# UNITED STATES ENVIRONMENT AL PROTECTION AGENCY WASHINGTON, 

# D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



# MEMORANDUM

Date: 6/29/2015

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

# Assays

PC Code: 035001 

# Decision No.: 457621 

# Petition No.: NA 

# Risk Assessment Type: NA TXR 

# No.: 0056198 

# MRID No.: See Table



# FROM: Greg Akerman, Ph.D. Immediate Office Health Effec ts Division (7509 P) 

# DP Barcode: D396168 

# Registration No.: NA 

# Regulatory Action: NA 

# Case No.: NA 

# CAS No.: 60-51-5 40 

# CFR: NA



# THROUGH: Jess Rowland 

# d--=->---'**Gl..c,. ~**  Deputy Director Health Effec t s Division



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

I. 

# ACTION REQUESTED Ver.Apr. 2010



# The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays
s ubmitted in response to the agency's Test Order for dimethoate: Test Order # EDSP-35001-38

.

Pa ge I of2

# Page 2 of 2 

# II. RESPONSE
Attached are the EDSP Tier 1 assay DERs for dimethoate.

III. MRID Table

Chemical: Dimethoate PC Code: 035001 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48670601 890.1150 Androgen Receptor Binding (Rat Prostate) 48616301, 48843501 890.1200 Aromatase Assay (Human Recombinant) 48616302 890.1250 Estrogen Receptor Binding NA 890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) 48616303
890.1350 Fish Short-Term Reproduction 48670603 890.1400 Hershberger (Rat) NA 890.1450 Female Pubertal (Rat) NA 890.1500 Male Pubertal (Rat) NA 890.1550 Steroidogenesis (Human Cell Line – H295R) 48616304 890.1600 Uterotrophic (Rat) NA NA= Not Applicable (requirement satisfied by ot her scientifically relevant information (OSRI))    



  



    

     

  







 

  













  

     

# EPA MRID Number 48670601 & 48670602 (range finder)

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# DER Template Version: 22 September 2011  



  

# EPA DP Barcode 402437

OECD Data Point 231 EPA MRID 48670601 & 48670602 (range finder)
EPA Guideline 890.1100 Amphibian Metamorphosis Assay (Frog)
  



 

  

# Dimethoate 



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# 98.1%
Common Name Dimethoate Chemical Name IUPAC Not Reported

CAS Name Phosphorodithioic acid, O,O-dimethyl S-[2-(methylamino)-2-oxoethyl]

ester
CAS No. 60-51-5 Synonyms None Reported
EPA PC Code 035001

  



 

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# John Marton 

 

# 10/24/12
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# Teri S. Myers 

 

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# Catherine Aubee, Biologist
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# Justin Housenger, Biologist
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# 05/27/15

   



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# 29-MAY-15 Digitally signed by JUSTIN HOUSENGER DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=JUSTIN HOUSENGER, dnQualifier=0000044455 Date: 2015.06.03 14:06:43 -04'00'    



  



    

     

  







 

  













  

     

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# Schneider, S.Z., T.Z. Kendall, and H.O. Krueger. 2012. Dimethoate: Amphibian Metamorphosis Assay. Unpublished study performed by Wildlife International, Ltd., Easton, Maryland. Laboratory report number 232A-132A. Study sponsored by Cheminova A/S, c/o Cheminova, Inc., Arlington, Virginia. Study completed May 4, 2012.

The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven

s creening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to intera ct 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

w ithin the assay, where available, and redundancy across the battery. Thus, the results of each individual

a ssay 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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# The 21-day assay of dimethoate on amphibian metamorphosis of African clawed frogs ( 

# Xenopus laevis

) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 per group, 20 per replicate) were exposed to dimethoate (98.1% purity) at nominal concentrations of 0 (negative control), 6.25, 25, and 100 mg a.i./L (highest concentration represents the guideline recommended maximum concentration). Mean-measured concentrations were <3.00 (<LOQ; control), 6.0, 24, and 100 mg a.i./L. The test system was maintained at 20.9 to 22.6°C and a pH of 8.0 to 8.3.

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control. The survival of tadpoles exposed to dimethoate was not significantly affected (p>0.05) and was determined to be 96% in the control group and 99% in each of the low and mid treatment concentrations and 100% in the highest treatment concentration. Three tadpoles in the control group died due to siphoning error. Behavioral signs were scattered amongst the control and treatment concentrations, and included lethargy, lying on bottom of the tank, surfacing, and erratic swimming. These effects were reported to be transient as all tadpoles appeared normal at test termination.

Dimethoate caused no significant acceleration or delay of median NF developmental stage observed at 7 or 21 days in any treatment group (p>0.05) relative to the negative control. Further, no asynchronous development was observed throughout the test. By test termination at Day 21, the median development stage was NF stage 57 for the control and the high treatment concentration and NF stage 58 for the low and middle treatment concentrations. At termination, late stage animals (>60 NF) comprised 6% of control tadpoles, and 8, 10, and 5% of tadpoles exposed to the low, mid, and high concentrations, respectively. Because the total number of organisms observed to be at late stage in the control were <20%, the animals >60 NF were not used in calculations of means and medians for the growth metrics presented below (e.g., snout-vent length, hind limb length, and wet body weight).

Dimethoate had no statistically significant effect on Day 7 and Day 21 normalized hind-limb length, snout-vent length or body weight up to and including the highest treatment concentration.    



  



    

     

  







 

  













  

     

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# DER Template Version: 22 September 2011 Limited effects on thyroid gland histopathology were observed at 100 mg a.i./L; these effects included slight increases in thyroid gland hypertrophy with 5/20 at grade 2 or 3 (0/20 in control) and increases in follicular cell height with 8/20 at grade 2 or 3 (3/20 control). Differences in severity scores of hypertrophy between the high treatment group and the negative control were statistically significant (study authors’ analysis), but differences in follicle cell height between the negative control and treatment groups were not. No other treatment-related changes were observed in thyroid gland histopathology.

Although there were minor deviations from the guideline, the study met all validity and performance criteria.

The assay  

  

  

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

 

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# Test organism NF stage at test initiation: 51 Test organism total length at test initiation (optional): Not reported Test type: flow-through    



  



    

     

  







 

  













  

     

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# 6.0 No No No No No No No
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100 No No No No No No 0 

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# Abbreviations: Diff. 

# Difference. NA 

# Not applicable.

1 

# A “yes” indicates a significant difference based o n 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 ofthe DER. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a boardcertified pathologist.

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# Hind-limb length is normalized to snout-vent lengt h (SVL).

4 

# Increases in the incidence and severity of thyroid gland hypertrophy was observed in the high treatment group, but all other diagnoses with remarkable effects (thyroid gland atrophy, follicular cell height increase, and follicular cell shape increase had no concentration-responsiveness in the severity scores. It was indicated in the histopathology report that most animals were classified as unremarkable for all endpoints listed.

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# This study was conducted following guidelines outlined in the U.S. Environmental Protection Agency Series 890- Endocrine Disruptor Screening Program Test Guidelines, OCSPP Number 890.1100: 

# Amphibian Metamorphosis (Frog)

; and the OECD Guidelines for Testing of Chemicals,    



  



    

     

  







 

  













  

     

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# DER Template Version: 22 September 2011 Guideline 231: 

# Amphibian Metamorphosis Assay . The following deviations
from OCSPP 890.1100 were noted:

1. Details on parental health were not provided.
2 . It was not reported if the best-single spawn method was used to select larvae, or if more than one spawn was evaluated. The study authors only reported that all tadpoles came from a single

spawn. 3. The loading rate during the acclimation period was not specified. However, the loading rate during the definitive test (2 tadpoles/L) was within guideline recommendations (  4 tadpoles/L). 4. The feeding rate during the definitive test was half of the recommended rates specified in OECD and OCSPP guidance. 5. Water hardness (142 mg/L as CaCO 3 

# ) was much higher than recommended in the guideline ( 40-48 mg/L as CaCO 3 

# ).

These deviations do not impact the interpretations o f the results of this study.

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# Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements were provided. This study was conducted in compliance with GLP Standards as published by the U.S. Environmental Protection Agency (40 CFR Parts 160 and 792); and OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17), with the following exceptions: periodic analyses of water for potential contaminants were not performed according to Good Laboratory Practice Standards, but were performed using a certified laboratory and standard U.S. EPA analytical methods; and, preliminary analyses of water iodide concentrations were not performed according to GLP Standards.

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# DER Template Version: 22 September 2011 OECD recommends describing water solubility, melting /boiling point stability in water and light, pKa, Pow or Kow, vapor pressure of test compound, expiration date.

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# Stable. Mean-measured concentrations yielded recoveries of 96 to 100% of
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# Stored at  20°CK L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page fof g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cSpecies common name:African clawed frog 

# EPA recommends African clawed frog (Xenopus laevis). Western [Africa] clawed frog Silurana (Xenopus) tropicalis may be used as an alternate species1



# ; however, a list of all of the necessary protocol deviations to accommodate this species is recommended for inclusion in the study report. The guideline recommends that the performance criteria used to support the reliability of the test be identified.Species scientific name: 

# Xenopus laevisSpecies strain (if stated): Not reported1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the EndocrineDisruptor Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention(OCSPP), Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page xof g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cWere parents maintained as inhouse stock?Yes 

# EPA recommends that larvae used in the assay be derived from in-house adults.Were parental acclimationconditions same as definitivetest?YesAcclimation period for parentalfrogs (if applicable):ContinuousDetails on parental feeding: At least twice dailyduring acclimation,larvae were fed SeraMicron® as asuspension in dilutionwater.Details on parental health:Not specifiedK L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page h yof g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cBest single spawn?Not specifiedThe study authors only reported that alltadpoles came from a single spawn. 

# EPA and OECD recommend that the best 2 – 3 individual spawns, with a minimum of 1500 larvae/spawn, be evaluated to identify the best single spawn, and that the larvae selected for testing originate from the best single spawn (i.e.,



# the spawns are not comixed)Number of spawns evaluated (ifapplicable):Not reportedNumber of eggs sampled perspawn:Not reportedNF stage at test initiation51



# EPA recommends that the definitive study be initiated with larvae at Nieuwkoop – Faber (NF) developmental stage 51 ( 

# 17 days post-fertilization).Age at test initiation:16 days postfertilization (dpf)Mean total length at testinitiation (if reported):Not reportedRange of total length at testinitiation (if reported):Not reportedK L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page h hof g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cWas the optional size selectionmethod used?Not reportedDetails on larval selection: All tadpoles wereobtained from a singlespawn and wereacclimated to testconditions. Tadpolesthat appeared normaland healthy wereselected for use oncethey reached NF stage51.Loading rate (rearing density): Not specified foracclimation period.During the definitivetest, the loading ratewas 2 tadpoles/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.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page h mof g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cType 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 North America,Montgomeryville, PAIodide concentration in diet (ifknown):Not reportedFrequency of feeding:two times/day duringacclimation and threetimes/day during thetest 

# EPA recommends that feeding occur at least twice per day.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page h zof g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cDetails on feeding regime: Tadpoles received halfof the recommendedamount of food.Day 0-4: 15 mg/animal/dayDay 5-7: 20 mg/animal/dayDay 8-10: 25 mg/animal/dayDay 11-14: 35 mg/animal/dayDay 15-21: 40 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).K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page h |of g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cType of exposure:Flow-through

# EPA recommends the use of a flowthrough system.Type of flow-through dilutionsystem (if applicable):Continuous-flow diluter



# Intermittent flow proportional diluters or continuous flow serial diluters are recommended.

2Flow-through rate (ifapplicable):~69 mL/minThis was calculated based on testchamber volume (10 L) and 10volume additions per day, and resultedin complete volume replacementapproximately every 2.4 hours. 

# Recommended flow-through rate is 25 mL/min (complete volume replacement ca. every 2.7 hrs).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page h }of g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cDetails on toxicant mixing forflow-through systems (ifapplicable):Fluid metering pumps wereused to deliver volumes of asingle test substance stocksolution to mixing chamberswhere the stock was mixedwith volumes of a single testsubstance stock solution tomixing chambers where thestock was mixed with dilutionwater prior to delivery to thetest chambers. The flow ofdilution water into eachmixing chamber wascontrolled using rotameters.After mixing, the test waterin each mixing chamber waspumped into test chambersusing a peristaltic pump.Test solutions were continuously stirredand protected from light. Pumps androtameters were calibrated prior to thetest and verified or recalibrated, ifnecessary, approximately weeklyduring the test. Flow rates werechecked weekly to verify that theyvaried by no more than ±10% of themean flow-rate for the replicates.

# 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%.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page h ~of g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cRenewal period for staticrenewal (if applicable):NA 

# If static renewal is used, EPA recommends 24-hr renewal; renewal period is recommended not to exceed 72

hours.Aeration?No



# EPA recommends maintaining dissolved oxygen concentrations >40% air saturation (>3.5 mg/L). Aeration may be maintained through bubblers. It is recommended to set bubblers at levels that do not cause stress on the tadpoles.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page h gof g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cSource of dilution water: Dilution water was freshwaterobtained from a wellapproximately 40 metersdeep located on-site. Wellwater was passed through asand-filter to removeparticles >25μm, pumpedinto a storage tank, andaerated with spray nozzles.Prior to use, water wasfiltered to 0.45 μm andpassed through a UVsterilizer. 

# 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

substr

ates of the iodine transporter of the thyroid gland (e.g., 

# fluoride, chlorate, perchlorate). OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?Yes. See Reviewer’sCommentsK L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page h fof g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cIodide supplementation inwater?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

# 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:12 L (37 x 22 x 17 cm)Fill volume:10 LK L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page h xof g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cAdditional details on exposuresystem:Volume in the test chamberswas maintained by anoverflow port on one end ofthe test chamber.

[ L b P V } n q Q _ _ L X ] S ^  L M V X  Q L P R M ] s Z L X L W M V X R c M R W c R T M Z V [ V c M q ] c M V _ j

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p T M V X O L P o Q R Y V P R T V U V W S _ _ V T Y L M R S T cHardness (mg/L as CaCO3) 132144142Measured weekly inthe control andhighest treatmentgroup. 

# EPA recommends hardness 40 to 48 mg/L as CaCO 3.pH8.08.38.2Measured weekly ineach test vessel.



# EPA recommends pH 7.5 ± 1, interreplicate and inter-treatment differentials should not exceed 0.5.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page m yof g h 

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p T M V X O L P o Q R Y V P R T V U V W S _ _ V T Y L M R S T cDissolved oxygen (mg/L)6.48.87.7Measured weekly ineach test vessel. 

# EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration >3.5 mg/L

(>40% air saturation).Temperature (°C)21.5 22.122.0Measured weekly ineach test vesseland continuously inone negative controlreplicate. 

# EPA recommends temperature 22±1 o 

# C; inter-replicate and inter-treatment differentials should not exceed 0.5

o 

# C.Iodide3 – 6 μg/L



# EPA recommends aquatic iodide range 0.5 – 10 μg/L (supplemental iodide should not exceed 2 μg/L).K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page m hof g h 

# DER Template Version: 22 September 2011

r L X L _ V M V X e R T R _ Q _ e L \ R _ Q _ e V L T e V L c Q X V _ V T M

p T M V X O L P o Q R Y V P R T V U V W S _ _ V T Y L M R S T cAmmoniaNot Reported 

# General recommendations for frequency of measurements: EPA recommends that water quality parameters be measured in a control and at one test item concentration at least weekly. In static renewal systems, water quality parameters, including ammonia, should be measured just prior to renewal. In addition, EPA recommends that DO be measured at each concentration at least weekly and that 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<0.50 mg/LPerchlorateNot ReportedChlorateNot ReportedAlkalinity (mg/L as CaCO3)176184181Measured weekly inthe control andhighest treatmentgroup.Specific Conductance(μS/cm)364 388375K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page m mof g h 

# DER Template Version: 22 September 2011

K j q M Q Y ] K V c R l T L T Y ` Y Y R M R S T L P N \ aV X R _ V T M L P s S T Y R M R S T c

[ L b P V ~ n U L T l V   R T Y R T l q M Q Y ] s S T Y R M R S T c u R ^ ` a a P R W L b P V v j

r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cWas a range-finder conducted? YesSubmitted as MRID 48670602:Schneider, S.Z., T.Z. Kendall, and H.O.Krueger. 2012. Dimethoate: AmphibianMetamorphosis Range Finding Assay.Unpublished study performed by WildlifeInternational, Ltd., Easton, Maryland.Laboratory report number 232A-134. Studysponsored by Cheminova A/S, c/oCheminova, Inc., Arlington, Virginia. Studycompleted March 23, 2012.All results reported in this DER.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page m zof g h 

# DER Template Version: 22 September 2011

r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cIf yes, what was the method fordetermining the highest testconcentration in the rangefinder?Based on the results ofacute toxicity testingwith 

# Xenopus, reportedwater solubility of 39.8μg/L at 25°C, and theguidelinerecommendations oftesting up to 100mg/L. 

# EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., <10% mortality), whichever concentration is lowest.Species: 

# Xenopus laevisLife stage:NF 51Test duration:14 daysK L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page m |of g h 

# DER Template Version: 22 September 2011

r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cAdditional details:The test wasconducted at nominalconcentrations of 0.81,2.7, 9.0, 30, and 100mg a.i./L. There wereno mortalities or overttoxicity observed in anycontrol or treatmentgroup. Since there wasno apparent treatmentrelated effect in anytreatment group, thehigh concentration forthe definitive test wasset as the guidelinerecommendedmaximum concentrationof 100 mg a.i./L.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page m }of g h 

# DER Template Version: 22 September 2011

[ L b P V g n K V ^ R T R M R O V q M Q Y ] s S T Y R M R S T c j

r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cTest duration:21 days 

# EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in thedefinitive test:Results of range-finderand guidelinerecommendations.



# EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., <10% mortality), whichever concentration is lowest.Reference study citation (ifapplicable):MRID- 48670601Separation of testconcentrations:0.25 

# EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page m ~of g h 

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r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cNumber of test concentrations: 3 

# EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean-measuredLimit of quantification (LOQ): 3.00 mg a.i./L 

# EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reportedFrequency of measurement: Days 0, 7, 12, 14, and21



# It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every week thereafter.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page m gof g h 

# DER Template Version: 22 September 2011

r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cNumber of replicates in control:4 

# EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA 

# EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per testitem treatment level:4



# EPA recommends 4 replicates.Number of larvae per treatmentat test initiation:80Was a solvent used?NoSolvent type (if applicable):NAK L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page m fof g h 

# DER Template Version: 22 September 2011

r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cMaximum solvent concentration(if applicable):NA 

# EPA recommends that the solvent not exceed 0.02 ml/L3 

# . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 

# .Was a positive control used?NoPositive control (if applicable):NAPositive control concentration(s)(if applicable):NAPhotoperiod:12 hrs light :12 hrs dark 

# EPA recommends photoperiod 12:12 (light:dark).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB(2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review.Aquatic Toxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic ToxicityTesting of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Serieson Testing and Assessment. No. 23. Paris, France.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page m xof g h 

# DER Template Version: 22 September 2011

r L X L _ V M V X t L P Q V u c v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cLight intensity at water’ssurface:0.680-1.660 Klux 

# EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:NA

[ L b P V f n q Q _ _ L X ] S ^ [ X V L M _ V T M s S T W V T M X L M R S T c R T M Z V ` _ a Z R b R L T e V M L _ S X a Z S c R c ` c c L ]  R M Z K R _ V M Z S L M V j

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u _ l L j R j { v e V L T s t u  v K V M L R P c S X U V _ L X w c o Q R Y V P R T V U V W S _ _ V T Y L M R S T cNegative Control0.00<LOQNAAll solutions appearedclear and colorless withno evidence ofprecipitates. 

# EPA and OECD recommend that test item concentrations be maintained at a coefficient of variation (CV) 

# 20%.Treatment 16.256.04.0Treatment 225244.6Treatment 31001003.1Abbreviations:CVCoefficient of variation.

N j k b c V X O L M R S T cK L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ K R _ V M Z S L M V M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48670601 & 48670602 (range finder)Page z yof g h 

# DER Template Version: 22 September 2011

i R S P S l R W L P N T Y a S R T M c nDay 7- NF stage, wet weight, snout-vent length (SVL), hind-limb length (HLL), and normalized HLLDay 21- NF stage, wet weight, SVL, HLL, normalized HLL, thyroid histopathologyDaily- mortality and clinical signs of toxicity

 V X V X L  u R T Y R O R Y Q L P v Y L M L a X S O R Y V Y Yes 

# 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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# Survival on Day 7 was 100% in the control and all treatment groups (Table 9). On Day 21, survival was 96.3% in the negative control and 98.8, 98.8, and 100% in the mean-measured 6.0, 24, and 100 mg a.i./L treatment groups, respectively.

  

 



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# 3.7

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24 80 0 0 60 1 1.2

100 80 0 0 60 0 0

Abbreviations: NA 

# Not applicable.

1 

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

2 

# The three mortalities in the control were incidenta l deaths during siphoning.

Median developmental stage on Day 7 was 53 in the negative control and 54, 53, and 53 in the mean-measured 6.0, 24, and 100 mg a.i./L treatment groups, respectively (Table 10). On Day 21,median development stage was 57 in the negative control and 58, 58, and 57 in the meanmeasured 6.0, 24, and 100 mg a.i./L treatment groups, respectively. No asynchronous development was observed.    



  



    

     

  







 

  













  

     

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# Negative Control 4 53 0 4 57 0

6.0 4 54 0 4 58 0

24 4 53 0 4 58 0

100 4 53 0 4 57 0

Abbreviations: NA 

# Not applicable.

T here were no treatment-related differences on SVL or body weight at Day 7 or 21. Mean Day 7 normalized HLL was 0.13 in the negative control and ranged from 0.12 to 0.14 in the treatment groups (Table 11). By Day 21, normalized HLL averaged 0.35 in the negative control and 0.35 to 0.36 in the treatment groups.    



  



    

     

  







 

  













  

     

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# Negative Control 4 2.05 0.17 0.13 4 8.39 1.07 0.35

6.0 4 2.25 0.13 0.14 4 8.50 0.65 0.36

24 4 2.03 0.08 0.13 4 8.79 0.74 0.36

100 4 1.96 0.07 0.12 4 8.54 0.48 0.35

Abbreviations: NA 

# Not applicable. SD 

# Standard deviation.

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Day 7 mean SVL ranged from 15.88 mm in the mean-measured 24 mg a.i./L treatment group to 16.58 mm in the mean-measured 6.0 mg a.i./L treatment group (Table 12). By Day 21, mean SVL ranged from 23.7 mm in the negative control to 24.1 mm in the mean-measured 24 mg a.i./L
treatment group.

Mean wet weight on Day 7 ranged from 0.285 g in the mean-measured 24 mg a.i./L treatment group to 0.333 g in the mean-measured 6.0 mg a.i./L treatment group (Table 12). On Day 21, mean wet weight ranged from 0.868 g in the mean-measured 100 mg a.i./L treatment group to 0.925 g in the mean-measured 24 mg a.i./L treatment group.    



  



    

     

  







 

  













  

     

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# Negative
Control 4 15.9 0.42 4 23.7 0.26 4 0.286 0.02 4 0.907 0.05

6.0 4 16.6 0.76 4 23.9 0.44 4 0.333 0.04 4 0.888 0.06

24 4 15.9 0.42 4 24.1 0.49 4 0.285 0.02 4 0.925 0.07

100 4 16.0 0.34 4 24.0 1.1 4 0.292 0.09 4 0.868 0.12

Abbreviations: NA 

# Not applicable. ND 

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# Standard deviation.

1 

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

Thyroid gross and histopathology data are reported in Tables 13, 14, and 15. Limited effects on thyroid gland histopathology were observed at 100 mg a.i./L; these effects included slight increases in thyroid gland hypertrophy with 5/20 at grade 2 or 3 (0/20 in control) and increases in follicular cell height with 8/20 at grade 2 or 3 (3/20 control). There were no indications of thyroid hyperplasia. For other diagnoses in which there were remarkable effects reported, there was generally no concentration responsiveness in the severity scoring and a higher incidence of effects in the control group than in the treatment groups.    



  



    

     

  







 

  













  

     

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# Negative
Control 0 20 16 20 17 NA NA NA NA

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# Negative
Control 0 20 20 20 16 20 18

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# DER Template Version: 22 September 2011Throughout the test, surfacing, loss of equilibrium, lethargy, and erratic swimming were observed in control and treatment group tadpoles. However,by test termination, all surviving tadpoles appeared normal and healthy (Table 16).

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[ ] aV T p T W R Y V T W VNegative ControlMissing eye, lethargy572%6.0Lethargy, lying on bottom of tank, erratic swimming597%24Lethargy598%100Lethargy6012%    



  



    

    

   







 

  













  

    


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# The study authors analyzed survival, developmental stage, body weight, SVL, normalized HLL, and the incidence and severity of thyroid abnormalities. Unless otherwise noted, the unit of statistical analysis was the replicate test chamber. Most endpoints were analyzed using two complementary statistical approaches. If responses appeared to be monotonic, a step-down Jonckheere-Terpstra trend test was used to evaluate trends in the ranks of replicate means to determine possible concentration responsive trends among treatment groups. All endpoints except for survival and developmental stage data also were analyzed by performing pair-wise comparisons using Dunnett’s multiple comparison test to determine which treatment groups differed statistically from the control group. Data for endpoints analyzed by Dunnett’s test were evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test ( Į =0.01). The data for this study passed the assumptions of normality and homogeneity of variance.

Survival, developmental stage, and histopathology severity scores were not amenable to the statistical methods used for analysis of other endpoints. In particular, the most suitable unit of statistical analysis for survival was the individual animal. Therefore, survival was analyzed using the Fishers Exact test and the histopathology severity scores were analyzed using the step-down Jonckheere-Terpstra trend test only. In accordance with recommendations of OECD TG 231 and OCSPP 890.1100 analysis of metamorphic stage was performed using the multi-quantile analysis developed by T. Springer and J. Green, and using the step-down Jonckheere-Terpstra trend test on stage quantiles in the replicate
test chambers.

Statistical tests used to evaluate treatment effects were performed at the confidence level of Į = 0.05
with SAS software.

There were no significant effects on growth or developmental endpoints. There were slight differences in gland size and maximum follicle size in the highest treatment group relative to the negative control group, but these differences were limited to a few individuals.    



  



    

    

   







 

  













  

    


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# The reviewer visually analyzed data for mortality because mortality was  3.7% in all treatment groups. Replicate median values for developmental stage were analyzed using the two-sided Mann-Whitney test (SAS v. 8.1) or the one-sided Jonckheere-Terpstra trend test if there were not visually apparent deviations from monotonicity. Data for SVL, normalized HLL (HLL:SVL), and weight were not consistent with a monotonic concentration-response. The data were tested for normality using Shapiro-Wilks test and for homogeneity of variance using Levene’s test (SAS). All continuous endpoints met the assumptions of normality and homogeneity of variance and were then analyzed using Dunnett’s test (SAS). No asynchronous development was observed. Day 21 replicate means for normalized HLL and all growth parameters were calculated by excluding all tadpoles at NF stage  61. There was no pattern noted among the occurrence of these individuals, with no tadpoles from the negative control and 4, 5, and 3 each from the 6.0, 24, and 100 mg a.i./L levels, respectively.

Unless otherwise indicated, effects were considered statistically significant at 

# p < 0.05. Any results determined to be statistically significant using a one-sided trend test were compared to a secondary

Į =0.025 to illustrate whether the choice of a one-sided versus two-sided trend test would have an impact on the conclusions.

%  



  





# Effects on thyroid gland histopathology observed at the high concentration included slight increases in thyroid gland hypertrophy with 5/20 at grade 2 or 3 (0/20 in control) and increases in follicular cell height with 8/20 at grade 2 or 3 (3/20 control). Differences in severity scores of hypertrophy between the high treatment group and the negative control were statistically significant (study authors’ analysis), but differences in follicle cell height between the negative control and treatment groups were not. No other treatment-related changes were observed in thyroid gland histopathology. There were no significant changes (p>0.05) observed for Day 7 or Day 21 for any growth or    



  



    

    

   







 

  













  

    


# EPA MRID Number 48675001

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# DER Template Version: 22 September 2011 developmental endpoint.K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ ` b L _ V W M R T M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48675001Page Õ Éof Ë Ì 

# DER Template Version: 22 September 2011

[ L b P V h g n K V O V P S a _ V T M L P L T Y [ Z ] X S R Y o X S c c r L M Z S P S l ] ¬ R c M S aL M Z S P S l ] N T Y a S R T M c1,2 R T M Z V ` e `  R M Z K R _ V M Z S L M V jTreatment

­ ®  ̄ ° ± 2 ± 3  ́ μ

¶ ® · °  ̧ 1 ® · ° o » 1⁄4 · 1⁄2 3⁄4 Ö × Ø · À · Ù Ú Û® ·  ̧ Á ° Ù ¿ Á °  ̄ · Ü 2  ̧ 1⁄2  ́ 2 ® Ý  ́ ·  ̧  ̄ Á Þ3 ß o Â  ̧ Å Þ 1⁄4 Ú  ̧Ú » o Ø · À · Ù Ú Û® ·  ̧ Á à Þ Â 1⁄4 Ú 2 1⁄2 á 1⁄4 Ú o o °  ̧ 1⁄2

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é ç ç · Å Á o ê ­ ë · o 3 Ö Ú μNegative Control53NA57NA0NA0NANANANANANA6.0540.1057.50.2010.00.101.420.99NANANANANo2453>0.9957.50.282.00.942.840.94NANANANANo10053>0.9957.50.32-2.00.940.00>0.99NANANANAYes4Statistical TestMann-WhitneyJonckheereTerpstraDunnett DunnettNA NA NAAbbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavilyweighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are consideredstatistically significant at 

# p< 0.05.3Hind-limb length is normalized to snout-vent length (SVL).4Increases in incidence and severity of thyroid gland hypertrophy was observed in the high treatment group, but all diagnoses with remarkable effects (thyroidgland atrophy, follicular cell height increase, and follicular cell shape increase had no concentration-responsiveness in the severity scores with more incidenceof effects in the control group than the treatment groups.    



  



    

    

   







 

  













  

    


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Negative Control -- -- -- -- -- -- -- -- 6.0 3.76 0.27 0.89 0.94 16.2 0.06 0.55 0.99
24 -0.31 >0.99 1.48 0.78 -0.52 >0.99 2.04 0.97
100 0.16 >0.99 1.19 0.87 2.10 0.98 -4.27 0.82
Statistical Test Dunnett Dunnett Dunnett Dunnett


# Abbreviations: Diff. 

# Difference. NA 

# Not applicable.

1 

# Unless otherwise indicated, effects are reported b ased on comparison to the negative (clean water) control.

2 

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

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# The reviewer’s results for individual endpoints were generally in agreement with those of the study
authors.

No pesticides or organic compounds were detected in the dilution water at levels considered toxic to 

# Xenopus laevis

tadpoles. However, the following analytes were detected: calcium (35.1 mg/L), chloride (3.5 mg/L), magnesium (13.0 mg/L), nitrate-nitrogen (0.55 mg/L), potassium (6.77 mg/L), sodium (18.5 mg/L), and sulfate (5.6 mg/L).    



  



    

    

   







 

  













  

    


# EPA MRID Number 48675001

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# DER Template Version: 22 September 2011 The in-life portion of the definitive toxicity test was conducted from December 15, 2011 to January 5,
2012.

< < < =

B

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# Cheminova A/S. 1980. Product Chemistry: Dimethoate Technical. Unpublished study submitted to EPA July 20, 1981 under 4787-7; CDL: 245604-A. US EPA MRID 00139284.

Nieuwkoop, P.D., Faber J. (eds.). 1994. 

# Normal Table of Xenopus laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis . Garland Publishing Co, New York, NY USA.

Boyd, C.E. 1979. 

# Water quality in warm water fish ponds . Agricultural Experiment Station, Auburn University, Auburn, Alabama.

Personal Communication Dr. Timothy Springer and Dr. John Green. November 30, 2010. Dr. Timothy Springer, Wildlife International, Ltd., Easton, MD 21601, USA. Dr. John Green, E.I. DuPont de Nemours and Company, Wilmington, DE 19898, USA.

The SAS System for Windows. 1999-2001. Version 8.2. SAS Institute, Inc., Cary, North Carolina.

Personal Communication Dr. Henry O. Krueger. 2011. Wildlife International, Ltd., Easton, MD 21601, USA.    



  



    

    

   







 

  













  

    


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*K L M L N O L P Q L M R S T U V W S X Y S T M Z V [ S \ R W R M ] S ^ ` b L _ V W M R T M S ` _ a Z R b R L T c d e V M L _ S X a Z S c R c ` c c L ]EPA MRID Number 48675001Page Ë Ìof Ë Ì 

# DER Template Version: 22 September 2011

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# DATA EVALUATION RECORD

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

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012
(Revisions to MRID 48616301 to include laboratory proficiency data; Main study was originally reviewed under TA 2-02-2012)

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

Arlington, VA 22202

Prepared by Dynamac Corporation

1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: David A. McEwen, B.S. Date: 11/30/2011
Secondary Reviewer Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 7/16/2012
Program Manager: Signature: Jack D. Early, M.S. Date: 7/25/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/25/2012

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

.

# The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

# DIMETHOATE I 035001 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page 1of17 OCSPP 890.1150/ OECD None 

# Primary Reviewer: Jessica Ryman, Ph.D., D.A.B.T 

# Health Effects Division Secondary Reviewer:**_G_re~g~A_k_er_m_a_n~,_P_h_.D_.** 

# __ _ 

# Health Effects Division

# Signa(-fT-;(~ Date: 

# '-5' Signature: Date: 

# <;:" /t r h r Template version 08/2011



# DATA EVALUATION RECORD

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



# PC CODE: 035001 

# DP BARCODE: D396168



# TXR#: 0056198 

# CAS No.: 60-51-5



# TEST MATERIAL (PURITY): Dimethoate (98.0% a.i.)



# SYNONYMS: Phosphorodithioic acid; o,o-dimethyl S-[2( dimethyl-amino )-2-oxoethyl] ester



# CITATION: Willoughby, 

# I.A (2011). Dimethoate Technical: Androgen Receptor Binding (Rat Prostate Cytosol). CeeTox, Kalamazoo, MI. Laboratory Study#: 9148V100373ARB, November 4, 2011. MRID 48616301. Unpublished.
Willoughby, 

# I.A. (2012). Supplemental Information

- Laboratory Proficiency Data for ERTA assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, MI. July 19, 2011. MRID 48843501. Unpublished.



# SPONSOR: Cheminova, Inc., P.O. Box 110566, One Park Drive, Suite 150, Durham, NC



# TEST ORDER#: EDSP-035001-38



# EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48616301), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct saturation and competitive binding experiments for dimethoate (98.0% a.i., Batch #611/A). 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 (R 1881 ). 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 of dimethoate (logarithmic increase from 

# 10

- 10 

# to 

# 10

- 3 

# M). Dimethyl sulfoxide (DMSO) was used as a vehicle at a final assay concentration of approximately 3.2%. The assay included dexamethasone as a weak positive control, and RI 881 as the ligand reference standard

. Three independent runs were conducted with 3 replicates per concentration per run.

Saturation binding data were not originally provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. The dissociation constant (Kct) for [3H]-Rl 881 was 1

. 080±0.690 nM , and the estimated Bmax was 1.071±0.167 fmol/l 00 μg protein for the single batch of prostate cytosol that was prepared. Only one of the three Kct values was within the

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None 

# range reported in the EPA validation program (0.685 to 1.57 nM), and all B max 

# values were below the recommended range (7 to 16 fmol/100 μg protein). The was a relatively large variation in R 2 

# and K d 

# estimates among runs.
In the competitive binding experiment, dimethoate had no significant effect on specific binding of the [ 3 

# H]-ligand (>75% bound) to the AR at all dimet hoate concentrations tested in all three runs. A log IC 50 

# and RBA could not be calcu lated. The estimated log IC 50 

# s for R1881 and dexamethasone were

− 9.3 and

− 4.4 M, respectively, and the m ean relative binding affinity (RBA) for dexamethasone was 0.0022%. Confidence in these numbers is high due to the small variation. However, it is noted that the variatio ns between Run 1 compared to Runs 2 and 3 are likely due to the concentration differences used for R1881 and dexamethasone. The curve for dexamethasone was not fully defined in the firs t run and therefore the calculation for the IC 50 

# may be affected. The range of concentrations fo r R1881 in the first run fully defined the curve, but the concentration range was off by more than an order of magnitude. This was considered a minor deviation, and the performance criteria were generally met. As all solvent control tubes were run at the beginning of each experiment, drif t in the assay from the beginning to the end of the run could not be determined.
Based on the results from the three runs, dimet hoate is classified as a Non-Binder in the Androgen Receptor Binding Assay.
The assay satisfies the EDSP Tier 1 Test Order require ments for an Androgen Receptor Binding Assay (OCSPP 890.1150).

COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance and Quality Assurance statements were provided.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None


# I. MATERIALS AND METHODS

A. MATERIALS
1. Test facility: CeeTox, Inc. Location: 4717 Campus Drive, Kalamazoo, MI 49008 Study Director: J.A. Willoughby, Sr. Other Personnel: D. Blakeman, C. Haines, and S. McColley Study Period: May 23, 2011 to November 04, 2011


# 2. Test substance: Dimethoate Description: White solid Source: Cheminova A/S Batch #: 611/A Purity: 98.0% Solubility: Not reported Volatility: Not reported Stability: Not reported Storage conditions: -20±10°C CAS #: 60-51-5 Molecular weight: 229.26 Structure: P S

S O

O CH

3



# CH

3 

# C H 2 

# N CH 3 

# O



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

# 4. Radioactive ligand: [ 3 

# H]-R1881 Supplier: Perkin-Elmer, Boston, MA Catalog and Batch #: NET590; Lot # 653698 Date of production: February 24, 2011 Date of use: September 10, 2011 to September 18, 2011 Radiochemical purity: >97% Specific activity: 85.1 Ci/mmol* *The SA adjusted 

# was 82.6 Ci/mol (10-Sept-2011), 82.5 Ci/mol (13-Sept-2011), and 92.5 Ci/mol (17-Sept-2011). Concentration in stock: 1.0 nM



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



# 6. Solvent/vehicle control: DMSO Justification for choice of solvent: Due to issues with pipetting and volat ilization, ethanol could not be used. Final concentration: 3.2%

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None


# B. METHODS
1. Preparation of Rat Ventral Prostate Cytosol: The rat ventral prostate tissue was purchased from Charles River La boratories (location not report ed). Male Sprague Dawley rats (number not reported) were castrated at 90 days of age and were euthanized the following day. The ventral prostate tissues we re collected and stored at approximately

− 80°C until use, and were processed as a batch and used for multiple studies.
The cytosol was prepared by adding low-salt TEDG buffer at pH 7.4 (inclusion of protease inhibitor was not reported) to th e ventral prostate tissues at 10 mL/g of tissue. The tissues were minced, homogenized at 4°C, and centrifuged for 30 min at 30,000 × g at 4°C. The supernatant was aliquoted (2-4 mL) in tubes and stored at

− 80°C until used. Protein concentration of the cytosol pr epared for this study was determined to be 6.2 mg/mL using the Bradford method (Bio-Rad).
2. Saturation Radioligand Binding Experiment: A saturation binding experiment measuring total and non-specific binding of [ 3 

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

TABLE 1. Summary of Conditions for Saturation Binding Experiment a 

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



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

# H]-R1881 (originally 85.1 Ci/mmol as manufactured on February 24, 2011) was adjusted for decay over time (adjusted specific activities were not reported), and seri al dilutions in low-salt TEDG+PMSF buffer were prepared to achieve th e final concentrations in cytosol of 0.25,0.50, 0.70, 1.0, 1.5, 2.5, 5.0, and 10 nM to determine total binding. To determine nonspecific binding, solutions of non-labeled R1881 were prepared in a similar manner to achieve concentrations that were 100-fold greater than each re spective radiolabeled concentration, resulting in final concentr ations in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1000 nM. In the absence of cytosol, th e radiation found in 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

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

# H]-R1881 was measured. For each batch of cytosol, the optimal protein concen tration was determined by calculating specific

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None 

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

TABLE 2. Saturation Binding Experiment Run a ,b 

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

# Tubes 25-48 d 

# Tubes 49-72 e 

# [ 3 

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

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

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

# H]-R1881 (μL) 0.25 0.25 25 10 7.5 0.50 0.50 50 10 15 0.70 0.70 70 10 21 1.0 1.0 100 10 30 1.5 1.5 150 10 45 2.5 2.5 250 100 7.5 5.0 5.0 500 100 15 10 10 1000 100 30 a Data were obtained from page 3 of the study report (MRID 48843501). b Each concentration was run in triplicate for a total of 72 samples. c Tubes 1-24 contained 50 μL of tr iamcinolone acetonide and 7.5-45 μL [ 3 

# H]-R1881. Samples were dried, and 300 μl of prostate cytosol were added. d Tubes 25-48 contained 50 μL of tr iamcinolone acetonide and 7.5-45 μL [ 3 

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

# H]-R1881 in a volume of 7.5-45 μL. Samples were dried, and 300 μl of prostate cytosol were added. e Tubes 49-72 contained only 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

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

# H]-R1881 without cytosol or other components to determine the total counts added.



# Following addition of triamcinolone acetonide, [ 3 

# H]-R1881, and/or R1881, the tubes were dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor for 20 hours at approximately 4°C. Samples were maintained at temperatures of approximately 4°C except during whole rack vortexing. To separa te bound from free R1881, hydroxyapatite (HAP) slurry was added to each tube and vortexed 5 times with 4-minute intervals over 20 minutes. The samples were then centrifuged, and the s upernatant was aspirate d and discarded. The samples were washed 4 times in 2 mL of ice cold TEDG+PMSF buffer, followed by vortexing and centrifugation for 3 minutes at 700 x g. Following the last wash and decanting of the Tris buffer pellets were then extracted by additional of 2 mL of ethanol. The samples were vortexed and centrifuged for 10 minutes at 1000 x g. Samples were maintained on ice at all times between vortexing . After the final centrifugation, the ethanol supernatants were decanted into scintillation vials that each contained 14-mL portions of scintillation cocktail, and the radiation was quantified by liquid scin tillation counting. A total of three runs were performed.
3. Competitive Binding Experiment: A summary of the assay conditions for the competitive binding experiment is presented in Table 3.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None



# TABLE 3. Summary of Conditions for Competitive Binding Experiment a 

# Source of receptor Rat prostate cytosol Concentration of radioligand 1 nM Optimization of receptor concentration Sufficient to bind 10-15% of radioligand b 

# Concentration of test substance (as serial dilutions) 10

− 10 

# to 10

− 3 

# M Incubation Temperature 4±2°C Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% (v/v) Protease Inhibitor (PI) c 

# 0.5% (v/v) DTT 1 mM Sodium Molybdate 1 mM a Data were obtained from page 15 of the main study report (MRID 48616301). b Stated in Study Report on page 15 c The recommended PI in th e Guideline is 1 mM PMSF. The PI was identified in the study protocol as Calbiochem Protease Inhibitor Cocktail, Set III, EDTA fre e. It is a mixture of six PIs that includes 4-(2- aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBS F has similar specificity to PMSF with greater stability at lower pH. 

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

# H]-R1881 (adjusted specific activity of 85.1 Ci/mmol) to the AR in the presence of incr easing concentrations of a test substance.
DMSO was used as the vehicle, and the solu bility of the test material at the top concentration of 10
− 3 

# M in 100% DMSO was determined by visual observation. Additionally, all test material solutions were examined for precipitation following the overnight incubation period.

The reviewer was able to calculate from the co mpetitive binding experiment that the amount of cytosolic protein used in the assay c ontained enough receptor to bind 8.7-9.7% of the [ 3 

# H]-R1881.

Dilutions of the test subs tance, reference standard (R1881), weak positive control (dexamethasone), and solvent control (DMSO) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of th ree independent runs on three different days, and each run contained three replicates at e ach concentration, resul ting in a total of 81 samples per run.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None



# TABLE 4. Competitor Final Molar (M) Con centrations in Competitive Binding Assay a, b



# Solvent Control Re ference standard c 

# Weak positive control d 

# Test Chemical None DMSO R1881 Dexamethasone Dimethoate Tubes 7-12 Tubes 13-18 and 19-33 e 

# Tubes 37-60 Tubes 61-84 Tubes 1-6 1×10

− 7.48 

# 1×10

− 6 

# 1×10

− 4 

# 1×10

− 3 

# 1×10

− 3 

# 1×10

− 8.48 

# 1×10

− 7 

# 1×10

− 5 

# 1×10

− 4 

# 1×10

− 4 

# 1×10

− 9.48 

# 1×10

− 8 

# 1×10

− 6 

# 1×10

− 5 

# 1×10

− 5 

# 1×10

− 10.48 

# 1×10

− 9 

# 1×10

− 7 

# 1×10

− 6 

# 1×10

− 6 

# 1×10

− 11.48 

# 1×10

− 10 

# 1×10

− 8 

# 1×10

− 7 

# 1×10

− 7 

# 1×10

− 12.48 

# 1×10

− 11 

# 1×10

− 9 

# 1×10

− 8 

# 1×10

− 8



# −− 1×10

− 10 

# 1×10

− 9 

# 1×10

− 9 

# -- 1×10

− 11 

# 1×10

− 10 

# 1×10

− 10 

# a Data were obtained from pages 53-58 of the study report. b Each concentration of each chemical was run in triplicat e for a total of 81 tubes per run. Tubes 7-84 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 

# H]-R1881. Samples were dried, and 300 μl of prostate cytosolwere added. Tubes 7-84 also contained 10 μL of the solvent control, reference standard (non-radiolabeled R1881), weak positive control, or test substance, with the exception of Tube s 13-18 that contained 30 μL of nonradiolabeled R1881 (used to evaluate non-specific binding). Tubes 1-6 contained only 30 μl of [ 3 

# H]-R1881. c The concentrations of R1881 used in the 1 st 

# competitive binding assay ranged from 1x10

− 7.48 

# to 1x10

− 12.48 

# , while the standard concentrations of 1x10

− 6 

# to 1x10

− 11 

# were used in the 2 nd 

# and 3 rd 

# assays. d The concentrations of dexamethasone used in the 1 st 

# competitive binding assay ranged from 1x10

− 4 

# to 1x10

− 11


# , while the standard concentrations of 1x10

− 3 

# to 1x10

− 10 

# were used in the 2 nd 

# and 3 rd 

# assays. e Tubes 13-18 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881.



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

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

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

# Swillens (1995) 2 

# . For the competitive binding assay, similar methods of nonlinear regression were used to fit a curve (for R1881, the positive control, and the test substance) to the H ill equation formula which incorporated log IC 50 

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

# K d 

# and B max 

# ) and competitive binding experiment (log IC 50 

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

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outlie rs when fitting data with nonlinear regression- a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics, Vol 7, pp 123-142. 2 Swillens, S. (1995) Interpretation of binding curves obtain ed with high receptor concentrations: practical aid for computer analysis. Molec. Pharmacol. 47(6):1197-1203.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None


# 5. Definitions

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

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

− 0.6 to

− 1.4.
Equivocal: The average lowest portion of curves across runs is between 50% and 75% radioligand binding ( i.e. radioligand displacement is at le ast 25% but less than 50%), or the Hill slope for the curve falls outside th e range for the weak positive control (

− 0.6 to

− 1.4).

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

Untestable: If the test compound is not soluble above 1×10
− 6 

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

B max 

# : maximal binding capacity K d 

# : dissociation constants IC 50 

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

# Relative Binding Affinity (%RBA): IC 50 

# of R1881 × 100 ÷ IC 50 

# of test substance Log RBA: Log 10 

# (IC 50 

# of R1881 ÷ IC 50 

# of test substance)

II. RESULTS

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

# ) for [ 3 

# H]-R1881 was 1.080±0.690 nM, and the estimated B max 

# was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cy tosol that was prepared. Only one of the three K d 

# values was within the range reported in the EPA validation program (0.685 to 1.57 nM); the mean value was above the recommended range. Confidence in these numbers is low according to the goodness of fit (R 2 

# = 0.880-0.986) and the la rge variation in K d 

# estimates among runs.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None



# TABLE 5. Saturation Binding Experiment of [ 3 

# H]-R1881 with Androgen R eceptor from Ra t Prostate Cytosol a 

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

# R 2 

# (unweighted) 0.880 0.986 0.893 0.880-0.986 B max 

# (nM) 0.010 0.008 0.011 0.010±0.002 B max 

# (fmol/100 μ g protein) 1.087 0.896 1.229 1.071±0.167 K d 

# (nM) 0.855 0.531 1.855 1.080±0.690 a Data were obtained from page 4 of the study report (MRID 48843501). b The range of R 2 

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

# Goodness of fit for curve calculated for specific binding.



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

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

# H]-R1881 to the androgen receptor. The data fits generally result ed in linear plots.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None 

# FIGURE 1. Binding of [ 3 

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

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None


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

# H]-R1881 to the Androgen Receptor.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None


# B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive binding experiments are summarized in Table 6 and shown graphically in Figures 3-5. Dimethoate was classified as a non-binder in all three runs si nce the percent radiolabeled R1881 bound to the AR was >75% at all concentrat ions of dimethoate tested. The estimated mean log IC 50 

# for the test material, dimethoate, coul d not be determined as it did not result in 50% inhibition (displacement) at a ny concentration. The estimated log IC 50 

# s for R1881 and the weak positive control (dexamethasone) were

− 9.3 and

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

# may be affected. The ra nge of concentrations for R1881 in the first run fully defined the curve, but the concentration range was off by more than an order of magnitude. In addition, all so lvent control tubes were run at the beginning of each experiment, preventing the reviewer fr om determining drift in the assay from the beginning to the end of the run.

TABLE 6. Competitive Binding Assay of Dimet hoate with AR from Rat Prostate Cytosol Parameter Assay 1 Assay 2 Assay 3 Mean±SE R 2 

# (unweighted) R1881 NR NR NR NA Dexamethasone NR NR NR NA Dimethoate NR NR NR NA Log IC 50 

# (M) a 

# R1881

− 9.9

− 9.0

− 9.0

− 9.3±0.30 b 

# Dexamethasone

− 4.3

− 4.4

− 4.6

− 4.4±0.09 b 

# Dimethoate NA NA NA NA IC 50 

# (M) b 

# R1881 1.26 × 10

− 10 

# 1.00 × 10

− 9 

# 1.00 × 10

− 9 

# 7.09 × 10

− 10 

# (±2.91) Dexamethasone 5.01 × 10

− 5 

# 3.98 × 10

− 5 

# 2.51 × 10

− 5 

# 3.83 × 10

− 5 

# (±0.73) Dimethoate NA NA NA NA Log RBA b 

# Dexamethasone

− 5.6

− 4.6

− 4.4

− 4.9±0.37 Dimethoate NA NA NA NA RBA (%) b 

# Dexamethasone 0.0003 0.0025 0.0040 0.0022±0.0011 Dimethoate NA NA NA NA

a Data were obtained from page 21 of the study report. b Values are reviewer-calculated from the log IC 50 

# values reported in the study report. For means expressed in scientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean value. R 2 

# Goodness of fit NA Not applicable NR Not reported RBA (%) relative binding affinity

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None 

# FIGURE 3. Percentage [ 3 

# H]-R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, or Dimethoate (Run 1).

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None 

# FIGURE 4. Percentage [ 3 

# H]-R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, or Dimethoate (Run 2).

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 15 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None 

# FIGURE 5. Percentage [ 3 

# H]-R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, or Dimethoate (Run 3).

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 16 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None


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

TABLE 7. Criterion a 

# Tolerance Limit(s) b 

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

# H]- R1881 added per assay tube (% binding) ≤ 15 8.7 to 9.7 X

Dimethoate Top (% binding) 80 to 115 103.5 to 108.8 X

R1881 fitted curve parameters Top (% binding) 82 to 114 96 to 103 X Bottom (% binding)

− 2.0 to 2.0 0 X Hill Slope

− 1.2 to

− 0.8

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

− 12 to 12 NA;

− 1 to 7 c 

# X Hill Slope

− 1.4 to

− 0.6

− 1.0 to

− 0.6 X Saturation Binding Experiment K d 

# (nM) 0.685 to 1.57 0.531 to 1.855 d 

# X e


# Non-specific binding (%) <20% 8.90-14% X a Data were obtained from pages 23-28 and 53, 55 and 57 of the study report. b These values represent ranges from the validation study. c The bottom plateau level for dexamethasone in Run #1 was reported as NA (not applicable) because lower concentrations were used due to an error in the stock preparation. d MRID 48843501 e One value was below the limit, one value was within the limit, and one value was above the limit. NR Not reported



# In the first run, the concentr ations for R1881 and dexameth asone were lower than the targeted (guideline) levels because of an e rror in the stock preparation. Therefore, nonspecific binding was not evaluated with 100X of cold (non-radiolabeled) R1881 but with 30X cold R1881, and the resulting curve for dexa methasone fit two of th e three criteria (Hill slope and Top plateau) but th e Bottom plateau was not achie ved for the first run. All performance criteria were met for the other two runs.
Additionally, the curve for the reference material showed that increasing concentrations of unlabeled R1881 displaced [ 3 

# H]-R1881 in a manner consiste nt with one-site binding, as indicated by a descent from 90% to 10% binding over approximately an 81-fold increase in concentration of R1881 (i.e., covering approxima tely 2 log units). Examination across the runs indicated consistency of the Hill slope , placement along the X-axis, and top and bottom plateaus.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: All three assays achie ved the non-binder criterion described in the guideline (>75% radioligand binding). There were no apparent alterations in radiolabeled R1881 binding at any of the lo wer concentrations test ed. Dimethoate was classified as non-binding based on the average of the three valid assays with a mean value of “0”`.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 17 of 17 DIMETHOATE / 035001 OCSPP 890.1150/ OECD None



# B. AGENCY COMMENTS: The saturation binding data were reported. The mean K d 

# for [ 3 

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

# was 1.071 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. Only one of the three K d 

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

Dimethoate was classified as a non-binder in all three competitive binding runs since the percent radiolabeled R1881 bound to the AR was > 75% at all concentrations of dimethoate tested. The estimated mean log IC 50 

# for the test material, dimethoate, could not be determined.
In the competitive binding experiment, the estimated average log IC 50 

# was − 9.3 M for R1881 and

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

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

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

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

 The protein concentration used in the assay was not reported.

# DATA EVALUATION RECORD

DIMETHOATE
Study Type: OCSPP 890.1200; Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-2-2012 (MRID 48616302)

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

Arlington, VA 22202

Prepared by Dynamac Corporation

1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 12/01/2011 Secondary Reviewer Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 12/02/2011
Program Manager: Signature: Jack D. Early, M.S. Date: 12/07/2011
Quality Assurance: Signature: Jack D. Early, M.S. Date: 12/07/2011

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

.

# The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

# DIMETHOATE I 

# 035001 

# Primary Reviewer: Vincent Chen M.S. 

# Health Effects Division Secondary Reviewer: Greg Akerman, Ph

. D. 

# Health Effects Division Aromatase (Human Recombinant) Assay (2011) I 

# Page I of 12 OCSPP 890.1200 I 

# OECD None

# Signature:**~** Date: 2-Y 

# Tv"-1 Z.UJ-.S

# Signature:**.7~** 7 

# 4.._ 

# Date: <4 /2 .::r 

# U>

- Template version 08 / 2011 

# DAT A EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890

. 1200



# PC CODE: 035001 

# DP BARCODE: 0396168



# TXR: 0056198 

# CAS: 60-51-5



# TEST MATERIAL (PURITY): Dimethoate (98.0% a.i

. )



# SYNONYMS:

IUPAC: CAS: 0,0-dimethyl S-methylcarbamoylmethyl phosphorodithioate 0 , 0-dimethyl S-[2-(methylamino)-2-oxoethyl] phosphorodithioate



# CITATION: Wilga, P

. (2011). Dimethoate Technical: Human Recombinant Aromatase Assay. CeeTox , Inc. [4717 Campus Drive , Kalama zo o , MI 49008 , US]. Laboratory Study No.: 9148V-100373AROM. 03 November 2011. MRID 48616302

. Unpublished.



# SPONSOR: Cheminova Inc ., P.O. Box 110566 , One Park Drive , Suite I 50, Research Triangle Park, NC 27709, US

Cheminova A/S, P .O

. Box 9 , DK-7620 Lemvig , DK



# TEST ORDER: CON-035001-3



# EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48616302) , dimethoate (98.0% a.i., batch # 611/A) was incubated with human recombinant aromatase and tritiated androstenedione**([l~- H(N)]-androst-4-ene-3,l**  

# 7-dione ; [3H]-ASDN) in dimethyl sulfoxide (DMSO) at logarithmic concentrations from 10

- 10 

# to 10

- 3 

# M for 15 minutes at 37°C to

assess its potential of dimethoate to inhibit aromatase activity.

Aro matase activity was determined by measuring the amount of tritiated water produced at the end of a I 5-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Three independent runs were conducted, and each run included a full activity control, a background activity control, a positive control series (10

- 10 

# to 1



# o

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

- 10 

# to 10

- 3 ) 

# with 3 repetitions per concentration.

The response of the full activity controls and background controls were acceptable for each run

. For the full activity control, aromatase activity averaged 0.212 ± 0

. 024 nmol

· mg# Aromatase (Human Recombinant) Assay (2011) / Page 2 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None 

# protein
− 1 

# ·min
− 1 

# across all three test runs. Activity in the background controls ranged 0.51% to 0.83% and averaged 0.63% of the full control activity.
For the positive control substance (4-OH ASDN), aromatase activity averaged 0.203±0.040 nmol·mg-protein
− 1 

# ·min
− 1 

# at the lowest tested concentration (10
− 10 

# M) and 0.002±0.000 nmol·mg-protein
− 1 

# ·min
− 1 

# at the highest tested concentration (10
− 5 

# M). The results were generally within the recommended ranges for the top of the curve, bottom curve, hill slope, log IC 50 

# , and coefficient of variation for replicates of each concentration.
For dimethoate, aromatase activity averaged 0.210±0.024 nmol·mg-protein
− 1 

# ·min
− 1 

# at the lowest tested concentration of 10
− 10 

# M and 0.181±0.021 nmol·mg-protein
− 1 

# ·min
− 1 

# at the highest tested concentration of 10
− 3 

# M. The average lowest portion of the response curve across runs was >75% activity at all concentrations of dimethoate and therefore an IC 50 

# could not be calculated. The estimated log IC 50 

# for the positive control 4OH-ASDN averaged

− 7.13 M. The average slope of the concentration re sponse curve for 4OH-ASDN was

− 1.02. Confidence in these numbers is high due to the small variation.
Based on the data from the average response curve, dimethoate is classified as a Non-inhibitor of aromatase activity in this assay.
The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Aromatase assay (OCSPP 890.1200).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.

# Aromatase (Human Recombinant) Assay (2011) / Page 3 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Dimethoate Description: White, granulated powder Source: Cheminova A/S Batch # (Expiration Date): 611/A (September 22, 2014) Purity: 98.0% a.i. Volatility: 1,107 mg/m 3 

# Storage Conditions:

− 80°C Stability: At least 10 years Solvent: DMSO Solubility (in Test Solvent): At least 10

− 3 

# M Highest Concentration Tested: 10 –3 

# M Stock Solution Preparation Methodology: Not specified; serial dilution assumed Molecular Weight: 229.3 CAS #: 60-51-5 Structure:



# 2. Non-Labeled Substrate: Androstenedione (ASDN) CAS # : 63-05-8 Source: Steraloids, Inc. (Cat. # L1712) Batch # (Expiration Date): L1712 (April 2016) Purity: 99.8%



# 3. Radiolabeled Substrate: [1 β

- 3 

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

# H]-ASDN) Source: Perkin Elmer (Cat. # NET-926) Batch # (Expiration Date): 619344 (January 10, 2012) Radiochemical Purity (Supplier): 97% Specific Activity: 26.3 Ci/mmol Radiochemical Purity (In-Lab Determination): Not provided



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


# 5. Solvent (Vehicle Control): Dimethyl sulfoxide (DMSO) Source: Sigma-Aldrich (Cat. # D2650) Lot # (Expiration Date): RNBB7617 (February 2013) Justification for Choice of Solvent: Acceptable vehicle by OCSPP guideline 890.1200 Concentration (% of Total Volume in Assay): 1% P O

K+ -O SCH 2



# CH 3 

# O

CO N H CH 3

# Aromatase (Human Recombinant) Assay (2011) / Page 4 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None



# 6. Test Microsomes: Human recombinant aromatase (CYP19) microsomes Source: BD Gentest TM 

# (Woburn, MA; Cat. # 456260) Lot # (Expiration Date): 03897 (February 2014) Protein Concentration: 7.4 mg/mL Cytochrome C Reduc tase Activity: 290 nmol/min/mg Aromatase Activity: 6.0 pmol/min/pmol P450



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

# H]-ASDN was prepared such that the final concentration of ASDN in the assay was approximately 100 nM ASDN, and the amount of tritium added to each incubation tube was approximately 0.1 μ Ci in a volume of 100 μ L.
Test chemical(s) stock solutions were prepar ed such that the total volume of each test chemical formulation used per assay was 1% (2 0 μL in 2 mL) of the total assay volume to minimize the potential for the solvent to inhi bit the enzyme. Selection of the solvent, DMSO, was based on the following reasons: i) DM SO was listed as an acceptable vehicle in the OCSPP 890.1200 Guidelines; ii) DMSO was le ss volatile than etha nol; and iii) DMSO was more accurate to pipette because of its density and viscosity.
A stock solution of the positive control substance, 4-OH ASDN, was formulated in DMSO. Fresh dilutions of the stock solution were prepar ed in the same solvent on the day of use. Dilutions were prepared such that the target concentrations of the pos itive control substance (0.1–10,000 nM; Table 4) were achieved by the addition of 20 μ L of the dilution for a final assay volume of 2 mL.
Human recombinant microsomes were purchased from BD Gentest TM 

# , and stored at

− 80±10oC for no longer than 2 months. Microsom es were portioned into individual vials based on the protein concentration of the batch (final dilution of 8 μg/mL) and stored at

− 80±10oC for no longer than 2 months.
Other assay components, such as sodium phosphate buffer, propylene glycol, and NADPH, are reported in Table 1.

TABLE 1. Assay Components and Conditions Assay Factor Values Sodium phosphate buffer (pH 7.4) 0.1M Microsomal Protein 0.004 mg/mL a NADPH 0.3 mM [3H]ASDN 100 nM Propylene Glycol 5% Temperature 37±2°C Incubation Time 15 min a The concentration of microsomal protein was optimized for microsomes that produce approximately 1200 pmol product/(min × mg protein) and 5 pmol product/pmol P450/min.



# 2. Suitability Assessments: The protein concentration and aromatase activity of the microsomes was supplied by the vendor. The minimum mean aromatase activity in the full

# Aromatase (Human Recombinant) Assay (2011) / Page 5 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None 

# activity control samples was determ ined to be 0.187 nmol·mg-protein -1 

# ·min -1 

# , which was greater than the minimum acceptable arom atase activity of 0.1 nmol·mg-protein -1 

# ·min -1



# . 3. Aromatase Assay: Each assay run contained 4 tube s for the full enzyme activity and background activity controls, respec tively, and a full concentrati on curve in triplicate for the positive control and test substance. The arom atase assay was generally conducted according to the procedures described in OCSPP 890.1200 (Section H, pp. 9-10).

The amount of 3 

# H 2 

# O in the aqueous fraction was quantified for each assay tube by liquid scintillation counting (LSC), and aromat ase activity was repo rted in units of nmol·mg-protein -1 

# ·min -1



# . 4. Demonstration of Proficiency: Proficiency data were not in cluded in the study. It was stated that prior to conducti ng this assay, a full-scale assay consisting of three independent runs was conducted using the positive control (4 -OH ASDN) and the four proficiency chemicals (econazole, fenarimol, nitrof en, and atrazine) listed in the OCSPP 890.1200 guideline. Proficiency was demonstrated wh en the positive control met the performance criteria stated in the guideline (see Table 2) and by the correct classification of the proficiency chemicals. It was stated that the data were ge nerated and maintained at the Performing Laboratory; however, the study report did not include actual results from the proficiency testing.

a. Positive Control:

TABLE 2. Performance Criteria for the Positive Control Parameter Lower Limit Criteria Upper Limit Criteria Actual Lower Limit Actual Upper Limit Slope -1.2 -0.8 –1.17 –0.92 Top (%) 90 110 95.68 101.21 Bottom (%) -5 +6 0.16 2.33 Log IC 50 

# (M) -7.3 -7.0 –7.18 –7.04 a Data were obtained from page 30 of the study report.



# b. Proficiency Chemicals:

TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations Econazole 24169-02-6 Inhibitor --- Fenarimol 60168-88-9 Inhibitor --- Nitrofen 1836-75-5 Inhibitor --- Atrazine 1912-24-9 Non-inhibitor ---



# 5. Determination of Aromatase Ac tivity with Test Chemical: The response of aromatase activity to the presence of eight concentrations of the test chem ical per run, in triplicate, was tested during three independent runs. After completion of the first run, the data were reviewed and solubility was vi sually assessed (presence of cl oudiness or a precipitate). No data adjustments were necessary.

# Aromatase (Human Recombinant) Assay (2011) / Page 6 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None

TABLE 4. Test Chemical Study Design for Each Test Run Sample Type Repetitions (Tubes) Description Reference or Chemical (M) Full Activity Control 4 All test components a 

# plus solvent vehicle N/A Bkgd Activity Control 4 Same as above without NADPH N/A 4-OH ASDN Conc 1 2 All test components plus 4-OH ASDN 1×10 -5 

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

# 4-OH ASDN Conc 3 2 All test components plus 4-OH ASDN 1×10 -6.5 

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

# 4-OH ASDN Conc 5 2 All test components plus 4-OH ASDN 1×10 -7.5 

# 4-OH ASDN Conc 6 2 All test components plus 4-OH ASDN 1×10 -8 

# 4-OH ASDN Conc 7 2 All test components plus 4-OH ASDN 1×10 -9 

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

# Test Chemical Conc 1 3 All test components plus test chemical 1×10 -3 

# Test Chemical Conc 2 3 All test components plus test chemical 1×10 -4 

# Test Chemical Conc 3 3 All test components plus test chemical 1×10 -5 

# Test Chemical Conc 4 3 All test components plus test chemical 1×10 -6 

# Test Chemical Conc 5 3 All test components plus test chemical 1×10 -7 

# Test Chemical Conc 6 3 All test components plus test chemical 1×10 -8 

# Test Chemical Conc 7 3 All test components plus test chemical 1×10 -9 

# Test Chemical Conc 8 3 All test components plus test chemical 1×10 -10 

# a The complete assay contained buffer, propylene glycol, microsomal protein, [ 3 

# H]ASDN, and NADPH



# C. DATA ANALYSIS

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

# ·min -1 

# ) and presented as a percent control for the positive co ntrol and test chemical. The following raw data and calculated endpoints for each r un were included in the report (Table 5).

TABLE 5. Raw and Calculated Data Raw/Calculated Data Included (X) DPM/mL for each portion of extract ed aqueous incubation mixture X Average DPM/mL for each aqueous portion (after extraction) X Total DPM for each aqueous portion (after extraction) X The total DPM present in the assay tube at initiation X The percentage of substrate converted to product X Total DPM after extraction corrected for background X Aromatase activity expressed in nmol/mg protein/min 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 graphica l displays were conducted using XLfit, v5.2.0.0 (IDBS, Guildford, UK). Basic stat istical analyses were performed on the data, which included means of replicates, standa rd deviation of the mean, standard error of the mean, and coefficient of variation.
The response curve was fitted by weighted leas t squares nonlinear regression analysis with weights equal to 1/Y. Model fits were carried out using a 4-parameter regression model (XLfit, v5.2.0.0, Fit Model 208, IDBS) and Tukey’s Bi -Weight statistical an alysis for outlier analysis. For each run, the individual percen t of control values were plotted versus logarithm (base 10) of the test chemical conc entration. The fitted concentration response curve was superimposed on the plot, with i ndividual plots prepared for each run. The average percent of control values versus loga rithm of test chemical concentration for the individual runs were included on the same graph with their respective fitted response curves.

# Aromatase (Human Recombinant) Assay (2011) / Page 7 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None 

# In addition, the average percent of control valu es for each run versus the logarithm of test chemical concentration were plotted on a separate graph along with the average concentration response curve across runs were superimposed on the same plot.
3. Interpretation of Results: Interpretation of the assay results was based on the average of three runs, using the categor ies presented in Table 6.

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

# Equivocal Average lowest portion of curves across runs is greater than 75% activity Non-inhibitor Data do not fit model ---


# II. RESULTS

A. CONTROL ACTIVITY: Aromatase activity in the full activity controls ranged from 0.181 to 0.249 nmol·mg-protein -1 

# ·min -1 

# for the 3 test runs, with a mean and standard deviation of 0.212±0.024 nmol·mg-protein -1 

# ·min -1 

# . Activity in the background controls ranged 0.51% to 0.83% and averaged 0.63% of the full control activity. The response of the full activity controls and background controls were acceptable for each run. Each full activity control was generally within 10% of the average for the full activity controls.

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.203± 0.040 nmol·mg-protein
− 1 

# ·min
− 1 

# at the lowest tested concentration (10
− 10 

# M) and 0.002±0.000 nmol·mg-protein
− 1 

# ·min
− 1 

# at the highest tested concentration (10
− 5 

# M). The mean aromatase activity of the positive control (expressed as % full control activity) for each concentration tested across a ll 3 runs is presented in Table 7, along with the overall standard deviation and % CV. Th e inhibition response curves for the positive control are shown in Figure 1. These results were generally within the recommended ranges for the top of the curve, bottom curve, hill slope, log IC 50 

# , and coefficient of variation for replicates of each concentration.

# Aromatase (Human Recombinant) Assay (2011) / Page 8 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None

TABLE 7. Effect of Dimethoate on Aromatase Activity (as Percent of Control) from Independent Runs a Chemical Conc. Log M # Runs Overall Mean Overall SD Overall SEM Overall %CV 4-OH ASDN (positive control) -5 3 1.01 0.13 0.05 13.31 -6 3 7.93 0.49 0.20 6.13 -6.5 3 20.47 1.13 0.46 5.53 -7 3 41.01 2.68 1.09 6.53 -7.5 3 69.67 4.31 1.76 6.19 -8 3 87.16 3.68 1.50 4.22 -9 3 99.73 2.90 1.18 2.90 -10 3 95.08 12.15 4.96 12.78 Dimethoate -3 3 87.90 15.73 6.42 17.90 -4 3 101.06 4.40 1.80 4.36 -5 3 102.94 2.82 1.15 2.74 -6 3 101.00 3.76 1.53 3.72 -7 3 99.94 3.28 1.34 3.28 -8 3 99.09 3.58 1.46 3.61 -9 3 101.03 4.40 1.80 4.36 -10 3 99.47 1.95 0.80 1.96 a Data were calculated by the reviewer using the data from i ndividual runs reported on pages 27-29 of the study report. SD Standard Deviation SEM Standard Error of the Mean CV Coefficient of Variance

# Aromatase (Human Recombinant) Assay (2011) / Page 9 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None 

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

C. TEST SUBSTANCE: For dimethoate, aromatase activity averaged 0.210±0.024 nmol·mg-protein
− 1 

# ·min
− 1 

# at the lowest tested concentration 10 -10 

# M and 0.181±0.021 nmol·mg-protein -1 

# ·min
− 1 

# at the highest tested concentration 10
− 3 

# M. The mean aromatase activity of dimethoate (expressed as % full c ontrol activity) for each concentration tested across all 3 runs is presented in Table 7, along with the overall standard deviation and %CV. Inhibition response curves for dimethoate from each run are shown in Figure 2, and the average inhibition response curve acro ss all runs is shown in Figure 3.

# Aromatase (Human Recombinant) Assay (2011) / Page 10 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None 

# FIGURE 2. Inhibition Response Curves for Dimethoate.

Note: Values enclosed in the diamond sy mbol represent outliers removed during th e regression analysis using Tukey’s BiWeight statistical analysis.

# Aromatase (Human Recombinant) Assay (2011) / Page 11 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None 

# FIGURE 3. Mean Inhibition Response Curve for Dimethoate

Note: Values enclosed in the diamond sy mbol represent outliers removed during th e regression analysis using Tukey’s BiWeight statistical analysis. 

# As shown in Figure 3, the mean inhibition re sponse curve for dimethoate was essentially flat; therefore, the slope was zero and an IC 50 

# was not calculated. The effect of 4OH-ASDN on inhibition of aromatase activity is pres ented in Table 8. The estimated log IC 50 

# for the positive control 4OH-ASDN averaged

− 7.13 M. The average slope of the concentration response curve for 4OH-ASDN was

− 1.02. Confidence in these numbers is high due to the small variation.

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

# (M) Dimethoate Not applicable 4-OH ASDN -7.04 -7.18 -7.18 -7.13 0.08 -1.13 Slope Dimethoate Not applicable 4-OH ASDN -1.17 -0.92 -0.97 -1.02 0.13 -12.97 a The mean, Standard Deviation (SD) and Coefficient of Variance (CV) were calculated by the reviewer. 

# Based on the data from the average response curv e and the criteria listed above in Table 7, the results support the c onclusion that dimethoate does not in hibit aromatase in this assay.

III. DISCUSSION AND CONCLUSIONS

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

# to 10 − 3 

# M resulted in essentially no decrease in aromatase activity. Aromatase activity in the presence of 10
− 3 

# M dimethoate was determined to be 87.0% of control activity. Based on the data inte rpretation criteria established for the assay, dimethoate was classified as a non-inhibitor of the aromatase enzyme.

B. AGENCY COMMENTS: Aromatase activity in the full activity controls ranged from 0.181 to 0.249 nmol·mg-protein
− 1 

# ·min -1 

# for the 3 test runs, with a mean and standard deviation of 0.212±0.024 nmol·mg-protein
− 1 

# ·min
− 1 

# . Activity in the background controls ranged 0.51% to 0.83% and averaged 0.63% of the full control activity. The response of the full activity controls and background controls were acceptable for each run. For the positive control substance (4-OH ASDN), aromat ase activity averaged 0.203±0.040 nmol·mg# Aromatase (Human Recombinant) Assay (2011) / Page 12 of 12 DIMETHOATE / 035001 OCSPP 890.1200 / OECD None 

# protein
− 1 

# ·min
− 1 

# at the lowest tested concentration (10
− 10 

# M) and 0.002±0.000 nmol·mgprotein
− 1 

# ·min
− 1 

# at the highest tested concentration (10
− 5 

# M). For dimethoate, aromatase activity averaged 0.210± 0.024 nmol·mg-protein
− 1 

# ·min
− 1 

# at the lowest tested concentration 10 -10 

# M and 0.181±0.021 nmol·mg-protein
− 1 

# ·min
− 1 

# at the highest tested concentration 10
− 3 

# M.
The average lowest portion of the response cu rve across runs was >75% activity at all concentrations of dimethoate. Because of th e flat response, the slope was zero, and an IC 50 

# was not calculated. The estimated log IC 50 

# for the positive control 4OH-ASDN averaged

− 7.13 M. The average slope of the concen tration response curve for 4OH-ASDN was

− 1.02. Confidence in these numbers is high due to the small variation. Based on the data from the average response curve, the results s upport the conclusion that dimethoate is a noninhibitor of the aromatase enzyme 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:

 Proficiency chemical da ta were not provided.

 Means and standard deviations for aromatase activity in nmol·mg-protein
− 1 

# ·min
− 1 

# for the full activity controls, background controls, and high and low concentrations for the positive control and test substance were not provided. The reviewers were able to calculate these values fr om the individual data.

 Overall means, standard deviations, standard errors and % coefficien ts of variation were not provided for the positive control and test substance (Table 7). The reviewers were able to calculate these values from the individual data.

 The mean, standard deviation, and % coefficien ts of variation were not provided for the positive control for the Hill Slope and log IC 50 

# . The reviewers were able to calculate these values from the individual data.

# DATA EVALUATION RECORD

DIMETHOATE
Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 2-2-2012 (MRID 48616304)

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 J. Sharpe-Kass, M.S. Date: 12/06/2011
Secondary Reviewer Signature: Rebecca L. Bryan, B.S. Date: 12/06/2011
Program Manager: Signature: Jack D. Early, M.S. Date: 12/06/2011
Quality Assurance: Signature: Jack D. Early, M.S. Date: 12/06/2011

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

.

# The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

# DIMETHOATE /03 5001 Estrogen Recept o r Transcriptional Activation (20 11) I 

# Pa ge I of 1 2 O CS PP 890.1300/ OE C D 455


# Primary Re v iewer: Sheila Healy, Ph

. D.

# Sign~n/e~~~ Health Effects Division Date·

. Seco ndary Review er: --'M'--=-i n=e=r-'-v=a-=-M =e= r'"""c=ad=o'"-'''-=P '""""

h=·=D-'-

. _____ 

# S igna

# tu.re:**~** Health E ffect s Divi s ion Date:**~** 

# M

- 1.J

Template version 08 / 2011



# DAT A EVALUATION RECORD

STU DY TYPE : Estrogen Receptor Transcriptiona l Activation (Human ce ll Line , HeLa-9903) ; OCSPP 890.1300; OECD 455.



# PC CODE: 035001 

# DP BARCODE: D 396168



# TXR#: 0056198 

# CAS No.: 60-51-5



# TEST MATERIAL (PURITY): Dimethoate (98% a.i.)



# SYN ONYMS: Phosphorodithioic acid, 0 , 0-dimethyl S-[2-(methy l -amino)

- 2

- oxoethyl] ester



# CITATION: Willoughby, 

# J.A. Sr., et.al. (2011). Dimethoate Technical: Estrogen Receptor Transcriptional Activation (Human Ce ll Line (HeLa-9903)). CeeTox , Kalamazoo , MI. Laboratory Study#: 9148V -l00 373ERTA , November 4 , 20 11. MRID 48616303. Unpublished

.


# SP ON S OR: Chemi no va A/S, P

. O. Box 9, DK-7620 Lemvig , Denmark



# TEST ORDER#: Not provided



# EXECUT IVE SUMMARY: In an estrogen receptor transcriptional activation assay (ERTA; MRID 48616303), hERa-HeLa-9903 ce ll s cultured 

# in vitro were exposed to dimethoate (98% a.i., Batch # 61 l/ 

# A) at concentrations increasing l ogarithmica ll y from 10

- 10 to 10- 3 

# Min DMSO (0.1 %). The assay was performed twice using 96

- well plates and each dimethoate concentration

was tested in 6 wells / plate in each assay. The reference chemica l s 17P-estradiol (strong estrogen), l 7a-estradiol (weak estrogen) , corticosterone (negative compound) and l 7amethyltestosterone (very weak agonist) were a l so tested

. Cells were exposed to the test agent for approximate l y 24 hour s to induce reporter (luciferase) gene products

. Luciferase expression in

was measured using a proprietary method

. Relative transcriptional activity was expressed as percent of the positive control (PC) 1 nM l 7P-estradiol) and compared to l 0 % of the PC maximal response (PC1 0). Cell viability (CV) was a l so monitored in a ll assay runs as percent of

ve hicle control. Laboratory proficiency was demon s trated b y testing the l 0 c h emicals li sted in the Guideline, h oweve r it was not clear if the same batch of ce ll s was used for the current assays.

Dimethoate was tested up to the limi t of cytotoxicity, l 0- 3 

# M. Cytotoxicity (76% CV) occurred in one of two runs, but was not observed in the preliminar y cytotoxicity and so lubili ty assay. In the main assays, the responsiveness of the cell s to the very weak positive control 17a-methyltestosterone was lower than the expected va lu es , indicating a decreased sensitivity of the assay to very weak agonists. At non-cytotoxic concentrations, 17a-methyltestosterone

# Estrogen Receptor Transc riptional Activation (2011) / Page 2 of 12 DIMETHOATE/035001 OCSPP 890.1300/ OECD 455 

# responses reached only 5.3-5.6% RPC. However, a dose-dependent trend was apparent for 17 α
methyltestosterone that reached 25-28% RPC at the cytotoxic (69-76% CV), highest tested concentration of 10 -5 

# M. In contrast, dimethoate r eached a maximum activity of 5.1% RPC without an apparent dose-dependent trend. The responses of dimethoate was also not comparable to the response of the weak estrogen control, 17 α -estradiol (116-134% RPC) and the RPC Max 

# < PC 10 

# in both assay runs. The negative co mpound corticosterone reached a maximum of 3% RPC, also without s howing a dose-dependent trend.

Because the RPC max 

# < PC 10 

# in two assay runs, dimethoate is considered negative for estrogen receptor transactivation in this test.
This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor Transcriptional Activation assay (OCSPP 890.1300).

COMPLIANCE: Signed and dated GLP, Quality Assurance and Data Confidentiality statements were provided.

# Estrogen Receptor Transcriptional Activation (2011) / Page 3 of 12 DIMETHOATE/035001 OCSPP 890.1300/ OECD 455


# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Dimethoate Description: White Solid Source: Cheminova A/S Lot/Batch #: 611/A Purity: 98.0% Solubility: Not reported (NR) Volatility: NR Stability: 10 years Storage conditions: Deep Freeze Vapor pressure: NR CAS #: 60-51-5 Structure:



# 2. Reference substances 17 β -estradiol (strong estrogen; positive control) Supplier: NR Catalogue and Batch #: NR Purity: NR CAS # : 50-28-2


# 17 α -estradiol (weak estrogen) Supplier: NR Catalogue and Batch #: NR Purity: NR CAS # : 57-91-0 

# Corticosterone (negative compound) Supplier: NR Catalogue and Batch #: NR Purity: NR CAS # : 50-22-6


# 17 α -methyltestosterone (very weak agonist) Supplier: NR Catalogue and Batch #: NR Purity: NR CAS # : 58-18-4


# 3. Vehicle(s) Solvent: DMSO. Supplier, lot and purity not reported Solvent control (final concentration): 0.1%



# B. METHODS

# Estrogen Receptor Transc riptional Activation (2011) / Page 4 of 12 DIMETHOATE/035001 OCSPP 890.1300/ OECD 455



# 1. Cell Culture: Stably-transfected hER α -HeLa-9903 cells were obtained from the Japanese Collection of Research Biores ources (JCRB) Cell Bank and were verified to be free of mycoplasma infection using DNA Fluorochrome an alysis by Bionique Tes ting Laboratories. 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 repor ted), in an incubator under 5% CO 2 

# at 37oC. Cells used in the range-finding study were from Pa ssage 35. For the assay runs new cells, frozen down at Passage 14, were cultured and upon reaching 75-90% confluence, cells were subcultured 5 times prior to e xposure to the test material.
2. Transcriptional Activation Assays: For each test, cells were plated in 96-well culture plates at a density of 1x10 4 

# cells/100 μL medium/well and a llowed to attach for at least 3 hours. The Growth media was replaced with media containing serial log dilutions of dimethoate in DMSO. Cells were incubated fo r 24±2 hours at 37±2°C. All concentrations were tested in replicates of 6/plate. Additionally, for each co ncentration 2 replicates/plate were prepared including the hER α antagonist ICI 182,780, to allow for the identification of non-specific induction of the luciferase gene. The total final concen tration of DMSO was 0.1%. Cytotoxicity was determined by a two-r ead propidium iodide (PI) uptake assay. Luciferase activity was determined using a proprietary method.
a. Preliminary Test: Proficiency data was provided in a separate report (MRID 48843501) and is summarized in the table below. However, it was not indicated if the specific batch of cells used for the current assays was tested.
b. Proficiency Chemicals: It was stated that all assays were performed by personnel that have demonstrated proficiency performing the assay as outlined in the guidelines. However, it was not stated that the cells taken from frozen stock were ru n through the proficiency test.

c. Reference Chemicals: To ensure the stability of th e response from the cell line, six concentrations of each of the following reference chemicals were included in each plate in the current assay, along w ith the test chemical: Reference Chemical CAS No. Concentration Range Class 17 β -estradiol 50-28-2 10 -14 

# to 10 -8 

# Strong estrogen 17 α -estradiol 57-91-0 10 -12 

# to 10 -6 

# Weak estrogen Corticosterone 50-22-6 10 -10 

# to 10 -4 

# Negative compound 17 α -methyltestosterone 58-18-4 10 -11 

# to 10 -5 

# Very weak agonist


# 3. Data analysis: To obtain the relative transcriptiona l activity to the 1 nM E2 positive control (PC), the luminescence signals from the concur rent plate were analy zed by subtracting the mean value of the vehicle control from each well value to normalize the data; each normalized value was then divided by the mean value of the normalized PC. The resulting value was multiplied by 100 in order to expres s relative transcriptional activity as a percentage of the PC. The RPC Max 

# for dimethoate was calculate d, but the software used was not reported. The test material was define d as negative for induci ng estrogen receptor transcriptional activation if the RPC Max 

# < PC 10 

# in 2 of 2 (or 2 of 3) runs. LogEC 50 

# 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%

# Estrogen Receptor Transc riptional Activation (2011) / Page 5 of 12 DIMETHOATE/035001 OCSPP 890.1300/ OECD 455 

# cytotoxicity or evidence of insolubi lity were excluded from analyses.

4. Definitions

EC 50 

# = concentration of agonist that induces a response ha lfway between the baseline (bottom) and maximum (top) response

PC 10 

# = concentration of a test chemical at whic h the response is 10% of the response induced by the positive control (E2 at 1 nM) in each plate

PC 50 

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

RPC Max 

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

PC Max 

# = concentration of a test chemical inducing the RPC Max



# II. RESULTS

A. PRELIMINARY TEST: The limit of solubility was determ ined by visual inspection of the test materials and controls afte r preparation of the fi nal 1x dosing solutions in culture media. A sample of the 1x dosing solution was placed into wells of a clear 96-well plate and an endoscope was used to assess precipitation. Cy totoxicity was monitored by a two-read PI uptake assay; PI is a light sensitive dye whic h cannot cross the plasma membrane of intact and viable cells. Cells were seeded into black-walled 96-well plate and exposed to dimethoate at logarithmi c concentrations of 10 -10 

# to 10 -3 

# M, or digitonin ( positive control for cytotoxicity). Following exposure, growth me dium was removed and cells were exposed to PI. Background-corrected fluor escence was calculated for each well and the % cell viability was reported. Based on these results summarized in Table 1, logarithmic concentrations of dimethoate from 10 -10 

# to 10 -3 

# M were selected for the assay.

TABLE 1. Preliminary Test for Solubility, Cytotoxi city, and Concentration-Selection for Dimethoate a 

# Concentration (M) % Viability b 

# Comments 10 -3 

# 90±3 No precipitation observed 10 -4 

# 95±3 No precipitation observed 10 -5 

# 96±5 No precipitation observed 10 -6 

# 90±5 No precipitation observed 10 -7 

# 91±2 No precipitation observed 10 -8 

# 96±4 No precipitation observed 10 -9 

# 94±5 No precipitation observed 10 -10 

# 96±4 No precipitation observed E2 1nM Not Tested VC c 

# Not Tested a Data were obtained from page 21 of the study report. b If viability is <80%, the concentration is considered cytotoxic. c VC = Vehicle control

# Estrogen Receptor Transc riptional Activation (2011) / Page 6 of 12 DIMETHOATE/035001 OCSPP 890.1300/ OECD 455 

# B. Positive and Negative Reference Chemicals
1. Proficiency Chemicals: Limited details of the profic iency chemicals were provided and are summarized in Table 2 below:

TABLE 2. Results of Proficiency Chemicals Test (from MRID 48843501) Compound CAS No. Conc. Range (M) Expected Response a 

# Laboratory Response Notes
Diethylstilbestrol (DES) 56-53-1 10 -14 

# to 10 -8 

# Positive Positive --- 17 α -Ethynyl estradiol (EE) 57-63-6 10 -14 

# to 10 -8 

# Positive Positive --- Hexestrol 84-16-2 10 -13 

# to 10 -7 

# Positive Positive --- Genistein 446-72-0 10 -12 

# to 10 -5 

# Positive Positive Cytotoxic at 0.01 b 

# , 0.1, and 1 mM Estrone 53-16-7 10 -12 

# to 10 -6 

# Positive Positive --- Butyl paraben 94-26-8 10 -11 

# to 10 -4 

# Positive Positive Cytotoxic at 0.1 b 

# and 1 mM 1, 3, 5-Tris (4- hydroxyphenyl) benzene c 

# 15797-52-1 10 -12 

# to 10 -5 

# Positive Positive Cytotoxic at 100μM. PC max 

# approx. 50% of PC. Binds to hER α and has ER antagonistic activity Dibutyl phthalate (DBP) 84-74-2 10 -11 

# to 10 -4 

# Negative d 

# Negative Cytotoxic at 1 mM Atrazine 1912-24-9 10 -11 

# to 10 -4 

# Negative Negative Cytotoxic at 1 mM b 

# Corticosterone 50-22-6 10 -10 

# to 10 -4 

# Negative Negative If not cytotoxic at 1 mM, then that should be the highest tested concentration a Positive = RPC max 

# ≥ 10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPC max 

# fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs b Cytotoxicity is expected to be close to 80% at th is concentration. c Compound selected to challe nge solubility and cytotoxicity d DBP is negative for ER α mediated transcriptio nal activation, but may not be negative for non-ER β mediated transcriptional activation. A positive result would indicate that the sy stem is detecting activity other than that due to pure ER α , and is therefore unacceptable.



# 2. Reference Chemicals: Performance measures derived from the concentration response curve ( e.g ., log PC 50, 

# log PC 10, 

# log EC 50, 

# Hill slope) for the concurrently run reference chemicals are included in Table 3. Th e transcriptional responsiveness of 17 α -estradiol was within the acceptable range. The log EC 50 

# for 17 β -estradiol was below the acceptable range in one out of two assays, and the Hill slope was high in that same assay. The log PC 50 

# and log PC 10 

# for the very weak agonist 17 α -methyltestosterone were not reported. The log PC 10 

# was not reached in either run due to cytotoxic ity observed at the highest concentration. The mean RPC Max 

# values for 17 α -methyltestosterone were 5.3% for the first run and 5.6 % for the second run, compared to 2.3% for the fi rst run and 3.4 % for the second run for corticosterone. However, if the RPC values for the highest tested cytotoxic concentration (that showed 69-76% CV) are considered, 17 α -methyltestosterone reached an RPC max 

# of 25- 28% and the PC 10 

# was reached at approximately 1.6x10 -6 

# M (estimated by the reviewer using non-linear regression). Therefore, while 17 α -methyltestosterone did not reach the expected activity levels according to the Guideline, it did reach the PC 10 

# in the presence of cytotoxicity and showed an apparent dose-dependent tre nd. On the other hand, the negative control corticosterone did not show a dose-dependent trend, even when the cytotoxic co ncentrations are included in the analysis.

# Estrogen Receptor Transc riptional Activation (2011) / Page 7 of 12 DIMETHOATE/035001 OCSPP 890.1300/ OECD 455



# TABLE 3. Performance Measures and Criteria for Reference Chemicals a


# Reference Chemical Parameter Expected Range Values Acceptable Run 1 Run 2 Yes No 17 β -estradiol Log PC 50 

# -11.4 to -10.1 -10.3 -10.5 X Log PC 10 

# <-11 -11.1 -11.5 X Log EC 50 

# -11.3 to -10.1 -10.0 -10.2 X Hill Slope 0.7 to 1.5 1.8 1.1 X Test range 10 -14 

# to 10 -8 

# M 10 -15 

# to 10 -8 

# M 10 -15 

# to 10 -8 

# M X 17 α -estradiol Log PC 50 

# -9.6 to -8.1 -8.7 -8.7 X Log PC 10 

# -10.7 to -9.3 -9.6 -10.7 X Log EC 50 

# -9.6 to -8.4 -8.6 -8.7 X Hill Slope 0.9 to 2.0 1.6 1.4 X Test range 10 -12 

# to 10 -6 

# M 10 -13 

# to 10 -6 

# M 10 -13 

# to 10 -6 

# M X Corticosterone Test range 10 -10 

# to 10 -4 

# M 10 -11 

# to 10 -4 

# M 10 -11 

# to 10 -4 

# M X 17 α -methyltestosterone Log PC 50 

# -6.0 to -5.1 NC NC X Log PC 10 

# -8.0 to -6.2 NC b 

# NC b 

# X Test range c 

# 10 -11 

# to 10 -5 

# M 10 -12 

# to 10 -6 

# M 10 -12 

# to 10 -6 

# M X a Data were obtained from pages 22-24 of the study report. b Estimated at -5.77 (run 1) and 5.76 (run 2) by the reviewer when including the highest tested (cytotoxic) concentration in the calculations. c Cytotoxicity was reported at 10 -5 

# M NC = Not Calculated - the chemical did not achieve 10% of the responsiveness of the positive control (calculated by the reviewer).



# C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls: Data for the vehicle and pos itive controls are included in Table 4. The mean response values (light units) for the vehicle control were 11325 and 11008 for the first and second run, respectively. The mean normalized values for the positive control were 231975 and 173117 for the first and second run, respectively. Light units corresponding to the PC 50 

# (50% of the maximum response) for E2 in this assay is 121650 and 92063 for the first and second run, respectively; the PC 10 

# (10% of the maximum response) is 33390 and 27219 for the first and second run, resp ectively, as calculated by the reviewer.

TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a 

# Sample Vehicle Control Positive Control b



# Runs Mean SD Mean SD Fold Induction c 

# 1 11325 2178 231975 41915 20 2 11008 1347 173117 14169 16 a Data were obtained from page 49-50 of the study report. Results were calculated by reviewer b Positive control was 17 β -estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC)



# 2. Test Material: Relative (to the PC) transcriptional activation at each concentration of dimethoate during the two valid assay runs is pr esented in Tables 5 and 6. A representative concentration-response curve depicting fold induc tion of relative transc riptional activation is presented in Figure 1 below. The mean RPC Max 

# for dimethoate was 3.2-5.1%, the PC Max 

# was

# Estrogen Receptor Transc riptional Activation (2011) / Page 8 of 12 DIMETHOATE/035001 OCSPP 890.1300/ OECD 455



# 10 -6 

# M and the PC 10 

# could not be calculated as the RPC Max 

# was less than 10%. Cytotoxicity was observed in the first valid run at 10 -3 

# M. However, no cytotoxicity was observed in the range-finding assay.

TABLE 5. Parameter for Dimethoate Relati ve Transcriptional Activation (RTA) a 

# Log EC 50 

# NA NA Hill Slope NA NA RPC Max 

# 3.2% 5.1% PC Max 

# 10 -6 

# 10 -6 

# PC 50 

# NA NA PC 10 

# NA NA a Data were obtained from pages 22-23 of the study report. NA= Not Applicable


# TABLE 6. Dimethoate RTA comp ared to reference compounds

Negative Dimethoate Positive References Molar Reference Very Weak Weak Strong Conc. Corticosterone 17 α -MT 17 α -estradiol 17 β -estradiol Run 1 1E-15 - - - - 0.7 1E-14 - - - - 0.4 1E-13 - - - 1.5 0.9 1E-12 - - 1.0 0 1.1 1E-11 1.3 - 0.8 1.3 11 1E-10 0.9 0.9 3.4 2.2 63 1E-09 1.7 0.4 0.8 21 139 1E-08 0.3 1.4 3.0 105 125 1E-07 2.3 1.0 4.5 116 - 1E-06 2.2 3.2 5.3 93 - 1E-05 -1.3 2.7 28 (76%) - - 1E-04 -1.9 (72%) 2.4 - - - 1E-03 - 0 (77%) - - - Run 2 1E-15 - - - - 0.6 1E-14 - - - - 1 1E-13 - - - - 2.1 1E-12 - - 0.2 0.5 3.2 1E-11 0.3 - -0.1 0.7 17 1E-10 0.3 0.5 0.2 1.1 80 1E-09 1.1 1.2 0.5 31 136 1E-08 0.6 1.9 2.7 25 126 1E-07 3.4 1.1 5.6 105 - 1E-06 2.5 5.1 4.3 134 - 1E-05 0.2 4.6 25 (69%) 95 (79.5%) - 1E-04 -2.6 (75%) 3.1 - - - 1E-03 - 1.1 - - -

Note: If cell viability < 80%, it is indicated in parenthesis.

# Estrogen Receptor Transc riptional Activation (2011) / Page 9 of 12 DIMETHOATE/035001 OCSPP 890.1300/ OECD 455



# 3. Performance Criteria: The concurrent reference chemical 17 α -estradiol included in each experiment fell within the acceptable range (Table 3). The log EC 50 

# for 17 β -estradiol was below the acceptable range in one out of two assays, and the Hill slope was high in that same assay. 17 α -methyltestosterone induced a maximal tr anscriptional activa tion of 28% of the positive control but only at cytotoxic concentrations; therefore a proper PC 10 

# and PC 50 

# could not be calculated for the very weak agonist. Based on calcula tions conducted by the reviewer on the raw data provided, the fold-induction corresponding to PC 10 

# of concurrent PC was greater than 1+2 standard devi ations of the fold-induction va lue of the concurrent VC. Variability was high and th e results were poorly re producible among runs.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The highest soluble concen tration of dimethoate was 10 -3 

# M, as identified in the preliminary assay. There was no cytotoxicity observed at any of the exposure concentrations of dimethoate tested except the highe st concentration (10 -3 

# M) in the first valid run.
Four runs were conducted; howev er, the first two runs were in valid because the data did not fit either the assay acceptance criteria or histor ical data for the performing laboratory. Data from these runs were not used to assess dime thoate's agonist activity at the human estrogen receptor alpha (hERa). The subsequent two runs of the assay using new cells were judged to be acceptable, and therefore, valid runs of the assay. In these two valid and independent runs of the transcriptional activation assay, dimethoate did not result in an in crease in luciferase activity at any of the concentrations assessed (RPC Max 

# <10%).

B. REVIEWER COMMENTS: Dimethoate was tested up to the limit of cytotoxicity, 10 -3 

# M. Cytotoxicity occurred in one of two runs, but was not observed in the preliminary cytotoxicity and solubility a ssay. The very weak agonist, 17 α -methyltestosterone, did not induce transcriptional activation equal to 50% or 10% of the positive co ntrol at non-cytotoxic concentrations; therefore a proper PC 10 

# and PC 50 

# could not be calculated. The reviewer noted that the maximum RPC of 17 α -methyltestosterone was 5.3% at 10 -4 

# M in the absence of cytotoxicity; however an RPC max 

# of 28% was reached at 10 -3 

# M in the presence of cytotoxicity (76% CV). Of additional concer n was the cytotoxicity observed at the highest concentrations of 17 α -estradiol and corticosterone. Cytot oxicity should not have occurred in any of the guideline recomme nded concentrations.

Because the RPC max 

# < PC 10 

# in two assay runs, dimethoate is considered negative for estrogen receptor transcriptional activati on in this test system.

C. STUDY DEFICIENCIES: The following study deficiencies were noted:

 17 α -methyltestosterone did not i nduced transcriptional activati on equal to 50% or 10% of the positive control in the absence of cytotoxicity; therefore proper PC 10 

# and PC 50 

# values could not be calculated.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 1 of 91 Data Requirement: EPA DP Barcode 402437
OECD Data Point 229
EPA MRID 48670603 EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test material: Dimethoate Purity: 98.1%
Common name
Chemical name: IUPAC: CAS name: Phosphorodithioic acid, O,O-dimethyl S-[2-(methylamino)-2- oxoethyl] ester.
CAS No.: 60-51-5 Synonyms:
EPA PC Code: 035001

Primary Reviewer: Joan Gaidos Signature: Senior Scientist, Cambridge Environmental, Inc. Date: 11/29/12

Secondary Reviewer: Teri S. Myers Signature: Senior Scientist, Cambridge Environmental, Inc Date: 11/30/12

Primary Reviewer: Amy Blankinship, Biologist
EPA/OPP/EFED/ERB3

Additional (and final) Reviewer: Justin Housenger, Biologist
EPA/OPP/EFED/ERB5 Date: 05/27/15

Date Evaluation Completed: 29-MAY-2015 Digitally signed by JUSTIN HOUSENGER DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=JUSTIN HOUSENGER, dnQualifier=0000044455 Date: 2015.06.03 13:58:39 -04'00' 

# AMY BLANKINSHI P Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.03 16:06:57 -04'00'

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 2 of 91 CITATION: Schneider, S.Z., K.H. Martin, T.Z. Kendall, and H.O. Krueger. 2012. Dimethoate; Fish Short-term Reproduction Assay with the Fathead Minnow ( Pimephales promelas ). Performed by Wildlife International, Ltd., Easton, Maryland, Report No.: 232A-131. Submitted by Cheminova A/S, Arlington, Virginia. Completion date May 4, 2012



# .

The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 3 of 91 EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay of dimethoate 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; 6 months old) were exposed to dimethoate (98.1% purity) at nominal concentrations of 0 (negative control), 4.0, 20, and 100 mg a.i./L; mean-measured concentrations were <LOQ (<1.00), 3.4, 18, and 96 mg a.i./L. The test system was maintained at 23.9 to 25.2 o 

# C and a pH of 7.8 to 8.4.

Adult male survival was 100% except at the lowest concentration in which one animal died (87.5% survival). Survival in females was 100% for all test levels. There were no clinical signs of toxicity reported with the exception of one male in the low treatment group observed to be lethargic and surfacing (the same fish that died) and another male in the middle treatment group observed to have a crooked spine. There were no notable observations with regards to behavior or changes in appearance, such as color/banding, or ovipositor appearance. The reviewer, however, noted that size of dorsal nape pad in males was higher in the control group (median score=5) relative to the dime thoate-treated groups (median scores = 3-4).

There were reductions, ranging from 11 to 17% of negative control values, in male body weight at the alltreatment concentrations which were significant at the mid and high concentrations (p<0.05; JonckheereTerpstra test) with the p-value in the low treatment concentration approaching significance (p = 0.057). Although the response was not strictly monotonic when examining the means, it did follow a concentration response when looking at the medians. There were no other significant differences for growth.

Spawning occurred at least every 4 days, and fecundity in negative control was 29.5 eggs/female/day/replicate (range 27.3-32.4); fertilization success in the negative control group averaged 98.3%. Both fecundity and fertility were significantly reduced by 80% and 3%, respectively, at the high concentration compared to the negative control (p<0.05, Dunnett’s test).

Male gonado-somatic index (GSI) was reduced 28-29% at the mid and high concentrations (p<0.05; Jonckheere-Terpstra test) compared to the negative control. Median male tubercle scores were reduced in a monotonic manner, with reductions of 17 and 21% at the mid and high concentrations compared to the negative

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 4 of 91 control values (p-values of 0.063 and 0.051, respectively ; Jonckheere-Terpstra test). There were no significant effects (p>0.05) on female GSI, and no female tubercles were observed.

There were no significant effects on male or female plasma vitellogenin (VTG) concentrations. Although not statistically significant, there was a 15 and 21% reduction in female VTG in the middle and high treatment group, respectively, compared to the negative control. Sex steroids (plasma 17 ȕ -estradiol and testosterone) were not measured during this study.

There were no significant differences (p>0.05) between histopathology severity scores or gonadal stages in treated groups compared to the negative controls. However, in male fish, there were two incidences (2/8) of minimal to mild increases in spermatogonia and primary spermatocytes with coinciding minimal to mild decreases in secondary spermatocytes and spermatids in the high treatment group. In females, there were two incidences (2/16) of mild to moderate increases in oocyte atresia in the high treatment group and one incidence (1/16) in the middle treatment group. This was observed with a one incidence of mild and moderate granulomatous inflammation in the low treatment concentration but no diagnoses at the middle and high treatment concentrations. Also noted was the presence of microsporidia in 3 females in the control group (that coincided with two incidences of granulomatous inflammation) and 2 females in the high treatment group that coincided with incidences of granulomatous inflammation and increased oocyte atresia.

All of the performance criteria were met except that dissolved oxygen concentrations fell to ca. 55% of saturation on Day 14 in the middle concentration and gentle aeration was added. Dissolved oxygen concentrations
remained above 60% of saturation for the remainder of the test.

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: 6 months Mean body weight at test initiation (if measured): Male 1.5 g; Female 0.9 g Mean length at test initiation (if measured): Not reported.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 5 of 91 Test Type (Flow-through, Static, Static Renewal): Continuous flow-throughData Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 6 of 91Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Dimethoate.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF3.4 No No No No NoNo No No No No NA NA NA NA18 NoNoYes3NoYesNoNoNoNoNoNA NA NA NA96YesYesYes3NoYesNoYes4Yes4NoNoNA NA NA NAAbbreviations:.E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, solvent control not included in this assay.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.3While not statistically significant (p>0.050), for male tubercle score, reductions of 17 and 21% at the 18 and 96 mg a.i./L treatment levels werenoted with p-values of 0.063 and 0.051, respectively (Jonckheere-Terpstra test).4Testes of 2 of 8 fish from high treatment group showed mild increase in spermatogonia and primary spermatocytes with minimal tomild decreasein secondary spermatocytes and spermatids. The ovaries of 2 of 16 fish in the high treatment group showed a mild to moderate increase inoocyte atresia, a single incidence of oocyte atresia was noted in the middle treatment group, and two incidences of granulomatous inflammationwere noted in the low treatment group. However, there were no statistically significant differences found between the severityscores or gonadalstages in the treatment groups as compared to the negative control.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 7 of 91 I. MATERIALS AND METHODS

GUIDELINE FOLLOWED: This study was conducted according to the U.S. EPA OCSPP 890.1350: “Fish Short-Term Reproductive Assay” and OECD 229 (2009). The following deviations were noted:

1. The 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. Additionally, the light intensity was reported to range from 616 to 1376 lux during exposure; OCSPP 890.1350 guideline requirements recommend light intensity 540 – 1080 lux (at water’s surface).

2. Dissolved oxygen concentrations in the test solutions remained at or above 60% of saturation (4.9 mg/L) for most of the exposure period. However, dissolved oxygen
concentrations fell to ca. 55% of saturation on Day 14, and gentle aeration was added. Dissolved oxygen concentrations remained above 60% of saturation for the remainder of the test. OCSPP 890.1350 guideline requirements recommend dissolved oxygen (DO) >4.9 mg/L (>60% air saturation).

3. Individual fish weights at study initiation were ± 30% for both males and females. EPA recommends that the subsample of fish weighed before the test be within ±20% of the
estimated mean for each sex.

Remaining OCSPP 890.1350 validity and performance criteria were met.

These deviations do not impact the interpretation of the results of the study.



# COMPLIANCE: Signed and dated No Data Confidentiality, GLP, and Quality Assurance statements were provided. This study was conducted in compliance with all pertinent U.S. EPA Good Laboratory Practice regulations with the following

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 8 of 91 exceptions: periodic analyses of water for potential contaminants were not performed according to Good Laboratory Practice Standards, but were performed using a certified laboratory and standard US EPA analytical methods.

A. TEST MATERIAL: Dimethoate; CAS# 60-51-5

Description: Solid

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



# Lot No./Batch No. : 60526-00

Purity: 98.1%

Impurities: None reported

Stability of Compound: Analytical verification of the test material from time 0, 7, 14, and 21 days yielded recoveries of 83.6% to 100% of nominal concentrations. Stock solution fortified with dimethoate at concentration of 800 mg a.i./L yielded a recovery of 88.3%. Pretest diluter verification samples collected 1 or 2 days prior to test initiation yield recoveries of 82.2-101% of nominal at the 4.0, 20 and 100 mg a.i./L test concentrations. The test material appears to be
stable under the test conditions at these treatment levels.

Storage Conditions of Test Chemicals: Stored at d 20 q C.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 9 of 91B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: Fathead Minnow 

# EPA recommends fathead minnow (Pimephales promelas).Species scientific name: 

# Pimephales promelasSpecies strain (if stated): Not reportedWere fish obtained from asingle laboratory stock?Yes 

# EPA recommends that fish be from a single laboratory stock.Were acclimation conditionssame as definitive test?Yes 

# EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive

test.Acclimation period:10 weeks 

# EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 10 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Mortalities during the 7 days prior topre-exposure period were <5%.Fish did not receive any treatment fordisease during acclimation period.Behavioral abnormalities or clinical signswere not reported. 

# EPA recommends that mortality during the 7 days prior to the pre-exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA recommends that the fish be rejected. If mortality is between 5-10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur during this extended acclimation period, EPA recommends that the fish not be used.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 11 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsType of food:Sera Vipan and brineshrimp nauplii ( 

# Artemia

salina) 

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

condition.Source of food:Sera, North AmericanFrequency of feeding:2 times/dayDetails on feeding:Fish not fed at least 12hours prior to testtermination.Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:6 months 

# EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months

old.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 12 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsMean weight of males at testinitiation (if determined):1.5 g Based on 10 males used to stockaquaria for pre-exposure phase. 

# EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for each sex.Range of individual weights(males) at test initiation (ifdetermined):Not reported Individual weights within ± 30% of theestimated mean.Mean weight of females at testinitiation (if determined):0.9 g Based on 10 females used to stockaquaria for pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):Not reported Individual weights within ± 30% of theestimated mean.Mean length of males at testinitiation (if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase: 14 days 

# EPA recommends a minimum of 14 days.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 13 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsWere pre-exposure conditionsidentical to the definitive test?Yes 

# EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:32Additional breeding groups weremaintained so breeding groups with aproven history of spawning could beselected for the exposure period. 

# EPA recommends that additional tanks set up at the beginning of pre-exposure will ensure that sufficient replicates with the correct sex ratio are available for the definitive test.Number of males per tank:2Number of females per tank:4Pre-exposure fecundity:Not reported The top 16 spawning groups from thepre-exposure period were selected forthe definitive test. 

# EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 14 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of spawns during preexposure:> two times in 7 daysand at least 15eggs/female/day 

# EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Details on pre-exposure:None.C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Continuous flow-through

# EPA recommends the use of a flowthrough system. As noted in the Corrections and Clarifications document 1 

# , the use of a static renewal system is not recommended for this assay.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 15 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem:Flow-through diluters Not further described 

# Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:6 volume additions/testvessel/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.Details on toxicant mixing forflow-through systems:Flow-splitting chambers were usedbetween diluter cells and the 4 replicatetest vessels.Treatment recoveries prior to test initiation(-1 to -2 days) were 82.2-101%.Flow splitting accuracy was ± 10%.

# 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%.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 16 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsAeration?Gentle aeration added onDay 14.Dissolved oxygen levels in the 20 mg a.i./Ltreatment group decreased to 

# ca. 55% ofsaturation. 

# EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (< 60% saturation).Source of dilution water: Well water. 

# EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?YesData Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 17 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsTest 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:12L (37 x 22 x 17 cmhigh). 

# EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Fill volume:10L; 14 cm depth 

# EPA recommends 10 L solution per tank.Spawning substrate material: Three inverted semicircular PVC pipes. 

# 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: 

# Ca. 10 cm lengthAdditional details on exposuresystem:None.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 18 of 91Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)23.9 25.224.6 Continuously 

# EPA recommends temperature 25±1 o 

# C; inter-replicate and inter-treatment differentials should not exceed 1 o 

# C.pH 7.88.48.1Weekly 

# EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)4.58.26.4Weekly 

# EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L asCaCO3)176 182 179 Weekly 

# EPA recommends total alkalinity > 20 mg/L as CaCO 3.Hardness (mg/L as CaCO3) 136 148 142 WeeklyTotal organic carbon (mgC/L)Not reported Not reported <1 Once 

# EPA recommends that total organic carbon in dilution water be < 2 mg/L.Unionized ammonia (Pg/L) Not reported Not reported Not reported None 

# EPA recommends that unionized ammonia in the dilution water be < 1

P g/L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 19 of 91ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsResidual chlorine (Pg/L) Not reported Not reported Not reported None 

# EPA recommends that residual chlorine in dilution water be < 10 P g/L.Conductivity (PS/cm) 348 376 362 Weekly 

# General recommendations for frequency of measurements: EPA recommends that temperature, pH, and dissolved oxygen be measured in all test tanks at least weekly and that hardness and alkalinity be measured in controls and in one tank at the highest test concentration at least weekly. In addition, continuous temperature monitoring of at least one tank is encouraged.Abbreviations:NANot applicable.1Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 20 of 91D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes 

# EPA recommends conducting a range-finder if 96-hour LC 50 

# data for the fathead minnow are unavailable.If yes, what was the method fordetermining the highest testconcentration in the rangefinder?Reported water solubilityof 39.8 g/L at 25qC andthe reported LC50 for fishranged from 6 to 133mg/L and guidelinerecommendations oftesting up to 100 mg/L. 

# EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is appropriate.Species:Not reportedLife stage:Not reported 

# EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in the

test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 21 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:14 days 

# EPA recommends a 96-hour exposure.Additional details:Nominal concentrationsof 0.81, 2.7, 9.0, 30and 100 mg a.i./L. 

# EPA recommends conducting a range-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentration-response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or 1/3 the derived 96-hr LC 50, 

# may be selected as the highest exposure concentration in the 21-

day test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 22 of 91Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days 

# EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in thedefinitive test:Guideline maximumconcentration of 100mg a.i./L.A spacing factor of 5 was selected forthe test concentrations. 

# EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96-hour LC 50 

# ), whichever concentration is lowest.Reference study citation (ifapplicable):Not reportedSeparation of testconcentrations:0 (control), 4.0, 20,and 100 mg a.i./L 

# EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or ten-fold) is scientifically acceptable 1 

# .Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 23 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of test concentrations:3 

# EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean measuredLimit of quantification (LOQ): <1.00 mg a.i./L 

# EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reported.Frequency of measurement: 0, 7, 14, and 21 days 

# It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two replicates per treatment level.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 24 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsWas the randomized completeblock design used?Yes 

# EPA recommends that all fish be randomly assigned to tanks during pre-exposure. Tanks are then ranked according to preexposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive test treatment and block first. Each block contains one replicate of each treatment, including controls.Number of replicates in control:4 

# EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):NA 

# EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per testitem treatment level:4 

# EPA recommends 4 replicates.Number of male fish perreplicate at test initiation:2 

# EPA recommends 2 males per replicate.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 25 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of female fish perreplicate at test initiation:4 

# EPA recommends 4 females per replicate.Was a solvent used?NoSolvent type (if applicable):NAMaximum solvent concentration(if applicable):NA 

# EPA recommends that the solvent not exceed 0.02 ml/L3 

# . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 

# .Was a positive control used?NoPositive control (if applicable):NA3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review.Aquatic Toxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Serieson Testing and Assessment. No. 23. Paris, France.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 26 of 91ParameterValue(s)Details or RemarksGuideline RecommendationsPositive control concentration(s)(if applicable):NAPhotoperiod:16 hrs light:8 hrs dark 

# EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’ssurface:616-1376 luxCool white fluorescents 

# EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:None.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 27 of 91Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Norflurazon.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl (dilutionwater only)<1.00 <LOQ NA 

# EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 

# 20%.Solvent control (ifapplicable)NA NA NATreatment 14.03.42.2± 0.07Treatment 220185.7± 1.1Treatment 3100961.7± 1.7Diluter StockNANANAAbbreviations:CVCoefficient of variation.LOQ=1.00 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 28 of 91E. ObservationsBiological Endpoints: Survival, fecundity, fertilization success,and clinical signs were observed daily. At test termination(Day 21),secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal stagingand 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 clinic al signs occur at least daily. At test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma vitellogenin, and plasma sex steroids (testosterone and 17ȕ 

# -estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal beha vior.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 29 of 91 II. RESULTS AND DISCUSSION

A. Results

Mean male survival values were 100, 87.5, 100 and 100% in the mean-measured 0 (control), 3.4, 18 and 96 mg a.i./L treatment levels, respectively, and female survival values were 100% at all treatment levels (Table 9). One male mortality and zero female mortalities were observed.

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

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

N 1 

# # Surviving % Survival N 1 

# # Surviving % Survival

Control (<LOQ) 8 8 100 16 16 100

3.4 8 7 87.5 16 16 100

18 8 8 100 16 16 100

96 8 8 100 16 16 100

Abbreviations: NA 

# Not applicable.

1 

# Total number of fish at test initiation. LOQ=1.00 mg a.i./L.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 30 of 91 Mean male body weight values were 2.00, 1.79, 1.67 and 1.72 g, and mean female body weight values were 1.05, 1.06, 1.04 and 1.06 g in the mean-measured 0 (control), 3.4, 18 and 96 mg a.i./L treatment levels, respectively (Table 10). Mean male body length values were 52, 51, 51, and 51 mm, and female body length values were 45, 44, 45 and 45 mm in the mean-measured 0 (control), 3.4, 18 and 96 mg a.i./L treatment levels, respectively.

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

Treatment (mg a.i./L)

[meanmeasured] Body Weight Length

Males Females Males Females

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

Control (<LOQ) 4 2.00 0.075 4 1.05 0.056 4 52 0.5 4 45 1.1

3.4 4 1.79 0.275 4 1.06 0.056 4 51 2.3 4 44 0.9

18 4 1.67 0.261 4 1.04 0.069 4 51 1.4 4 45 0.8

96 4 1.72 0.070 4 1.06 0.102 4 51 1.3 4 45 1.7

Abbreviations: NA 

# Not applicable. ND 

# Not determined. SD 

# Standard deviation. LOQ=1.00 mg a.i./L.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 31 of 91 Mean fecundity values were 29.5, 29.6, 31.3 and 6.0 eggs/female/day, and fertilization success was 98.3, 96.8, 97.0 and 95.0% in the 0 (control), 3.4, 18 and 96 mg a.i./L treatment levels, respectively (Table 11).

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

.
Treatment (mg a.i./L)

[meanmeasured] Fecundity 1 

# ±SD Fertilization Success (%) 2 

# ±SD

Control (<LOQ) 29.5 2.19 98.3 0.55

3.4 29.6 7.44 96.8 1.19

18 31.3 6.99 97.0 2.10

96 6.0 3.27 95.0 1.81

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=1.00 mg a.i./L.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 32 of 91 Median male tubercle scores were 24, 22, 20 and 19 in the mean-measured 0 (control), 3.4, 18 and 96 mg a.i./L treatment levels, respectively (Table 12). None of the surviving females were found to have
tubercles.

Median male fatpad scores were 4.4, 3.4, 3.6 and 3.4 in the mean-measured 0 (control), 3.4, 18 and 96 mg a.i./L treatment levels, respectively. According to the study authors, there were no apparent treatment related changes in size or shape of male fatpads in any treatment group compared to the negative controls. No females were noted with a fatpad (score 1 = no fatpad visible).

Table 12: Nuptial Tubercle Score in Fathead Minnow ( P imephales promelas)

.

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

n Median
Tubercle Score n Median
Tubercle Score

Control (<LOQ) 4 24 4 0

3.4 4 22 4 0

18 4 20 4 0

96 4 19 4 0

Abbreviations: NA 

# Not applicable. ND 

# Not determined. SD 

# Standard deviation. LOQ=1.00 mg a.i./L.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 33 of 91 Mean male GSI was 1.75, 1.42, 1.25 and 1.27%, and mean female GSI was 14.5, 13.5, 14.1 and 16.6% in the mean-measured 0 (control), 3.4, 18 and 96 mg a.i./L treatment levels, respectively (Table 13).

Table 13: Gonado-Somatic Index (GSI) in Fathead Minnow ( P imephales promelas)

.

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

n Mean GSI 1 

# (%) ±SD n Mean GSI 1 

# (%) ±SD

Control (<LOQ) 4 1.75 0.189 4 14.5 0.94

3.4 4 1.42 0.444 4 13.5 1.83

18 4 1.25 0.178 4 14.1 1.21

96 4 1.27 0.210 4 16.6 4.57

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=1.00 mg a.i./L.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 34 of 91 Median male gonadal stages were 2.5, 2, 2 and 2.3, and female gonadal stages were 2.5, 2.5, 2.5 and 35 in the mean-measured 0 (control), 3.4, 18 and 96 mg a.i./L treatment levels, respectively (Table 14).

Table 14: Gonadal Staging in Fathead Minnow ( P imephales promelas)

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

n Median Stage 1 

# n Median Stage 2



# Control (<LOQ) 4 2.5 

# 4 2.5



# 3.4 4 2 

# 4 2.5



# 18 4 2 

# 4 2.5



# 96 4 2.3 4 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-dev elopment, 3=late development, 4=late development/hydrated, 5=post-ovulatory. LOQ=1.00 mg a.i./L.

In male fish, there were two incidences of minimal to mild increases in spermatogonia and primary spermatocytes with coinciding minimal to mild decreases in secondary spermatocytes and spermatids in the high treatment group (Table 15). In females, there were two incidences of mild to moderate increases in oocyte atresia in the high treatment group and one incidence of in the middle treatment group (Tables 17- 18). There were also two incidences of granulomatous inflammation in the low treatment group.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 35 of 91 Table 15: Gonadal Histopathology in Male Fathead Minnow ( P imephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Diagnostic Observations 1



# Severity 

# Increased Proportion of Spermatogonia Presence of
Testis-Ova Increased
Testicular Degeneration Duct
Mineralization Interstitial cell (Leydig) hypertrophy/hyperplasia

n Incidence n Incidence n Incidence n Incidence n Incidence

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

1 8 0 NA NA NA NA NA NA 8 0

2 8 0 NA NA NA NA NA NA 8 0

3 8 0 NA NA NA NA NA NA 8 0

4 8 0 NA NA NA NA NA NA 8 0

3.4 0 7 7 NA NA NA NA NA NA 7 7

1 7 0 NA NA NA NA NA NA 7 0

2 7 0 NA NA NA NA NA NA 7 0

3 7 0 NA NA NA NA NA NA 7 0

4 7 0 NA NA NA NA NA NA 7 0

18 0 8 8 NA NA NA NA NA NA 8 8

1 8 0 NA NA NA NA NA NA 8 0

2 8 0 NA NA NA NA NA NA 8 0

3 8 0 NA NA NA NA NA NA 8 0

4 8 0 NA NA NA NA NA NA 8 0

96 0 8 6 NA NA NA NA NA NA 8 7
1 8 2 NA NA NA NA NA NA 8 1
2 8 0 NA NA NA NA NA NA 8 0

3 8 0 NA NA NA NA NA NA 8 0

4 8 0 NA NA NA NA NA NA 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=1.00 mg a.i./L. NA = Not applicableData Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 36 of 91Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( 

# P imephales promelas).Treatment(mg a.i./L)[meanmeasured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceControl(<LOQ)0NA NA NA NA NA NA88881NANANANANANA80802NANANANANANA80803NANANANANANA80804NANANANANANA80803.40NA NA NA NA NA NA77771NANANANANANA70702NANANANANANA70703NANANANANANA70704NANANANANANA7070Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 37 of 91Treatment(mg a.i./L)[meanmeasured]Additional Diagnostic Observations1SeverityDecreasedProportion ofSpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence180NA NA NA NA NA NA88881NANANANANANA80802NANANANANANA80803NANANANANANA80804NANANANANANA8080960NA NA NA NA NA NA86871NANANANANANA81812NANANANANANA81803NANANANANANA80804NANANANANANA80801Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. SeeAppendix E of the test guideline for reference.LOQ=1.00 mg a.i./L.NA = Not applicable

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Page 38 of 91 Table 17: Gonadal Histopathology in Female Fathead Minnow ( P imephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1



# Severity Increased Oocyte
Atresia Perifollicular Cell Hyperplasia/ Hypertrophy Decreased
Yolk Formation Aggregates of macrophages,

multifocal

n Incidence n Incidence n Incidence n Incidence

Control (<LOQ) 0 16 16 NA NA NA NA NA NA



# 1 16 0 NA NA NA NA NA NA



# 2 16 0 NA NA NA NA NA NA


# 3 16 0 NA NA NA NA NA NA



# 4 16 0 NA NA NA NA NA NA

3.4 

# 0 16 16 NA NA NA NA NA NA



# 1 16 0 NA NA NA NA NA NA



# 2 16 0 NA NA NA NA NA NA


# 3 16 0 NA NA NA NA NA NA



# 4 16 0 NA NA NA NA NA NA

18 

# 0 16 15 NA NA NA NA NA NA



# 1 16 0 NA NA NA NA NA NA



# 2 16 0 NA NA NA NA NA NA


# 3 16 0 NA NA NA NA NA NA



# 4 16 1 NA NA NA NA NA NA

96 

# 0 16 14 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 

# 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=1.00 mg a.i./L

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Page 39 of 91 Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow ( P imephales promelas)

.

Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1



# Severity Interstitium/multifocal
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 16 16 NA NA

1 NA NA NA NA 16 0 NA NA
2 NA NA NA NA 16 0 NA NA
3 NA NA NA NA 16 0 NA NA
4 NA NA NA NA 16 0 NA NA
3.4 0 NA NA NA NA 16 14 NA NA

1 NA NA NA NA 16 1 NA NA
2 NA NA NA NA 16 1 NA NA
3 NA NA NA NA 16 0 NA NA
4 NA NA NA NA 16 0 NA NA
18 0 NA NA NA NA 16 16 NA NA

1 NA NA NA NA 16 0 NA NA
2 NA NA NA NA 16 0 NA NA
3 NA NA NA NA 16 0 NA NA
4 NA NA NA NA 16 0 NA NA

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Page 40 of 91 Treatment (mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1



# Severity Interstitium/multifocal
inflammation Egg Debris in
Oviduct Granulomatous
Inflammation Decreased PostOvulatory
Follicles

n Incidence n Incidence n Incidence N Incidence

96 0 NA NA NA NA 16 16 NA NA

1 NA NA NA NA 16 0 NA NA
2 NA NA NA NA 16 0 NA NA
3 NA NA NA NA 16 0 NA NA
4 NA NA NA NA 16 0 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. 2 Endpoints at the high treatment level were not statistically analyzed due to high mortality. LOQ=1.00 mg a.i./L NA = Not applicable

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Page 41 of 91 Mean male VTG was 120, 140, 170 and 40 ng/mL, and mean female VTG was 1807000, 2262000, 1543000 and 1436000 ng/mL in the mean-measured 0 (control), 3.4, 18 and 96 mg a.i./L treatment levels, respectively (Table 19).

Table 19: Plasma Vitellogenin in Fathead Minnow ( P imephales promelas)

.

Treatment (mg a.i./L) [mean-measured] Plasma Vitellogenin (VTG)

Males Females

n Mean (ng/mL plasma) ±SD 1 

# n Mean (ng/mL plasma) ±SD 1



# Control (<LOQ) 4 120 55 

# 4 1807000 593000



# 3.4 4 140 103 

# 4 2262000 493000



# 18 4 170 191 

# 4 1543000 429000



# 96 4 40 25 4 1436000 695000

Abbreviations: NA 

# Not applicable. ND 

# Not determined. SD 

# Standard deviation. LOQ=1.00 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 42 of 91Mean plasma testosterone and plasma 17ȕ-estradiol in male and females was not measured (Table 20).Table 20: Plasma Sex Steroids in Fathead Minnow ( 

# P imephales promelas). Not measured.Treatment(mg a.i./L)[mean-measured]Plasma Testosterone (T)Plasma 17ȕ-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDControl (<LOQ) NA NA NA NANA NA NA NA NA NA NA NA3.4 NA NA NA NA NA NANA NA NA NA NA NA18 NA NA NA NA NA NANA NA NA NA NA NA96 NA NA NA NA NA NANA NA NA NA NA NAAbbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=1.00 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 43 of 91There were no notable observations with regards to behavior or changes in appearance, specifically color/banding, ovipositor appearance, orsize of dorsal nape pad in the control or treated groups (Table 21). There was one incidental appearance of a male with a crooked spine inthe 18 mg a.i./L treatment group, and one male appeared lethargic on Day 15 in the 3.4 mg a.i./L treatment group. All other fish in thenegative controls and treatment groups appeared normal, and no overt signs of toxicity were observed during the test period.Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow ( 

# P imephales promelas).Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceTypenIncidenceControl (<LOQ)None8 0None16 03.4 Lethargic,surfacing,mortality81 None 16018 Crookedspine81 None16096 None80None160LOQ=1.00 mg a.i./L.

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Page 44 of 91 B. Study Author’s Analysis and Conclusions

The study author analyzed survival, wet weight, total length, 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 controls.

Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant effects were detected for p d 0.05 (SAS, version 8.2). Survival data were analyzed using Fishers Exact test, and histopathology scores and stages were analyzed using step-down Jonckheere-Terpstratrend test. For the other endpoints, if the results were consistent with a monotonic concentrationresponse, Jonckheere-Terpstra test was used. All endpoints except survival and histopathology severity scores were analyzed by performing pair-wise comparisons using Dunnett’s Multiple Comparison 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 non-normality orunequal variance were indicated (p<0.01), a non-parametric analysis was performed (Wilcoxon ranksum test) on the ranks of the data. Male GSI, which did not pass the assumptions of homogeneity of variance and log transformation did not correct the condition therefore, the non-parametric Wilcoxon rank-sum test was used. Measurements of vitellogenin are inherently variable and boxplots were log transformed VTG values were used to identify potential outliers (Tukey’s method). These methods appear to be consistent with the methods recommended in the guideline.

There were no significant differences in survival at any treatment level compared to the negative controls (p>0.05; Fishers Exact test; Table 9). There was a slight but significant decreasing trend in male wet weight at the 18 and 96 mg a.i./L treatment levels (p d 0.05; Jonckheere-Terpstra test). However, the study author determined that the differences were not concentration-responsive, and Dunnett’s test indicated the differences were not statistically significant compared to the negative control (p>0.05). There was a statistically significant reduction in fecundity in the 96 mg a.i./L treatment group which was considered treatment related (p d 0.05; Dunnett’s test). There was also a

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

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Page 45 of 91 significant decrease in fertility in the 96 mg a.i./L treatment group (p d 0.05; Jonckheere-Terpstra and Dunnett’s tests); however, the fertility in this group still met the guideline requirements for control fertility (95%). There was a slight but significant decreasing trend in male GSI in the 96 mg a.i./L treatment group (p d 0.05; Jonckheere-Terpstra test). However, the differences were not concentrationresponsive and were affected by one low replicate; the Wilcoxon Rank-Sum test indicated no statistically significant effects on GSI among males in any treatment groups compared to the negative control (p>0.05). There were no statistically significant differences between histopathology severity scores or gonadal stages in treated groups compared to the negative controls. There were no statistically significant differences in vitellogenin concentrations in males or females. Sex steroid
concentrations 17 ȕ -estradiol (E2) and testosterone (T) were not measured.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via Toxstat 3.5. Female body weight and male and female length, male and female VTG, female gonadal somatic index (GSI), and fecundity and fertility data were not consistent with a monotonic concentration-response. All data were tested for normality using Shapiro-Wilks test and for homogeneity of variance using Levene’s test. Data which met the assumptions of normality and homogeneity of variance and were consistent with a monotonic trend were then analyzed using the parametric Dunnett’s test. Male body length and VTG were neither monotonic nor parametric and were analyzed using the Mann-Whitney U test. Male body weight and male GSI exhibited a linear concentration-response pattern up to only the middle concentration; both the mean and median top concentration responses were higher than the middle concentration response. The reviewer selected results from the concentration-dependent Jonckheere-Terpstra test for male body weight and GSI endpoints because the response range (min/max) for the negative control values does not overlap with the middle and highest concentration response ranges, and these levels do not differ greatly from each other (means display similar trend as medians). None of the surviving females were found to have tubercles. Unless otherwise indicated, effects were considered statistically significant at p<0.05. These analyses were conducted using SAS ® 

# (SAS Institute, Cary, NC; version 8.1). Both the study author’s and reviewer relied on the mean-measured concentrations to discuss effects in this study.

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Page 46 of 91 Conclusions:

Survival was unaffected by treatment for males and was 100% for females. Growth was reduced, as evidenced by 11 to 17% reductions from the negative control in male body weight at all treatment levels (p<0.05; Jonckheere test); however, the response was not strictly monotonic. Developmental effects included a 28-29% reduction in male GSI, compared to the negative control, at the 18 and 96 mg a.i./L treatment levels (p<0.05; Jonckheere test). Both fecundity and fertility were reduced by 80% and 3%, respectively, at the 96 mg a.i./L treatment level compared to the negative control (p<0.05, Dunnett’s test). The median male tubercle scores were NOT significantly reduced, butexhibited monotonic reductions of 17 and 21% at the 18 and 96 mg a.i./L treatment levels (pvalues of 0.063 and 0.051, respectively; Jonckheere). There were no statistically significant differences between histopathology severity scores or gonadal stages in treated groups compared to the negative controls. However, in male fish, there were two incidences (2/8) of minimal to mild increases in spermatogonia and primary spermatocytes with coinciding minimal to mild decreases in secondary spermatocytes and spermatids in the high treatment group (Table 15). In females, there were two incidences (2/16) of mild to moderate increases in oocyte atresia in the high treatment group and one incidence (1/16) in the middle treatment group. There were no notable observations with regards to behavior or changes in appearance, such as color/banding or ovipositor appearance. The reviewer noted that the size of the dorsal nape pad in males was higher in the control (median value: 5), relative to all treated groups (median values: 3-4). There were no clinical signs of toxicity for any treatment group compared to the controls.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 47 of 91Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( 

# P imephales promelas)in the FSTRA with Dimethoate.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.P% Diff.p% Diff.pNegative Control(<LOQ)24 NA0 NANo0 NANA NA NA NA3.4 22 0.282 -18.71 0.171 No 10.00 0.889 NA NA NA NA1820 0.0613-28.86 0.033No 32.00 0.780 NA NA NA NA9619 0.0513-27.71 0.018 Yes4-68.00 0.152 NA NA NA NAStatistical Test Jonckheere Jonckheere NA MannWhitneyNANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3Not statistically-significant (p>0.050), but a decreasing trend was observed with reductions of 17 and 21% at the middle and high concentrationwith p values very close to 0.050.4Testes of 2 of 8 fish from high treatment group showed mild increase in spermatogonia and primary spermatocytes with minimal to mild decreasein secondary spermatocytes and spermatids. The ovaries of 2 of 16 fish in the high treatment group showed a mild to moderate increase in oocyteData Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 48 of 91atresia, a single incidence of oocyte atresia was noted in the middle treatment group, and two incidences of granulomatous inflammation were notedin the low treatment group. However, there were no statistically significant differences found between the severity scores or gonadal stages in thetreatment groups as compared to the negative control.LOQ=1.00 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 49 of 91Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( 

# P imephales promelas)in the FSTRA with Dimethoate.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercleScoreGSIGonadalStagingand Histo.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.PMedianp% Diff.pEffect?(Yes/No)% Diff.p%Diff.p%Diff.pControl(<LOQ)0 NA 0 NA 0 NA 0 NA No0 NA NA NA NA NA3.40.34 1.00 -1.58 0.381 0 NA -6.90 0.903 No 25.18 0.544 NA NA NA NA186.19 0.934 -1.35 0.498 0 NA -2.93 0.991 No -14.60 0.846 NA NA NA NA96-79.75<0.001-3.430.0230 NA 14.48 0.539 Yes -20.50 0.682 NA NA NA NAStatistical TestDunnett’sDunnett’sNA Dunnett’s NA Dunnett’s NA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.LOQ=1.00 mg a.i./L.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

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Page 50 of 91 Table 24: Growth Endpoints 1,2 

# in the Fish Short-Term Reproduc tion Assay (FSTRA) with Dimethoate.

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 0 NA 0 NA



# 3.4 -10.85 0.057 0.48 0.999 -2.39 0.547 -0.56 0.984



# 18 -16.83 0.015 -0.95 0.995 -2.39 0.547 0.56 0.984



# 96 -14.46 0.006 1.43 0.983 -2.39 0.547 1.12 0.894



# Statistical Test Jonckheere Dunnett’s Dunnett’s Dunnett’s

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=1.00 mg a.i./L.



# E. Study Deficiencies

There were some deviations described in Section I. Materials and Methods of this DER regarding failure to report certain water characteristics and meet environmental criteria:

1. The 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. Additionally, the light intensity was reported to range from 616 to 1376 lux during exposure; OCSPP 890.1350 guideline requirements recommend light intensity 540 – 1080 lux (at water’s surface).

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

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Page 51 of 91 2. Dissolved oxygen concentrations in the test solutions remained at or above 60% of saturation (4.9 mg/L) for most of the exposure period. However, dissolved oxygen
concentrations fell to ca. 55% of saturation on Day 14 and gentle aeration was added. Dissolved oxygen concentrations remained above 60% of saturation for the remainder of the test. OCSPP 890.1350 guideline requirements recommend dissolved oxygen (DO) >4.9 mg/L (>60% air saturation).

3. Individual fish weights at study initiation were ± 30% for both males and females. EPA recommends that the subsample of fish weighed before the test be within ±20% of the
estimated mean for each sex.

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

F. Reviewer’s Comments

The reviewer’s and the study authors’ results were in general agreement. Both the study authors’ and reviewer’s analysis detected statistically significant reductions (p<0.05) in male body weight and GSI, fecundity, and fertility. The study authors maintained that male body weight and male GSI were not concentration-dependent responses and relied on tests that did not detect statistical significance for these endpoints (p>0.05). The reviewer believes that the reductions from negative control observed for these endpoints are suggestively concentration-dependent, particularly given their magnitude and linear pattern up to the middle concentration, and failure of their median response ranges to overlap with that of the negative control (except for body wt at low concentration). The reviewer also detected a trend toward a significant reduction in male tubercle scores at the middle and high test concentrations (p = 0.063 and 0.051); the study authors reference the mean tubercle scores, rather than the median scores. The study author and reviewer came to similar conclusions regarding fecundity and fertility and no other statistically significant differences were noted in the other endpoints.

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Page 52 of 91 The reviewer notes that fatpad scores appear to be lower in the treated conditions, relative to the negative
control:

Fatpad score
Female Control 3.4 18 96

1 1 1 1 1
2 1 1 1 1
3 1 1 1 1

4 1 1 1 1
5 1 1 1 1
6 1 1 1 1

7 1 1 1 1
8 1 1 1 1
9 1 1 1 1
10 1 1 1 1

11 1 1 1 1
12 1 1 1 1
13 1 1 1 1

14 1 1 1 1
15 1 1 1 1
16 1 1 1 1

Median 1 1 1 1
Mean

Male Control 3.4 18 96

1 5 4 3 3
2 3 3 4 3

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Page 53 of 91 3 4 3 4 4
4 4 3 4 3
5 5 3 5 4
6 5 4 3 3
7 5 4 3 4
8 4 3 3 3

Median 4.5 3 3.5 3
Mean 4.4 3.4 3.6 3.4

III. REFERENCES

U.S. Environmental Protection Agency. 2009. Series 890-Endocrine Disrupter Screening Program Test Guidelines, OCSPP Number 890.1350: Fish Short-Term Reproduction Assay.

Organization for Economic Cooperation and Development. 2009. OECD Guidelines for Testing of Chemicals, Guideline 229. Fish Short Term Reproduction Assay. Adopted 7 September 2009.

Cheminova A/S. 1980. Product Chemistry: Dimethoate Technical. Unpublished study submitted to EPA July 20, 1981 under 4787-7; CDL: 245604-A. US EPA MRID 00139284.

APHA, AWWA, WEF. 1998. Standard Methods for the Examination of Water and Wastewater. 20th Edition, American Public Health Association. American Water Works Association. Water Environment Federation, Washington, D.C.

Organization for Economic Cooperation and Development. 2006. Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application. OECD ENV/JM/MONO(2006)
18.

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Page 54 of 91 The SAS System for Windows. 1999 - 2001. Version 8.2. SAS Institute, Inc. Gary, North Carolina.

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Page 55 of 91 APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.941 0.361 2.703 0.092 USE PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 1.05 0.06 0.03 5.42 0.96, 1.14 Dose1 4 1.05 0.06 0.03 5.28 0.96, 1.14 Dose2 4 1.04 0.07 0.03 6.65 0.93, 1.15 Dose3 4 1.06 0.10 0.05 9.73 0.90, 1.23

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.07 0.97 1.09 . . Dose1 1.05 1.00 1.11 100.48 -0.48 Dose2 1.05 0.95 1.11 99.05 0.95 Dose3 1.07 0.95 1.16 101.43 -1.43

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 0.08 0.970
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.05 . 1.05 . . . . .

. Dose1 1.05 0.999 1.05 0.603 . . . .

.

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Page 56 of 91 Dose2 1.04 0.995 1.05 0.638 0.991 . . .

. Dose3 1.06 0.983 1.05 0.657 0.997 0.962 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 0.22 0.974
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 1.07 . . Dose1 1.05 0.887 0.616 Dose2 1.05 0.889 0.471 Dose3 1.07 0.889 0.593

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

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 0.08 0.970
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.05 . -1.05 . . . . .

. Dose1 -1.05 0.999 -1.05 0.597 . . . .

. Dose2 -1.04 0.995 -1.05 0.631 0.991 . . .

. Dose3 -1.06 0.983 -1.06 0.508 0.997 0.962 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 0.22 0.974
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -1.07 . . Dose1 -1.05 0.887 0.384 Dose2 -1.05 0.889 0.529 Dose3 -1.07 0.889 0.407

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.949 0.469 5.278 0.015 USE NON-PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 2.01 0.08 0.04 3.79 1.88, 2.13 Dose1 4 1.79 0.27 0.14 15.37 1.35, 2.22 Dose2 4 1.67 0.26 0.13 15.84 1.25, 2.09 Dose3 4 1.72 0.07 0.03 3.97 1.61, 1.82

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 2.00 1.93 2.10 . . Dose1 1.87 1.39 2.02 89.15 10.85 Dose2 1.68 1.41 1.91 83.17 16.83

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 58 of 91 Dose3 1.70 1.65 1.81 85.54 14.46

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 2.29 0.130
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 2.01 . 2.01 . . . . .

. Dose1 1.79 0.318 1.79 0.085 . . . .

. Dose2 1.67 0.079 1.69 0.027 0.825 . . .

. Dose3 1.72 0.142 1.69 0.027 0.953 0.986 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 7.83 0.050
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 2.00 . . Dose1 1.87 0.156 0.042 Dose2 1.68 0.067 0.014 Dose3 1.70 0.067 0.006

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose1

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 59 of 91 3 12 2.29 0.130
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -2.01 . -1.79 . . . . .

. Dose1 -1.79 0.318 -1.79 0.961 . . . .

. Dose2 -1.67 0.079 -1.79 0.972 0.825 . . .

. Dose3 -1.72 0.142 -1.79 0.977 0.953 0.986 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 7.83 0.050
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -2.00 . . Dose1 -1.87 0.156 0.958 Dose2 -1.68 0.067 0.986 Dose3 -1.70 0.067 0.994

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 60 of 91 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.956 0.590 1.404 0.289 USE PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 44.75 1.26 0.63 2.81 42.75, 46.75 Dose1 4 44.50 0.58 0.29 1.30 43.58, 45.42 Dose2 4 45.00 0.82 0.41 1.81 43.70, 46.30 Dose3 4 45.25 1.89 0.95 4.18 42.24, 48.26

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 45.00 43.00 46.00 . . Dose1 44.50 44.00 45.00 99.44 0.56 Dose2 45.00 44.00 46.00 100.56 -0.56 Dose3 44.50 44.00 48.00 101.12 -1.12

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 0.27 0.846
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 44.75 . 44.88 . . . . .

. Dose1 44.50 0.984 44.88 0.642 . . . .

. Dose2 45.00 0.984 44.88 0.677 0.939 . . .

. Dose3 45.25 0.894 44.88 0.696 0.828 0.992 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 0.73 0.867
MannWhit - testing each trt median signif. different from control

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 61 of 91 Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 45.00 . . Dose1 44.50 0.656 0.268 Dose2 45.00 1.000 0.594 Dose3 44.50 1.000 0.559

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

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 0.27 0.846
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -44.75 . -44.63 . . . . .

. Dose1 -44.50 0.984 -44.63 0.642 . . . .

. Dose2 -45.00 0.984 -45.00 0.492 0.939 . . .

. Dose3 -45.25 0.894 -45.25 0.382 0.828 0.992 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 0.73 0.867
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -45.00 . .

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 62 of 91 Dose1 -44.50 0.656 0.732 Dose2 -45.00 1.000 0.406 Dose3 -44.50 1.000 0.441

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.946 0.435 5.179 0.016 USE NON-PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 52.25 0.50 0.25 0.96 51.45, 53.05 Dose1 4 51.00 2.45 1.22 4.80 47.10, 54.90 Dose2 4 51.00 1.15 0.58 2.26 49.16, 52.84 Dose3 4 51.00 1.41 0.71 2.77 48.75, 53.25

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 52.00 52.00 53.00 . . Dose1 51.50 48.00 53.00 97.61 2.39 Dose2 51.00 50.00 52.00 97.61 2.39 Dose3 51.50 49.00 52.00 97.61 2.39

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 0.65 0.597
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 63 of 91 Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 52.25 . 52.25 . . . . .

. Dose1 51.00 0.547 51.00 0.164 . . . .

. Dose2 51.00 0.547 51.00 0.175 1.000 . . .

. Dose3 51.00 0.547 51.00 0.181 1.000 1.000 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 2.36 0.501
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 52.00 . . Dose1 51.50 0.884 0.381 Dose2 51.00 0.177 0.140 Dose3 51.50 0.180 0.100

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

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 0.65 0.597
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 64 of 91 Ctrl -52.25 . -51.31 . . . . .

. Dose1 -51.00 0.547 -51.31 0.869 . . . .

. Dose2 -51.00 0.547 -51.31 0.894 1.000 . . .

. Dose3 -51.00 0.547 -51.31 0.907 1.000 1.000 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 2.36 0.501
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -52.00 . . Dose1 -51.50 0.884 0.619 Dose2 -51.00 0.177 0.860 Dose3 -51.50 0.180 0.900

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.952 0.514 0.253 0.858 USE PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 1807000 593126.2 296563.1 32.82 863203.9, 2750796 Dose1 4 2262000 492948.3 246474.1 21.79 1477609, 3046391

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 65 of 91 Dose2 4 1543250 428629.9 214314.9 27.77 861204.2, 2225296 Dose3 4 1436500 695457.9 347728.9 48.41 329871.3, 2543129

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1859000 1058000 2452000 . . Dose1 2186500 1772000 2903000 125.18 -25.18 Dose2 1572500 1092000 1936000 85.40 14.60 Dose3 1181000 928000.0 2456000 79.50 20.50

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 1.71 0.217
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1807000 . 2034500 . . . . .

. Dose1 2262000 0.544 2034500 0.795 . . . .

. Dose2 1543250 0.846 1543250 0.331 0.316 . . .

. Dose3 1436500 0.682 1436500 0.244 0.215 0.993 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 3.46 0.326
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 1859000 . . Dose1 2186500 0.494 0.807 Dose2 1572500 0.678 0.232 Dose3 1181000 0.678 0.096

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 66 of 91 Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 1.71 0.217
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1807000 . -1762188 . . . . .

. Dose1-2262000 0.544 -1762188 0.630 . . . .

. Dose2-1543250 0.846 -1762188 0.665 0.316 . . .

. Dose3-1436500 0.682 -1762188 0.684 0.215 0.993 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 3.46 0.326
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -1859000 . . Dose1 -2186500 0.494 0.193 Dose2 -1572500 0.678 0.768 Dose3 -1181000 0.678 0.904

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

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 67 of 91 test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.906 0.100 3.745 0.041 USE NON-PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 125.00 55.68 27.84 44.54 36.40, 213.60 Dose1 4 137.50 99.79 49.90 72.58 -21.29, 296.29 Dose2 4 165.00 194.34 97.17 117.78 -144.23, 474.23 Dose3 4 40.00 23.09 11.55 57.74 3.25, 76.75

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 135.00 50.00 180.00 . . Dose1 150.00 20.00 230.00 110.00 -10.00 Dose2 95.00 20.00 450.00 132.00 -32.00 Dose3 40.00 20.00 60.00 32.00 68.00

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 0.91 0.467
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 125.00 . 142.50 . . . . .

. Dose1 137.50 0.997 142.50 0.672 . . . .

. Dose2 165.00 0.924 142.50 0.707 0.985 . . .

.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 68 of 91 Dose3 40.00 0.598 40.00 0.204 0.628 0.435 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 3.92 0.270
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 135.00 . . Dose1 150.00 0.889 0.614 Dose2 95.00 0.780 0.442 Dose3 40.00 0.152 0.054

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

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 0.91 0.467
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -125.00 . -116.88 . . . . .

. Dose1 -137.50 0.997 -116.88 0.625 . . . .

. Dose2 -165.00 0.924 -116.88 0.660 0.985 . . .

. Dose3 -40.00 0.598 -116.88 0.679 0.628 0.435 . .

.

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***

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 69 of 91 NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 3.92 0.270
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -135.00 . . Dose1 -150.00 0.889 0.386 Dose2 -95.00 0.780 0.558 Dose3 -40.00 0.152 0.946

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.899 0.078 2.395 0.119 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 14.50 0.92 0.46 6.35 13.04, 15.96 Dose1 4 13.50 1.83 0.91 13.54 10.59, 16.41 Dose2 4 14.08 1.25 0.63 8.90 12.08, 16.07 Dose3 4 16.60 4.55 2.28 27.42 9.36, 23.84

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 14.20 13.80 15.80 . . Dose1 14.05 10.90 15.00 93.10 6.90 Dose2 14.10 12.60 15.50 97.07 2.93 Dose3 15.30 12.80 23.00 114.48 -14.48

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 70 of 91 *****************************************************************************

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 1.10 0.386
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 14.50 . 14.67 . . . . .

. Dose1 13.50 0.903 14.67 0.622 . . . .

. Dose2 14.08 0.991 14.67 0.657 0.989 . . .

. Dose3 16.60 0.539 14.67 0.676 0.363 0.529 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 1.45 0.693
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 14.20 . . Dose1 14.05 0.580 0.234 Dose2 14.10 0.678 0.330 Dose3 15.30 0.678 0.680

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

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 71 of 91 3 12 1.10 0.386
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -14.50 . -14.00 . . . . .

. Dose1 -13.50 0.903 -14.00 0.694 . . . .

. Dose2 -14.08 0.991 -14.08 0.713 0.989 . . .

. Dose3 -16.60 0.539 -16.60 0.178 0.363 0.529 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 1.45 0.693
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -14.20 . . Dose1 -14.05 0.580 0.766 Dose2 -14.10 0.678 0.670 Dose3 -15.30 0.678 0.320

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 72 of 91 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.979 0.951 6.057 0.009 USE NON-PARAMETRIC TESTS

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*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 1.75 0.19 0.09 10.63 1.45, 2.05 Dose1 4 1.42 0.45 0.22 31.40 0.71, 2.13 Dose2 4 1.25 0.18 0.09 14.07 0.97, 1.52 Dose3 4 1.27 0.21 0.10 16.51 0.93, 1.60

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.78 1.50 1.95 . . Dose1 1.43 0.98 1.86 81.29 18.71 Dose2 1.21 1.08 1.48 71.14 28.86 Dose3 1.33 0.97 1.44 72.29 27.71

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 2.83 0.083
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.75 . 1.75 . . . . .

. Dose1 1.42 0.271 1.42 0.071 . . . .

. Dose2 1.25 0.061 1.26 0.016 0.803 . . .

. Dose3 1.27 0.073 1.26 0.016 0.852 1.000 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 6.42 0.093

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 73 of 91 MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 1.78 . . Dose1 1.43 0.346 0.124 Dose2 1.21 0.067 0.029 Dose3 1.33 0.067 0.018

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 2.83 0.083
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -1.75 . -1.42 . . . . .

. Dose1 -1.42 0.271 -1.42 0.972 . . . .

. Dose2 -1.25 0.061 -1.42 0.981 0.803 . . .

. Dose3 -1.27 0.073 -1.42 0.984 0.852 1.000 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 6.42 0.093
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -1.78 . . Dose1 -1.43 0.346 0.876

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 74 of 91 Dose2 -1.21 0.067 0.971 Dose3 -1.33 0.067 0.982

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) )

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

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 0.00 0.00 0.00 . . , . Dose1 4 0.00 0.00 0.00 . . , . Dose2 4 0.00 0.00 0.00 . . , . Dose3 4 0.00 0.00 0.00 . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.00 0.00 0.00 . . Dose1 0.00 0.00 0.00 . . Dose2 0.00 0.00 0.00 . . Dose3 0.00 0.00 0.00 . .

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value . . . 1 . . . . . .

.

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

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 75 of 91 Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 0.00 . . . . . . .

. Dose1 0.00 . . . . . . .

. Dose2 0.00 . . . . . . .

. Dose3 0.00 . . . . . . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 0.00 1.000
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 0.00 . . Dose1 0.00 1.000 . Dose2 0.00 1.000 . Dose3 0.00 1.000 .

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

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value . . . 1 . . . . . .

.

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 76 of 91 Ctrl 0.00 . . . . . . .

. Dose1 0.00 . . . . . . .

. Dose2 0.00 . . . . . . .

. Dose3 0.00 . . . . . . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 0.00 1.000
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 0.00 . . Dose1 0.00 1.000 . Dose2 0.00 1.000 . Dose3 0.00 1.000 .

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams Dose1 Jonckheere Dose1
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.949 0.474 0.443 0.727 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 23.25 3.97 1.98 17.07 16.94, 29.56 Dose1 4 22.00 2.74 1.37 12.45 17.64, 26.36 Dose2 4 18.75 3.33 1.66 17.76 13.45, 24.05 Dose3 4 19.75 4.94 2.47 25.02 11.89, 27.61

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 77 of 91 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 23.75 18.50 27.00 . . Dose1 22.00 19.00 25.00 94.62 5.38 Dose2 19.75 14.00 21.50 80.65 19.35 Dose3 18.75 15.00 26.50 84.95 15.05

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 1.15 0.369
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 23.25 . 23.25 . . . . .

. Dose1 22.00 0.938 22.00 0.388 . . . .

. Dose2 18.75 0.274 19.25 0.104 0.639 . . .

. Dose3 19.75 0.456 19.25 0.107 0.839 0.982 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 2.84 0.417
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 23.75 . . Dose1 22.00 0.678 0.282 Dose2 19.75 0.283 0.061 Dose3 18.75 0.346 0.051

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

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 78 of 91 Jonckheere >highest dose (no sign. differences)

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 1.15 0.369
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -23.25 . -20.94 . . . . .

. Dose1 -22.00 0.938 -20.94 0.868 . . . .

. Dose2 -18.75 0.274 -20.94 0.893 0.639 . . .

. Dose3 -19.75 0.456 -20.94 0.906 0.839 0.982 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 2.84 0.417
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -23.75 . . Dose1 -22.00 0.678 0.718 Dose2 -19.75 0.283 0.939 Dose3 -18.75 0.346 0.949

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for fish screen study - Dimethoate

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 79 of 91 ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.975 0.913 1.846 0.193 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 29.50 2.19 1.09 7.41 26.02, 32.98 Dose1 4 29.60 7.44 3.72 25.13 17.76, 41.44 Dose2 4 31.33 6.99 3.50 22.33 20.19, 42.46 Dose3 4 5.98 3.27 1.64 54.80 0.76, 11.19

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 29.15 27.30 32.40 . . Dose1 31.75 19.50 35.40 100.34 -0.34 Dose2 30.70 23.60 40.30 106.19 -6.19 Dose3 5.50 3.10 9.80 20.25 79.75

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 19.60 <.001
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 29.50 . 30.14 . . . . .

. Dose1 29.60 1.000 30.14 0.651 . . . .

. Dose2 31.33 0.934 30.14 0.687 0.969 . . .

. Dose3 5.98 <.001 5.98 <.001 <.001 <.001 . .

.

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 80 of 91 *****************************************************************************

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 8.67 0.034
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 29.15 . . Dose1 31.75 0.780 0.668 Dose2 30.70 0.780 0.722 Dose3 5.50 0.067 0.031

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

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 19.60 <.001
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -29.50 . -24.10 . . . . .

. Dose1 -29.60 1.000 -24.10 0.951 . . . .

. Dose2 -31.33 0.934 -24.10 0.964 0.969 . . .

. Dose3 -5.98 <.001 -24.10 0.970 <.001 <.001 . .

.

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*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 8.67 0.034

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 81 of 91 MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -29.15 . . Dose1 -31.75 0.780 0.332 Dose2 -30.70 0.780 0.278 Dose3 -5.50 0.067 0.969

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.965 0.748 1.296 0.321 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 4 98.33 0.55 0.28 0.56 97.45, 99.20 Dose1 4 96.78 1.19 0.59 1.23 94.88, 98.67 Dose2 4 97.00 2.10 1.05 2.17 93.66, 100.34 Dose3 4 94.95 1.81 0.91 1.91 92.06, 97.84

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 98.45 97.60 98.80 . . Dose1 96.55 95.60 98.40 98.42 1.58 Dose2 97.90 93.90 98.30 98.65 1.35 Dose3 94.90 92.80 97.20 96.57 3.43

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 3.27 0.059

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 82 of 91 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 98.33 . 98.33 . . . . .

. Dose1 96.78 0.381 96.89 0.125 . . . .

. Dose2 97.00 0.498 96.89 0.133 0.997 . . .

. Dose3 94.95 0.023 94.95 0.005 0.374 0.283 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 7.25 0.064
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl 98.45 . . Dose1 96.55 0.156 0.042 Dose2 97.90 0.343 0.093 Dose3 94.90 0.067 0.006

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

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 3.27 0.059
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 83 of 91 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -98.33 . -96.76 . . . . .

. Dose1 -96.78 0.381 -96.76 0.955 . . . .

. Dose2 -97.00 0.498 -96.76 0.967 0.997 . . .

. Dose3 -94.95 0.023 -96.76 0.973 0.374 0.283 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups

Degrees of Freedom TestStat P-value 3 7.25 0.064
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

Ctrl -98.45 . . Dose1 -96.55 0.156 0.958 Dose2 -97.90 0.343 0.907 Dose3 -94.90 0.067 0.994

INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR13 ( F testosterone (ng/mL) )

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

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

*** BASIC SUMMARY STATISTICS

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 84 of 91 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl . . . . . Dose1 . . . . . Dose2 . . . . . Dose3 . . . . .

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 3.27 0.059
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

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

Jonckheere Dose1

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 3.27 0.059
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

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

Jonckheere Dose1
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR14 ( M testosterone (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 85 of 91 Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl . . . . . Dose1 . . . . . Dose2 . . . . . Dose3 . . . . .

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 3.27 0.059
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

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

Jonckheere Dose1

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 3.27 0.059
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 86 of 91 Level Median MannWhit p-value Jonckheere p-value

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

Jonckheere Dose1
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR15 ( F 17b-estradiol (ng/mL) )

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

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl . . . . . Dose1 . . . . . Dose2 . . . . . Dose3 . . . . .

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 3.27 0.059
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

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

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 87 of 91 Jonckheere Dose1

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 3.27 0.059
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

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

Jonckheere Dose1
test for fish screen study - Dimethoate ANALYSIS RESULTS FOR VARIABLE VAR16 ( M 17b-estradiol (ng/mL) )

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

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

*** BASIC SUMMARY STATISTICS

Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval

Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl . . . . . Dose1 . . . . . Dose2 . . . . . Dose3 . . . . .

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests

# Data Evaluation Record on the Fish Shor t-Term Reproduction Assay with Dimethoate

EPA MRID Number 48670603

Page 88 of 91 Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 3.27 0.059
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend

Level Median MannWhit p-value Jonckheere p-value

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

Jonckheere Dose1

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test

Numerator df Denominator df F-stat P-value 3 12 3.27 0.059
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value

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

Jonckheere Dose1Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 89 of 91EndpointMonotonic? Parametric? 890.1350 EFEDCommentsFemale bodyweightNo Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectMale body weight Strictlylinear to themiddle dose,but top dosefor mean andmedian arehigher thanthe middledoseresponse.However, theresponserange(min/max) forcontrol doesnot overlapwith middleand highestlevels andthese levelsdonot differgreatly fromeach other.Means displaysimilar trendas medians.No Jonckheere:Dose 1 p=0.042Dose 2 p=0.014Dose 3 p=0.006Jonckheere:Dose 1 p=0.042Dose 2 p=0.014Dose 3 p=0.006890.1350 and EFED same conclusions;Jonckheere reveals significant 11 to 17%reduction in male body weight at alltreatment levels, relative to the negativecontrolFemale bodylengthNo Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectMale body length NoNoMannWhitney:n.s. p>0.05MannWhitney:n.s. p>0.05890.1350 and EFED same conclusions, noeffectFemale VTG NoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, noeffectMale VTGNoNoMannWhitney:n.s. p>0.05MannWhitney:n.s. p>0.05890.1350 and EFED same conclusions, noeffectData Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 90 of 91EndpointMonotonic? Parametric? 890.1350 EFEDCommentsFemale GSI NoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale GSIStrictlylinear to themiddle dose,but top dosefor mean andmedian arehigher thanthe middledoseresponse.However, theresponserange(min/max) forcontrol doesnot overlapwith middleand highestlevels andthese levelsdo not differgreatly fromeach other.Means displaysimilar trendas medians.NoJonckheere:Dose 1 p=0.124Dose 2 p=0.029Dose 3 p=0.018Jonckheere:Dose 1 p=0.124Dose 2 p=0.029Dose 3 p=0.018890.1350 and EFED same conclusions;Jonckheere reveals significant 28-29%reduction in male GSI at the middle andhighest treatment levels, relative to thenegative controlFemale tuberclescoreNA NA NA NA No scoreMale tuberclescoreYes, medianvalues arelinearYes Jonckheere:Dose 2 p=0.063Dose 3 p=0.051William’s:Dose 2 p=0.104Dose 3 p=0.107890.1350 and EFED same conclusions.Jonckheere’s revealsNOsignificant effect,but middle and highest dose p-valuessuggest a strong trend toward a significanteffect.FecundityNoYesDunnett’s:Dose 3 p<0.001Dunnett’s:Dose 3 p<0.001890.1350 and EFED same conclusions,significant 80% reduction from negativecontrol at the highest dose level.Data Evaluation Record on the Fish Short-Term Reproduction Assay with DimethoateEPA MRID Number 48670603Page 91 of 91EndpointMonotonic? Parametric? 890.1350 EFEDCommentsFertilityNoYesDunnett’s:Dose 3 p=0.023Dunnett’s:Dose 3 p=0.023890.1350 and EFED same conclusions,significant 3% reduction from negativecontrol at the highest dose level.F testosterone NANANANANAM testosterone NANANANANAF estradiol NANANANANAM estradiol NANANANANA

# DATA EVALUATION RECORD

DIMETHOATE
Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-2-2012 (MRID 48616304)

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

Arlington, VA 22202

Prepared by 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: 12/07/2011 Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 12/07/2011
Program Manager: Signature: Jack D. Early, M.S. Date: 12/07/2011
Quality Assurance: Signature: Jack D. Early, M.S. Date: 12/07/2011

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

# The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and re dundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potent ial to interact with the E, A or T pathways, a Weight of Evidence ( WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

# DIMETHOATE I 035001



# Pri mary Reviewer Gregory Akerman 

# Health Effects Divi s ion Seco ndary R ev iewer: John Liccione, Ph

. D. 

# Health E ffect s Divi s ion S t e roido ge nesi s Assay (2011) I 

# P age I of 15 O CS PP 890.1550 / OE C D No ne

# Signature:~**Date: _~**  

# Signature:**Dat ~,,.~** **----~-------/----,,-** 

# Template ve r sio n 08/2011



# DATA EVALUAT I ON RECORD



# STU DY TYPE: Steroidogenesis Assay (H295R Ce ll s); OCSPP 890.1550



# PC CODE: 035001 

# DP BARCODE: D396168



# TXR#: 0056198 

# CAS No.: 60-51-5



# TEST MATERIAL (PURITY): Dimethoate (98% a.i.)



# SYN ONYMS: 0,0-dimethyl S-[2-(methylamino)-2-oxoethy l] phosphorodithioate



# CITAT ION: Wagner, 

# H. et al. (2011). Dimethoate Technica l : H295R Steroidogenesis Assay. CeeTox, Inc., 4717 Campus Drive, Kalamazoo, MI. Laboratory Study No. 9148V-100373STER, Nov 3, 20 11. MRID 48616304. Unpub li shed.



# SP ON S OR: Cheminova A/S, PO Box 9, DK-7620 Lemvig, Denmark.



# TEST ORDER#: Not provided



# EXECUT IVE SUMMARY: In an 

# in vitro steroidogenesis assay (MRID 48616304 , H295R cells cultured in vitro in 24-well plates were incubated with dimethoate (98.0% purity , Batch No. 611/A) at logarithmic concentrations from of 10- 1 0 

# to 10 -4 Min triplicate for 48 ± 

# 2 hours

. Dimethyl sulfoxide ( DMSO) was used as the vehic l e, at a fina l concentration of 0.05%

.
Testostero n e and estradiol concentrations were measured by HPLC-MS / MS

. Four independent assay runs were conducted, with three acceptab l e assay runs completed and reported. A Quality Control (QC) plate was run concurrently with eac h 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.

Guideline recommendations and requirements were met , including adequate production of testosterone and estradiol, acceptable reproducibility ( lo w %CV), and appropriate induction and inhibition with positive contro l s. Cytotoxicity greater than 20% was not o b served.

Dimethoate was tested at concentrations up to 10- 5 

# M to l 0- 4 

# M with no ev id ence of cytotoxicity in the 

# in vitro steroi do ge n esis assay (precipitation was observed in the 1 

# o- 8 

# M wells in one assay run). Three individual assay run s were comp l eted, and testosterone a nd estradio l concentrations

were determined after H295R cells were incubated for 48 ± 

# 2 hours. Estradio l production was statistica ll y decreased in one of three independent assays at 10- 8 

# and 10- 7 

# M on l y, with no dose

# Steroidogenesis Assay (2011) / Page 2 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None 

# response being observed. This decrease in es tradiol production was not interpreted to be biologically relevant.
Based on the hormone responses in each of the th ree independent runs, dimethoate treatment did not result in statistically signifi cant and reproducible alterations in testosterone or estradiol production.
The assay satisfies the EDSP Tier 1 Test Order requi rements for a Steroidogenesis assay (OCSPP 890.1550).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.

# Steroidogenesis Assay (2011) / Page 3 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None


# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc. Location: 4717 Campus Drive, Kalamazoo, MI Study Director: Hilary Wagner Other Personnel: Coleen Toole, Director Of Project Management Study Period: May 23,2011 to November 3, 2011



# 2. Test Substance: Dimethoate Description: White solid Batch # (expiration date): 611/A (September 22, 2014) Purity: 98.0% Solvent used: DMSO Stability: 10 years Storage conditions: -20 ± 10  ̊ C CAS #: 60-51-5 Molecular weight: 229.3 Structure: 

# P S

S O

O CH

3



# CH

3 

# C H 2 

# N H CH 3 

# O



# 3. Positive Control: Forskolin Description: White powder Source: Sigma Aldrich, St. Louis, MO Lot # (expiration date): 097K50653V (May, 2016) and 109K50571V (July, 2016) Purity: 99% and 98%, respectively Solvent used: DMSO Storage conditions: CAS #: Room temperature 66575-29-9 Molecular weight: 410.5 

# 4. Negative Control: Prochloraz Description: White powder Source: Sigma Aldrich, St. Louis, MO Lot # (expiration date): SZE6220X (August 8, 2012) Purity: 99.1% Solvent used: DMSO Storage conditions: Room temperature CAS: Molecular weight: 67747-09-5 376.67



# 5. Other Materials: 22R-Hydroxycholesterol Description: White powder Source: Sigma Aldrich, St. Louis, MO Lot # (expiration date): 089K4132 (retest date July, 2012) and 060M4098 (retest date June, 2013) Purity: 99.0% Solvent used: Ethanol Storage conditions: Room temperature CAS: Molecular weight: 17954-98-2 402.65

# Steroidogenesis Assay (2011) / Page 4 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None



# 6. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) Description: e.g. technical, nature, color, molecular weight Source: Sigma Aldrich, St. Louis, MO Lot #: RNBB7617 Purity: Not reported Storage conditions: Not reported CAS #: 67-68-5 Justification for choice of solvent: Test guideline recommendation

Final concentration: (% volume in assay) 0.05%



# 7. Stock Medium: Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture Source: Sigma Aldrich, St. Louis, MO Lot #: 021M8304, exp. date not reported Sodium bicarbonate: Sigma Aldrich, St. Louis, MO, Lot No. 068K0105 Nu-Serum: Becton Dickinson, Lot No. 81515; tested for background hormone concentrations ITS+ premix: Becton Dickinson, Lot Nos. 05245 and 09233



# 8. Test Cells: H295R human adrenocortical carc inoma cells (ATCC CLR-2128; Lot No. 7635054). The hormone concentrations in und iluted NuSerum for testosterone and estradiol were determined to be 3754 pg/ mL and 3846 pg/mL, respectively. H295R cells were grown for five passages and frozen in liquid nitrogen. Subsequently, cells were thawed and cultured for seven or eight additi onal passages prior to use in the assay. The specific cell passage identifier for ea ch assay run was not reported. The following performance criteria were met (indicated by an “x”): X Cell passage identifier. 7.5 – 8.5 X Cells frozen down at passage 5 X Frozen cells cultured for 7–8 additional pa ssages (minimum of 4 passages required) X Total number of passages does not exceed 10



# B. METHODS

1. Pre-Test Information
a. Hormone Assay Interference Test: A chemical hormone assay interference test was not conducted because testosterone and estrad iol concentrations were determined by HPLC-MS/MS.

b. Hormone Extraction: Hormone extraction efficiency was determined using [ 2 

# H 5 

# ]-testosterone and [ 2 

# H 5 

# ]-estradiol as internal standards in the methyl tert-butyl ether (MTBE) liquid-liquid extraction so lution. Recoveries for testosterone and estradiol QC data ranged from 88.7% to 103.7% and 84.6% to 104.4%, respectively, with the percent precision and accuracy within acceptable guideline ranges.
c. Laboratory Proficiency Test: No laboratory proficiency test data were provided. 2. Test Solutions: Dimethoate was dissolved in DMSO to make a 200 mM stock and then serially diluted 1:10 with DM SO. 22R-Hydroxycholesterol wa s dissolved in ethanol (no source, identification, or physic al property details provided) to make a 40 mM stock, and

# Steroidogenesis Assay (2011) / Page 5 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None 

# diluted with supplemented medium to a final concentration of 10 μ M. Dimethoate was diluted 1:2000 in supplemented medium containing 10 μ M 22R-hydroxycholesterol to prepare master mix solutions. Forskolin and pr ochloraz were dissolved in DMSO to make 100 mM solutions and serially diluted in DMSO . Forskolin and prochloraz were diluted 1:2000 in supplemented medium containing 10 μ M 22R-hydroxycholesterol. These dilutions yielded final concentrations of 1 μ M and 10 μ M for forskolin, 0.1 μ M and 1 μ M for prochloraz, and 0.0001 to 100 μ M for dimethoate on addition to the cell culture plate wells. The final concentration of DMSO in th e medium was held cons tant at 0.05% (v/v), and the final ethanol concentration in the supplemented medi um was 0.025% (v/v).
3. Cell Plating and Preincubation: Cells were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham with 15 mM HEPES, sodium bicarbonate, ITS+Premix, and 2.5% Nu-Serum in a 5% CO 2 

# incubator at 37 ± 2°C. H295R cells were grown for five passages, frozen in liquid nitrogen, and subsequently thawed and cultured for seven or eight additional passages prior to use in the assay. The culture medium was supplemented with 10 μ M 22R-hydroxycholesterol at the time of plating, dosing, and harvesting. The concentration of 22R-hydroxycholestero l (lower than the recommended 20–40 μ M concentration) was selected based on labor atory proficiency experiments previously conducted at the test facility. Th e cells were plated into well s of 24-well cell culture plates at a density of approximately 300,000 cells/mL. The seeded plates were placed into a 5% CO 2 

# incubator at 37 ± 2°C for approximately 24 hours prior to chemical exposure to achieve approximately 50-60% confluency.
4. Exposure: The cells were checked microscopi cally for good attachment and proper morphology, and the medium was removed and replaced with 1 mL of medium that contained 10 μ M 22R-hydroxycholesterol and each serial dilution of the test compound or DMSO in triplicate according to the schematic presented in Table 1.

TABLE 1. Dosing Schematic for the Exposure of H295R Ce lls to Dimethoate (Final Concentrations in μM).a 1 2 3 4 5 6 A DMSO DMSO DMSO 0.1 0.1 0.1 B 100 100 100 0.01 0.01 0.01 C 10 10 10 0.001 0.001 0.001 D 1 1 1 0.0001 0.0001 0.0001 a Data were obtained from page 16 of the study report. Dosing was calculated based on a to tal volume of 1 mL per well.



# A concurrent QC plate was included with each of the three independent runs of the test chemical plates to demonstrate the assay’s res ponse to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testoste rone and estradiol production). The QC plate was prepared a nd dosed in the same manner as the test compound plate with forskolin or prochloraz according to the schematic presented in Table 2. Due to the high background produc tion of testosterone, background hormone production was calculated from three wells with cells on the QC plate that received no 22Rhydroxycholesterol.

# Steroidogenesis Assay (2011) / Page 6 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None

TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Co ncentrations in μM).a 1 2 3 4 5 6 A Blank b 

# Blank Blank Background c 

# Background Background B DMSO DMSO DMSO DMSO + MeOH d 

# DMSO + MeOH DMSO + MeOH C Forskolin 1μM Forskolin 1μM Forskolin 1μM 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 to tal volume of 1 mL per well. b Blank wells received medium contai ning 10μM 22R-hydroxycholesterol. c Background wells received media without 22R-hydroxycholesterol. d MeOH (70% methanol) was added to these wells for 30 mi nutes at room temperature following medium removal.



# Following dosing, the plates were incubated for 48 ± 2 hours under the conditions previously described. The medium from each well was removed, split into two aliquots, and frozen at

− 80 ± 2°C until hormone analysis.
5. Cell Viability/Cytotoxicity Assay: Cell viability was m onitored by the MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl te trazolium bromide] assay (Mosman, 1983) 1 

# after exposure. On the QC plates, wells designated to receive methanol (control wells for cell death measurements) were rinsed twice with phosphate-buffered saline (PBS), and incubated with methanol for 30 minutes at room temperature. After the methanol incubation, the methanol-treated wells were ri nsed again with PBS three times. Following media removal or media removal and metha nol treatment, 0.5 mL of a 0.5 mg/mL MTT (Sigma, Cat#M5655, Lot Nos. MKBD8254V and MKBGI61IV) solution in supplemented medium containing 10 μ M 22R-hydroxycholesterol was adde d to each well of the test chemical and QC plates. The plates were incubated at 37 ± 2°C in a 5% CO 2 

# incubator for 3 hours. After incubation, the MTT solution was removed and 0.5 mL of isopropanol was added to each well. Plates were subsequently incubated at room temperature for 20 minutes while shaken. After this incubation, absorban ce at 570 nm and 650 nm were measured with a Packard Fusion plate reader (location not reported). The absorbance at 650 nm was subtracted from the absorbance at 570 nm to calculate the MTT viability value for each well. The difference in cell viability was dete rmined by comparison of treated wells to the solvent control wells. A gr eater than 20% reduction in cel l viability was considered evidence of cytotoxicity according to the guideline.
6. Hormone Measurement System: HPLC-MS/MS.

Testosterone and estradiol le vels were measured by HPLC -MS/MS (OpAns, LLC, Durham, NC). Testosterone and estradiol were ex tracted from H295R-supplemented medium by liquid-liquid extraction with MT BE after the addition of [ 2 

# H 5 

# ]-testosterone and [ 2 

# H 5 

# ]-estradiol as internal standards. Extr acts were analyzed by HPLC-MS/MS with positive ion multiple reaction monitoring. Th e method detection limit was 100 pg/mL for testosterone and 10 pg/mL for estradiol. The resulting minimum basal production levels were 500 pg/mL for testosterone and 25 pg/mL for estradiol. Calib ration standards for testosterone and estradiol over the validated ranges of 100 to 100,000 pg/mL and 10 to 10,000 pg/mL, respectively, were used for each an alytical run. Replicate quality control

1 Mosman T. (1983). Rapid colorimetric assay for gr owth and survival: application to proliferation and cytotoxicity. J. Immunol. Methods . 100:45-50.

# Steroidogenesis Assay (2011) / Page 7 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None 

# (QC) samples spiked in supplemented medi um at three concentrations (300, 8,000, and 80,000 pg/mL for testosterone and 30, 800, and 8,000 pg/mL for estradiol) were incorporated in each run. QC samples were prepared prior to sample receipt and stored at -80  ̊ C with the study samples until analysis.
The following performance criteria were met (indicated by an “x”): X Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol) X Spiked sample recovery acceptable for two concentrations of testosterone and estrad iol (mean measured amount from triplicate samples within 30% of nominal concentration) NA Hormone cross-reactivity (antibody-based assays only; ≤ 30% of basal production of the respective hormone) NR Solvent control within 75% range be low maximum response on standard curve X Test compound tested for interfer ence with meas urement system



# C. DATA ANALYSIS: Normality of the data was evaluated with Shapiro-Wilk's test, and homogeneity of the variances between the tr eatment groups was evaluated with Levene's test. If the p-values >0.05 in both tests, statistical signi ficance between each treatment group and the control group was evaluated with Dunnett's test . If the p-values were ≤ 0.05 in either the normality or the homogeneity test, a log transformation was performed on the data to attempt to approximate a normal distributi on. If, following the transformation, p-values were >0.05 in both the normality and homogeneity tests, Dunnett's test was performed. If the p-values ≤ 0.05 in either the normality or the hom ogeneity test after log transformation, the non-transformed data set was analyzed with the nonparametric Kruskal-Wallis test followed by Dunn's test to evaluate statistical significance. The statis tical analyses of the data were considered appropriate.
The guideline recommendations th at the results be normalized to the mean solvent control (SC) for each 24-well plate used to test a gi ven chemical with the following equation were met: Relative change = (hormone concentrati on in each well) ÷ (mean SC hormone concentration).
The raw data for testosterone and estradiol concentrations, and fold change over SC were reported as mean ± SD. A si gnificant difference compared to the SC was set at p ≤ 0.05. Data were expected to exhib it a dose-response profile at non-cytotoxic doses. Only noncytotoxic concentrations (> 80% viability) were included. Results at concentrations that demonstrated cloudiness or a prec ipitate were also not include d. For forskolin induction of testosterone, relative change was calculated both with and without subtracting the background hormone production from the forsko lin-treated wells and blank and solvent control wells. This was done because of the high background production of testosterone and the small dynamic range for forskolin i nduction. Background hormone production was calculated from three wells with cells on the QC plate that received no 22Rhydroxycholesterol at the time of exposure.

II. RESULTS

# Steroidogenesis Assay (2011) / Page 8 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None


# A. TEST COMPOUND: The basal hormone production for the SC (DMSO) was greater than 500 and 40 pg/mL for testosterone and estrogen (in the presence of 22R -hydroxycholesterol. The % CVs of replicate wells of the solven t control within a pl ate were 0.23-5.22% for absolute testosterone concentrations and 3.38- 4.87% for absolute estradiol concentrations. The between-plate CVs for solvent controls based on absolute concentration were 7.4% for testosterone and 20% for estrad iol. Guideline recommendations were met. Precipitation was observed at the 100 μ M concentration in Run 1. The hi ghest concentra tion that could be analyzed was 10 μ M in Run 1 and 100 μ M in Runs 2 and 4.
No reproducible alteratio ns in testosterone or estradiol production were demonstrated by the results of the three independent runs with di methoate in the steroi dogenesis assay (Table 3 and Figures 1–6). Slight, but statistically significant, decr eases in estradiol (0.01 and 0.1 μ M; p ≤ 0.05) production were observed in a sing le assay run, but we re not considered biologically relevant. Under the conditions of this study, dimethoate did not alter testosterone or es tradiol production.

TABLE 3. Mean (±SD) Hormone Concentrations A fter Treatment with Dimethoate for 48 Hours. a 

# Nominal Concentration (μM) Run 1 Run 2 Run 4 Run 1 Run 2 Run 4 Mean b 

# ± SD b 

# Statistical Significance Testosterone (pg/mL) Fold Difference DMSO 2443 2758 2419 NR NR NR NR NR 0.0001 2489 2697 2338 1.02 0.98 0.97 0.99 0.03 0.001 2406 2625 2306 0.98 0.95 0.95 0.96 0.02 0.01 2576 2699 2272 1.05 0.98 0.94 0.99 0.06 0.1 2809 2765 2261 1.15 1.00 0.93 1.03 0.11 1 2626 2765 2402 1.07 1.00 0.99 1.02 0.04 10 2519 2740 2338 1.03 0.99 0.97 1.00 0.03 100 2428 2841 2282 NA 1.03 0.94 0.99 NC Estradiol (pg/mL) Fold Difference DMSO 162 242 231 NR NR NR NR NR 0.0001 163 226 218 1.01 0.93 0.95 0.96 0.04 0.001 165 235 222 1.01 0.97 0.96 0.98 0.03 0.01 171 240 214 1.05 0.99 0.93* 0.99 0.06 p ≤ 0.05, Trial 3 only 0.1 177 237 210 1.09 0.98 0.91* 0.99 0.09 p ≤ 0.05, Trial 3 only 1 169 242 225 1.04 1.00 0.98 1.01 0.03 10 166 252 227 1.02 1.04 0.98 1.01 0.03 100 165 249 226 NA 1.03 0.98 1.01 NC a Data were obtained from page 29-30 and 55-57 of the study report. b Calculated by the reviewers. NR Not reported (SC = 1.0) NA Not applicable; not calculated due to pr ecipitation observed at time of exposure. NC Not calculated, n = 2.

# Steroidogenesis Assay (2011) / Page 9 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None 

# FIGURE 1. Change in Testosterone Producti on Relative to Dimethoate Concentration (Run 1). a



# a The 100 μ M concentration was excluded due to precipitation. 

# FIGURE 2. Change in Testosterone Producti on Relative to Dimethoate Concentration (Run 2).

# Steroidogenesis Assay (2011) / Page 10 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None 

# FIGURE 3. Change in Testosterone Producti on Relative to Dimethoate Concentration (Run 4).

FIGURE 4. Change in Estradiol Production Relative to Dimethoate Concentration (Run 1). a



# a The 100 μ M concentration was excluded due to precipitation.

# Steroidogenesis Assay (2011) / Page 11 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None 

# FIGURE 5. Change in Estradiol Production Relative to Dimethoate Concentration (Run 2).

FIGURE 6. Change in Estradiol Production Relative to Dimethoate Concentration (Run 4).

* Significantly different from the solvent control at p ≤ 0.05.



# B. CYTOTOXICITY: It was reported that treatmen t with dimethoate did not induce cytotoxicity of greater than 20% (the ma ximum permitted by the guideline) during any of the three steroidogenesis assays. Cell viability on the test and QC plates (relative to control) ranged from 92.6 – 103.3% and 96.2 – 106.4%, re spectively (Table 4 and Figures 7–9). The inclusion of methanol with the cells in DMSO only yielded cell viability measurements of 5.6 – 7.7%.

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TABLE 4. Mean (±SD) MTT Cell Viability Results After Treatment with Forsko lin, Prochloraz, or Dimethoate for 48 Hours.a
Compound Concen. (μM) Cell Viability - Run 1 Cell Viabilit y - Run 2 Cell Viability - Run 4 Mean SD Mean SD Mean SD Blank Na 106.4 2.12 99.5 1.94 98.8 3.66 Background Na 102.6 4.12 98.9 1.22 99.8 0.63 SC + Methanol Na 7.7 2.04 5.6 1.92 5.7 2.45 Forskolin 1 b 

# 103.9 2.52 103.5 2.13 104.1 0.87 Forskolin 10 98.7 4.11 101.8 1.23 101.7 1.74 Prochloraz 0.1 100.8 0.76 96.9 1.68 98.3 2.14 Prochloraz 1 99.0 0.86 96.2 0.35 99.5 0.94 Dimethoate 0.0001 94.7 4.70 96.6 0.65 96.6 2.30 Dimethoate 0.001 95.1 1.88 97.2 3.39 98.1 2.51 Dimethoate 0.01 100.2 0.62 98.1 1.89 99.7 0.89 Dimethoate 0.1 103.3 3.05 98.5 3.10 99.1 0.80 Dimethoate 1 92.6 3.99 97.4 0.47 99.9 4.96 Dimethoate 10 95.0 1.90 98.0 2.34 97.6 0.80 Dimethoate 100 100.0 2.85 99.4 0.81 101.4 2.27 a Data were obtained from page 23-24 of the study report. b Forskolin concentration was 3.33 μM in Run 1.



# FIGURE 7. MTT Cell Viability Results (Run 1). a



# a The 100 μ M concentration was excluded due to precipitation.

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# FIGURE 8. MTT Cell Viability Results (Run 2).

FIGURE 9. MTT Cell Viability Results (Run 4).

C. QC PLATE: Concentrations of testosterone an d estradiol, and the fold induction or inhibition in the presence of forskolin and prochloraz, on the QC plates are shown in Table 5. Due to a protocol deviation in Run No. 1, the final forskolin concentrations were 3.33 μ M and 10 μ M. The minimum basal hormone produc tion levels for testosterone (500 mg/mL) and estradiol (25 pg/mL) were met in both blank and SC wells. Testosterone induction in the presence of 10 μM forskolin wa s 1.55- to 1.88-fold, which is slightly less than the required ≥ 2-fold requirement; however, after subtraction of the background from the forskolin wells, the testosterone induction in the presence of 10 μM forskolin was 2.03- to 2.57-fold. Estradiol induction in the pres ence of 10 μM forskolin was greater than the ≥ 7.5-fold requirement.

# Steroidogenesis Assay (2011) / Page 14 of 15 DIMETHOATE / 035001 OCSPP 890.1550/ OECD None


# Cells incubated with 10 μ M forskolin induced testosterone production by 2.03- to 2.57-fold and induced estradiol production by 10.39- to 13.56-fold. Cells incubated with 1 μ M prochloraz inhibited testosterone production by 0.43- to 0.49-fold and inhibited estradiol production by 0.26- to 0.43-fold. These induc tion and inhibition results met the guideline requirements.
The variability (%CV) between the runs (calculated by the re viewer) based on the absolute hormone concentrations in the SC were 12% fo r testosterone and 18% for estradiol, within the recommended limit of ≤ 30%. The %CVs within each ru n for the QC plates were 1.42- 5.00% for testosterone and 2.17-5.23% for estradiol.

TABLE 5. Hormone Concentrations (Mea n ± SD) Following Treatment with Fo rskolin or Prochloraz for 48 Hours. a



# Compound Conc. ( μ M) Run 1 Run 2 Run 4 Run 1 Run 2 Run 4 Mean b 

# ± SD b


# Testosterone (pg/mL) Fold Difference Background na 1181 1298 1315 0.47 0.44 0.56 0.49 0.06 Blank na 2476 3069 2323 0.98 1.01 1.00 1.00 0.02 DMSO na 2523 2955 2330 1.00 1.00 1.00 1.00 0 Forskolin c 

# 1 3307 4304 3164 1.31 1.46 1.36 1.38 0.08 Forskolin 10 3909 5558 3702 1.55 1.88 1.59 1.67 0.18 Prochloraz 0.1 2172 2279 1768 0.86 0.77 0.76 0.80 0.06 Prochloraz 1 1159 1260 1137 0.46 0.43 0.49 0.46 0.03 Estradiol (pg/mL) Fold Difference Background na 71 48 79 0.44 0.21 0.35 0.33 0.12 Blank na 160 234 230 0.99 1.04 1.04 1.02 0.03 DMSO na 162 225 222 1.00 1.00 1.00 1.00 0 Forskolin c 

# 1 1936 1479 1571 11.9 6.59 7.07 8.53 2.95 Forskolin 10 2201 2333 2314 13.6 10.4 10.4 11.4 1.83 Prochloraz 0.1 154 177 161 0.95 0.79 0.73 0.82 0.11 Prochloraz 1 69 58 75 0.43 0.26 0.34 0.34 0.09 a Data were obtained from page 25-26 of the study report. b Calculated by the reviewers. c Forskolin concentration was 3.33 μM in Run 1. d When the background is subtracted, the valu es are 1.58-, 1.81-, and 1.82-fold difference for Runs 1, 2, and 3, respectively, with a mean of 1.74-fold difference. e When the background is subtracted, the valu es are 2.03, 2.57, and 2.35-fold difference for Runs 1, 2, and 3, respectively, with a mean of 2.32-fold difference.



# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Dimethoate demonstrated a statistically significant reduction of estradiol concentration in one of three assay runs. This effect was not observed at the highest concentrations of dimethoate tested, and was not dose dependent. No effect on estrad iol concentrations was observed in the other two assay runs, and statistically significant cha nges in testosterone concentra tions were not observed in any of the assay runs. Based on these results, dime thoate was not considered to be positive in the steroidogenesis assay.

B. AGENCY COMMENTS: Dimethoate was tested at concentrations up to 100 μ M (10 μ M in the first assay run due to precipitation at 100 μ M) with no evidence of cytotoxicity in the in vitro steroidogenesis assay. Three individual assay runs were completed, and testosterone

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# and estradiol concentrations were determin ed after H295R cells were incubated with dimethoate, forskolin (inducer), and prochlor az (inhibitor) for 48 ± 2 hours. The guideline requirements for CV ≤ 30% between replicate wells within a plate and between plates were met for all three assay runs. Basal hormone production was acceptable based on the method detection limits determined with the HPLC-M S/MS method used for this study. Estradiol production was statistically decr eased in one of three inde pendent assays at 0.01 and 0.1 μ M only, but no dose response was observed. This decrease in estradiol production was not interpreted to be biologically relevant. Based on the hormone responses in each of the three independent runs, dimethoate treatment did not result in a statistically significant reproducible alteration in testos terone or estr adiol production.

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:

 Documentation/details supporting proficienc y testing/training for pertinent staff involved in the study are reporte dly on file but were not available for review with the study.

 The study report states that proficienc y testing was conducted according to the guideline US EPA OCSPP 890.1300 (Estrogen Receptor Transcriptional Activation), instead of the appropriate guideline (US EPA 890.1550).

 The specific cell passage identifier for each assay run was not reported.

 EC 50 

# ranges for forskolin and proc hloraz were not reported.

 Validity of the solvent control in terms of its position within the hormone standard curve ranges was not reported.