UNITED ST A TES ENVIRONMENT AL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: 6/29/2015 

SUBJECT: 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 de6 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 

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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 

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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'

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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 number 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, 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). 

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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 (Ę4.7%) and Day 21 (Ę4%) at high treatment level relative to the negative control (p<0.05). Additionally, 

Day 21 snout vent length (SVL) was significantly increased (Ė6%) 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 greater than NF stage 60 (late stage) which may confound 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 satisfies the EDSP Tier 1 Test Order requirements for an 

Amphibian Metamorphosis Assay (OCSPP Guideline 890.1100). 

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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 Effects1,2 in the Amphibian 

Metamorphosis Assay (AMA) with Methomyl. 

Treatment

(mg a.i./L) 

[measured]

NF Developmental 

Stage 

Hind Limb 

Length3,4

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 

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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 CaCO3) exceeded the 

recommended range (40-48 mg/L as CaCO3). 

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%.

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Storage Conditions of 

Test Chemicals: Stored under ambient conditions 

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B. Test Organism

Table 2: General Information About the Test Species and Parental Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species 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 Reported 

1 U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor 

Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf). 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as in-house stock? 

Yes EPA recommends that larvae used in the 

assay be derived from in-house adults.

Were parental acclimation conditions same as definitive test? 

Yes

Acclimation period for parental frogs (if applicable):

NA Parental frogs were continuously 

maintained under conditions similar to those employed in the definitive test. 

Details on parental feeding: Parental frogs were fed 

~1 tablespoon of #3 frog food pellets per tank twice a day. 

Food source is Zeigler Bros., Inc. 

Details on parental health: Parental frogs were in 

good health, displaying no abnormalities or signs of illness. 

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Table 3: Larval Selection and Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Best 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 (if applicable): 

2 

Number of eggs sampled per spawn:

All eggs were sampled on a given day.

Embryonic viability was checked daily. 

NF stage at test initiation 51 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): 

NA 

Range of total length at test initiation (if reported): 

NA 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was the optional size selection method used? 

No 

Details on larval selection: When a sufficient 

number of the pre-exposure population 

reached NF 51, larvae were transferred to a pooling tank. Larvae requiring >17 days to reach NF 51 were not used in the test. If >50% of the larvae in a given clutch required >17 days to reach NF 51, an alternate clutch was used. All tadpoles were from a single clutch. 

In order to guarantee at least 400 stage 51 acceptable larvae at test setup, clutch sizes of ~1,500 embryos were given preference.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Loading 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, PA

Iodide concentration in diet (if known):

Not Provided 

Frequency of feeding: 2 times/day EPA recommends that feeding occur at least 

twice per day.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding regime: Same as EPA guidelines Day 0-4 : 30 mg/animal/day 

Day 5-7 : 40 mg/animal/day Day 8-10 : 50 mg/animal/day Day 11-14 : 70 mg/animal/day Day 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).

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C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a flow-through 

system.

Type of flow-through dilution system (if applicable):

Continuous flow serial diluter

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate (if applicable):

25 mL/min Flow-rate provided a complete volume 

replacement approximately every 2.7 hours. 

Recommended flow-through rate is 25 

mL/min (complete volume replacement ca. 

every 2.7 hrs).

2 Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems (if applicable):

Modified Benoit Mini-Diluter 

Mixing chamber was a continuous mix chamber in which stock solution and dilution water were supplied. Baffle ensured complete mixing since the concentration desired as at or below the limit of water solubility. Concentration prepared in mixing chamber represents the highest concentration, and the remaining concentrations were obtained by sequential 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 static renewal (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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of dilution water: Treated municipal tap 

water

Water was prepared by passing tap water through a 4 filter system: a multimedia filter to remove suspended solids in the feed water; a 10” pre-treatment filter (5μm) to 

remove any additional solids; a 3.6 ft3

activated virgin carbon treatment filter to remove chlorine, ammonia, and higher molecular weight organics; and a 5μm polishing filter to remove any carbon particles from the carbon treatment phase.

EPA recommends natural or reconstituted 

water; it is recommended that natural water 

be sterilized with UV and tested for 

pesticides, heavy metals, and other 

possible contaminants, including known 

substrates of the iodine transporter of the 

thyroid gland (e.g., fluoride, chlorate, 

perchlorate). OECD accepts any water in 

which the test species show control survival 

at least as good as indicated in the test 

guideline.

Was dilution water analyzed for pesticides, heavy metals, and other contaminants? 

Yes (pesticides, PAHs, PCBs, and heavy metals) 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Iodide supplementation in water?

No If reconstituted water is used or if 

background levels of iodide in natural water 

are less than 0.5 

μg/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 exposure system: 

None 

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Table 5: Summary of Water Quality Characteristics in the Test System. 

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Hardness (mg/L as CaCO3) 

134 152 142 

Weekly EPA recommends hardness 40 to 48 mg/L as CaCO3.

pH 6.9 7.2 NA 

3x 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 4.8 

3x Weekly EPA recommends dissolved oxygen (DO) >3.5 mg/L 

(>40% air saturation). OECD recommends DO concentration 

>3.5 mg/L (>40% air saturation).

Temperature 21.9 22.4 22.2 

Daily EPA recommends temperature 22±1oC; inter-replicate and 

inter-treatment differentials should not exceed 0.5oC.

Iodide (μg/L) 2 5.1 3.6 Semi-annually EPA recommends aquatic iodide range 0.5 – 10 

μg/L 

(supplemental iodide should not exceed 2 

μg/L).

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Ammonia (mg/L) 0 0.04 0.02 Bi-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.

Fluoride Not Reported Perchlorate Not Reported Chlorate Not Reported Other NA NA NA NA 

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 D. Study Design and Additional Experimental Conditions

Table 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? YesIf yes, what was the method for determining the highest test concentration in the range-finder? 

Historical toxicity data provided in a similar species 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 laevis Life stage: 10 Test duration: 4 days

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: A 4-day range-finding 

study was conducted under static conditions at nominal concentrations of 0 (negative control), 0.1, 0.5, 1.0, 5.0, 10.0, 25.0, and 50.0 mg a.i./L. The LC50 and 

EC50 values were 2.67 

and 0.80 mg a.i./L, respectively.

No morbidity was observed at 0.51 mg a.i./L, which was the highest range-finding test concentration without 

adverse effects. Therefore, 0.51 mg a.i./L 

was set as the highest concentration in the definitive test with a 0.33x dilution series. 

However, abnormal swimming behavior was observed after 24 hrs in the definitive study at 0.51 mg a.i./L. Since this compromised the collection of data for the AMA study, the study was terminated at the direction of the Sponsor and re-initiated using 0.168 mg a.i./L as the 

highest test conc. The 0.33x series was maintained: 0, 0.0183, 0.056, 0.168 mg a.i./L. 

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Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Test duration: 21 days EPA recommends that the duration of the 

definitive test be 21 days. 

Method for selecting the highest test concentration in the definitive test:

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 (if applicable):

NA 

Separation 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Are nominal concentrations adjusted for purity? 

No 

Indicate the type of values presented for measured concentrations: 

Mean-measured concentrations

Limit of quantification (LOQ): 0.0417 mg a.i./L The lowest test concentration is 0.0183 

mg 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./L Frequency 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates in solvent control (if applicable): 

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 item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at test initiation: 

80 

Was a solvent used? No Solvent type (if applicable): NA Maximum 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

.

3 Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. Aquatic 

Toxicology, 76, pp.69–92. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a positive control used? No Positive control (if applicable): NA Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 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 were 

clear and colorless with no precipitate throughout the study. 

4 OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on 

Testing and Assessment. No. 23. Paris, France. 

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Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Methomyl. 

Treatment ID

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0.00 <0.0124 NA 

Based on 

mean-measured 

concentrations. 

EPA and OECD recommend that test item 

concentrations be maintained at a coefficient of 

variation (CV) 

≤20%.

Treatment 1 0.0183 0.0170 4.3 Treatment 2 0.056 0.0519 2.0 Treatment 3 0.168 0.186 1.2 

Abbreviations: CV Coefficient of variation. 

 E. Observations

Biological Endpoints: Day 7: NF stage, wet weight, SVL, HLL, normalized HLL 

Day 21: NF stage, wet weight, SVL, HLL, normalized HLL, thyroid histopathology 

 Daily: mortality, clinical signs 

Were raw (individual) data provided? Yes

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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 (days 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 larval selection); and thyroid 

gland gross pathology and histopathology (day 21). Note the histopathology section of the test guideline also includes thyroid gross pathology observations.

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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 71 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 12 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 10th and 90th

percentiles of developmental stage in negative control tadpoles were 62 and 65, respectively. There was 

no asynchronous development. 

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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. 

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

Treatment

(mg a.i./L) 

[mean-measured]

Hind Limb Length (HLL) 

Day 7 Day 211

n Mean

(mm) 

±SD 

HLL: 

SVL2 n Mean(mm) 

±SD

HLL: 

SVL2

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. 

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Table 12: Larval Growth in Xenopus laevis.

Treatment

(mg a.i./L)

[meanmeasured]

Snout-Vent Length (SVL) Body Weight1

Day 7 Day 212 Day 7 Day 212

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. 

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Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus laevis.

Treatment (mg 

a.i./L) 

[measured]

Diagnostic Observations1

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.

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Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis. 

Treatment 

(mg a.i./L) 

[mean-measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 -- 20 -- 20 -- 20 -- 20 -- 

1 20 -- 20 -- 20 -- 20 -- 20 -- 

2 20 -- 20 -- 20 -- 20 -- 20 -- 3 20 -- 20 -- 20 -- 20 -- 20 -- 

0.0170 0 20 -- 20 -- 20 -- 20 -- 20 -- 

1 20 -- 20 -- 20 -- 20 -- 20 -- 

2 20 -- 20 -- 20 -- 20 -- 20 -- 3 20 -- 20 -- 20 -- 20 -- 20 -- 

0.0519 0 18 -- 18 -- 18 -- 18 -- 18 -- 

1 18 -- 18 -- 18 -- 18 -- 18 -- 

2 18 -- 18 -- 18 -- 18 -- 18 -- 3 18 -- 18 -- 18 -- 18 -- 18 -- 

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Treatment 

(mg a.i./L) 

[mean-measured]

Additional Qualitative Observations1

Severity

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell Height 

(Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.186 0 20 -- 20 -- 20 -- 20 -- 20 -- 

1 20 -- 20 -- 20 -- 20 -- 20 -- 

2 20 -- 20 -- 20 -- 20 -- 20 -- 3 20 -- 20 -- 20 -- 20 -- 20 -- 

1 Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference. 

No signs of clinical toxicity were observed during the study (Table 15). 

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Table 15: Clinical Signs in Xenopus laevis.

Treatment 

(mg a.i./L) 

[measured]

Clinical Signs 

Type n Incidence 

Negative Control NA 20 0 

0.0170 NA 20 0 

0.0519 NA 20 0 

0.186 NA 20 0 

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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 pd0.05 (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 parametric 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. 

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 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. 

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Table 16: Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with Methomyl (DPX-X1179) Technical. 

Treatment

(mg a.i./L) 

[mean-measured]

NF Developmental Stage Hind Limb Length3 Asynchronous 

Development 

Thyroid Gross and 

Histopathology 

Day 7 Day 214 Day 7 Day 214 Day 7 Day 214 Day 214

Median p Median p 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff. 

p 

Treatment-Related 

Effects? (Yes/No) 

Negative Control 54 -- 63.5 -- -- -- -- -- -- -- -- -- No 0.0170 54 1.0 63.5 1.0 1.18 0.36 0.0 0.50 -- -- -- -- No 0.0519 54 1.0 63.5 1.0 -2.35 0.17 -2.0 0.24 -- -- -- -- No 0.186 54 1.0 63 1.0 -4.71 0.02 -4.0 0.046 -- -- -- -- No Statistical Test Mann-Whitney Mann-Whitney Jonckheere Jonckheere -- -- -- Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily weighted 

by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at 

p < 0.05. 

3 Hind-limb length is normalized to snout-vent length (SVL). 4 Values are for late stage individuals because all tadpoles were at NF stage >60 at Day 21. 

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Table 17: Growth Endpoints1,2 in the AMA with Methomyl (DPX-X1179) Technical. 

Treatment

(mg a.i./L) 

[measured]

Snout-Vent Length Body Weight 

Day 7 Day 213 Day 7 Day 213

% 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 developmental 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, 

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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. 

Page 41 of 241
DATA EVALUATION RECORD 

METHOMYL 

Study Type: OCSPP 890.1150, Androgen Receptor 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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 42 of 241
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).

Page 43 of 241
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- 10 to 10-3 M) of methomyl (99.4% a.i., Lot 

# JUL06LP276). 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 the EPA validation program, but the Bmax 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 ICso was - 9.15 M for RI 881 

and - 4.41 M for dexamethasone. Compared to Rl881 , the mean RBA for the dexamethasone 

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Androgen 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 all of the three criteria were met, except for the 

top % binding in the third run which was higher than the tolerance limits. Although, the 

performance criteria for both R1881 and dexamethasone were not achieved across the three runs, 

based on the estimated IC50s for R1881 and the weak positive control, 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 [3H]-ligand bound to the AR was >75% at all concentrations of methomyl in all 

three runs. Therefore, a log IC50 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 requirements for an Androgen Receptor Binding 

assay (OCSPP 890.1150). 

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

statements were provided. 

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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 # : 50-02-2 

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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 received, the prostates were stored frozen (−60 to 

−80oC) until processed into cytosol. 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 supernatants were pooled, discarding 

the resulting pellets. Protein concentration of the cytosol was determined to be 6 mg/mL. 

Cytosol was divided into aliquots (amount 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 [3H]-R1881 was performed to demonstrate that 

the AR was present in reasonable concentrations and had the appropriate affinity for the 

R1881 ligand. The conditions for the saturation binding experiment are summarized in 

Table 1. 

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat 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 specific 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 binding, solutions of non-labeled R1881 were 

prepared in a similar manner to achieve concentrations that were 100-fold greater than each 

Page 47 of 241
Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 5 of 15 

METHOMYL / 090301 OCSPP 890.1150/ OECD None

respective radiolabeled concentration, resulting in final concentrations 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 [3H]-R1881 was measured. It was stated that 

the cytosol was used undiluted at a concentration of approximately 6 mg/mL, which has 

previously been shown to bind to approximately 23% of 0.25 nM [3H]-R1881 when assayed. 

Each run contained three concurrent replicates at each concentration, 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 molar 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, hydroxyapatite (HAP) slurry was added to each 

tube and vortexed once every 5 minutes for 20 minutes. The samples were then centrifuged, 

and the supernatant was discarded. The samples 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 presence of increasing 

concentrations of a test substance. A summary of the assay conditions for the competitive 

binding experiment is included in Table 3. 

Page 48 of 241
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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 μM 

a Data were obtained from pages 12 and 14 of the study report. 

b The study reports that the prostate cytosol was diluted in cold TEDG + PMSF buffer 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 reference standard (R1881) and positive control 

(dexamethasone). It was reported that the results from the saturation 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 substance, 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 independent runs on three 

different days. Each run included triplicate sets of the blanks (solvent controls) and 1 μM 

R1881 (non-specific binding, NSB) at the beginning and end of each run, along with 

triplicate tubes of the reference standard, the weak 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. 

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TABLE 4. Competitor Final Molar (M) Concentrations in Competitive Binding Assay a b

Solvent Control Reference standard Weak positive control Test Chemical None, 

Water or Ethanol Hot Mix 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 triplicate 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, separation of bound from free R1881 (washing with 

buffer and extraction with ethanol), followed by scintillation counting of bound [3

H]-R1881 

were similar to those used in the saturation binding experiment. A total of three runs were 

performed as described. 

4. Data Analysis: The maximal binding capacity (Bmax), dissociation constant (Kd), and 

inhibition concentration (IC50) were calculated using nonlinear 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 nonlinear 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 least 25% but less than 50%), or the 

curve falls outside the range for the weak positive control (−0.6 to −1.4). 

Non-Binder: The average lowest portion of curves across runs is greater than 75% activity 

(i.e. less than 25% displacement of radioligand), or the data do not fit the model. 

Page 50 of 241
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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 

Bmax: maximal binding capacity

Kd: dissociation constant

IC50: Concentration of the test substance at which 50% of radioligand is displaced from the AR 

by the competitor

Relative Binding Affinity (RBA): (IC50 of R1881 ÷ IC50 of test substance) × 100 

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The mean Kd for [3H]-R1881 was 0.903 nM (±0.060), and the 

estimated average Bmax was 4.5 fmol/100 μg protein (±0.08) for the single batch of prostate 

cytosol that was prepared. The Kd was within the range reported in the EPA validation 

program, but the Bmax was below the EPA values (7 to 16 fmol/100 μg protein). Confidence 

in these numbers is high according to the goodness of fit (R2 = 0.821-0.872) and the small 

variation among runs. 

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

Parameter Run 1 b Run 2 b Run 3 b Mean ± SE c

R2 (unweighted) 0.865 0.872 0.821 0.821-0.865 

Bmax (nM) 0.282 0.275 0.265 0.274 ± 0.005 

Bmax (fmol/100 μg protein) 4.7 4.6 4.4 4.5 ± 0.08 

Kd (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 concurrent replicates of each run. 

c The R2

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

R2

 Goodness of fit for curve calculated for specific binding 

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

H]-R1881 to the 

androgen receptor. 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 (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. 

Page 51 of 241
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METHOMYL / 090301 OCSPP 890.1150/ OECD None

FIGURE 1. Binding of [3H]-R1881 to the Androgen Receptor during the Saturation 

Binding Experiment.

FIGURE 2. Scatchard Plot of the Binding of [3H]-R1881 to the Androgen Receptor.

Page 52 of 241
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METHOMYL / 090301 OCSPP 890.1150/ OECD None

B. COMPETITIVE BINDING EXPERIMENT: Competitive binding experiment 

parameters are presented in Table 6 and shown 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 IC50 and RBA for methomyl could not be determined. Since the 

percent [3H]-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 IC50 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 Methomyl with AR from Rat Prostate Cytosol a

Parameter Run 1b Run 2b Run 3b Mean ± SEc

R2 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 IC50 (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 

IC50 (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 concurrent replicates within each run. 

c The range of r2 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. 

R2

 Goodness of fit. 

RBA Relative binding affinity

Page 53 of 241
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FIGURE 3. Percentage R1881 Bound to the Androgen Receptor in the Presence of Test 

Compounds (Run 1).

FIGURE 4. Percentage R1881 Bound to the Androgen Receptor in the Presence of Test 

Compounds (Run 2).

Page 54 of 241
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 Androgen Receptor in the Presence of Test 

Compounds (Run 3).

FIGURE 6. Mean Percentage R1881 Bound to the Androgen Receptor in the Presence of 

 Methomyl in Three Competitive Binding Assays. 

Page 55 of 241
Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 13 of 15 

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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 Kd (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 was 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 R1881 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 parameters (Top and Bottom % binding and Hill slope) were met for the 

second run. However, the goodness of fit of the 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 higher than the tolerance limits. 

The study reported that these variances in the performance criteria did not warrant 

performing another run since the values were only slightly outside of the suggested range. 

All runs were within or very close to the suggested performance criteria and therefore the 

assays were considered valid by the laboratory. Furthermore, the IC50s calculated were 

comparable between runs and within the expected ranges for R1881 and dexamethasone (a 

weak binder to the androgen receptor). 

The non-specific binding was <1% and the curve for the reference material showed that 

increasing concentrations of unlabeled 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. 

Page 56 of 241
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 consistent 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 experiment demonstrated that the 

androgen receptor was present in a reasonable concentration and functioning with 

appropriate affinity for the R1881 ligand. The mean Kd for [3H]-R1881 was 0.903 nM 

(±0.060), and the estimated average Bmax was 4.5 fmol/100 μg protein (±0.08) for the single 

batch of prostate cytosol that was prepared. The Kd was within the range reported in the 

EPA validation program, but the Bmax was below the EPA values (7-16 fmol/100 μg 

protein). Confidence in these numbers is high according to the goodness of fit (R2

 = 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 [3H]-R1881; all other NSB values were ≤20%.

In the competitive binding experiments, the estimated average log IC50 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 dexamethasone 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 parameters (Top and Bottom % binding and Hill slope) 

were met for the second run. However, the goodness of fit of the 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 higher than the tolerance limits. 

The non-specific binding was <1% and the curve for the reference material showed that 

increasing concentrations of unlabeled 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 criteria for both R1881 and dexamethasone were not achieved 

across the three runs, based on the estimated IC50s 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 competitive binding assays. It is unlikely that 

information from additional competitive binding runs would change the interpretation of this 

study. 

Page 57 of 241
Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 15 of 15 

METHOMYL / 090301 OCSPP 890.1150/ OECD None

Since the percent [3H]-R1881 bound to the AR was >75% (87-118% binding) at all 

concentrations of methomyl in all three runs, methomyl is classified 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 Bmax (fmol/100 μg protein) for the prostate cytosol in the saturation 

binding experiment was lower than the EPA guideline values. 

 The performance criteria (Top and Bottom percent binding and/or Hill slope) for R1881 

and dexamethasone were not achieved across the three runs in the competitive binding 

experiment. 

Page 58 of 241
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 59 of 241
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).

Page 60 of 241
METHOMYL I 103301 

Aromatase (Human Recombinant) Assay (2011) I Page I of 12 

OCSPP 890.1200 I 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: ---------,,..-,-,.--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.) 

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

CITATION: Snajdr, S. I. (2012). Methomyl (DPX-Xl 179) Technical: In vitro aromatase 

Inhibition using human recombinant microsomes. E.I. du Pont de Nemours and 

Company, DuPont Haskell Global Centers for Health & Environmental 

Sciences, P.O. Box 50, Newark, Delaware, 19714. Laboratory Report No.: 

DuPont-32081. January 5, 2012. MRID 48672002. Unpublished. 

SPONSOR: 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- io 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- 10 to 10- 5 M) using a known inhibitor, 4-hydroxyandrostenedione ( 4-0H ASDN), and the 

methomyl series (10- 10 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 activity controls (water 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- 1 

Page 61 of 241
Aromatase (Human Recombinant) Assay (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 was 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 response 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 requirements for an Aromatase assay (OCSPP 

890.1200). 

COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance, and Quality 

Assurance statements were provided. 

Page 62 of 241
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; ([3H]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); deionized 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 applicable 

Page 63 of 241
Aromatase (Human Recombinant) Assay (2011) / Page 4 of 12 

METHOMYL / 103301 OCSPP 890.1200 / OECD None

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

Source: GentestTM, 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-labeled 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 added to each incubation tube was 0.05 μCi. 

Methomyl was formulated in deionized water as it is readily soluble in water and the assay 

is run in an aqueous solution. No indication of precipitation of the 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 positive control substance 

(10−10 to 10−5 M; Table 4) were achieved by the addition of 20 μL of the dilution for a final 

assay volume of 2 mL. The final concentration of DMSO in the assay was 1%. 

Human recombinant microsomes were purchased from GentestTM, and were received on dry 

ice. Upon receipt, the microsomes were thawed rapidly in a water bath at approximately 

37 oC, placed on ice, aliquoted, 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 

[3H]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. Aromatase 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 water for methomyl and DMSO for the 

positive control (4-OH ASDN), each assay run contained 8 tubes for the full enzyme 

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METHOMYL / 103301 OCSPP 890.1200 / OECD None

activity (4 tubes with only water as the solvent, and 4 tubes with 1% DMSO) and 8 tubes for 

the background activity controls (4 with water, 4 with 1% DMSO). Two tubes of each full 

activity and background control were run at the beginning of the assay, and two of each 

control were run at the end of the assay. A full concentration curve in duplicate for the 

positive control, and a full concentration curve in triplicate for the test substance were 

established. The aromatase assay was conducted generally according to the procedures 

described in OCSPP 890.1200 (Section h, pp. 9-10). 

The amount of 3

H2O 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 assays were performed by personnel 

that demonstrated proficiency in conducting the assay as outlined in the test guideline. 

However, the actual data from the proficiency testing were not included 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 inhibitor 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 replicates within each sample type and concentration was 

<15%. 

(2) Demonstration of Proficiency of New Technician for Conducting Assay (when 

applicable): The positive control data for slope, top 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 limit criteria of 110% (Table 2). 

TABLE 2. Performance Criteria for the Positive Control a

Parameter Lower Limit Criteria Upper Limit Criteria 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 IC50 (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. 

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Aromatase (Human Recombinant) Assay (2011) / Page 6 of 12 

METHOMYL / 103301 OCSPP 890.1200 / OECD None

b. Proficiency Chemicals: Assays to classify proficiency 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 Activity 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 (Table 4). Solubility was visually assessed (presence of 

cloudiness or a precipitate), and no precipitation was observed. Although four independent 

experimental runs were conducted, the full activity control in only water in Run 2 had 

aromatase activity of approximately 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 lowest 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 components") contained buffer, propylene glycol, microsomal protein, ASDN/[3H]-

ASDN substrate and NADPH. 

c 4-OH ASDN solvent was DMSO. 

d Methomyl solvent was deionized water. 

Page 66 of 241
Aromatase (Human Recombinant) Assay (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 included in the report (Table 5). 

TABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) 

DPM/mL for each portion of extracted aqueous incubation mixture X 

Average DPM/mL for each aqueous portion (after extraction) X 

Total DPM for each aqueous portion (after extraction) X 

The total DPM present in the assay tube at initiation X 

The percentage of substrate converted to product X 

Total DPM after extraction corrected for background X 

Aromatase activity expressed in nmol·mg-protein−1·min−1 X 

Average aromatase activity in the full activity control tubes X 

Percentage of control activity remaining in the presence of various inhibitor concentrations X 

DPM Disintegrations per minute

2. Statistical Methods: Statistical analyses and displays such as summary tables, graphical 

displays, analysis of variance, and multiple comparisons were conducted Origin 8.5.1 

(OriginLab Corp., Northampton, MA). The response curve was fitted by weighted least 

squares nonlinear regression analysis with weights equal to 1/Y. In each run, the model 

parameters were estimated along with their standard errors. The variances (squared standard 

errors) for each parameter were compared using 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 IC50) based on whether the corresponding F-test was significant or 

not. Statistical significance was at the 0.05 significance level. Normality of the underlying 

data from each run was assessed from the residuals from the model. 

Assay drift was assessed by comparing the % full 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 categories presented in Table 6. 

TABLE 6. Interpretation 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% activityb

Equivocal 

Average lowest portion of curves across runs is greater 

than 75% activityb

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 cross 50%, the chemical is typically 

determined to be untestable in the aromatase assay. 

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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 all 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 control are shown in Figures 1 and 2. 

TABLE 7. Effect of Methomyl on Aromatase Activity (as percent of control) from Independent Runsa

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 control activity) for each concentration tested 

across all 3 runs is presented in Table 7 (presented above), along with the overall standard 

deviation and % CV. As a percent of control, aromatase activity for methomyl was 

Page 68 of 241
Aromatase (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 for 4-OH ASDN From Each Test Run (Runs 1, 

3, 4).

Page 69 of 241
Aromatase (Human Recombinant) Assay (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).

Page 70 of 241
Aromatase (Human Recombinant) Assay (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 model; therefore, log IC50 and Hill slope estimates were not determined 

for methomyl. For 4-OH ASDN, the estimated log IC50 averaged −7.2 and the Hill slope 

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

TABLE 8. Effect of Methomyl on Aromatase Activity (as Percent of Control) From Independent Runsa

Chemical Run 1 Run 2 Run 3 Mean SD %CVb

Log IC50 (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 methomyl 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 aromatase 

Page 71 of 241
Aromatase (Human Recombinant) Assay (2011) / Page 12 of 12 

METHOMYL / 103301 OCSPP 890.1200 / OECD None

inhibition in all three successful independent runs. The mean estimated log IC50 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 averaged 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 between 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 background 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 IC50, 

and % CV. The estimated log IC50 for 4-OH ASDN averaged −7.2 M and the slope was 

−1.0. 

For methomyl, aromatase activity at the lowest and the highest tested concentrations 

(10−10 and 10−3 M) were 105.11% and 92.93% of the control, respectively. 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 of the full activity control; 

therefore, these data could not be fit to the 4-parameter nonlinear 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 does 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 data were not provided. 

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

Page 72 of 241
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 73 of 241
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).

Page 74 of 241
METHOMYL/090301 

Estrogen Receptor Bindi ng (Rat Uterine Cytosol) (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 

Sign a tu re: ------Jc..._..;;...:'-b-~----==--­

Date: ---~-=-t-L-.:.._ __ 

Signature: _..,,,.'--..L-.,.+-~---­

Da te: ----""'--'-.L.:.--'----

Template version 08/2011 

DATA EVALUATION RECORD 

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

OCSPP 890.1250 

PC CODE: 090301 DP BARCODE: 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- 10 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. 

Page 75 of 241
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 14 

METHOMYL/090301 OCSPP 890.1250/ OECD None

In the competitive binding experiments, the three reference chemicals performed as expected. 

The average log IC50 was −9.16 M for 17β-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 [3H]-ligand (85-105% 

binding). 

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

ER in the three runs; therefore, a log IC50 and RBA could not be calculated 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 requirement for an Estrogen Receptor Binding 

Assay (OCSPP 890.1250). 

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

statements were provided. 

Page 76 of 241
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

3

CH CH3

CH3

3. Non-labeled ligand: 17β-Estradiol 

 Supplier: Sigma Aldrich, St Louis, MO 

 Catalog #: E8875 

 Batch #: 21k1267 

 Purity: 99% 

 CAS #: 50-28-2 

4. Radioactive ligand: [

3

H]-17β-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 #: 68-22-4 

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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, positive 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 Laboratories, 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 the cytosol, uteri were thawed on ice and 

homogenized at 0.1g of tissue per 1.0 mL ice-cold TEDG (Tris, EDTA, DTT, glycerol) + 

PMSF (phenylmethylsulfonyl fluoride) buffer. After homogenization, uteri were 

centrifuged for 10 min at 2500 × g at 4 oC. The resulting supernatants were transferred and 

centrifuged for 60 minutes at 105000 × g, discarding the resulting pellets. Protein 

concentration of the cytosol was determined to be 3.5 mg/mL 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 [3H]-17β-estradiol was performed to 

demonstrate that the ER was present in reasonable 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. 

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On the day of the assay, the specific activity of the stock solution [3H]-17β-estradiol was

adjusted for decay over time, and serial dilutions 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 17β-estradiol were prepared in a similar manner to achieve concentrations that 

were 100-fold greater than each respective radiolabeled 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 testing serial amounts of protein 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-concurrent runs. Each run 

included three replicates of each test substance at each concentration, resulting in the 72 

samples depicted in Table 2. 

TABLE 2. Saturation Binding Experiment Run a

Total binding b Non-specific 

binding c

Radioligand alone d

 Assay Components 

Tubes 1-24 Tubes 25-48 Tubes 49-72 

350 μL 300 μL --- TEDG + PMSF buffer 

50 μL 50 μL 50 μL [

3

H]-17β-estradiol (8 serial dilutions) e

--- 50 μL --- Non-labeled 17β-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]-17β-estradiol bound to ER 

c Non-specific binding = [3

H]-17β-estradiol and 100-fold greater non-labeled bound to ER 

d Total [3

H]-17β-estradiol alone for dpm determination at each concentration 

e Final concentrations of [3

H]-17β-estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. 

f Final concentrations of non-labeled 17β-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 added 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]-17β-estradiol, followed by vortexing, and 

centrifugation for 10 min at 1000 x g. An aliquot of supernatant was radioassayed by 

scintillation counting. The temperature was maintained 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. 

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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 performed as methomyl is readily soluble in water. On the day of 

the assay, the specific activity of the stock solution [3

H]-17β-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 protein 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 radioactivity added. Serial dilutions of the test 

substance, positive control (19-norethindrone), negative control (octyltriethoxysilane), and 

reference material (non-labeled 17β-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 determine total radioactivity, resulting in a total of 114 samples. 

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

Methomyl 

Positive control Negative control Reference Chemical 

19-norethindrone Octyltriethoxysilane Non-labeled 

17β-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, positive control, negative control, solvent control, or 

non-labeled 17β-estradiol; 390 μL of TEDG + PMSF buffer with [3

H]-17β-estradiol; and 100 μL of uterine 

cytosol (with ER), for a total of 500 μL. 

c Each concentration of each chemical was run in triplicate, for a total of 138 tubes per run; tubes 76-99 were 

skipped in sequence. 

d Solvent is water for methomyl and ethanol for the reference chemicals (<2%) 

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The procedures for assay incubation, separation of bound from free estradiol (washing with 

buffer and extraction with ethanol), followed by scintillation counting of bound [3H]-17βestradiol were similar to those used in the saturation binding experiment. 

C. DATA ANALYSIS: For the saturation binding experiment, total binding and non-specific 

binding data were modeled via non-linear regression using Origin 8.5.1 (OriginLab Corp., 

Northampton, MA. For the competitive binding assay, similar methods of nonlinear 

regression were used to fit a curve (for 17β-estradiol, the positive control, and the test 

substance) to the Hill equation formula which incorporated log IC50 as a parameter to be 

estimated. For parameters reported from the saturation binding experiment (Kd and Bmax) and 

competitive binding experiment (log IC50 and RBA), mean 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 material is based on the average of 

three runs. Each run was first individually 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 estradiol 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 data is above 75% (i.e., 

<25% of the radiolabeled estradiol has been displaced from the ER) or a 

binding curve cannot be fitted and the lowest average percent binding among 

concentration groups in the data is above 75%. 

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

test chemical concentration of 10−6

M and either a binding curve can be fit but 

≤50% of the radiolabeled estradiol has been displaced from the ER or a 

binding curve cannot be fit and the lowest average percent binding among 

concentration groups in the data is >50%. 

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

above. 

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

Run Classification Numerical Value 

Interactive 2 

Equivocal 1 

Not interactive 0 

Equivocal up to the limit of concentrations tested “missing” 

The values for each run were then averaged across runs and the chemical classified using the 

following ranges: 

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Test Material Classification Numerical Range 

Interactive average ≥1.5 

Equivocal 0.5≥ average <1.5 

Not interactive average <0.5 

Equivocal up to the limit of concentrations tested “missing” 

b. Descriptors for receptor binding: 

Bmax: maximum specific binding number (fmol ER/100 μg cytosol protein) measures the 

concentration of active receptor sites

Kd: dissociation constant (nM), measures the affinity of the receptor for its natural ligand

IC50: concentration of the test substance (M) at which 50% of the radioligand is displaced 

from the receptor

Relative Binding Affinity (RBA %): (IC50 of 17β-estradiol ÷ IC50 of test substance) × 100 

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: The saturation binding experiment 

parameters are presented in Figure 1 and Table 5. The mean Kd for [3H]-17β-estradiol was 

0.081 ± 0.002, and the mean estimated Bmax was 32.5 ± 2.25 fmol/100μg protein for the 

prepared at uterine cytosol. The Kd for each run was within the expected range of 0.03 to 

1.5 nM. The data produced a linear Scatchard plots (r2 = 0.88-0.91); Table 2. Confidence in 

these numbers is high due to the goodness of fit and the small variation among runs. 

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FIGURE 1. Binding of [3H]-17β-estradiol to the Estrogen Receptor during the Saturation 

Binding Experiment. 

FIGURE 2. Scatchard Plot of the Binding of [3H]-17β-estradiol to the Estrogen Receptor. 

Page 83 of 241
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TABLE 5. Saturation Binding Experiment of 17β-estradiol with Estrogen Receptor from Rat Uterine 

Cytosol a

Parameter Run 1b Run 2b Run 3b Mean ± SE c

R2 (unweighted) 0.913 0.880 0.889 0.880-0.913 

Bmax (nM) 0.020 0.024 0.024 0.023± 0.001 

Bmax (fmol/100 μg protein) 28.0 35.1 34.5 32.5 ± 2.25 

Kd (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 concurrent replicates of each run. 

c The range of R2 is reported and the mean and SE are reported for the other parameters. 

R2

 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 performed as expected. The average log IC50 was −9.16 M 

for 17β-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 [3H]-ligand (85-105% binding). 

Confidence in these numbers is high due to the small variation. Additional runs were 

unnecessary. 

Methomyl had no effect on specific binding (88-113%) of the [3H]-ligand indicating there 

was no competitive interactions with the ER. Therefore, a log IC50 and RBA could not be 

calculated for the methomyl. Based on the results of the three valid runs, methomyl is 

classified as not interactive with the estrogen receptor under the conditions of this assay 

(Table 7). 

FIGURE 3. Percentage E2 Bound to the Estrogen Receptor in the Presence of Estradiol.

Page 84 of 241
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FIGURE 4. Percentage E2 Bound to the Estrogen Receptor in the Presence of 

Octyltriethoxysilane. 

FIGURE 5. Percentage E2 Bound to the Estrogen Receptor in the Presence of 19-

Norethindrone. 

Page 85 of 241
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FIGURE 6. Percentage E2 Bound to the Estrogen 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) 17β-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 IC50 (M) 17β-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 

IC50 (M)d 17β-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 r2

, 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. r2

 is more appropriately expressed as a range, as opposed to a mean.

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TABLE 7. Binding Classification of Methomyl with Estrogen 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 competitive binding assay functioned 

properly, each run was evaluated using the following criteria. 

TABLE 8. Criterion a Tolerance 

Limit(s) Value Yes No 

17β-estradiol fitted curve parameters 

Loge 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 (log10(M)−1) −1.1 to −0.7 −1.0 to −0.9 X 

Weak Positive control (19-norethindrone) fitted curve parametersb

Loge residual SD NA ≤2.14 

Top (% binding) NA 96 to 103 

Bottom (% binding) NA −22.1 to 3.0 

Hill Slope (log10(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]-17β-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-norethindrone. Acceptance criteria were only 

defined for norethynodrel, which cannot be obtained commercially. The values reported were considered 

acceptable as they show 19-norethindrone to be an acceptable weak positive control.

NA Not applicable 

The top % binding for 17β-estradiol was only 76% for Run 1. However, the curve for the 

reference material showed that increasing concentrations of unlabeled 17β-estradiol 

displaced [3

H]-17β-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 receptor binding assay. 

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B. AGENCY COMMENTS: In the saturation binding experiment, the maximum binding 

capacity (Bmax) was 32.5 ± 2.25 fmol/100 μg protein and the dissociation constant (Kd) was 

0.081 ± 0.022 nM. For the competitive binding experiment, performance criteria were 

generally met for 17β-estradiol (with the exception top % binding for Run 1); the log PC50, 

log PC10, log EC50, and Hill Slope for the reference chemicals 17β-estradiol and 

octyltriethoxysilane all performed within the 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 similarly to the other two runs. The average log 

IC50 was −9.16 M for 17β-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 IC50 and RBA could not be calculated for methomyl. Based on the 

results of the three valid runs, methomyl is classified as not interactive with the ER as 

specific binding was >75% at all concentrations tested. 

C. STUDY DEFICIENCIES: The following deficiency was noted that is not considered to 

have had an adverse impact on the results, interpretation or conclusions of this study:

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

111%).

Page 88 of 241
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 89 of 241
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).

Page 90 of 241
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 Signaturey: ~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 

# JUL06LP276) at logarithmically increasing concentrations from 10- 108 to 10-3s 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 

very 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 

RPCMax < PC1 0 in two assay runs, methomyl was considered negative for estrogen receptor 

transcriptional activation in this test system. 

Page 91 of 241
Estrogen Receptor Transcriptional 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. 

 

Page 92 of 241
Estrogen Receptor Transcriptional 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

3CH CH3

CH3

2. Reference substances 

 17β-estradiol (strong estrogen; positive control)

 Supplier: Sigma-Aldrich, St. Louis, MO 

 Catalog and Lot #: E8875; 110M0138V 

 Purity: 100% 

 CAS # : 50-28-2 

 

 17α-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 

 

 17α-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) and DMSO (reference compounds) 

Solvent control 

(final concentration): 

Water – NA; DMSO – 0.1% 

Page 93 of 241
Estrogen Receptor Transcriptional Activation (2012) / Page 4 of 14 

METHOMYL/090301 OCSPP 890.1300/ OECD 455 

B. METHODS

1. Cell Culture: Stably-transfected hERα-HeLa-9903 cells were obtained from the Japanese 

Collection of Research Bioresources 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 red, supplemented with kanamycin (60 mg/L) 

and 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS; source not 

reported), in an incubator under 5% CO2 at 37oC. Upon reaching 75-90% confluency, cells 

were subcultured at least twice prior 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 1×104 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 transcriptional activation of the estrogen receptor was determined as 

described in CeeTox Standard Operating Protocol (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 

10-3.8M was conducted to determine the appropriate concentration range and to determine 

concentrations resulting in insolubility 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 tested 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. 

Page 94 of 241
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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 --- 

17α-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 0.01b, 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 0.1b and 1 mM 

1,3,5-Tris(4-hydroxyphenyl)benzene c 15797-52-1 10−12 to 10−5 Positive Cytotoxic at 100 μM. PCMax

approx. 50% of PC. Binds to 

hERα 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 = RPCMax ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs 

Negative = RPCMax 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 ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional 

activation. A positive result would indicate that the system is detecting activity other than that due to pure ERα, 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 with the test chemical: 

Reference Chemical CAS No. Concentration Range Class 

17β-estradiol 50-28-2 10−15 to 10−8 Strong estrogen 

17α-estradiol 57-91-0 10−13 to 10−6 Weak estrogen 

Corticosterone 50-22-6 10−11 to 10−4 Negative compound 

17α-methyltestosterone 58-18-4 10−12 to 10−5 Very weak agonist 

3. Data analysis: To obtain the relative transcriptional activity to the 1 nM E2 positive control 

(PC), the luminescence signals from the concurrent plate were analyzed by subtracting the 

mean value of the vehicle control from each well value to normalize the data; each 

normalized value was then divided by the mean value of the normalized PC. The resulting 

value was multiplied by 100 in order to express relative transcriptional activity as a 

percentage of the PC. The test material was defined as negative for inducing estrogen 

receptor transcriptional activation if the RPCMax < PC10 in at least 2 of 2 runs. Log EC50 and 

Hill slope values are calculated only if a positive response is observed. Coefficients of 

variation (CV) were calculated for the luminescence data triplicates. Concentrations 

showing >20% cytotoxicity or evidence of insolubility were excluded from analyses. 

4. Definitions 

EC50 = concentration of agonist that induces a response halfway between the baseline 

(bottom) and maximum (top) response 

PC10 = concentration of a test chemical at which the response is 10% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

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PC50 = 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 

RPCMax = 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 

PCMax = concentration of a test chemical inducing the RPCMax

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, logarithmically increasing concentrations from 10−10.8 to 

10−3.8 M were selected for the assay. 

TABLE 1. Preliminary Test for Solubility, Cytotoxicity, and Concentration-Selection for Methomyla

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. 

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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 --- 

17α-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 0.01b, 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 0.1b and 1 mM 

1,3,5-Tris(4-hydroxyphenyl)benzene c 15797-52-1 10−12 to 10−5 Positive Cytotoxic at 100 μM. PCMax

approx. 50% of PC. Binds to 

hERα 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 = RPCMax ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs 

Negative = RPCMax 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 ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional 

activation. A positive result would indicate that the system is detecting activity other than that due to pure ERα, and is 

therefore unacceptable. 

In the proficiency tests, the reference chemicals 17β-estradiol, 17α-estradiol and 17αmethyltestosterone 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 Reference Chemicals in the Proficiency test

Reference Chemical 

Parameter

Acceptable 

Range 

Values Acceptable 

Run 1 Run 2 Run 3 Yes No 

17β-estradiol

 Log PC50 −11.4 to −10.1 −9.6 −11.3 −10.6 Run 1 

 Log PC10 <−11 −11.5 −12.5 −12.1 X 

 Log EC50 −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 

17α-estradiol

 Log PC50 −9.6 to −8.1 −8.3 −9.4 −8.7 X 

 Log PC10 −10.7 to −9.3 −9.3 −10.5 −9.9 X 

 Log EC50 −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 

17α-methyltestosterone

 Log PC50 −6.0 to −5.1 −6.2 NC −5.2 Run 1, 2 

 Log PC10 −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 three estrogenic reference chemicals 

are shown in Figures 1 – 3, and the values derived from the concentration-response curves 

(e.g., log PC50, log PC10, log EC50, Hill slope) for the concurrently run reference materials 

are presented in Table 3. 

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METHOMYL/090301 OCSPP 890.1300/ OECD 455 

Although all four performance criteria were generally met for 17β-estradiol, 17α-estradiol 

and corticosterone, the available data indicate that both runs were invalid due to the lack of 

responsiveness of the cells to 17α-methyltestosterone. For 17α-methyltestosterone, the 

mean RPCMax 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 highest concentration in 

the first run and, in both runs, the maximum response of the cells to both 17β-estradiol and 

17α-estradiol exceeded (149-170%) the response of the PC. 

TABLE 3. Performance Criteria for Reference Chemicalsa

Reference Chemical 

Parameter Acceptable Range Values Acceptable 

Run 1 Run 2 Yes No 

17β-estradiol

 Log PC50 −11.4 to −10.1 -10.5 -10.7 X 

 Log PC10 <−11 -11.5 -11.8 X 

 Log EC50 −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 

17α-estradiol

 Log PC50 −9.6 to −8.1 -8.8 -8.5 X 

 Log PC10 −10.7 to −9.3 -9.8 -9.4 X 

 Log EC50 −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 

17α-methyltestosterone

 Log PC50 −6.0 to −5.1 NA b NA X 

 Log PC10 −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; 17α-methyltestosterone did not achieve 50% activation at up to 10−5 M. 

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FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of 17β-Estradiol 

Compared to the Positive Control. 

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FIGURE 2. Fold Induction of Relative Transcription Activation (RTA) of 17α-Estradiol 

Compared to the Positive Control. 

 

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Estrogen Receptor Transcriptional Activation (2012) / Page 11 of 14 

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FIGURE 3. Fold Induction of Relative Transcription Activation (RTA) of 17αMethyltestosterone 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 value 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 PC50 (50% of the maximum response) for E2 in this 

assay is 244258 for the first run and 173310 for the second run, and the PC10 (10% of the 

maximum response) is 48852 for the first run and 34662 for the second run.

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 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 data obtained from pages 31-32 of the study report. 

b Positive control was 17β-estradiol (E2) at 1 nM. 

c Fold-induction = (mean TA of PC)/(mean TA of VC) 

2. Test Material: Relative (to the PC) transcriptional activation at each concentration of the 

test chemical during the two assay runs is presented in Table 5. The concentration-response 

curves depicting fold induction of relative transcriptional activation is presented in Figure 4 

below. The mean RPCMax was 3.1% for the first run and 3.7% for the second, and the 

associated PCMax was 10−6.8 M for the first run and 10−6.8 M for the second run. Because the 

RPCMax < PC10 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 EC50b NA NA 

Hill Slopeb NA NA 

RPCMax 3.1 3.7 

PCMax 10−6.8 10−6.8

PC50 NA NA 

PC10 NA NA 

a Data were obtained from pages 21-22 of the study report 

NA = Not Applicable

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Estrogen Receptor Transcriptional Activation (2012) / Page 13 of 14 

METHOMYL/090301 OCSPP 890.1300/ OECD 455 

FIGURE 4. Fold Induction of Relative Transcription Activation (RTA) of Methomyl 

Compared to the Positive Control. 

3. Performance Criteria: While the log PC50, log PC10, log EC50, 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. 

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Estrogen Receptor Transcriptional 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 activation assay, methomyl did 

not result in an increase in luciferase activity (RPCMax <10%) at any of the viable 

concentrations tested; methomyl is not an agonist of human estrogen receptor alpha (hERα) 

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 concentrations, the RTA of methomyl was ≤3.7%, indicating it 

is negative for estrogenic activity under 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 17α-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 

RPCMax < PC10 in two assay runs, methomyl was considered 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, 

17α-methyltestosterone. 

 Certificates of Analysis were not provided for the reference chemicals. 

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

Page 104 of 241
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'

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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). 

 

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

EPA MRID Number 48701401 

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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 24.8o

C and a pH 

of 7.88 to 8.33. 

Adult male and female survival was unaffected by treatment 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 negative 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. 

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

EPA MRID Number 48701401 

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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, Static Renewal): Continuous flow-through 

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Table 1: Summary of Reproductive and HPG Effects1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Methomyl. 

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity 

Fert. 

Success 

Tubercle Score GSI Gonadal Histo. Plasma VTG Plasma T Plasma E2 

M F M F M F M F M F M F 

0.0947 No No No No No No No No No No NA NA NA NA

0.312 Yes Yes No No No Yes No No No No NA NA NA NA

1.03 Yes Yes No No No Yes Yes Yes No No NA NA NA NA

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. M Male. NA Not applicable. T Testosterone. VTG Vitellogenin.

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. 

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

EPA MRID Number 48701401 

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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.

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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. 

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B. Test organism: 

Table 2: General Information About the Test Species and Acclimation.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead Minnow EPA recommends fathead minnow 

(Pimephales promelas). Species scientific name: Pimephales promelas

Species strain (if stated): Not reported Were fish obtained from a single laboratory stock? 

Yes In-house stock. EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same 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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: There were no mortalities during the 7 

days prior to pre-exposure period. Fish did not receive any treatment for disease during acclimation period. Behavioral abnormalities or clinical signs were 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 brine 

shrimp 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, UT 

Frequency of feeding: 3 times/day 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding: Fish not fed 12 hours 

prior to test termination. 

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: Ca. 6 months EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months 

old.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Mean weight of males at test initiation (if determined): 

Not reported Based on 10 males used to stock 

aquaria 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 (if determined): 

Not reported Individual weights within ± 20% of the 

estimated mean. 

Mean weight of females at test initiation (if determined): 

Not reported Based on 10 females used to stock 

aquaria for pre-exposure phase. 

Range of individual weights (females) at test initiation (if determined): 

Not reported Individual weights within ± 10% of the 

estimated mean. 

Mean length of males at test initiation (if determined): 

Not reported 

Mean length of females at test initiation (if determined): 

Not reported 

Duration of pre-exposure phase: 14 days EPA recommends a minimum of 14 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Were pre-exposure conditions identical 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: 36 Additional breeding groups were 

maintained to account for a potential lack of spawning and/or mortality during 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: 2 Number of females per tank: 4 Pre-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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of spawns during preexposure: > two times in 7 days and at least 15 

eggs/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. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Proportional flow-through EPA recommends the use of a flowthrough system. As noted in the 

Corrections and Clarifications document1 , 

the use of a static renewal system is not 

recommended for this assay.

1 U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor 

Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf). 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of flow-through dilution system: 

Flow-through diluters Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: 10 volume additions/test 

vessel/day. 8.4 cycles/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.

2 Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems: 

A proportional diluter was used to produce nominal concentrations. Flowsplitting chambers were used between diluter cells and the 4 replicate test vessels. Treatment recoveries prior to test initiation were 101-109% of the applied. Flow splitting accuracy was not reported; however proper operation of the diluter was 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).

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of dilution water: Blended moderately hard 

well water and reverse osmosis demineralized well water. 

Dilution water was aerated, heated, filtered and UV sterilized prior to delivery to 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 analyzed for pesticides, heavy metals, and other contaminants? 

Yes 

Test 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 cm 

long x 22 cm high.

EPA recommends the use of 18 L test 

chambers (e.g., 40 x 20 x 20 cm).

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Fill volume: 12 cm depth EPA recommends 10 L solution per tank. Spawning substrate material: Stainless steel screened 

trays. 

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 reported Additional details on exposure system: 

 None. 

Table 5: Summary of Water Quality Characteristics in the Test System.

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Temperature (°C) 24.2 24.8 24.5 Continuously 

EPA recommends temperature 25±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 1oC.

pH 7.88 8.33 7.6 Weekly EPA recommends pH 6.5 to 9.0.

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Dissolved oxygen (mg/L) 5.17 8.24 6.7 Weekly EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3) 148 162 155 Weekly 

EPA recommends total alkalinity > 20 

mg/L as CaCO3.

Hardness (mg/L as CaCO3) 136 154 145 Weekly Total organic carbon (mg C/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 

Pg/L. 

Residual chlorine (

Pg/L) Not reported Not reported Not reported None 

EPA recommends that residual chlorine 

in dilution water be < 10 

Pg/L. 

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Conductivity (

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: NA Not applicable. 1 Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels. 

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D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or 

Remarks 

Guideline Recommendations 

Was a range-finder conducted? No EPA recommends conducting a range-finder if 96-hour LC50 data for the 

fathead minnow are unavailable. 

If yes, what was the method for determining the highest test concentration in the rangefinder? 

NA EPA recommends that the highest test concentration be selected based on 

toxicity data for other fish studies or species, if available. Otherwise, either the 

solubility limit of the test compound or 100 mg/L (whichever is lower) is 

appropriate.

Species: NA Life 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. 

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Parameter Value(s) Details or 

Remarks 

Guideline Recommendations 

Additional details: None. EPA recommends conducting a range-finder with five test concentrations plus a 

control (six total treatment levels), with four females and two males per 

exposure tank (36 fish total). The number of mortalities that occur may be used 

to develop a concentration-response curve. 

Based upon the results, the highest concentration that does not result in 

increased mortality or signs of overt morbidity compared to controls, or 1/3 the 

derived 96-hr LC50, may be selected as the highest exposure concentration in 

the 21-day test.

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Test duration: 21 days EPA recommends that the duration of the 

definitive test be 21 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Method for selecting the highest test concentration in the definitive test:

1.0 mg a.i./L Based on existing acute methomyl 

aquatic toxicity data for fathead minnows 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 LC50), whichever 

concentration is lowest.

Reference study citation (if applicable):

Not reported 

Separation of test concentrations:

Ca. 3 fold EPA suggests that a concentration 

separation of between 0.33 (or three-fold) 

and 0.1 (or ten-fold) is scientifically 

acceptable1 . 

Number of test concentrations: 3 EPA recommends a minimum of 3 

concentrations and a control, plus solvent 

control if appropriate.

Are nominal concentrations adjusted for purity? 

Yes 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Indicate the type of values presented for measured concentrations: 

Mean measured 

Limit 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was the randomized complete block 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 solvent control (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 test item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a solvent used? No Solvent type (if applicable): NA Maximum 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? No Positive control (if applicable): NA Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 16 hrs light: 

8 hrs dark 

EPA recommends photoperiod 16:8 

(light:dark).

3 Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. Aquatic 

Toxicology, 76, pp. 69–92. 

4 OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing 

and Assessment. No. 23. Paris, France. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Light intensity at water’s surface: 

634.4-900.9 lux Fluorescents EPA recommends light intensity 540 – 

1080 lux (at water’s surface).

Additional details: The test solutions were reported as 

clear and colorless with no visible precipitates, films or undissolved test substance. 

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Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Methomyl. 

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Mean Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Control (dilution water only) 

<0.0124 <LOQ NA 

EPA recommends that test item 

concentrations be maintained at a coefficient 

of variation (CV) 20%. Solvent control (if 

applicable) 

NA NA NA 

Treatment 1 0.090 0.0947 1.9 Treatment 2 0.30 0.312 1.2 Treatment 3 1.0 1.03 3.5 Diluter Stock NA NA NA 

Abbreviations: CV Coefficient of variation. LOQ=0.0124 mg a.i./L. 

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E. Observations

Biological Endpoints: Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination (Day 21), 

secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, 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, fertilization success, secondary sex characteristics, and other clinical signs occur at least 

daily. At test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, 

plasma vitellogenin, and plasma sex steroids (testosterone and 17-estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of 

gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. 

Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal behavior. 

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II. RESULTS AND DISCUSSION A. Results 

There were no treatment-related mortalities observed in the study, and no mortalities in the controls; however there were two incidental mortalities (Table 9). 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. Also 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. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

N1 # Surviving % Survival 

N1 # Surviving % Survival 

Control (<LOQ) 8 8 100 15-162 15 100 

0.0947 7-83 8 100 15-162 15 100

0.312 8-93 8 100 16 16 100

1.03 8 8 100 16 16 100

Abbreviations: NA Not applicable. 1 Total number of fish at test initiation. 2 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. 

3 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. 

LOQ=0.0124 mg a.i./L. 

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Mean 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, 1.732 and 

1.752 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 author did 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 Weight Length 

Males Females Males Females 

n 

Mean

(g) 

±SD n 

Mean

(g) 

±SD n 

Mean

(mm) 

±SD n 

Mean

(mm) 

±SD 

Control (<LOQ) 4 4.374 0.533 4 1.867 0.079 4 NA NA 4 NA NA 

0.0947 4 4.089 0.328 4 1.834 0.12 4 NA NA 4 NA NA 

0.312 4 3.947 0.348 4 1.732 0.18 4 NA NA 4 NA NA 

1.03 4 3.795 0.167 4 1.752 0.23 4 NA NA 4 NA NA 

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

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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]

Fecundity1 ± 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.

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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. 

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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 GSI1

(%) 

±SD n 

Mean GSI1

(%) 

±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.

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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 Stage1 n Median Stage2

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. 

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Table 15: Gonadal Histopathology in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[meanmeasured]

Diagnostic Observations1

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.

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Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Control (<LOQ) 0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 4 NA NA NA NA NA NA NA NA NA NA 

0.0947 

0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 4 NA NA NA NA NA NA NA NA NA NA 

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased 

Proportion of 

Spermatogonia 

Increased Vascular 

or Interstitial 

Proteinaceous Fluid 

Asynchronous 

Gonad 

Development 

Altered Proportions 

of Spermatocytes 

or Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.312 

0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 4 NA NA NA NA NA NA NA NA NA NA 

1.03 

0 NA NA NA NA NA NA NA NA NA NA 

1 NA NA NA NA NA NA NA NA NA NA 

2 NA NA NA NA NA NA NA NA NA NA 3 NA NA NA NA NA NA NA NA NA NA 4 NA NA NA NA 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.

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

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Table 17: Gonadal Histopathology in Female Fathead Minnow (Pimephales promelas). 

Treatment 

(mg a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

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. 

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Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow (Pimephales 

promelas).

Treatment 

(mg a.i./L) 

[meanmeasured]

Additional Diagnostic Observations1

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. 

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

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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.

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

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Mean 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) 

Males Females Males Females 

n 

Mean

(ng/mL 

plasma) 

±SD N 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD 

Control (<LOQ) NA NA NA NA NA NA NA NA NA NA NA NA

0.0947 NA NA NA NA NA NA NA NA NA NA NA NA

0.312 NA NA NA NA NA NA NA NA NA NA NA NA

1.03 NA NA NA NA NA NA NA NA NA NA NA NA

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

LOQ=0.0124 mg a.i./L. 

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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 (Table 21). In the 1.03 mg a.i./L treatment group, there was one male that displayed irregular respiration (Day 17) and one female that showed a loss of equilibrium (Day 21). There were also two females in the 

control 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 mg a.i./L treatment were observed to have flared operculum at test termination. All other fish in the negative controls and treatment groups appeared normal, and no overt signs of toxicity were observed during the test period. 

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Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs 

Males Females 

Type n Incidence Type n Incidence 

Control (<LOQ) None 8 0 Vertical banding 15 2 

0.0947 None 7 0 None1 15 0 

0.312 None 9 0 None1 16 0 

1.03 Flared operculum 8 3 

Loss of equilibrium 16 1 

Flared operculum 16 1 

1 On Day 21, one female in the low treatment and 2 in the middle treatment were coded as “DC” in the study report, however, the definition of “DC” 

was not provided. 

LOQ=0.0124 mg a.i./L. 

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

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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 pd0.05 (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 homogeneity 

tests 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 

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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 significant 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 treated 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 control or treated groups. There were no treatment-related clinical signs of 

toxicity. 

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Table 22: Reproductive and HPG Endpoints1,2 for Male Fathead Minnow (Pimephales promelas) in the FSTRA with Methomyl. 

Treatment 

(mg a.i./L) 

[mean-measured]

Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG3 Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Negative Control (<LOQ) 

36 NA 0 NA No 0 NA NA NA NA NA 

0.0947 38 0.56 -4.57 0.98 No -34.8 0.89 NA NA NA NA 

0.312 32 0.28 4.95 0.97 No 10.9 0.89 NA NA NA NA 

1.03 30 0.09 1.71 1.0 Yes 116.03 0.89 NA NA NA NA 

Statistical Test Jonckheere-Terpstra Dunnett’s NA Mann-Whitney NA NA Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. NA Not applicable.

1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding 

histopathology may be heavily weighted by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 This 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 an 

increase in male VTG from the population of fish at this treatment compared to the control. 

LOQ=0.0124 mg a.i./L. 

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Table 23: Reproductive and HPG Endpoints1,2 for Female Fathead Minnow (Pimephales promelas) in the FSTRA with Methomyl. 

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity Fert. Success 

Tubercle 

Score 

GSI 

Gonadal 

Staging 

and Histo. 

Plasma VTG Plasma T Plasma E2 

% 

Diff. 

p 

% 

Diff. 

P Median p 

% 

Diff. 

p 

Effect? 

(Yes/No) 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff. 

p 

Control (<LOQ) 

0 NA 0 NA 0 NA 0 NA No 0 NA NA NA NA NA 

0.0947 -12.6 0.15 2.17 0.32 0 NA 0.00 0.56 No 37.9 0.15 NA NA NA NA 

0.312 -23.3 0.02 -1.63 0.021 0 NA -15.4 0.011 No 23.2 0.53 NA NA NA NA 

1.03 -77.2 0.0002 -33.2 0.0002 0 NA -21.0 0.0065 Yes -7.64 0.95 NA NA NA NA 

Statistical Test 

JonckheereTerpstra Jonckheere- Terpstra NA Jonckheere- Terpstra NA Dunnett’s NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin.

1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regarding 

histopathology may be heavily weighted by the expert opinion of a board-certified pathologist. 

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

LOQ=0.0124 mg a.i./L. 

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

EPA MRID Number 48701401 

Page 49 of 51 

Table 24: Growth Endpoints1,2 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. 

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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-monotonic 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 control and treated fish (5 fish in the control; 3 fish in 

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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. 

Page 155 of 241
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

Analysis ID: 00-8527-2873 Endpoint: Fecundity CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.0947 1.13 2.68 16 6 0.5525 CDF Non-Significant Effect

0.312 2.1 2.68 16 6 0.1373 CDF Non-Significant Effect

1.03* 6.96 2.68 16 6 <0.0001 CDF Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 29.7% 0.312 1.03 0.5669

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

Total 4857.938 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.883 11.3 0.8294 Equal Variances

Distribution Shapiro-Wilk W Normality 0.928 0.841 0.2252 Normal Distribution

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

Fecundity Summary

0 Negative Control 4 53.8 44.9 62.6 51.5 50 62 2.78 10.3% 0.0%

0.0947 4 47 32 62 49 34 56 4.71 20.0% 12.6%

0.312 4 41.3 25.9 56.6 42 31 50 4.84 23.5% 23.3%

1.03 4 12.3 -1.15 25.7 12 3 22 4.21 68.7% 77.2% Centered Untransformed

 Rankits

-2

-4

-6

-8

-10

-12

-14

0

2

4

6

8

10

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

Fecundity

 Group

Reject Null

Reject Null

0

10

20

30

40

50

60

70

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 156 of 241
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

Analysis ID: 20-4554-5955 Endpoint: Fecundity CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.0947 1.02 1.64 1 -2 0.1536 Asymp Non-Significant Effect

0.312* 2.06 1.64 2 -2 0.0197 Asymp Significant Effect

1.03* 3.56 1.64 2 -2 0.0002 Asymp Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.0947 0.312 0.1719

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

Total 4857.938 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.883 11.3 0.8294 Equal Variances

Distribution Shapiro-Wilk W Normality 0.928 0.841 0.2252 Normal Distribution

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

Fecundity Summary

0 Negative Control 4 53.8 44.9 62.6 51.5 50 62 2.78 10.3% 0.0%

0.0947 4 47 32 62 49 34 56 4.71 20.0% 12.6%

0.312 4 41.3 25.9 56.6 42 31 50 4.84 23.5% 23.3%

1.03 4 12.3 -1.15 25.7 12 3 22 4.21 68.7% 77.2% Centered Untransformed

 Rankits

-2

-4

-6

-8

-10

-12

-14

0

2

4

6

8

10

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

Fecundity

 Group

0

10

20

30

40

50

60

70

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 157 of 241
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

Analysis ID: 06-8901-7753 Endpoint: FemaleBodyWt CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.0947 0.284 2.68 0.307 6 0.9837 CDF Non-Significant Effect

0.312 1.18 2.68 0.307 6 0.5236 CDF Non-Significant Effect

1.03 1.01 2.68 0.307 6 0.6324 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 16.4% 1.03 >1.03 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

Total 0.3637237 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 2.93 11.3 0.4019 Equal Variances

Distribution Shapiro-Wilk W Normality 0.978 0.841 0.9496 Normal Distribution

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

FemaleBodyWt Summary

0 Negative Control 4 1.87 1.74 1.99 1.89 1.75 1.94 0.0396 4.25% 0.0%

0.0947 4 1.83 1.64 2.03 1.86 1.68 1.94 0.0607 6.62% 1.74%

0.312 4 1.73 1.45 2.02 1.77 1.5 1.9 0.0898 10.4% 7.22%

1.03 4 1.75 1.39 2.11 1.74 1.49 2.04 0.113 12.9% 6.17% Centered Untransformed

 Rankits

-0.1

-0.2

-0.3

0.0

0.1

0.2

0.3

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

FemaleBodyWt

 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

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 158 of 241
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

Analysis ID: 17-4347-5156 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Dunnett T3 Multiple Comparison Test

DF P-Type

Negative Control 0.0947 0 4.43 2.56 3 >0.05 CDF Non-Significant Effect

0.312 2.83 3.31 2.34 5 0.0869 CDF Non-Significant Effect

1.03 1.76 3.77 5.86 4 0.3459 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 45.0% 1.03 >1.03 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

Total 54.28437 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Mod Levene Equality of Variance 6.24 5.95 0.0085 Unequal Variances

Variances Levene Equality of Variance 7.16 5.95 0.0052 Unequal Variances

Distribution Shapiro-Wilk W Normality 0.912 0.841 0.1235 Normal Distribution

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

FemaleGSI Summary

0 Negative Control 4 13 11.2 14.8 13 12 14 0.577 8.88% 0.0%

0.0947 4 13 13 13 13 13 13 0 0.0% 0.0%

0.312 4 11 9.7 12.3 11 10 12 0.408 7.42% 15.4%

1.03 4 10.3 5.69 14.9 9.8 7.5 14 1.44 28.0% 21.0% Centered Untransformed

 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

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

FemaleGSI

 Group

Reject Null

Reject Null

0

5

10

15

20

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 159 of 241
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

Analysis ID: 19-7827-7497 Endpoint: FemaleGSI CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.0947 8 NA -2 0.5571 Exact Non-Significant Effect

0.312* 2.28 1.64 3 -2 0.0114 Asymp Significant Effect

1.03* 2.48 1.64 4 -2 0.0065 Asymp Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.0947 0.312 0.1719

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

Total 54.28437 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Mod Levene Equality of Variance 6.24 5.95 0.0085 Unequal Variances

Variances Levene Equality of Variance 7.16 5.95 0.0052 Unequal Variances

Distribution Shapiro-Wilk W Normality 0.912 0.841 0.1235 Normal Distribution

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

FemaleGSI Summary

0 Negative Control 4 13 11.2 14.8 13 12 14 0.577 8.88% 0.0%

0.0947 4 13 13 13 13 13 13 0 0.0% 0.0%

0.312 4 11 9.7 12.3 11 10 12 0.408 7.42% 15.4%

1.03 4 10.3 5.69 14.9 9.8 7.5 14 1.44 28.0% 21.0% Centered Untransformed

 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

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

FemaleGSI

 Group

0

2

4

6

8

10

12

14

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 160 of 241
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

Analysis ID: 16-2853-4104 Endpoint: FemaleMedianTubercleScore CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Mann-Whitney U Two-Sample Test

DF P-Type

Negative Control 0.0947 8 NA 1 6 1.0000 Exact Non-Significant Effect

0.312 8 NA 1 6 1.0000 Exact Non-Significant Effect

1.03 8 NA 1 6 1.0000 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 1.03 >1.03 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

Total 0 15

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

FemaleMedianTubercleScore Summary

0 Negative Control 4 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 0 0 0 0 0 Centered Untransformed

 Rankits

0.0E+00

2.5E-01

5.0E-01

7.5E-01

1.0E+00

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

FemaleMedianTubercleScore

 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

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 161 of 241
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

Analysis ID: 11-3914-0061 Endpoint: FemaleVTG CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.0947 2.08 2.72 2E+06 6 0.1462 CDF Non-Significant Effect

0.312 1.18 2.72 2E+06 5 0.5289 CDF Non-Significant Effect

1.03 0.42 2.72 2E+06 6 0.9529 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 49.5% 1.03 >1.03 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

Total 1.59613E+13 14

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 0.934 11.3 0.8172 Equal Variances

Distribution Shapiro-Wilk W Normality 0.929 0.833 0.2630 Normal Distribution

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

FemaleVTG Summary

0 Negative Control 4 3.58E+6 2.03E+6 5.14E+6 3580000 2.71E+6 4.46E+6 4.89E+5 27.3% 0.0%

0.0947 4 4.94E+6 3.22E+6 6.66E+6 5010000 3.61E+6 6.13E+6 5.39E+5 21.8% -37.9%

0.312 3 4.41E+6 3.10E+6 5.72E+6 4410000 3.89E+6 4.94E+6 3.04E+5 11.9% -23.2%

1.03 4 3.31E+6 1.88E+6 4.74E+6 3240000 2.44E+6 4.31E+6 4.49E+5 27.1% 7.64% Centered Untransformed

 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

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

FemaleVTG

 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

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 162 of 241
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

Analysis ID: 15-1782-6111 Endpoint: Fertility CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Mann-Whitney U Two-Sample Test

DF P-Type

Negative Control 0.0947 9.5 NA 3 6 0.7429 Exact Non-Significant Effect

0.312 12 NA 0 6 0.2286 Exact Non-Significant Effect

1.03* 16 NA 0 6 0.0286 Exact Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 24.3% 0.312 1.03 0.5669

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

Total 4856 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 25.1 11.3 <0.0001 Unequal Variances

Distribution Shapiro-Wilk W Normality 0.8 0.841 0.0027 Non-normal Distribution

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

Fertility Summary

0 Negative Control 4 92 81.3 103 95 82 96 3.37 7.32% 0.0%

0.0947 4 94 91.7 96.3 93.5 93 96 0.707 1.5% -2.17%

0.312 4 90.5 88.9 92.1 90 90 92 0.5 1.1% 1.63%

1.03 4 61.5 21.8 101 68.5 28 81 12.5 40.5% 33.2% Centered Untransformed

 Rankits

-5

-10

-15

-20

-25

-30

-35

0

5

10

15

20

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

Fertility

 Group

0

20

40

60

80

100

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 163 of 241
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

Analysis ID: 17-5007-2084 Endpoint: Fertility CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.0947 0.461 1.64 3 -2 0.3224 Asymp Non-Significant Effect

0.312* 2.03 1.64 4 -2 0.0213 Asymp Significant Effect

1.03* 3.54 1.64 4 -2 0.0002 Asymp Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.0947 0.312 0.1719

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

Total 4856 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 25.1 11.3 <0.0001 Unequal Variances

Distribution Shapiro-Wilk W Normality 0.8 0.841 0.0027 Non-normal Distribution

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

Fertility Summary

0 Negative Control 4 92 81.3 103 95 82 96 3.37 7.32% 0.0%

0.0947 4 94 91.7 96.3 93.5 93 96 0.707 1.5% -2.17%

0.312 4 90.5 88.9 92.1 90 90 92 0.5 1.1% 1.63%

1.03 4 61.5 21.8 101 68.5 28 81 12.5 40.5% 33.2% Centered Untransformed

 Rankits

-5

-10

-15

-20

-25

-30

-35

0

5

10

15

20

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

Fertility

 Group

0

20

40

60

80

100

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 164 of 241
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

Analysis ID: 19-9910-1693 Endpoint: MaleBodyWt CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.0947 1.1 2.68 0.697 6 0.5743 CDF Non-Significant Effect

0.312 1.64 2.68 0.697 6 0.2811 CDF Non-Significant Effect

1.03 2.23 2.68 0.697 6 0.1098 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 15.9% 1.03 >1.03 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

Total 2.348266 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 3.08 11.3 0.3790 Equal Variances

Distribution Shapiro-Wilk W Normality 0.889 0.841 0.0539 Normal Distribution

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

MaleBodyWt Summary

0 Negative Control 4 4.37 3.53 5.22 4.57 3.59 4.77 0.266 12.2% 0.0%

0.0947 4 4.09 3.57 4.61 4.21 3.61 4.33 0.164 8.02% 6.52%

0.312 4 3.95 3.39 4.5 3.99 3.49 4.32 0.174 8.81% 9.76%

1.03 4 3.79 3.53 4.06 3.79 3.63 3.97 0.0835 4.4% 13.2% Centered Untransformed

 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

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

MaleBodyWt

 Group

Reject Null

Reject Null

0

1

2

3

4

5

6

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 165 of 241
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

Analysis ID: 07-6773-6472 Endpoint: MaleBodyWt CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.0947 12 NA -2 0.1714 Exact Non-Significant Effect

0.312* 36 NA -2 0.0463 Exact Significant Effect

1.03* 70 NA -2 0.0215 Exact Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.0947 0.312 0.1719

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

Total 2.348266 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 3.08 11.3 0.3790 Equal Variances

Distribution Shapiro-Wilk W Normality 0.889 0.841 0.0539 Normal Distribution

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

MaleBodyWt Summary

0 Negative Control 4 4.37 3.53 5.22 4.57 3.59 4.77 0.266 12.2% 0.0%

0.0947 4 4.09 3.57 4.61 4.21 3.61 4.33 0.164 8.02% 6.52%

0.312 4 3.95 3.39 4.5 3.99 3.49 4.32 0.174 8.81% 9.76%

1.03 4 3.79 3.53 4.06 3.79 3.63 3.97 0.0835 4.4% 13.2% Centered Untransformed

 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

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

MaleBodyWt

 Group

0

1

2

3

4

5

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 166 of 241
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

Analysis ID: 13-6902-2141 Endpoint: MaleGSI CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.0947 0.307 2.68 0.524 6 0.9798 CDF Non-Significant Effect

0.312 0.333 2.68 0.524 6 0.9747 CDF Non-Significant Effect

1.03 0.115 2.68 0.524 6 0.9989 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 40.0% 1.03 >1.03 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

Total 0.9496938 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 4.48 11.3 0.2137 Equal Variances

Distribution Shapiro-Wilk W Normality 0.926 0.841 0.2132 Normal Distribution

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

MaleGSI Summary

0 Negative Control 4 1.31 0.631 1.99 1.13 1.03 1.95 0.214 32.6% 0.0%

0.0947 4 1.25 0.864 1.64 1.24 1.01 1.53 0.122 19.5% 4.57%

0.312 4 1.38 1.01 1.74 1.45 1.05 1.56 0.114 16.6% -4.95%

1.03 4 1.34 1.17 1.5 1.35 1.2 1.44 0.0524 7.84% -1.71% Centered Untransformed

 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

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

MaleGSI

 Group

Reject Null

Reject Null

0.0

0.5

1.0

1.5

2.0

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 167 of 241
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

Analysis ID: 14-4243-4245 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Dunnett Multiple Comparison Test

DF P-Type

Negative Control 0.0947 0.483 2.68 11.1 6 0.9301 CDF Non-Significant Effect

0.312 0.785 2.68 11.1 6 0.7757 CDF Non-Significant Effect

1.03 1.33 2.68 11.1 6 0.4356 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 31.8% 1.03 >1.03 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

Total 545.4375 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 2.72 11.3 0.4362 Equal Variances

Distribution Shapiro-Wilk W Normality 0.909 0.841 0.1122 Normal Distribution

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

MaleMedianTubercleScore Summary

0 Negative Control 4 35 30.7 39.3 36 31 37 1.35 7.74% 0.0%

0.0947 4 37 24.1 49.9 37.5 29 44 4.06 22.0% -5.71%

0.312 4 31.8 23 40.5 33 24 37 2.75 17.3% 9.29%

1.03 4 29.5 20.3 38.7 29.5 24 35 2.9 19.7% 15.7% Centered Untransformed

 Rankits

-2

-4

-6

-8

0

2

4

6

8

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

MaleMedianTubercleScore

 Group

Reject Null

Reject Null

0

10

20

30

40

50

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 168 of 241
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

Analysis ID: 13-4172-9067 Endpoint: MaleMedianTubercleScore CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

Negative Control 0.0947 -0.147 1.64 2 -2 0.5586 Asymp Non-Significant Effect

0.312 0.592 1.64 4 -2 0.2770 Asymp Non-Significant Effect

1.03 1.36 1.64 5 -2 0.0867 Asymp Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 1.03 >1.03 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

Total 545.4375 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 2.72 11.3 0.4362 Equal Variances

Distribution Shapiro-Wilk W Normality 0.909 0.841 0.1122 Normal Distribution

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

MaleMedianTubercleScore Summary

0 Negative Control 4 35 30.7 39.3 36 31 37 1.35 7.74% 0.0%

0.0947 4 37 24.1 49.9 37.5 29 44 4.06 22.0% -5.71%

0.312 4 31.8 23 40.5 33 24 37 2.75 17.3% 9.29%

1.03 4 29.5 20.3 38.7 29.5 24 35 2.9 19.7% 15.7% Centered Untransformed

 Rankits

-2

-4

-6

-8

0

2

4

6

8

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

MaleMedianTubercleScore

 Group

0

5

10

15

20

25

30

35

40

45

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 169 of 241
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

Analysis ID: 12-7263-9993 Endpoint: MaleVTG CETIS Version: CETISv1.8.7

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:

Species: Pimephales promelas Brine:

Source: Lab In-House Culture

Analyst:

Age:

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

Mann-Whitney U Two-Sample Test

DF P-Type

Negative Control 0.0947 9 NA 0 6 0.8857 Exact Non-Significant Effect

0.312 9 NA 0 6 0.8857 Exact Non-Significant Effect

1.03 9 NA 0 6 0.8857 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 347.0% 1.03 >1.03 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

Total 439018600 15

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

Variances Bartlett Equality of Variance 8.63 11.3 0.0347 Equal Variances

Distribution Shapiro-Wilk W Normality 0.821 0.841 0.0052 Non-normal Distribution

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

MaleVTG Summary

0 Negative Control 4 2.87E+3 -2.36E+3 8.10E+3 1310 1.07E+3 7.80E+3 1.64E+3 115.0% 0.0%

0.0947 4 1.87E+3 -7.56E+2 4.50E+3 1650 1.00E+2 4.09E+3 8.26E+2 88.2% 34.8%

0.312 4 3.18E+3 -2.94E+3 9.30E+3 1990 1.30E+2 8.62E+3 1.92E+3 121.0% -10.9%

1.03 4 6.20E+3 -1.00E+4 2.24E+4 1370 5.60E+2 2.15E+4 5.10E+3 165.0% -116.0% Centered Untransformed

 Rankits

-2000

-4000

-6000

0

2000

4000

6000

8000

10000

12000

14000

16000

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

MaleVTG

 Group

0

5000

10000

15000

20000

25000

0 N 0.0947 0.312 1.03

Graphics

000-516-187-1 CETISTM v1.8.7.7 Analyst:________ QA:________

Page 170 of 241
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 171 of 241
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).

Page 172 of 241
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 

Signat

Da

ure: 

te: 

~~ 

cpQ.Jf~L---­

Signature: ___....,..__...._:_=+-~--­

Date: "'2(i/J£1t? 

Template version I0/2011 

DATA EVALUATION RECORD 

STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 

PC CODE: 103801 DP BARCODE: 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 

activity, methomyl (99.4% a.i., lot # JUL06LP276) in deionized water was administered daily 

via oral gavage to six 56-day old (approximate age), castrated male Sprague Dawley rats at dose 

levels of 0 (vehicle), 0.25, 0.5, or 1.0 mg/kg/day. An androgenic positive control group 

consisted of 6 castrated rats exposed to 0.4 mg/kg/day testosterone propionate (TP) in corn oil 

with 1 % ethanol by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, methomyl (99.4% a.i., lot # JUL06LP276) in 

deionized water was administered daily via oral gavage to six 56-day old, castrated male Sprague 

Dawley rats at dose levels of 0 (vehicle), 0.25, 0.5, or 1.0 mg/kg/day in conjunction with a daily 

dose of TP at 0.4 mg/kg/day in corn oil with 1 % ethanol by s.c. injection. The anti-androgenic 

positive control group consisted of 6 castrated rats dosed with 0.4 mg/kg/day TP in corn oil with 

1 % ethanol by s.c. injection and flutamide (FT) in 0.1 % Tween® 80 in 0.5% methylcellulose at 3 

mg/kg/day by oral gavage. TP alone was used as the anti-androgenic negative control. 

For both components of the assay, the animals were dosed for 10 consecutive days and 

necropsied approximately 24 hours after the final dose administration to determine weights of 

liver and the five androgen-dependent tissues. Body weights, body weight gain and food 

consumption were measured throughout the dosing period. 

Page 173 of 241
In vivo Hershberger Assay (2011) / Page 2 of 12 

METHOMYL/090301 OCSPP 890.1400/ OECD 441

All animals survived until scheduled termination. There were no clinical signs of toxicity and no 

treatment-related gross pathological findings. 

In the androgen agonist assay, there were no effects on body weights, body weight gains, or food 

efficiency in the methomyl treated groups. Overall food consumption was increased (p<0.05) in 

the 0.5 mg/kg/day group (9%), but the effect was not dose dependent. Rats dosed with TP 

(positive control) had increases (p<0.05) in overall body weight 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 significant increases in all five accessory sex organ 

weights (p<0.05). The performance criteria indicated that the assay was performing as expected. 

In the anti-androgen assay, there were no effects on body weights, body weight gains, food 

consumption, or food efficiency in the methomyl groups or positive control (TP + FT) group 

when compared to the TP control. There was 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-adverse 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 requirements for a Hershberger assay (OCSPP 

890.1400). 

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were 

provided. 

Page 174 of 241
In vivo Hershberger Assay (2011) / 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

CH3 3CH

CH3

Page 175 of 241
In vivo Hershberger Assay (2011) / 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 approximately 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 quarantined 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 androgenic 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. Additionally, 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 significant decrease in two or more target organ weights of the 

treated groups (test substance + TP) compared to the TP-only control group. For both 

assays, the animals were dosed for 10 consecutive days and necropsied approximately 24 

hours after the final dose administration 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 through 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 computerized randomization, stratified 

by body weight, to the test groups noted in Table 1. Statistical analysis indicated that there 

were no significant differences in group body weight means at study initiation. 

Furthermore, the body weight of each animal was within ±20% of the overall mean. 

Page 176 of 241
In vivo Hershberger Assay (2011) / 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 (+TPb) 0 6 

Low (+TPb) 0.25 6 

Mid (+TPb) 0.5 6 

High (+TPb) 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 reductions (>10%) in cholinesterase had been 

established to occur, but not high enough to induce clinical signs of toxicity. In acute oral 

neurotoxicity (HL-1998-01080; MRID number not provided) and relative sensitivity (MRID 

46646401) studies, 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 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 cholinesterase activity were 

observed in male (up to 25% and 43%, respectively) and female (up to 30% and 38%, 

respectively) rats (MRID 46646401, and reversibility study HL-1997-00641; MRID number 

not provided). There were no clinical signs of toxicity 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 

(MRID 46646401). 

6. (a) Dose Preparation: Dose formulations for the test substance were prepared daily 

(method of preparation not reported). Dose formulations for TP were prepared once prior to 

study initiation by dissolving TP in ethanol and diluting in corn oil; the final concentration 

of ethanol in corn oil was 1%. TP solution was 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 day 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 and 8 days. Concentration verifications were 

conducted near the beginning of the study in the 0.25, 0.5, and 1.0 mg/kg dose formulations. 

Page 177 of 241
In vivo Hershberger Assay (2011) / 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 variation 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 storage 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 suspension, homogeneity measurements are not 

required. 

7. Dosage administration: Test formulations were administered to the animals daily via oral 

gavage (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw, and 

flutamide was administered via oral gavage at 5 mL/kg bw. Dose volumes 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 significant, 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 clinical signs of 

toxicity were conducted twice daily. 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 daily food efficiency was determined using the 

food consumption and body weight data. 

Page 178 of 241
In vivo Hershberger Assay (2011) / 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 vesicles, LABC, Cowper’s 

gland, and glans penis) were excised, trimmed free of adhering tissue, and weighed fresh 

(unfixed) according to the standard operating procedures detailed in the U.S. EPA Guideline 

(OCSPP 890.1400). Additionally, the liver was weighed. 

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until 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 presented in Table 2. Body weights in all 

methomyl-treated groups were comparable to controls throughout the duration of the assay. 

There was a statistically significant increase (p<0.05) in mean body weight gain (↑23%) 

over the duration of the study in rats dosed with TP compared to controls. 

TABLE 2. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Androgen 

Agonist Assay a

Study Day 

Dose (mg/kg/day) 

Vehicle Control 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

Body 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. 

Page 179 of 241
In vivo Hershberger Assay (2011) / Page 8 of 12 

METHOMYL/090301 OCSPP 890.1400/ OECD 441

TABLE 3. Selected Group Mean Body Weights and Cumulative 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

Body 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 assay and anti-androgen assay are presented in 

Tables 4 and 5, respectively. For the androgen 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 compared to the vehicle control; however, mean food efficiency 

was comparable to the vehicle control. Average 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 consumption 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 compared to the negative control 

(not statistically significant). 

TABLE 4. Food Consumption and Efficiency 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<0.05 

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METHOMYL/090301 OCSPP 890.1400/ OECD 441

TABLE 5. Food Consumption and Efficiency in the Anti-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 CONCENTRATIONS (OPTIONAL): Serum hormone 

concentrations were not determined. 

E. ORGAN WEIGHTS: Accessory sex organ weights and 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-treated groups. Liver weights in the treatment 

groups were comparable to the vehicle control. 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 increases (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 performance criteria in the Guideline; all %CV 

values were less than the maximum recommended values for each organ.

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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 weights for the anti-androgen 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 accessory 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<0.05) 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-dose groups were compared to the performance 

criteria in the Guideline; all %CV values were less than the maximum recommended values 

for each organ. 

Page 182 of 241
In vivo Hershberger Assay (2011) / Page 11 of 12 

METHOMYL/090301 OCSPP 890.1400/ OECD 441

TABLE 7. Accessory Sex Organ Weights (mg) and Liver Weights (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 reviewers 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 bioassay 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 androgen-dependent tissues, consistent with an 

androgen receptor agonist. Rats administered FT showed effects consistent with an 

androgen receptor antagonist. When administered to rats receiving a TP as a reference 

agonist, FT administration decreased organ weights for all androgen-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 treatment-related gross pathological findings. 

In the androgen agonist assay, there were no treatment-related effects on body weights, body 

weight gains, or food efficiency 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 corresponding effect in the 1 mg/kg/day group. Compared to the 

vehicle control, rats in the positive control group (TP) had increases (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 significant 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 the positive control group had accessory sex organ 

Page 183 of 241
In vivo Hershberger Assay (2011) / 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 glands; 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 effects 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 weight. However, there were 

no significant organ weight increases in the remaining four target accessory sex organs at 

any dose. Rats in the positive control group (TP + FT) had accessory sex organ weight 

decreases (p<0.05) as follows: 69% in seminal 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 was performing as expected. 

The %CV values for the androgenic and anti-androgenic 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-androgenicity under conditions of this assay. 

C. STUDY DEFICIENCIES: None

Page 184 of 241
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 185 of 241
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).

Page 186 of 241
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-"-LDATA 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. 

Page 187 of 241
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 during the study. All clinical chemistry parameters 

in the treated groups were similar to control. 

The dose levels tested in this assay are considered 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 requirements for a Female Pubertal Assay 

(OCSPP 890.1450). 

COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance, and Quality 

Assurance statements were provided. 

 

Page 188 of 241
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

3CH CH3

CH3

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 Laboratories, Inc., Portage, MI 

 Housing: Two/cage in in plastic cages with non-heat-treated Aspen bedding.

 Diet: Harlan Laboratories 2016CM Teklad Global Certified 16% protein rodent diet, ad libitum 

Phytoestrogen content average 11.0 ppm total isoflavones (genistein+daidzein+glycitein)

 Water: Municipal water, reverse-osmosis 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 Table 

Page 189 of 241
Female Pubertal Assay (2012)/ Page 4 of 15 

METHOMYL/ 090301 OCSPP 890.1450/ OECD None

1 using a randomization procedure designed to ensure equal stratification of body weights. 

Littermates were not assigned to the same treatment group. 

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of 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 results of previously-conducted studies with 

methomyl that have documented the doses required to achieve decreased 

acetylcholinesterase activity and associated clinical toxicity. These previous studies include 

an acute neurotoxicity study (MRID 44487501, DuPont Haskell Laboratories, 1998; HL1998-01080)1

, a reversibility study with carbamate 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 deionized water. The test substance formulations 

were divided into aliquots for daily dispensation and stored refrigerated. Dose formulations 

were stirred continuously during preparation and dosing, and were visually inspected for 

homogeneity at first dosing. Concentration analyses were performed on all dose levels. 

During a previous study4, homogeneity and stability analyses were performed; however, 

results were not reported in this study. Stability and homogeneity analyses were not 

conducted in the current study. Samples for concentration analysis were collected from the 

middle stratum of each dosing formulation (including the control group) prepared during the 

in-life phase of the study.

 

1DuPont Haskell Laboratories. Acute Oral Neurotoxicity Study in Rats (HL-1998-01080). Unpublished Report, 1998. 2 DuPont Haskell Laboratories. Reversibility Study with Carbamate Insecticides in Rats (HLR-1997-00641). Unpublished 

Report, 1997. 

3 DuPont Haskell Laboratories. Methomyl (DPX-X1179) Technical: Comparison of Cholinesterase Activity in Adult and 

Preweanling Rats (DuPont-15433). Unpublished Report, 2005. 

4 Freer, S.D. (2011) An Analytical Method Validation and Stability Study of Methomyl (DPX-X1179) Technical in Aqueous 

Formulations (Study No. WIL-189240). WIL Research Laboratories, LLC, Ashland, OH.

Page 190 of 241
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 variation between nominal and actual dosage to the 

animals was acceptable. 

6. Dosage Administration: All doses were administered once daily by oral gavage, from 

PND 22 through PND 42, in a volume of 5 mL/kg of body weight. Individual dosages were 

based on the most recently recorded body weights 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 animals cycling were 

assessed using Chi-square analysis. Estrous cycle length and day of first estrus were 

analyzed using a one-way analysis of variance (ANOVA) followed by Dunnett’s test when 

intergroup differences were indicated by ANOVA. Dichotomous pathology data were 

analyzed with pairwise Fisher’s exact test. Tests were conducted at the p≤0.05 significance 

level. 

The data that were statistically analyzed included organ weights (liver, kidneys, pituitary 

and adrenals), organ weight to final body weight ratios, daily body weights, weight gains, 

serum chemistries, hormones and vaginal opening data. Each endpoint was tested for 

homogeneity of variance using Levene’s test. If this was positive (p≤0.01), then log or 

square root transformations were applied to the data; if these were positive, then a nonparametric test was used. Organ weights, age at vaginal opening and weight at vaginal 

opening data, when parametric, were analyzed using analysis of covariance (ANCOVA) 

followed by Dunnett’s test using PND 21 body weights 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 test. Dichotomous histopathology data were 

analyzed using a Fisher’s exact test. 

Mean values were presented with standard deviations and coefficient of variation. Analyses 

were conducted using SAS® (SAS Institute, Inc., Cary, NC) version 9.2 (or higher) software. 

Page 191 of 241
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, followed 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 homogeneity of variance using Levene’s test. If that test was 

significant at p=0.01, then a log transform was applied and Levene’s test conducted on the 

transformed data. If that test was still significant, then the square root transformation was 

applied to the raw data and Levene’s test conducted again. If the test was still significant, 

then a nonparametric test, as described below, was used to analyze the data. 

If the variances were homogeneous, then an analysis of variance (ANOVA) was 

conducted, on the raw or transformed data, as appropriate. The statistical model contained 

a factor for treatment group and a blocking factor 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 substance groups when compared with the vehicle 

control. If the transformations were unsuccessful in making the 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 groups with the vehicle 

control. Since these are pre-planned pairwise 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 subject to the following analyses 

if they met the homogeneity of variance criteria: 1) Analysis of covariance (ANCOVA) 

with Dunnett’s test. The model 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. 

When an animal did not attain 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-sided at the 0.05 significance 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 considered appropriate by the reviewers. 

C. METHODS

1. Mortality and Clinical Examinations: All animals were examined twice daily for 

mortality and moribundity. Clinical examinations 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 weight changes were calculated for each 

corresponding interval and also for PND 22-42. 

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3. Vaginal Opening (VO): Beginning on PND 22, all animals were examined daily for onset 

of VO. The appearance of a small “pin hole”, 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 opening was observed, vaginal lavages 

were performed daily, and the slides were evaluated microscopically to determine the stage 

of estrus of each female until necropsy. The age 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 sacrifice, 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 may 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, and all sacrifices were completed by 1300 

hours. Immediately following decapitation, trunk blood samples were taken for T4, TSH, 

and serum chemistry analyses. The samples were stored at ≤−20° C. Necropsies were 

performed on all females found dead or that survived to the scheduled sacrifice. Any tissues 

with gross lesions were saved in 10% neutral-buffered formalin for possible future 

histopathologic examination. 

a. Hormone Analysis: Total thyroxine (T4) was analyzed using an electrochemiluminescent 

assay. Thyroid stimulating hormone (TSH) levels were analyzed using the [125I]rTSH kit 

obtained from Izotop, Institute of Isotopes Ltd. (Budapest, Hungary); the assay system relies 

on magnetic separation for quantitative 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. 

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 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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) Albumin globulin ratio (A/G Ratio, by calculation) 

 Alanine aminotransferase (ALT/also SGPT) Bile acids 

 Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase 

 Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in female 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 subjected 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 based on guideline 890.1450 

+ Histopathological examination required 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 fluid in the lumen (blotted weight). 

The ovaries (left or right not specified), uterus, and kidneys were fixed in 10% buffered 

formalin, rinsed, and stored in 70% ethanol prior 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% ethanol 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 thyroid, kidney, ovary, and uterus were routinely 

processed into paraffin blocks, sectioned, stained with hematoxylin and eosin, and examined 

microscopically. 

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Thyroid sections 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 were noted. At least two sections of the thyroid were examined. Five 

random sections of an ovary were evaluated for follicular development and any 

abnormalities/lesions (such as atrophy). The ovary 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, tertiary, and atretic follicles and growth phase, 

mature, and regressive corpora lutea. Uterine 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 the estrous cycle at the time of 

necropsy was taken into account in the final histological assessment, as ovarian and uterine 

cellular changes are dependent 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 weights, 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%). 

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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 presented in Table 3. No effects on 

organ weights were observed during the study. 

 Guideline criteria were met for organ weights in the controls, except the mean adrenal 

weight (33.6 mg) was slightly outside the acceptable range of 38.34-48.84 mg. 

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TABLE 3. Organ Weights at Necropsya

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 provided in Table 4. 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. 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 Cyclicitya

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 1b

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 2c 

a Data were obtained from pages 46 and 47 of the study report. 

b One female was in diestrus, but insufficient data to determine cycling status. 

c Two females were in diestrus, but insufficient data to determine cycling status. 

N Number of animals 

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F. HORMONE LEVELS AND CLINICAL CHEMISTRY: Mean hormone levels and 

clinical chemistry values are presented in Table 5. Serum T4 and TSH levels were not 

affected by treatment. The creatinine and urea nitrogen clinical chemistry parameters of the 

treated groups were similar to the control group. In addition, the study report provided 

normal ranges for the clinical chemistry measures, 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 T4 level in the controls. 

TABLE 5. Hormone Levels and Clinical Chemistrya

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 T4, 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 report. 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 treatment-related findings in the thyroid gland. 

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TABLE 6. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment 

Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

Gradeb Incidence Gradeb 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 findings in the ovaries, uterus, or kidneys. 

Page 199 of 241
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TABLE 7. Incidence of Histopathological Lesions of the Ovaries, Uterus and Kidneya

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 orally 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-related 

macroscopic 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 microscopic 

alterations 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 differences in mean age at vaginal opening, body weight at 

vaginal opening, or final body weights in the treated groups relative to controls. 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. 

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There were no treatment-related microscopic findings 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 cholinesterase 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 the results, interpretation or conclusions of this 

study: 

 The mean CV value for age at vaginal opening (10.54%) in the controls exceeded the 

performance criteria maximum (6.52%).

 The control group mean for adrenal gland weight (33.6 mg) was less than the 

performance criteria minimum (38.34 mg). 

Page 201 of 241
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 202 of 241
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).

Page 203 of 241
METHOMYL/ 090301 

Primary Reviewer: Karlyn Middleton 

Health Effects Division 

Secondary 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 Pubertal Assay; OCSPP 890.1500 

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 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. 

Following 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, pituitary, 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 similar in the treated groups to the control group. Serum testosterone levels were similar in 

the treated groups to the control group. 

Page 204 of 241
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-adverse clinical signs of cholinesterase inhibition at doses ≥2.0 

mg/kg/day. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Male Pubertal Assay (OCSPP 

890.1500). 

COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance, and Quality 

Assurance statements were provided. 

 

Page 205 of 241
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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

3CH

O

O

N S

CH3

CH3

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 Laboratories, Inc., Portage, MI 

 Housing: Two/cage in plastic cages with non-heat-treated Aspen bedding. 

 Diet: Harlan Laboratories 2016CM Teklad Global Certified 16% protein rodent diet, ad libitum 

Phytoestrogen content average 11.0 ppm total isoflavones (genistein+daidzein+glycitein)

 Water: Municipal water, reverse-osmosis 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 Table 

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1 using a randomization procedure designed to ensure equal stratification of body weights. 

Littermates were not assigned to the same treatment group. 

TABLE 1. Study Designa

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 results of previously-conducted studies with 

methomyl that have documented the doses required to achieve decreased 

acetylcholinesterase activity and associated clinical toxicity. These previous studies include 

an acute neurotoxicity study (Dupont Haskell Laboratories, 1998; HL-1998-01080, MRID 

44487501)1

, a reversibility study with carbamate insecticides (Dupont Haskell Laboratories, 

1997; HL-1997-00641)2

, and a sensitivity study comparing cholinesterase activity in adult 

versus preweanling rats (Dupont Haskell 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 prepared approximately weekly 

by mixing the appropriate amounts of test substance in deionized water. The test substance 

formulations were divided into aliquots for daily dispensation, stored refrigerated, and 

stirred continuously. Concentration analyses 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 stability 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. Acute Oral Neurotoxicity Study in Rats (HL-1998-01080). Unpublished Report, 1998. 2 DuPont Haskell Laboratories. Reversibility Study with Carbamate Insecticides in Rats (HLR-1997-00641). Unpublished 

Report, 1997. 

3 DuPont Haskell Laboratories. Methomyl (DPX-X1179) Technical: Comparison of Cholinesterase Activity in Adult and 

Preweanling Rats (DuPont-15433). Unpublished Report, 2005. 

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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/kg of body weight. Individual dosages were 

based on the most recently recorded body weights to provide the correct mg/kg/day dose. 

Dosing was performed between 0700 and 0900 hours daily. 

7. Statistics: Data statistically analyzed included the organ weights, 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, then a nonparametric test was used to analyze the data. If the 

variances were homogeneous, then an analysis of variance (ANOVA) was conducted. The 

statistical model contained a factor for treatment 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 article groups when compared 

with the vehicle control. If the variances 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-sided (p=0.05) looking for significant differences 

from the vehicle control. In addition, organ weights, age and weight at PPS were subject to 

the following analyses if they met the homogeneity of variance criteria: 1) Analysis of 

covariance (ANCOVA) with Dunnett's test, using 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 coefficient 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 Method Validation and Stability Study of Methomyl (DPX-X1179) Technical in Aqueous 

Formulations (Study No. WIL-189240). WIL Research Laboratories, LLC, Ashland, OH. 

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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 variance using Levene’s test. If that test 

was significant at p=0.01, then a log transform was applied and Levene’s test conducted 

on the transformed data. If that test was still significant, then the square root 

transformation was applied to the raw data and Levene’s test conducted again. If the test 

was still significant, then a nonparametric test, as described below, was used to analyze 

the data. 

If the variances were homogeneous, then an analysis of variance (ANOVA) was 

conducted, on the raw or transformed data, as appropriate. The statistical model 

contained a factor for treatment group and a blocking factor 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 substance groups when compared with the vehicle 

control. If the transformations were unsuccessful in making the 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 groups with the vehicle 

control. Since these are pre-planned pairwise 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 preputial separation data were subject to the following 

analyses if they met the homogeneity of variance criteria: 1) Analysis of covariance 

(ANCOVA) with Dunnett’s test. The model 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 significance 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 considered appropriate by the reviewers. 

C. METHODS

1. Mortality and Clinical Examinations: All males were observed twice daily, once in the 

morning and once in the afternoon, for moribundity and mortality. Individual clinical 

observations were recorded daily (prior to test substance administration during the treatment 

period) through the day of euthanasia. Each animal was observed for signs of toxicity at 

approximately 45 minutes after dose administration. 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 weight changes were calculated for each 

corresponding interval and for the overall treatment period (PND 23-53). 

3. Preputial Separation (PPS): Beginning on PND 30, all animals were examined daily for 

onset of PPS. The appearance of a partial or a persistent thread of tissue between the glans 

and prepuce was recorded. Age and weight at on the day of completion of PPS were 

recorded. In addition, if any animal within a group displayed incomplete separation 

(persistent threads) for >3 days, a separate second analysis was conducted with the age at 

which incomplete separation was observed. 

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4. Sacrifice and Pathology: On the day before euthanasia (PND 52), rats were moved to a 

holding room separate from where the necropsies were to be performed. Dosing was 

performed in the holding room on the day of termination (PND 53) and the rats were moved 

to the necropsy room one at a time for terminal procedures. Beginning 2 hours following 

dosing, animals were euthanized randomly across all groups by decapitation without 

anesthesia, and all sacrifices were completed by 1300 hours. Immediately following 

decapitation, trunk blood samples were taken for T4, 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-buffered formalin for potential histopathologic 

examination. 

a. Hormone Analysis: Serum concentrations of T4 and testosterone were analyzed with an 

electrochemiluminescent assay on the Cobas e411®. Serum TSH levels were analyzed with 

the [125I]rTSH kit obtained from Izotop, Institute of Isotopes Ltd. (Budapest, 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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) Albumin globulin ratio (A/G Ratio, by calculation) 

 Alanine aminotransferase (ALT/also SGPT) Bile acids 

 Aspartate aminotransferase (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 subjected to histological 

examination. 

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 UROGENITAL OTHER 

XX Testes (left and right separately)*+ XX Thyroid*+

XX Epididymides (left and right separately)*+ X Liver* 

X Seminal vesicle plus coagulating gland (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 based on guideline 890.1500 

+ Histopathological examination required based on guideline 890.1500 

The right testis, right epididymis, and kidneys were weighed prior to fixation. Following 

weighing, the testis and epididymis were fixed in Bouin’s solution for no more than 24 

hours. The thyroid (with parathyroid) was collected with the trachea 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% ethanol until histological processing. All other 

tissues listed above were placed in 10% neutral-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 routinely processed into paraffin blocks, sectioned, 

stained with hematoxylin and eosin, and examined 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 termination, 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 perforation in the esophagus 

and yellow contents in the thoracic cavity. The cause of death for the vehicle control group 

animal was not determined, but a dark red discoloration of the lungs was noted at necropsy. 

B. Clinical Signs of Toxicity: Daily clinical observations are summarized in Table 2. Red 

material around the mouth or ear was observed infrequently during the daily examinations.

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TABLE 2. Incidence of Daily Clinical Observationsa

Observation 

Dose Level (mg/kg/day) 

Vehicle Control Methomyl 

0.5 mg/kg/day

Methomyl 

1.0 mg/kg/day 

# Observedb # Examined # Observedb # Examined # Observedb # 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 animals with that observation at least once. 

C. General Growth and Preputial Separation: Body weights, body weight gains, age at 

attainment of PPS and weight at day of PPS are presented in Table 3. There were no 

treatment-related effects on final body weights, overall body weight gain, or age or body 

weight at PPS. 

The mean % CV for weight at age of attainment of PPS in the control group (9.55%) was 

above the performance criteria maximum of 7.57%, but the mean weight was within the 

acceptable range. 

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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 necropsy are presented in Table 4. There were no 

significant effects observed for organ weights, with one exception. The LABC muscle 

weight in the 1.0 mg/kg/day group was significantly decreased (p≤0.0374) compared to 

control, but this decrease was considered related to a decreased (↓3.21%) mean body weight, 

and was not biologically significant 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 performance criteria minimum value 

(2.242 g), but the % CV value for the kidneys was within the acceptable range in the 

performance criteria. 

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TABLE 4. Organ Weights at Necropsya

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 T4, TSH, and testosterone were not 

affected by treatment, and the two serum chemistry parameters evaluated were not affected 

by treatment (Table 5). 

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Guideline criteria for serum T4, 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 Chemistrya

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 T4, 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 the 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 treatment-related findings in the thyroid gland or kidneys. Mild 

seminiferous tubular degeneration 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 spermatids 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. 

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TABLE 6. Incidence of Histopathological Lesions of the Thyroid Gland a

Treatment 

Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

Gradeb Incidence Gradeb 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 the high-dose animal was determined to be 

caused by an intubation error; the cause of the death for the 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 final body weights, overall body weight gain, or 

the age or body weight at PPS. There were no significant effects observed for organ 

weights. 

Serum T4, TSH, and testosterone were not affected by treatment. No clinical chemistry 

findings were noted. At the scheduled euthanasia, no treatment-related macroscopic 

findings were noted in the thyroid gland or kidneys. Mild seminiferous tubular degeneration 

was noted in the right testis of one animal in the 1.0 mg/kg/day group, characterized as 

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random mild degeneration of spermatocytes and round spermatids within the seminiferous 

tubules. The right epididymis in the same animal 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-adverse 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 maximum 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 value 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 criteria acceptance range. 

Page 217 of 241
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 218 of 241
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).

Page 219 of 241
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. Because 

Page 220 of 241
Steroidogenesis Assay (2012) / Page 2 of 11 

METHOMYL / 090301 OCSPP 890.1550/ OECD None

all other guideline requirements for basal concentrations and hormone induction and inhibition 

were met for testosterone and estradiol, these results were deemed acceptable. 

The %CVs within each plate for the SC on the test chemical plates analyzed for hormone 

production ranged from 3.12 to 12.37% for testosterone and 0.78 to 12.45% for estradiol. The 

between-plate %CVs for the test plates were 6.93% and 7.0%, respectively, for testosterone and 

estradiol. These values are within the Guideline limit of ≤30%. 

Precipitation was not observed at any tested methomyl concentration, and methomyl was not 

found to be cytotoxic to the H295R cells at concentrations up to 100 μM. 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 μM (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 treatment did not result in 

statistically significant and reproducible alterations in testosterone or estradiol production. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis assay 

(OCSPP 890.1550). 

COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance, and Quality 

Assurance statements were provided. 

 

Page 221 of 241
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

3CH CH3

CH3

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 applicable 

Page 222 of 241
Steroidogenesis 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-Serum. Stock dosing media contained 

90% charcoal-stripped media and 10% unstripped media, to adjust the basal 

concentration of testosterone. Adjusted background hormone concentrations 

were not reported by the performing laboratory 

ITS+ premix: BD Bioscience, Cat # 35452, Lot # 14793 

8. Test Cells: H295R human adrenocortical carcinoma cells (ATCC CLR-2128; Lot # 

58333130). Cells were stored frozen in liquid nitrogen for no longer than 24 months. From 

an original ATCC batch, H295R cells were grown for 5 passages and subsequently frozen in 

liquid nitrogen. Cells started from frozen batches 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% CO2. 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 

17β-estradiol by H295R cells was conducted by HPLC/MS/MS using internal standards to 

measure extraction and quantitation 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 HPLC/MS/MS analyses. For estradiol analysis, 

samples were extracted with methylene chloride after addition of the internal standard 

Page 223 of 241
Steroidogenesis Assay (2012) / Page 5 of 11 

METHOMYL / 090301 OCSPP 890.1550/ OECD None

(D4-estradiol, Cambridge Isotope Laboratories, Andover, MA). The methylene chloride 

was evaporated to dryness, followed by estradiol solubilization with 100 mM sodium 

bicarbonate buffer (pH 10.5), and derivatization with dansyl chloride. For testosterone 

analysis, samples were aliquoted onto a 96-well protein-precipitation plate with acetonitrile, 

and the internal standard (D2-testosterone, Cambridge Isotope Laboratories, Andover, MA) 

was added. After shaking and then allowing the 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 prepared 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 water, yielding a range of 100 nM to 100 mM. 

Forskolin and prochloraz solutions were prepared 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 thawed 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 incubated for 24 hours at 37°C in a 5% CO2 atmosphere. 

4. Exposure: The cells were checked microscopically for good attachment and proper 

morphology, and the medium was removed and replaced with 1 mL of a mastermix of each 

concentration of methomyl, positive control, or solvent in medium with charcoal stripped 

Nu-Serum. Cells were exposed in triplicate according 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 total 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 response to forskolin (an inducer of testosterone 

and estradiol production) and prochloraz (an inhibitor of testosterone and estradiol 

production). The QC plate was prepared and dosed in the same manner with either forskolin 

or prochloraz according to the schematic presented in Table 2. 

Page 224 of 241
Steroidogenesis Assay (2012) / Page 6 of 11 

METHOMYL / 090301 OCSPP 890.1550/ OECD None

TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM)a

 1 2 3 4 5 6 

A Blankb

 Blank Blank Blank + MeOHc Blank + MeOH Blank + MeOH 

B DMSO DMSO DMSO DMSO + MeOH DMSO + MeOH DMSO + MeOH 

C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochloraz 0.1μM Prochloraz 0.1μM Prochloraz 0.1μM 

D Forskolin 10μM Forskolin 10μM Forskolin 10μM Prochloraz 1μM Prochloraz 1μM Prochloraz 1μM 

a Data were obtained from page 16 of the study report. Dosing was calculated based on a total volume of 1 mL per well. 

b Blank wells received medium only. 

c MeOH = 70% methanol was added to these wells for 30 minutes 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 removed, 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, Eugene, OR) immediately after removal of the 

culture medium. The plate wells were rinsed twice with 300 μL of phosphate buffered 

saline (PBS) (with Ca2+ and Mg2+). 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 binding 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 the average calcein AM fluorescence for each 

sample was divided by its EthD-1 fluorescence 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 protein 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 for 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 below maximum response on standard curve 

NA Test compound tested for interference with measurement 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: 

Page 225 of 241
Steroidogenesis Assay (2012) / Page 7 of 11 

METHOMYL / 090301 OCSPP 890.1550/ OECD None

Relative change = (hormone concentration in each well) ÷ (mean SC hormone 

concentration). 

Data were expressed as mean ± standard deviation. All doses exhibiting 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 probability plots or any other appropriate statistical method (e.g., 

Shapiro-Wilk's test). If the data were not normally distributed, the data were transformed to 

approximate a normal distribution. If the data were normally distributed or approximately 

normally distributed, differences between chemical 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 considered significant at p<0.05. 

II. RESULTS

A. TEST COMPOUND: The hormone concentrations after 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 methomyl concentration. 

The %CVs within each plate for the SC on the test chemical plates analyzed for hormone 

production ranged from 3.12 to 12.37% for testosterone and 0.78 to 12.45% for estradiol. 

The between-plate %CVs for the test plates 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 production was observed at any tested methomyl 

concentration. There were significant (p<0.05) decreases in mean testosterone levels at 

methomyl concentrations of 0.01 μM (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-sequential concentrations. 

Page 226 of 241
Steroidogenesis Assay (2012) / Page 8 of 11 

METHOMYL / 090301 OCSPP 890.1550/ OECD None

TABLE 3. Mean (±SD) Hormone Concentrations Following 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 Testosterone Production Relative to the Solvent 

Control at Methomyl Concentrations from 0.0001 to 100 μM, across all 

Three Runs. 

Page 227 of 241
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 μM, 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 Cell Viability Results after Treatment with Forskolin, Prochloraz, or 

Methomyl for 48 Hours. a

Compound Concen. 

(μM) 

Cell Viability – Run 1 Cell Viability – 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 control 

Page 228 of 241
Steroidogenesis Assay (2012) / Page 10 of 11 

METHOMYL / 090301 OCSPP 890.1550/ OECD None

C. QC PLATE: The hormone concentrations after exposure to the reference chemicals, SC, 

and blank samples, as well as the fold-difference change relative to SC (individual and mean 

± SD) for the three assay runs are presented 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 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 MDL as per the Guideline recommendation). 

Estradiol production in the presence of 10 μM forskolin met 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 testosterone 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 increase in the induction of testosterone production. 

Testosterone and estradiol inhibition were both greater than 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 (calculated by reviewer) based on the absolute 

concentrations of the SC were 20.9% for testosterone and 23.5% for estradiol; within the 

recommended limit of ≤30% and acceptable. The %CVs within 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 Treatment 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 quantification (LLQ) was 100 pg/mL for 

testosterone and 5 pg/mL for estradiol. 

Page 229 of 241
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 changes in testosterone or 17β-estradiol synthesis relative to vehicle 

control in three independent experimental runs. Positive control chemicals, forskolin and 

prochloraz, were evaluated concurrently to verify the test system performance. As expected, 

forskolin showed effects consistent with testosterone and 17β-estradiol induction and 

prochloraz showed effects consistent with testosterone and 17β-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 induction or inhibition of steroid biosynthesis. 

B. AGENCY COMMENTS: Precipitation was not observed at any tested methomyl 

concentration, and methomyl was not found to be cytotoxic to the H295R cells at 

concentrations up to 100 μM. Viability across all of the test plate runs ranged from 89.1-

101.9% relative to SC. 

Three independent test runs of the steroidogenesis assay were 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 μM 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 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 μM (0.89-fold) and 100 μM (0.83-

fold). The effect was consistent across all three run, but it was not dose-dependent, as it 

occurred only at two non-sequential concentrations. Methomyl 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 assay proficiency and validation tests for the performing 

laboratory were reported as completed and sufficient, no data were included in the 

study report to support 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 only 1.76-1.80-fold; slightly less 

than the ≥2-fold requirement. 

Page 230 of 241
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 231 of 241
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).

Page 232 of 241
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-[[(methylamino)carbonyl]oxy]ethanimidothioate; (DPX-Xl 179) 

CITATION: Snajdr, S. (2011). Methomyl (DPX-Xl 179) Technical: 3-Day Uterotrophic Assay 

for Detecting Estrogenic 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 

group. Uterine weights in the methomyl treated groups were comparable to the controls. 

Page 233 of 241
In vivo Uterotrophic Assay (2011) / Page 2 of 9 

METHOMYL/090301 OCSPP 890.1600/ OECD 440

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 weights were in the expected range. 

The dose levels selected for this study are 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 this assay. Methomyl is 

negative in the uterotrophic assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a uterotrophic assay (OCSPP 

890.1600). 

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were 

provided. 

Page 234 of 241
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. (Manager); 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: 17α-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

CH3 3CH

CH3

Page 235 of 241
In 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 genistein 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 arrival 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 five days of the post-ovariectomy acclimation 

period for evidence of conversion out of diestrus. The dose administration period was from 

approximate PND 63 through 65. Rats were euthanized approximately 24 hours after the 

last dose and necropsied for uterine weight measurements. 

3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the 

test groups noted in Table 1. Statistical analysis indicated that there 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 

17α-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 reductions (>10%) in cholinesterase had been 

established to occur, but not high enough to induce clinical signs of toxicity. In acute oral 

neurotoxicity (HL-1998-01080; MRID 44487501) and reversibility (HL-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 observed at dose levels of 2.0 mg/kg/day and greater. At dose 

Page 236 of 241
In 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% and 43%) and female (up to 30% and 38%) rats 

(HL-1998-01080 and MRID 46646401). There were no clinical signs of toxicity 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 prepared once prior to initiation 

of treatment by dissolving the appropriate amounts 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 measurements. 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 variation between nominal and actual dosage to the 

animals was acceptable. Because the test substance dose formulation was not a suspension, 

homogeneity measurements 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 significant, 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. 

 

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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 minutes after dosing (time of peak-effect for 

cholinesterase inhibition). 

2. Body Weight: Animals were weighed at randomization, study initiation, 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 administration of the test substance), 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 ovarian 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 and 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-related clinical signs of toxicity in 

any treatment group, including the EE positive control. One rat in the vehicle control group 

had black discharge from one eye each dosing day. 

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In vivo Uterotrophic Assay (2011) / Page 7 of 9 

METHOMYL/090301 OCSPP 890.1600/ OECD 440

TABLE 2. Incidence of Clinical Observations in the Estrogen 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 weights 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 significant. 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 consumption 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 significant. 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 group. 

Page 239 of 241
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 positive control group showed 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%, respectively, 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. 

Page 240 of 241
In vivo Uterotrophic Assay (2011) / Page 9 of 9 

METHOMYL/090301 OCSPP 890.1600/ OECD 440

TABLE 5. Uterine Weights from Estrogen 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: The 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 the 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 any treatment group. Body 

weights, food consumption, and food efficiency 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 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 uterine 

weights for the EE group were increased (p<0.001 and p<0.05, respectively) by 213% and 

166%, respectively. The increased uterine weights were in the expected range. 

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

negative in the uterotrophic assay. 

C. STUDY DEFICIENCIES: None 

Page 241 of 241