates analyzed for hormone production ranged from 3.12 to 12.37% for testos terone and 0.78 to 12.45% for estradiol. The between-plate %CVs for the test pl ates were 6.93% and 7.0%, respectively, for testosterone and estradiol. These values are within the Guideline limit of ≤ 30%.

No significant effect on estradiol produc tion was observed at a ny tested methomyl concentration. There were sign ificant (p<0.05) decrea ses in mean testosterone levels at methomyl concentrations of 0.01 μ M (0.89-fold) and 100 μM (0.83-fold), and the effect was consistent across all three run. However, the effect was not dose-dependent, as it occurred only at two non-sequentia l concentrations.Steroidogenesis Assay (2012) / Page 8 of 11 METHOMYL / 090301 OCSPP 890.1550/ OECD None



### TABLE 3. Mean (±SD) Hormone Concentrations Fo llowing Treatment with Methomyl for 48 Hours. a
Nominal Concentration (μM) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean ± SD Statistical Significance

Testosterone (pg/mL) Fold Difference
DI Water 1440 1540 1006 — — — — — NA
0.0001 1310 1373 909 0.910 0.892 0.904 0.902 0.055 NA
0.001 1387 1400 971 0.963 0.909 0.966 0.946 0.044 NA
0.01 1297 1323 906 0.900 0.859 0.901 0.887 0.060 0.022
0.1 1557 1427 982 1.081 0.926 0.976 0.994 0.083 NA
1 1363 1373 970 0.947 0.892 0.965 0.934 0.077 NA
10 1547 1463 1019 1.074 0.950 1.013 1.012 0.073 NA
100 1217 1230 845 0.845 0.799 0.840 0.828 0.053 0.0007 Estradiol (pg/mL) Fold Difference
DI Water 36.23 29.50 31.20 — — — — — NA
0.0001 43.26 26.70 29.96 1.194 0. 905 0.960 1.020 0.192 NA
0.001 34.68 27.36 31.98 0.957 0. 928 1.025 0.964 0.079 NA
0.01 34.83 28.33 27.66 0.961 0. 960 0.887 0.936 0.100 NA
0.1 37.66 31.23 36.16 1.040 1. 059 1.159 1.086 0.090 NA
1 39.74 28.20 32.23 1.090 0. 956 1.033 1.018 0.085 NA
10 34.53 25.40 30.76 0.953 0. 861 0.986 0.933 0.072 NA
100 38.73 26.56 33.40 1.069 0. 901 1.071 1.013 0.126 NA a Data were obtained from page 28 of the study report. NA not applicable



## FIGURE 1. Mean (±SD) Change in Testost erone Production Relative to the Solvent Control at Methomyl Concentrations from 0.0001 to 100 μ M, across all Three Runs.Steroidogenesis Assay (2012) / Page 9 of 11 METHOMYL / 090301 OCSPP 890.1550/ OECD None


## FIGURE 2. Mean (±SD) Change in Estradiol Production Relative to the Solvent Control at Methomyl Concentrations from 0.0001 to 100 μ M, across all Three Runs.

B. CYTOTOXICITY: Methomyl was not found to be cytotoxic to the H295R cells (>80% viability; Table 4). Viability across all of the test plate runs ranged from 89.1-101.9% relative to SC.

TABLE 4. Mean Percent (±SD) MTT Ce ll Viability Results aft er Treatment with Fors kolin, Prochl oraz, or Methomyl for 48 Hours. a

Compound Concen. (μM) Cell Viability – Run 1 Cell Viabilit y – Run 2 Cell Viability – Run 3 Mean SD Mean SD Mean SD Blank NA NR NR NR NR NR NR Background NA 100 NR 100 NR 100 NR SC + Methanol NA NR NR NR NR NR NR Forskolin 1 NR NR NR NR NR NR Forskolin 10 NR NR NR NR NR NR Prochloraz 0.1 NR NR NR NR NR NR Prochloraz 1 NR NR NR NR NR NR Methomyl 0.0001 100.2 NR 99.2 NR 100.8 NR Methomyl 0.001 101.8 NR 97.7 NR 96.1 NR Methomyl 0.01 101.7 NR 98.0 NR 96.9 NR Methomyl 0.1 101.6 NR 101.9 NR 99.9 NR Methomyl 1 100 NR 101.3 NR 97.7 NR Methomyl 10 95.2 NR 96.5 NR 92.8 NR Methomyl 100 94.9 NR 93.5 NR 89.1 NR a Data were obtained from pages 48-49 of the study report. NA = Not applicable NR = not reported SC = Solvent controlSteroidogenesis Assay (2012) / Page 10 of 11 METHOMYL / 090301 OCSPP 890.1550/ OECD None



## C. QC PLATE: The hormone concentrations after expos ure to the reference chemicals, SC, and blank samples, as well as the fold-differenc e change relative to SC (individual and mean ± SD) for the three assay runs are presente d in Table 5. The minimum basal hormone production levels for testosterone (500 pg/mL ) were met in both blank and SC wells for testosterone. The minimum basal hormone prod uction levels for estradiol were below the recommended concentration of 40 pg/mL, except in the blank and DMSO cells in Run 1. Although the basal estradiol production was gene rally less than 40 pg/mL, all values were >12.5 pg/mL (2.5-fold greater than the MD L as per the Guideline recommendation). Estradiol production in the pr esence of 10 μM forskolin me t induction requirements ( ≥ 7.5- fold). However, induction of testosterone production was only 1.76- to -1.80-fold, slightly less than the ≥ 2-fold requirement. Induction of test osterone production in the presence of 1 μM forskolin was 1.59- to 1.63-fold, indicating that the ten-fold increase in forskolin concentration resulted in only a minor increas e in the inductio n of testosterone production. Testosterone and estradiol i nhibition were both greater th an the required 50% in the presence of 1 μM prochloraz. Because all other guideline requirements for basal concentrations and hormone induction and inhibition were met for testosterone and estradiol, these results were deemed acceptable.

The variability (%CV) between the runs (cal culated by reviewer) based on the absolute concentrations of the SC were 20.9% for test osterone and 23.5% for estradiol; within the recommended limit of ≤ 30% and acceptable. The %CVs w ithin each run for the QC plates for testosterone and estradiol ranged from 0.05-4.19% and 0.095-16.02%, respectively.

TABLE 5. Hormone Concentrations Following Treat ment with Forskolin or Prochloraz for 48 Hours. a

Compound Conc. (μM) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean ± SD
Testosterone (pg/mL) Fold Difference Background NA 1767 1560 1067 — — — — —
DMSO NA 1537 1490 1025 — — — — — Forskolin 1 2450 2687 1670 1.594 1.600 1.629 1.676 0.133 Forskolin 10 2740 2383 1800 1.783 1.803 1.756 1.713 0.187 Prochloraz 0.1 1257 1310 884 0.818 0.879 0.863 0.853 0.047 Prochloraz 1 535 641 436 0.348 0.430 0.425 0.401 0.044 Estradiol (pg/mL) Fold Difference Blank NA 51.23 36.2 30.43 — — — — —
DMSO NA 46.30 34.13 29.6 — — — — — Forskolin 1 1109 790.6 630.6 23.95 23.16 21.31 22.81 2.365 Forskolin 10 2367 1277 1162 51.13 37.42 39.27 42.13 6.773 Prochloraz 0.1 40.13 26.23 20.96 0.867 0.769 0.708 0.771 0.090 Prochloraz 1.0 5.880 2.123 4.527 0.099 0.062 0.229 0.105 0.096 a Data were obtained from page 29 of the study report. . The lower limit of quantifi cation (LLQ) was 100 pg/mL for testosterone and 5 pg/mL for estradiol.Steroidogenesis Assay (2012) / Page 11 of 11 METHOMYL / 090301 OCSPP 890.1550/ OECD None



## III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Methomyl did not cause statistically significant or biologically relevant ch anges in testosterone or 17 β -estradiol synthesis relative to vehicle control in three independent experimental runs . Positive control chemicals, forskolin and prochloraz, were evaluated concur rently to verify the test syst em performance. As expected, forskolin showed effects consis tent with testosterone and 17 β -estradiol induction and prochloraz showed effects consistent with testosterone and 17 β -estradiol inhibition. Performance criteria for a valid assay were met. Under the conditions of this study, the test substance was judged to be negative for the i nduction or inhibition of steroid biosynthesis.

B. AGENCY COMMENTS: Precipitation was not observe d at any tested methomyl concentration, and methomyl was not found to be cytotoxic to the H295R cells at concentrations up to 100 μ M. Viability across all of the te st plate runs ranged from 89.1- 101.9% relative to SC.

Three independent test runs of the steroidogenesis assay we re conducted with methomyl. Although the basal hormone production levels for estradiol (>12.5 pg/mL) was below the Guideline specified 40 pg/mL and the induction of testosterone production (1.59- to 1.93- fold) by forskolin at 10 μ M was slightly below the recommended 2-fold level, the performance criteria for the assay were generally met.

Within- and between-plate %CVs met the Guideline requirements of ≤ 30% on all test and QC plates.

No significant effect on estradiol produc tion was observed at a ny tested methomyl concentration. Across the three runs, there were significant (p<0.05) decreases in mean testosterone levels at methomyl concentrations of 0.01 μ M (0.89-fold) and 100 μM (0.83- fold). The effect was consistent across a ll three run, but it was not dose-dependent, as it occurred only at two non-seque ntial concentrations. Methom yl was therefore considered negative in the steroidogenesis assay.

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

 Although the steroidogenesis assa y proficiency and validati on tests for the performing laboratory were reported as completed and su fficient, no data were included in the study report to s upport this claim.

 The minimum basal estradiol production levels were generally below the recommended concentration of 40 pg/mL.

 Induction of testosterone production by forskolin was onl y 1.76-1.80-fold; slightly less than the ≥ 2-fold requirement.

# DATA EVALUATION RECORD

METHOMYL

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-09-2012 (MRID 48617701)

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: 01/12/2012

Secondary Reviewer Signature: David A. McEwen, B.S. Date: 01/13/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 01/19/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 01/19/2012

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).METHOMYL/090301 

### In vivo Uterotrophic Assay (2011) 

### I Page I of 9 

### OCSPP 890.l 0 / OECD 440



## Primary Reviewer: Karlyn Middleton 

## Signature:**_.,..L__....,__,~~--** 

## Health Effects Division Date:**~-----'~-+-~-** 

## Secondary Reviewer:**-"J-=-e=ss"""""'R~ow_:_:_:_:la=n=do.__** 

### _________ 

## Signature: __._,,.__=--"'-'==--------'= Health Effects Division Date:**--~~** Template version 09/2011


## DATA EVALUATION RECORD

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440



## PC CODE: 090301 

## DP BARCODE: D39626 l



## TXR#: 0056254 

## CAS#: 16752-77-5



## TEST MATERIAL (PURITY): Methomyl (99.4% purity)



## SYNONYMS: Methyl N-[[(methy lamino )carbonyl]oxy]ethanimidothioate; (DPX-Xl 179)



## CITATION: Snajdr, S. (2011). Methomyl (DPX-Xl 179) Technical: 3-Day Uterotrophic Assay for Detecting Estroge nic Activity. DuPont Haskell Global Centers for Health & 

### Environmental Sciences, Newark

, DE. Laboratory Study ID: DuPont-32084, November 8

, 2011. MRID 48617701.



## SPONSOR: E.I. du Pont de Nemours and Company

, Wilmington, DE



## TEST ORDER#: EDSP-090301 -62



## EXECUTIVE SUMMARY: In a uterotrophic assay (MRID 48617701) conducted to screen for potential estrogenic activity, methomyl (99.4% a.i.

, lot # JUL06LP276) in water was administered daily via oral gavage to groups of six ovariectomized female Sprague Dawley rats
at dose levels of 0 (vehicle), 0.25, 0.5 or 1.0 mg / kg / day on post-natal days (PND) 63-65 (approximate). A positive control group was treated with l 7a-ethynyl estradiol (EE) by daily oral gavage at a dose level of 0.1 mg / kg / day on PND 63-65. All animals were terminated and necropsied approximately 24 hours after the final dose administration to determine wet and blotted uterine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in
any treatment group. Body weights, food consumption, and food efficiency in the methomyl treated groups were comparable to the control throughout the study. Jn the positive control
group, body weights were decreased (p < 0.05) by l 0% on Day 4 compared to the vehicle control
group, resulting in an overall weight loss (p < 0.05) of -4

. 0 g , compared to the vehicle control
group (23 .1 g weight gain). Overall food consumption and efficiency were decreased (p < 0.05) by 28% and 128%

, respectively

, in the positive control group compared to the vehicle control
g roup

. Uterine weights in the methomyl treated gro ups were comparable to the controls.In vivo Uterotrophic Assay (2011) / Page 2 of 9 METHOMYL/090301 OCSPP 890.1600/ OECD 440


## The mean blotted vehicle control uterine weig ht was <0.031% of the mean vehicle control terminal body weight, which meets the Guide line requirement (<0.04%). Absolute wet and blotted uterine weights for the EE group were increased (p<0.001 and p<0.05, respectively) by 213% and 166%, respectively. The increased uter ine weights were in the expected range.

The dose levels selected for this study ar e adequate based on red blood cell and brain cholinesterase inhibition seen at dose levels ≥ 2.0 mg/kg/day in the toxicology database.

No statistically significant changes were seen in uterine weight in th is assay. Methomyl 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 GLP Compliance a nd Quality Assurance statements were provided.In vivo Uterotrophic Assay (2011) / Page 3 of 9 METHOMYL/090301 OCSPP 890.1600/ OECD 440



## I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: DuPont Haskell Global Centers for Health & Environmental Sciences
Location: Newark, DE Study Director: S. Snajdr Other Personnel: Wang, D (Research Chemist); Singh, B (Senior Research Pathologist); Jepson, G. (Man ager); and Malley, L (Senior Research Toxicologist) Study Period: May 16 to November 8, 2011



## 2. Test Substance: Methomyl Description: Solid Source: E.I. du Pont de Nemours and Company Lot #: JUL06LP276, expires May 18, 2016 Purity: Solubility: 99.4 % a.i. 58 g/L in water Stability: Stable in deionized water for up to 8 days (MRID 48617702) CAS #: 16752-77-5 Structure:



## 3. Reference Estrogen: 17 α -ethynyl estradiol (EE) Supplier: Sigma Aldrich, St. Louis, MO Lot/Batch #: 028K1411, expiration date not reported Purity: 99 % CAS # : 57-63-6



## 4. Solvent/Vehicle Control (Methomyl): Deionized water Supplier: Test facility Lot/Batch #: Not applicable Rationale (if other than water): Not applicable Final concentration: Not applicable



## 5. Solvent/Vehicle Control (EE): Corn oil with 1% ethanol Supplier: Not reported Lot/Batch #: Not reported Rationale (if other than water): Not provided Final concentration: Not applicable N

S O N H O

CH 3 CH

3

CH 3In vivo Uterotrophic Assay (2011) / Page 4 of 9 METHOMYL/090301 OCSPP 890.1600/ OECD 440



## 6. Test Animals: Species: Rat (ovariectomized females) Strain: Sprague Dawley (CrL:CD[SD]) Age/weight at dose initiation: PND 63 (approximate)/ 251.1 – 310.9 g females Source: Charles River Laboratory (Kingston, NY) Housing: 2/cage in solid bottom cages with alpha cellulose bedding (Shepherd’sTM ALPHA-dri® bedding) Diet: Harlan Teklad 2016 certified feed (Harlan Teklad, Madison, WI)

, ad libitum . A portion of feed was collected for phytoestrogen content analysis; however, because feed excludes alfalfa and soybean meal, the study author concluded that phytoestrogen content was below 350 μg/g ge nistein equivalents without analysis. Water: Filtered tap water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 18-26 oC 30-70% Not provided 12 hrs light/ 12 hrs dark Acclimation period: 6 days



## B. STUDY DESIGN

1. In-Life Dates: Start: May 17, 2011 End: May 20, 2011

2. Study Design: Sexually mature female rats were ovariectomized on PND 49 (by the supplier), at least 14 days prior to initiation of dosing. Upon arri val at the test facility, the rats were acclimated for at least 6 days prior to initiation of dosing. All rats were evaluated for vaginal cytology daily during the last fi ve days of the post-ovariectomy acclimation period for evidence of conversion out of diestr us. The dose administration period was from approximate PND 63 through 65. Rats were euthanized approximately 24 hours after the last dose and necropsied for ut erine weight measurements.

3. 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 between group mean weights at study initiation. Furthermore, the body weight of each animal on Day 1 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 0 6 Low 0.25 6 Mid 0.5 6 High 1.0 6 17 α -ethynyl estradiol (EE), Reference Estrogen 0.1 6 a Data were obtained from page 13 of the study report.



## 4. Dose Selection Rationale: The dose levels were chosen with the objective of having midand high-dose levels in which adverse reduc tions (>10%) in cholinesterase had been established to occur, but not high enough to induc e clinical signs of toxi city. In acute oral neurotoxicity (HL-1998-01080; MRID 44487501) a nd reversibility (H L-1997-00641; MRID number not provided) studies, toxicologically adverse clinical signs of cholinesterase inhibition, accompanied by marked decreases in brain and red blood cell cholinesterase activity (46-57%), were observe d at dose levels of 2.0 mg/kg/ day and greater. At doseIn vivo Uterotrophic Assay (2011) / Page 5 of 9 METHOMYL/090301 OCSPP 890.1600/ OECD 440


## levels of 0.5-0.75 mg/kg/day, adverse reductions of brain and red blood cell cholinesterase activity were observed in male (up to 25% a nd 43%) and female (up to 30% and 38%) rats (HL-1998-01080 and MRID 46646401). There were no cl inical signs of t oxicity or adverse effects on red blood cell, plasma, or brain cholinesterase activity in male rats at a dose level of 0.25 mg/kg/day (HL-1998-01080).

5. Dose Preparation and Analysis: Dose formulations were pr epared once prior to initiation of treatment by dissolving the appropriate amount s of test substance in deionized water and adjusting for purity. The reference estrogen, EE, was initially dissolved in ethanol and then mixed with corn oil; the final concentration of ethanol was 1% . Doses were adjusted daily based on individual body weight measuremen ts. Concentration verifications were conducted near the beginning of the study in the 0.25, 0.5, and 1.0 mg/kg dose formulations. In a concurrent study (MRID 48617702), methomyl (lot # JUL06LP276) was determined to be stable in deionized water for up to 8 days at room temperature.

Results of Dose Analysis

Homogeneity: not applicable; methomyl is soluble in water at the concentrations used for dosing.

Stability (% of nominal): 102-103% after 3 days at room temperature; 101-102% after 8 days at room temperature (MRID 48617701)

Concentration (% of nominal): 102.6-103.0%

The analytical data indicated that the varia tion between nominal and actual dosage to the animals was acceptable. Because the test su bstance dose formulation was not a suspension, homogeneity measuremen ts are not required.

6. Dosage Administration: Animals were administered the methomyl or EE or vehicle daily via oral gavage for three consecutive days in a dose volume of 5 mL/kg body weight for the test substance and vehicle or a dose volume of 2 mL/kg body weight for EE. Dose volumes were adjusted daily based on the concurrent body weight measurement.

7. Statistics: Body weights, body weight gains, food consumption, food efficiency, and organ weights were analyzed using Levene’s test for homogeneity and Shapiro-Wilk test for normality. Significance was noted at p < 0.05. For parameters for which the homogeneity and normality tests were not found to be signi ficant, analyses were conducted using oneway ANOVA followed by Dunnett’s test; otherwise, the data were transformed (using log, square-root, or rank order) to achieve normality and/or variance homogeneity. With the exception of uterine weights, all statistical analyses used two- tailed tests. The statistical analyses were considered adequate.In vivo Uterotrophic Assay (2011) / Page 6 of 9 METHOMYL/090301 OCSPP 890.1600/ OECD 440



## C. METHODS

1. Clinical Examinations: Cage-side checks for mortality, moribundity and abnormal behavior and/or appearance were conducted twice daily. Rats were also examined for acute signs of toxicity approximately 30-60 minut es after dosing (time of peak-effect for cholinesterase inhibition).

2. Body Weight: Animals were weighed at randomi zation, study initiati on, daily throughout the dosing period and at termination.

3. Food Consumption and Efficiency: Food consumption was measured for each cage by subtracting the amount of food remaining in the cage from the amount supplied, accounting for spillage (only spillage >25 g was recorded). Values were reported as group mean daily food consumption (g/animal/day). Mean daily food efficiency was calculated from the food consumption and body weight data.

4. Necropsy and Measurement of Uterine Weight: On approximately PND 66 (approximately 24 hours after final administratio n of the test substa nce), all surviving animals were euthanized by exsanguination under isoflurane anesthesia and subjected to a gross necropsy. Dissection of the uterus was performed according to the U.S. EPA Guideline. Briefly, the entire uterus from ova rian stump to cervix was removed and quickly dissected free of fat and connective tissue. The “wet” uterus (i.e., containing the luminal fluid) was weighed. Subsequently, the uterus was nicked, blotted to express luminal fluid, and weighed again.

5. Microscopic Examination (Optional): The uterus (including uterine horns and cervix), vagina, and ovarian stumps were collected a nd placed in 10% neutral-buffered formalin. However, microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination.

2. Clinical Signs of Toxicity: There were no test substance-rela ted clinical signs of toxicity in any treatment group, including the EE positive contro l. One rat in the vehicle control group had black discharge from one eye each dosing day.In vivo Uterotrophic Assay (2011) / Page 7 of 9 METHOMYL/090301 OCSPP 890.1600/ OECD 440



### TABLE 2. Incidence of Clinical Obser vations in the Estr ogen Agonist Assay a



### Observation Dose (mg/kg/day)

Vehicle Control 0.25 0.5 1.0 Reference Estrogen EE (0.1) # Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined 

### Black discharge 1 6 0 6 0 6 0 6 0 6

a Data were obtained from Table 5 on page 30 of the study report.



## B. BODY WEIGHT AND WEIGHT GAIN: Body weight and body weight gain data are presented in Table 3. Body wei ghts were slightly decreased in all methomyl treated groups compared to controls, yielding decreased body weight gains ( ↓ 18-24%) for the overall study (Days 1-4); however, the decreases were not statistically signifi cant. In the positive control group, body weights were decreased (p<0.05) by 10% on Day 4 compared to the negative control group, resulting in a mean overall body weight loss (p<0.05) of -4.0 g, compared to the vehicle control group (23.1 g gain).

TABLE 3. Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a

Study Day Dose (mg/kg/day)

Vehicle Control 0.25 0.5 1.0 Reference Estrogen EE (0.1) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD
1 6 287.2 12.6 6 284.2 18.7 6 287. 2 15.7 6 285.1 13.6 6 284.3 18.7 2 6 293.9 14.6 6 287.9 17.6 6 292. 6 15.7 6 288.5 14.5 6 278.5 21.6 3 6 300.6 16.6 6 293.5 18.3 6 297. 7 17.3 6 295.0 14.5 6 276.7 19.2 4 6 310.3 17.2 6 302.1 19.3 6 306.1 16.9 6 302.7 14.3 6 280.3* ( ↓ 10) 22.9

Body Weight Gain (Days 1-4) 6 23.1 6.3 6 17.9 5.0 6 18.9 3.0 6 17.6 4.9 6 -4.0* 10.1
a Data were obtained from Tables 1 and 2 on pages 26 and 27 of the study report. Percent differences from controls are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from vehicle control at p<0.05



## C. FOOD CONSUMPTION: Overall food consumption and food efficiency data are presented in Table 4. Food c onsumption in all methomyl treatment groups was comparable to the vehicle control group over the duration of the study. Mean food efficiency was decreased in all methomyl treatment groups compared to the control ( ↓ 16-20%); however, the decreases were not statistically significan t. Overall food consumption and efficiency were decreased (p<0.05) by 28% and 128%, re spectively, in the pos itive control group compared to the vehi cle control group.In vivo Uterotrophic Assay (2011) / Page 8 of 9 METHOMYL/090301 OCSPP 890.1600/ OECD 440



### TABLE 4. Food Consumption and Efficiency in the Estrogen Agonist Assay a

Study Day Dose (mg/kg/day)

Vehicle Control 0.25 0.5 1.0 Reference Estrogen EE (0.1) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD Food Consumption (g/animal/day) Overall (Day 1-4) 6 23.5 1.4 6 22.1 1.1 6 23. 0 2.0 6 22.7 1.7 6 16.9* ( ↓ 28) 1.6

Food Efficiency (g body weight gain/g food consumption per day) Overall (Day 1-4) 6 0.327 0.084 6 0.268 0.066 6 0.276 0.050 6 0.260 0.075 6 -0.093* ( ↓ 128) 0.218

a Data were obtained from Tables 3 and 4 on pages 28 and 29 of the study report. Percent differences from controls are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05



## D. PATHOLOGY: The rats were evaluated for estrous cycle stage each dosing day during the study. All rats in the vehicle control and methomyl treatments groups were found to be in diestrus during Days 1-4. All rats in the pos itive control group show ed cytological markers indicative of proestrus on Day 3 and indicative of estrus on Day 4.

1. Uterine Weights: Uterine weight data are presented in Table 5. Uterine weights in the methomyl treatment groups were comparable to the controls. The mean blotted vehicle control uterine weight was 0.031% of the mean vehicle control terminal body weight, which meets the Guideline requirement (<0.04%).

Absolute wet and blotted uterus weights for the EE group were increased (p<0.001 and p<0.05, respectively) by 213% and 166%, respec tively, compared to the control. The increased uterine weights were within the expected range.

At necropsy, no ovarian tissue was observed in the gross evaluation of the ovarian stumps of the rats in all dose groups.In vivo Uterotrophic Assay (2011) / Page 9 of 9 METHOMYL/090301 OCSPP 890.1600/ OECD 440



### TABLE 5. Uterine Weights from Estr ogen Agonist Assay in SD Rats a

Parameter Dose (mg/kg/day)

Vehicle Control 0.25 0.5 1.0 Reference Estrogen EE (0.1) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD
Terminal BW (g) 6 310.3 17.2 6 302.1 19.3 6 306. 1 16.9 6 302.7 14.3 6 280.3* ( ↓ 10) 22.9

Wet, absolute (mg) 6 97.6 10.1 6 102.5 16.0 6 107. 7 17.9 6 92.2 21.9 6 305.2** ( ↑ 213) 71.6

Wet, relative (%) 6 0.032 0.004 6 0.034 0.005 6 0. 035 0.007 6 0.030 0.007 6 0.110* 0.032

Blotted, absolute (mg) 6 95.5 9.6 6 99.3 16.1 6 104. 0 17.3 6 89.7 20.8 6 254.1* ( ↑ 166) 39.0

Blotted, relative (%) 6 0.031 0.004 6 0.033 0.005 6 0. 034 0.007 6 0.030 0.007 6 0.092* 0.019

a Data were obtained from Table 6 on page 31 of the study report. Percent differences from controls were calculated by the reviewer and included in parentheses. BW Body weight N Number of animals in the group SD Standard Deviation * Significantly different from vehicle control at p<0.05. ** Significantly different from vehicle control at p<0.001.



## 2. Microscopic Examination (Optional): Microscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: T he test substance, methomyl, did not induce changes in any parameters consistent with the potential to act as an estrogen agonist in ovariectomized adult female rats. Under th e conditions of this study, methomyl did not induce estrogenic effects in this Uterotrophic assay when administered at up to 1 mg/kg/day for 3 consecutive days.

B. AGENCY COMMENTS: The dose levels tested were adequate since they were selected based on the results of the available studies . All animals survived until scheduled termination. No clinical signs of toxicity were observed in an y treatment group. Body weights, food consumption, and food efficien cy in the methomyl treated groups were comparable to the controls throughout the duration of the study. In the positive control group, body weights were decreased (p<0.05) by 10% on Day 4 compared to the control group.Overall food consumption and efficiency were decreased (p<0.05) by 28% and 128%, respectively, in the positive control group compared to the negative control group. Uterine weights in the methomyl treated groups were co mparable to the controls. The mean blotted vehicle control uterine weight was <0.031% of the mean vehicle control terminal body weight, which meets the Guideline requirement (<0.04%). Absolute wet and blotted uterine weights for the EE group were increased (p <0.001 and p<0.05, respectively) by 213% and 166%, respectively. The increased uterine we ights were in the expected range.

No statistically significant changes were seen in uterine weight in th is assay. Methomyl is negative in the uterotrophic assay.

C. STUDY DEFICIENCIES: None