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



# MEMORANDUM

Date: 6/29/2015 OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



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

PC Code: 036101 

# Decision No.: 461007, 464740 

# Petition No.: NA Risk Assessment Type: NA TXR No.:**04~r7.:t'(** 

# MRID No.: See Table 

# DP Barcode: D398779, D401753 

# Registration No.: NA Regulatory Action: NA CaseNo.: NA CAS No.: 1582-09-8 

# 40 CFR: NA

FROM: Greg 

# Akerman, Ph.D.

# ~tfJ:-­ Immediate Office Health Effects Division (7509P)



# THROUGH: Jess Rowland

# \__.._~ Deputy

# Directo~- Health Effects Division



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



# I. ACTION REQUESTED Ver.Apr. 2010



# The Pesticide Re-evaluation Division (PRO) of OPP 

# has requested that the Health Effects Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays submitted in response to the agency's Test Order for trifluralin: Test Order# CON-036101-26.

Page 1 of2

# Page 2 of 2 

# II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for trifluralin.

III. MRID Table

Chemical: Trifluralin PC Code: 036101 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48619701 890.1150 Androgen Receptor Binding (Rat Prostate) 48619702 890.1200 Aromatase Assay (Human Recombinant) 48619703 890.1250 Estrogen Receptor Binding 48619704 890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) 48619705

890.1350 Fish Short-Term Reproduction 48619706 890.1400 Hershberger (Rat) 48619707 890.1450 Female Pubertal (Rat) 48674001 890.1500 Male Pubertal (Rat) 48674002 890.1550 Steroidogenesis (Human Cell Line – H295R) 48619708 890.1600 Uterotrophic (Rat) 48619709

# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ    ԢԭԞԡԭԟԹՊԻՇԼԽ  

ԬԢԠԡԡԹՌԹԭՇՁՆՌ  

ԢԭԞԪԯԦԡ   

ԢԭԞԤՍՁԼԽՄՁՆԽ   
      ԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊ ՈՀՇՋՁՋԞՋՋԹՑ
ԣՊՇԿ       

ԠՇՅՅՇՆԫԹՅԽ  ԱՊՁԾՄՍՊԹՄՁՆ
ԠՀԽՅՁԻԹՄԫԹՅԽ  ԦԲԭԞԠ   ԡՁՆՌՊ ՇԫԫԼՁՈՊՇՈՑՄ
ՌՊՁԾՄՍՇՊՇՅԽՌՀ ՑՄԺԽՆՒԽՆԹՅՁՆԽ
 ԠԞ԰ԫԹՅԽ  ԫՇՌՊԽՈՇՊՌԽԼ

ԠԞ԰ԫՇ   ԰ՑՆՇՆՑՅՋ  ԫՇՆԽՈՊՇՎՁԼԽԼ

ԢԭԞԭԠԠՇԼԽ 

ԧՇՀՆԪԹՊՌՇՆ 

ԱԽՊՁ԰ԪՑԽՊՋ     



ԠԹՌՀԽՊՁՆԽԞՍԺԽԽ      

ԞՆՁՌԹԲՄՄԹԿԹԼԼՁ ԫՇՄՇՆԿԽՊՏՁՌՀԢԭԞ   



ԯՇԺՁՆ԰ՌԽՊՆԺԽՊԿ   



 Digitally signed by ROBIN STERNBERG DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN STERNBERG, dnQualifier=0000039126 Date: 2015.06.02 12:59:16 -04'00'    

       



   



    

    







 





    





 

   



# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

# ՇԾ  



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ     !

"

# 

# ԠՇԹԼՑԨԨԨԩԥՍՌԻՀՁՆՋՇՆԱԞԪԹՊՁՆՇԢԩԪԻԠՄՑՅՇՆՌԹՆԼ ԧԱՀՇՅԹՋԱՊՁԾՄՍՊԹՄՁՆԱՀԽ
ԞՅՈՀՁԺՁԹՆ ԪԽՌԹՅՇՊՈՀՇՋՁՋ ԞՋՋԹՑ ԲՋՁՆԿ ՌՀԽ ԞԾՊՁԻԹՆ ԠՄԹՏԽԼ ԣՊՇԿ 

# ԵԽՆՇՈՍՋ ՄԹԽՎՁՋ  ԲՆՈՍԺՄՁՋՀԽԼ ՋՌՍԼՑ ՈԽՊԾՇՊՅԽԼԺՑԱՇՐՁԻՇՄՇԿՑԢՆՎՁՊՇՆՅԽՆՌԹՄԯԽՋԽԹՊԻՀԹՆԼԠՇՆՋՍՄՌՁՆԿ ԱՀԽԡՇՏԠՀԽՅՁԻԹՄԠՇՅՈԹՆՑԪՁԼՄԹՆԼ ԪՁԻՀՁԿԹՆ  ԩԹԺՇՊԹՌՇՊՑ ՊԽՈՇՊՌ ՆՍՅԺԽՊ  ԰ՌՍԼՑ ՋՈՇՆՋՇՊԽ Լ ԺՑ ԡՇՏ ԞԿՊՇ԰ԻՁԽՆԻԽՋ ԩԩԠ
ԦՆԼՁԹՆԹՈՇՄՁՋԦՆԼՁԹՆԹ

ԱՀԽԲ԰ԢԭԞԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊ԰ ԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅ
Ԣԡ԰ԭԱՁԽՊ ՋԻՊԽԽՆՁՆԿԺԹՌՌԽՊՑ ՁՋԻՇՅՈՊՁՋԽԼՇԾԽՄԽՎԽՆ ՋԻՊԽԽՆՁՆԿԹՋՋԹՑՋՁՆՌԽՆԼԽԼՌՇՁԼԽՆՌՁԾՑԹԻՀԽՅՁԻԹՄ0ՋՄՁՃԽՄՑ ԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՁԽՁՌՋՈՇՌԽՆՌՁԹՄՌՇՁՆՌԽՊԹԻՌՏՁՌՀ ՌՀԽԽՋՌՊՇԿԽՆԹՆԼՊՇԿԽՆՇՊՌՀՑՊՇՁԼ
ԢԞՇՊԱՈԹՌՀՏԹՑՋԱՀԽՊՇԺՍՋՌՆԽՋՋՇԾՌՀԽԱՁԽՊԺԹՌՌԽՊՑՁՋԺԹՋԽԼՇՆՌՀԽ
ՋՌՊԽՆԿՌՀՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՌՇՁԼԽՆՌՁԾՑՈՇՌԽՆՌՁԹՄ ԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՏՁՌՀԻՇՅՈՄԽՅԽՆՌԹՊՑԽՆԼՈՇՁՆՌՋՏՁՌՀՁՆ
ՌՀԽԹՋՋԹՑՏՀԽՊԽԹՎԹՁՄԹԺՄԽԹՆԼ ՊԽԼՍՆԼԹՆԻՑԹԻՊՇՋՋՌՀԽԺԹՌՌԽՊՑ ԱՀՍՋՌՀԽ ՊԽՋՍՄՌՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑ

ՋՀՇՍՄԼՆՇՌԺԽԻՇՆՋՁԼԽՊԽԼՁՆՁՋՇՄԹՌՁՇՆԺՍՌ ՊԹՌՀԽՊՋՀՇՍՄԼԺԽԻՇՆՋ ՁԼԽՊԽԼՁՆՌՀԽԻՇՆՌԽՐՌՇԾՇՌՀԽՊԹՋՋԹՑՋՁՆՌՀԽ ԺԹՌՌԽՊՑԹՋՏԽՄՄԹՋԬՌՀԽՊ԰ԻՁԽՆՌՁԾՁԻԹՄՄՑԯԽՄԽՎԹՆՌԦՆԾՇՊՅԹՌՁՇՆ
Ԭ ԰ԯԦԦՆՇՊԼԽՊՌՇԼԽՌԽՊՅՁՆԽՁԾԹԻՀԽՅՁԻԹՄՀԹՋ ՌՀԽՈՇՌԽՆՌՁԹՄՌՇՁՆՌԽՊԹԻՌՏՁՌՀՌՀԽԢԞՇՊԱՈԹՌՀՏԹՑՋԹԴԽՁԿՀՌՇԾԢՎՁԼԽՆԻԽ
ԴՇԢԽՎԹՄՍԹՌՁՇՆՇԾԱՁԽՊԹՋՋԹՑ
ՊԽՋՍՄՌՋՁՆԻՇՅԺՁՆԹՌՁՇՆՏՁՌՀՌՀԽԾՁՆԼՁՆԿՋՁՆՌՀԽԬ԰ԯԦՋՀՇՍՄԼԺ ԽՍՆԼԽՊՌԹՃԽՆ
ՊԽԾԽՊՌՇՌՀԽԴՇԢԡՇԻՍՅԽՆՌ

$ % & ' ( ) % * + , - 

# ԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋՁՆՌՀՁՋԡԢԯՌԽՅՈՄԹՌԽԹՊԽՇԾԾԽՊԽԼ ԹՋԹԿԽՆԽՊԹՄՊԽԾԽՊԽՆԻԽՌՇ
ԹՁԼՁՆՈՊԽՈԹՊԹՌՁՇՆՇԾՌՀԽԡԢԯԱՀԽՈՍՊՈՇՋԽՇԾՌՀԽՋԽՊԽԻՇՅՅԽՆԼԹ ՌՁՇՆՋՁՋՆՇՌՌՇՋԽՊՎԽԹՋՋՍԺՋՌՁՌՍՌԽԾՇՊ
ՌՀԽԱԽՋՌԤՍՁԼԽՄՁՆԽՋՆՇՊՌՇՈՊՇՎՁԼԽԹՆՑԿՍՁԼԹՆԻԽՇՆՀՇՏՌՀԽՋՌՍԼՑՋՀՇՍՄԼԺԽԻՇՆԼՍԻՌԽԼ    

       



   



    

    







 





    





 

   



# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

# ՇԾ  



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ   !



" #



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!





 %



# ԱՀԽԼԹՑԹՋՋԹՑՇԾՌՊՁԾՄՍՊԹՄՁՆ
ՈՍՊՁՌՑՇՆԹՅՈՀՁԺՁԹՆՅԽՌԹՅՇՊՈՀՇՋՁՋՇԾՌՀԽԞԾՊՁԻԹՆԻՄԹՏԽԼԾՊՇԿ 

# ԵԽՆՇՈՍՋ

ՄԹԽՎՁՋ ՏԹՋԻՇՆԼՍԻՌԽԼՍՆԼԽՊԾՄՇՏՌՀՊՇՍԿՀԻՇՆԼՁՌՁՇՆՋԞՅՈՀՁԺՁԹՆՄԹՊՎԹԽԹՌԫՁԽՍՏՃՇՇՈԣԹԺԽՊ
ԫԣՋՌԹԿԽ ԻՇՆՌՊՇՄԹՆԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՊԽՈՄՁԻԹՌԽՏԽՊԽԽՐՈՇՋԽԼՌՇ ԹՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՋՇՄՎԽՆՌԻՇՆՌՊՇՄ
 ՅԩԩԼՁՅԽՌՀՑՄԾՇՊՅԹՅՁԼԽԡԪԣԹՆԼՆՇՅՁՆԹՄՌՊՁԾՄՍՊԹՄՁՆԻՇՆԻԽՆՌՊԹ ՌՁՇՆՋՇԾԹՆԼՅԿ ԹՁԩ ԱՀԽ ՊԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼ ՌՁՅԽՏԽՁԿՀՌԽԼ ԹՎԽՊԹԿԽ 
ԱԴԞ ՅԽ ԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ՏԽՊԽ  ԩԬԮԻՇՆՌՊՇՄՋԹՆԼՅԿԹՁԩԱՀԽՌԽՋ ՌՋՑՋՌԽՅՏԹՋՅԹՁՆՌԹՁՆԽԼԹՌՌՇոԠ
ԹՆԼԹՈԥՇԾՌՇ

ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼՁՆՌՀՁՋԡԢԯԹՄՄԽԾԾԽԻՌՋԹՊԽՊԽՈՇՊՌԽ ԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ

ԫՇՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼՅՇՊՌԹՄՁՌՁԽՋՇՊԻՄՁՆՁԻԹՄՋՁԿՆՋՏԽՊԽՇԺՋԽՊՎԽԼԼՍՊՁՆԿՌՀՁՋՋՌՍԼՑԬՆԡԹՑՇՆԽՌԹԼՈՇՄԽՁՆ ՌՀԽՀՁԿՀՌՊԽԹՌՅԽՆՌԿՊՇՍՈՏԹՋՇԺՋԽՊՎԽԼՊԽՋՌՁՆԿԹՌՌՀԽՋՍՊԾԹԻԽԦ ՆՌՀԽՋԹՅԽՎԽՋՋԽՄՇՆԽՌԹԼՈՇՄԽՏԹՋՇԺՋԽՊՎԽԼ
ՏՁՌՀ ԹԺՆՇՊՅԹՄ ՋՍՋՈԽՆՋՁՇՆ ԹՆԼ ԹՈՈԽԹՊԽԼՌՇ ՀԹՎԽ ՁՆՌԽՊՆԹՄ ԺՄԽԽԼՁՆԿ  ՇՆԡԹՑՋ  ԹՆԼ  ԹՆԼՏԹՋ ՇԺՋԽՊՎԽԼ ՊԽՋՌՁՆԿՇՆՌՀԽՋՍՊԾԹԻԽՇՆԡԹՑԱՀԽՋԽՇԺՋԽՊՎԹՌՁՇՆՋՏԽՊԽՆՇՌԻ ՇՆՋՁԼԽՊԽԼՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԹՆԼՅԹՑ
ՀԹՎԽԺԽԽՆԼՍԽՌՇՈՀՑՋՁԻԹՄՀԹՊՅՌՇԹՋՁՆԿՄԽՌԹԼՈՇՄԽԞՌՌԽՋՌՌԽՊ ՅՁՆԹՌՁՇՆՋՈՁՆԹՄԻՍՊՎԹՌՍՊԽ
ԺԽՆՌՌԹՁՄՏԹՋՆՇՌԽԼ
ՁՆԺՇՌՀԻՇՆՌՊՇՄՋԹՆԼՁՆԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՌՀԽՈԽՊԻԽՆՌԹԿԽՇԾ ԹԾԾԽԻՌԽԼՌԹԼՈՇՄԽՋՊԹՆԿԽԼԾՊՇՅՌՇ

ԡԹՑ  ԺՇԼՑ ՏԽՁԿՀՌ ԹՆԼ ՋՆՇՍՌՎԽՆՌ ՄԽՆԿՌՀ 
԰Գԩ ՏԽՊԽ ՋՁԿՆՁԾՁԻԹՆՌՄ Ց ՊԽԼՍԻԽԼ 
Ո ԺՑ  ԹՆԼ 
ՊԽՋՈԽԻՌՁՎԽՄՑԹՌՌՀԽՀՁԿՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄՏՀՁՄԽԡԹՑ԰ԳԩՏԹՋՋ ՁԿՆՁԾՁԻԹՆՌՄՑՁՆԻՊԽԹՋԽԼ
ՈԺՑԹՌՌՀԽ
ՅՁԼՌՊԽԹՌՅԽՆՌՄԽՎԽՄՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԡԹՑԺՇ ԼՑՏԽՁԿՀՌԹՆԼ԰ԳԩՏԽՊԽԻՇՅՈԹՊԹԺՄԽ
Ո 
ԺԽՌՏԽԽՆՌՊՁԾՄՍՊԹՄՁՆՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԹՆԼՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ

ԱՊՁԾՄՍՊԹՄՁՆՀԹԼՆՇԽԾԾԽԻՌ
Ո ՇՆԡԹՑՆՇՊՅԹՄՁՒԽԼ
ԾՇՊՋՆՇ ՍՌՎԽՆՌՄԽՆԿՌՀՀՁՆԼՄՁՅԺՄԽՆԿՌՀ
ԥԩԩԡԹՑ  ՆՇՊՅԹՄՁՒԽԼԥԩԩՏԹՋ ՋՁԿՆՁԾՁԻԹՆՌՄՑ ՁՆԻՊԽԹՋԽԼ 
Ո ՁՆՌՀԽՋ ՇՄՎԽՆՌԻՇՆՌՊՇՄ ԹՆԼՄՇՏ ԹՆԼՅՁԼՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄՋ ԺՑ  ԹՆԼ  ՊԽՋՈԽԻՌՁՎԽՄՑ ԻՇՅՈԹՊԽԼՏՁՌՀՌՀԽ ՆԽԿԹ ՌՁՎԽ ԻՇՆՌՊՇՄ ԡԹՑ ՆՇՊՅԹՄՁՒԽԼԥԩԩՏԹՋ ՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼ
ՈԺՑԹՌՌՀԽՀՁԿՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄԻՇՅՈԹՊԽԼՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԱՀԽՊԽ
ՏԽՊԽՆՇԽԾԾԽԻՌՋՇՆԡԹՑՇՊԡԹՑԫԣԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԫՇԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌՏԹՋՇԺՋԽՊՎԽԼ    

       



   



    

    







 





    





 

   



# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

# ՇԾ  



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ԱՀԽՊԽՏԽՊԽՆՇԹՈՈԹՊԽՆՌՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՇՆՌՀՑՊՇՁԼՀՁՋ ՌՇՈԹՌՀՇՄՇԿՑԣՇՄՄՁԻՍՄԹՊԻԽՄՄՀԽՁԿՀՌԹՆԼԹՊԽԹ

ՏԽՊԽՆՇՌԼԽՌԽՊՅՁՆԽԼ

Ԟ ՌՇՌԹՄ ՇԾ  ՁՆԼՁՎՁԼՍԹՄՋ ՏԽՊԽ ԽՐԻՄՍԼԽԼ ԾՊՇՅ ՌՀԽ ԹՆԹՄՑՋՁՋ ՇԾ ԡԹ Ց  ԿՊՇՏՌՀ ԽՆԼՈՇՁՆՌՋ ԺԽԻԹՍՋԽ ԫԣ ՋՌԹԿԽ ԽՐԻԽԽԼԽԼ
՟ՄԹՌԽՋՌԹԿԽՠՌՀԽՋԽՁՆԻՄՍԼԽԼՇՆԽՌԹԼՈՇՄԽԾՊՇՅՌՀԽ ՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԾՇՍՊԾՊՇՅՌՀԽՋՇՄՎԽՆՌԻՇՆՌՊՇՄ
ՋԽՎԽՆԹՆԼՆՁՆԽՌԹԼՈՇՄԽՋՁՆՌՀԽ ՄՇՏԹՆԼՅՁԼՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋ ՊԽՋՈԽԻՌՁՎԽՄՑԹՆԼՆՇՆԽՁՆՌՀԽ ՀՁԿՀՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄԴՀՁՄԽՌՀԽՇԻԻՍՊՊԽՆԻԽՇԾՄԹՌԽՋՌԹԿԽՌԹԼՈՇՄԽՋՏԹՋՆՇՌԹՆԹՄ ՑՒԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՌՀԽՈԹՌՌԽՊՆՇԾՇԻԻՍՊՊԽՆԻԽՁՋ ԹՌ ՄԽԹՋՌ ՋՍՈԽՊԾՁԻՁԹՄՄՑ ԻՇՆՋՁՋՌԽՆՌ ՏՁՌՀ ՌՀԽ ՇԺՋԽՊՎԹՌՁՇՆՋ ՇԾ ԿՊԽԹ ՌԽՊ ԡԹՑ  ՆՇՊՅԹՄՁՒԽԼ ԥԩԩ ՁՆ ՌՀԽ ՄՇՏԽՊ ՌՏՇ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄՋ 
ՋՍԿԿԽՋՌՁՆԿ ԹԼՎԹՆԻԽԼ ԼԽՎԽՄՇՈՅԽՆՌ ԺՍՌ ՋՅԹՄՄԽՊ ԡԹՑ  ՆՇՊՅԹՄՁՒԽԼ ԥԩԩ ԹՌ ՌՀԽ ՀՁԿՀԽՋՌ
ՌՊԽԹՌՅԽՆՌՄԽՎԽՄՏՀԽՆԻՇՅՈԹՊԽԼՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ

ԞՄՄՈԽՊԾՇՊՅԹՆԻԽԹՆԼՎԹՄՁԼՁՌՑԻՊՁՌԽՊՁԹՏԽՊԽՅԽՌՁՆՌՀՁՋՋՌՍԼՑՏՁ ՌՀՌՀԽԽՐԻԽՈՌՁՇՆՌՀԹՌՌՀԽԻՇԽԾԾՁԻՁԽՆՌՇԾՎԹՊՁԹՌՁՇՆ ԠԳ ԾՇՊ ՌՀԽ ԱԴԞՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՇԾ ՌՀԽ ՀՁԿՀՌՊԽԹՌՅԽՆՌ Մ ԽՎԽՄՏԹՋ   ԰ՁՆԻԽ ՌՀԽԠԳ ԾՇՊ ՌՀԽ ՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՏԹՋ ՂՍՋՌ ՇՎԽՊ ՌՀԽ ՈԽՊԾՇՊՅԹՆԻԽ ԻՊՁՌԽՊՁՇՆ ՎԹՄՍԽ ՇԾ  

#  ՁՌ ԼՁԼ ՆՇՌ ՁՅՈԹԻՌ ՌՀԽ ՁՆՌԽՊՈՊԽՌԹՌՁՇՆՇԾՌՀԽՋՌՍԼՑ

԰ՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌՋՇՄՎԽՆՌԽԾԾԽԻՌՋՏԽՊԽՇԺՋԽՊՎԽԼԻՇՅՈԹՊԽ ԼՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԣՇՊԡԹՑՆՇՊՅԹՄՁՒԽԼ ԥԩԩԹՋՁԿՆՁԾՁԻԹՆՌՁՆԻՊԽԹՋԽ
ՈՏԹՋՇԺՋԽՊՎԽԼՁՆՌՀԽՋՇՄՎԽՆ ՌԻՇՆՌՊՇՄՏՀՁԻՀՏԹՋՋՁՅՁՄԹՊՁՆՅԹԿՆՁՌՍԼԽՌՇ
ՌՀԽՁՆԻՊԽԹՋԽՇԺՋԽՊՎԽԼՁՆՌՀԽՄՇՏԹՆԼՅՁԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԴՀԽՆ ԻՇՅՈԹՊԽԼՌՇՌՀԽՋՇՄՎԽՆՌԻՇՆՌՊՇՄՌՀԽՊԽՏԽՊԽ ՆՇԼՁԾԾԽՊԽՆԻԽՋԹՌՌՀԽՄՇՏԹՆԼՅՁԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԾՇՊԡԹՑՆ ՇՊՅԹՄՁՒԽԼԥԩԩՀՇՏԽՎԽՊԡԹՑՆՇՊՅԹՄՁՒԽԼ ԥԩԩԹՌՌՀԽՀՁԿՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼ
Ո ԞԼԼՁՌՁՇՆԹՄՄՑՌՀԽՊԽՏԽՊԽՋՁԿՆՁԾՁԻԹՆՌՁՆԻՊԽԹՋԽՋ ՈՁՆՌՀԽՋՇՄՎԽՆՌԻՇՆՌՊՇՄ ՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇ ՄՁՆԡԹՑ԰ԳԩԹՆԼԡԹՑԺՇԼՑՏԽՁԿՀՌԣՇՊ
ԺՇՌՀՇԾՌՀԽՋԽԽՆԼՈՇՁՆՌՋՌՀԽՄՇՏԹՆԼՅՁԼՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՏԽՊԽ ԹՄՋՇՁՆԻՊԽԹՋԽԼՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ ՆՇՌԹՄՄԽԾԾԽԻՌՋՋՁԿՆՁԾՁԻԹՆՌԹՌՈՏՀՁՄԽՌՀԽՀՁԿՀՌՊԽԹՌՅԽ ՆՌՄԽՎԽՄՏԹՋԼԽԻՊԽԹՋԽԼՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄԠՇՅՈԹՊՁՋՇՆՋՌՇՌՀԽՋՇՄՎԽՆՌԻՇՆՌՊՇՄՋՀՇՏԽԼՋՁԿՆՁԾՁԻԹՆՌ ՊԽԼՍԻՌՁՇՆՋ
ՈԾՇՊԡԹՑ԰ԳԩԹՆԼԡԹՑ
ԺՇԼՑՏԽՁԿՀՌՁՆՌՀԽՀՁԿՀՌՊԽԹՌՅԽՆՌԿՊՇՍՈԤՁՎԽՆՌՀԽՋՁԿՆՁԾՁԻԹՆՌ ԽԾԾԽԻՌ
ՈԹՌԹՄՄՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋԾՇՊԡԹՑ
ՆՇՊՅԹՄՁՒԽԼԥԩԩՏՀՁԻՀԻԹՆՆՇՌԺԽԽՐՈՄԹՁՆԽԼԻՇՅՈՄԽՌԽՄՑԺՑՋՇՄՎԽ ՆՌՁՆՌԽՊԾԽՊԽՆԻԽՌՀԽՊԽՁՋԽՎՁԼԽՆԻԽՌՇՋՍԿԿԽՋՌ ՌՀԹՌՌՊՁԾՄՍՊԹՄՁՆՅԹՑԹԾԾԽԻՌԼԽՎԽՄՇՈՅԽՆՌԹՄԽՆԼՈՇՁՆՌՋԹՋՋՇԻՁԹՌԽԼ ՏՁՌՀՊԽԼՍԻԽԼԿՊՇՏՌՀՁՆՌԹԼՈՇՄԽՋԽՐՈՇՋԽԼՌՇՌՀԽ ՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋԹՆԼԻՇՆԼՁՌՁՇՆՋՇԾՌՀՁՋՋՌՍԼՑ    

       



   



    

    







 





    





 

   



# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

# ՇԾ  



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ԱՀԽԹՋՋԹՑ     



 

# ՌՀԽԢԡ԰ԭԱՁԽՊԱԽՋՌԬՊԼԽՊՊԽՉՍՁՊԽՅԽՆՌՋԾՇՊԹՆԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԬԠ԰ԭԭ ԤՍՁԼԽՄՁՆԽ

 

     

 



# ԱԽՋՌՇՊԿԹՆՁՋՅԫԣՋՌԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԫԣ ԱԽՋՌՇՊԿԹՆՁՋՅՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՇՈՌՁՇՆԹՄԫՇՌՋ ՈԽԻՁԾՁԽԼ ԱԽՋՌՌՑՈԽԾՄՇՏՌՀՊՇՍԿՀ

 

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#  ԫՇ ԫՇ ԫՇ 3  

# ԫՇ ԫՇ ԫՇ

 ԫՇ ԫՇ ԫՇ 3  

# ԫՇ ԫՇ ԫՇ

 ԫՇ ԫՇ ԫՇ 3  

# ԫՇ ԫՇ ԫՇ

ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԡՁԾԾ 

# ԡՁԾԾԽՊԽՆԻԽ ԫԞ 

# ԫՇՌԹՈՈՄՁԻԹԺՄԽ

 

# Ԟ՟ՑԽՋՠ ՁՆԼՁԻԹՌԽՋԹՋՁԿՆՁԾՁԻԹՆՌԼՁԾԾԽՊԽՆԻԽԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆ ԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄՍՆՄԽՋՋ
ՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼ

 

# ԱՀԽԻՊՁՌԽՊՁԹԾՇՊՋՁԿՆՁԾՁԻԹՆԻԽԹՊԽԼԽՋԻՊՁԺԽԼՁՆՌՀԽԯԽՎՁԽՏԽՊ0ՋԞ ՆԹՄՑՋՁՋԹՆԼ԰ՌԹՌՁՋՌՁԻԹՄԳԽՊՁԾՁԻԹՌՁՇՆՋԽԻՌՁՇՆՋՇԾ ՌՀԽԡԢԯԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏ ԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼ ԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ

 

#  ԥՁՆԼՄՁՅԺՄԽՆԿՌՀՁՋՆՇՊՅԹՄՁՒԽԼՌՇՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ
԰Գԩ    

       



   



    

    







 





    





 

   



# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

# ՇԾ  



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ   

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#  ԱՀՁՋ ՋՌՍԼՑ ՏԹՋ ԻՇՆԼՍԻՌԽԼ ԾՇՄՄՇՏՁՆԿ ԿՍՁԼԽՄՁՆԽՋ ՇՍՌՄՁՆԽԼ ՁՆ ԲՆՁ ՌԽԼ ԰ՌԹՌԽՋ ԢՆՎՁՊՇՆՅԽՆՌԹՄ ԭՊՇՌԽԻՌՁՇՆ ԞԿԽՆԻՑ 
Բ԰ԢԭԞ  ԢՆԼՇԻՊՁՆԽ ԡՁՋՊՍՈՌ ՇՊ ԰ԻՊԽԽՆՁՆԿ ԭՊՇԿՊԹՅ ԱԽՋՌ ԤՍՁԼԽՄՁՆԽՋ ԬԠ԰ԭԭ  ԞՅՈՀՁԺՁԹՆ ԪԽՌԹՅՇՊՈՀՇՋՁՋ 
ԣՊՇԿ ԞՋՋԹՑ ԢԭԞ Ԡ ԬԻՌՇԺԽՊ  ԹՆԼ

 ԬՊԿԹՆՁՒԹՌՁՇՆ ԾՇՊ ԢԻՇՆՇՅՁԻ ԠՇՇՈԽՊԹՌՁՇՆ ԹՆԼ ԡԽՎԽՄՇՈՅԽՆՌ 
ԬԢԠԡ   ԬԢԠԡԤՍՁԼԽՄՁՆԽԾՇՊՌՀԽԱԽՋՌՁՆԿՇԾԠՀԽՅՁԻԹՄՋԱՀԽԞՅՈՀՁԺՁԹՆԪԽՌԹ ՅՇՊՈՀՇՋՁՋ ԞՋՋԹՑԬԢԠԡԞԼՇՈՌԽԼ԰ԽՈՌԽՅԺԽՊԱՀԽԾՇՄՄՇՏՁՆԿԼԽՎՁԹՌՁ ՇՆՋԹՆԼ

ԼԽԾՁԻՁԽՆԻՁԽՋՏԽՊԽՆՇՌԽԼ

 ԱՀԽԻՇԽԾԾՁԻՁԽՆՌՇԾՎԹՊՁԹՌՁՇՆ
ԠԳԾՇՊՌՀԽԱԴԞՅԽԹՋՍՊԽԼԻՇՆԻԽՆ ՌՊԹՌՁՇՆՇԾՌՀԽՀՁԿՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄ
ՏԹՋՏՀՁԻՀԽՐԻԽԽԼՋՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆՎԹՄՍԽՇԾ ) 

#   ԩԹՊՎԹՄՎՁԹԺՁՄՁՌՑՏԹՋՆՇՌԹՋՋԽՋՋԽԼ
ԽԿԺԽՋՌՋՁՆԿՄԽՋՈԹՏՆՇ ՈՌՁՇՆԹՄՋՁՒԽՋԽՄԽԻՌՁՇՆ  ԣՄՇՏՊԹՌԽՁՆՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ
չՅԩՅՁՆԽՐԻԽԽԼԽԼՌՀԽ ՊԽԻՇՅՅԽՆԼԽԼՅԹՐՁՅՍՅԾՄՇՏՊԹՌԽ ՇԾՅԩՅՁՆ԰ՌՍԼՑ ԹՍՌՀՇՊՋ ՊԽՈՇՊՌԽԼՌՀԹՌ ՈՊԽՎՁՇՍՋ ՋՌՍԼՁԽՋ ՀԹՎԽ  ՋՀՇՏՆՌՀՁՋ ՊԹՌԽՌՇ ՀԹՎԽ ՆՇ

ԽԾԾԽԻՌՇՆ 

# ԵԽՆՇՈՍՋ ԼԽՎԽՄՇՈՅԽՆՌ  ԱՀԽԫԣՋՌԹԿԽՇԾՌՀԽՌԹԼՈՇՄԽՋՍՋԽԼՁՆՌՀԽՊԹՆԿԽԾՁՆԼՁՆԿՋՌՍԼՑ ՏԹՋՆՇՌՊԽՈՇՊՌԽԼ  ԴԹՌԽՊ ՀԹՊԼՆԽՋՋ 
ՅԿԩ ԹՋԠԹԠԬ  

#  ԹՆԼ Ոԥ 
ԾԽՄՄ ՇՍՌՋՁԼԽ ՇԾՌՀԽ ՊԽԻՇՅՅԽՆԼԽԼ ՊԹՆԿԽՋ
ՅԿԩԹՋԠԹԠԬ  

# չ
 ԱՀԽ ՁՆՎԽՋՌՁԿԹՌՇՊՋ ԼՁԼ ՆՇՌ ԼԽՅՇՆՋՌՊԹՌԽ ՌՀԹՌ ԺԹԻՃԿՊՇՍՆԼ ՄԽՎԽՄՋ ՇԾ ՁՇԼՁԼԽ ՁՆ ՌՀԽ ՌԽՋՌՏԹՌԽՊ ՏԽՊԽ ՏՁՌՀՁՆ ՌՀԽ ՊԹՆԿԽ ՊԽԻՇՅՅԽՆԼԽԼ ՁՆ ՌՀԽ ՌԽՋՌ ԿՍՁԼԽՄՁՆԽ 


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# Կԩ ԞՄՌՀՇՍԿՀ ՁՇԼՁԼԽ ՏԹՋ ՅԽԹՋՍՊԽԼՁՌՏԹՋԺԽՄՇՏՌՀԽԩԬԡ
ՅԿԩՌՀԽԹՆԹՄՑՌՁԻԹՄՅԽՌ ՀՇԼՏԹՋՆՇՌՋԽՆՋՁՌՁՎԽԽՆՇՍԿՀ
ՌՇԼԽՌԽՊՅՁՆԽՏՀԽՌՀԽՊՁՌՏԹՋՏՁՌՀՁՆՌՀԽՊԽԻՇՅՅԽՆԼԽԼՊԹՆԿԽԫՇՁ ՇԼՁԼԽՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՏԹՋԿՁՎԽՆ

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# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

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# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

ԾՇՄՄՇՏՁՆԿԤՇՇԼԩԹԺՇՊԹՌՇՊՑԭՊԹԻՌՁԻԽ԰ՌԹՆԼԹՊԼՋԬԢԠԡ԰ԽՊՁԽՋՇՆԭ ՊՁՆԻՁՈՄԽՋ ՇԾԤՇՇԼԩԹԺՇՊԹՌՇՊՑԭՊԹԻՌՁԻԽԹՆԼԠՇՅՈՄՁԹՆԻԽԪՇՆՁՌՇՊՁՆԿԫՍՅԺԽՊ ԬԢԠԡ ԭՊՁՆԻՁՈՄԽՋ ՇՆ ԤՇՇԼ ԩԹԺՇՊԹՌՇՊՑ ԭՊԹԻՌՁԻԽ 
ԹՋ ՊԽՎՁՋԽԼ ՁՆ  ԢԫԳԪԠԠԥԢԪ
ԹՆԼԲ԰ԢԭԞԣԦԣԯԞԤԩԭՋԱՁՌՄԽԠԣԯԭԹՊՌ  ԣԽԼԽՊԹՄ ԦՆՋԽԻՌՁԻՁԼԽ ԣՍՆԿՁԻՁԼԽ ԹՆԼ ԯՇԼԽՆՌՁԻՁԼԽ ԞԻՌ 
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ԱԴԞ ԻՇՆԻԽՆՌ ՊԹՌՁՇՆՋ

ՍՋՁՆԿ ՌՀԽ ՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԾՊՇՅ ԡԹՑՋ  ԱՀԽ ԠԳ 
 ՏԹ Ջ ԻԹՄԻՍՄԹՌԽԼԺԹՋԽԼՍՋՁՆԿՌՀԽՋՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆԾՊՇՅՌՀԽՋԹՅԽՌՁՅ ԽՈԽՊՁՇԼԹՆԼ ՌՀԽ ԱԴԞ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԱՀԽ ԠԳ ՊԹՆԿԽԼ ԾՊՇՅ  ՌՇ  ԱՀԽ ԱԴԞ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՑՁԽՄԼԽԼՇՎԽՊԹՄՄՊԽԻՇՎԽՊՁԽՋՇԾՌՇՇԾՆ ՇՅՁՆԹՄ

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#   ԫՇՌՊԽՈՇՊՌԽԼ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԢԭԞՊԽԻՇՅՅԽՆԼՋԞԾՊՁԻԹՆԻՄԹՏԽԼԾՊՇԿ ԵԽՆՇՈՍՋՄԹԽՎՁՋԴԽՋՌԽՊՆ#ԞԾՊՁԻԹ%ԻՄԹՏԽԼ ԾՊՇԿ԰ՁՄՍՊԹՆԹ
ԵԽՆՇՈՍՋՌՊՇՈՁԻԹՄՁՋՅԹՑԺԽ ՍՋԽԼԹՋԹՆԹՄՌԽՊՆԹՌԽՋՈԽԻՁԽՋ 

# ՀՇՏԽՎԽՊԹ ՄՁՋՌՇԾԹՄՄՇԾՌՀԽՆԽԻԽՋՋԹՊՑՈՊՇՌՇԻՇՄ ԼԽՎՁԹՌՁՇՆՋՌՇԹԻԻՇՅՅՇԼԹՌԽՌՀՁՋՋՈԽԻՁԽՋՁՋ ՊԽԻՇՅՅԽՆԼԽԼԾՇՊՁՆԻՄՍՋՁՇՆՁՆՌՀԽՋՌՍԼՑ ՊԽՈՇՊՌԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽ ՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹՍՋԽԼՌՇՋՍՈՈՇ ՊՌՌՀԽ ՊԽՄՁԹԺՁՄՁՌՑՇԾՌՀԽՌԽՋՌԺԽՁԼԽՆՌՁԾՁԽԼ԰ՈԽԻՁԽՋՋԻՁԽՆՌՁԾՁԻՆԹՅԽ 

# ԵԽՆՇՈՍՋՄԹԽՎՁՋ԰ՈԽԻՁԽՋՋՌՊԹՁՆ
ՁԾՋՌԹՌԽԼԫՇՌՋՈԽԻՁԾՁԽԼԲ԰ԢՆՎՁՊՇՆՅԽՆՌԹՄԭՊՇՌԽԻՌՁՇՆԞԿԽՆԻՑ
ԢԭԞ
ԠՇՊՊԽԻՌՁՇՆՋԹՆԼԠՄԹՊՁԾՁԻԹՌՁՇՆՋՇՆԱԽԻՀՆՁԻԹՄԞՋՈԽԻՌՋՇԾՌՀԽԱԽՋՌԤՍՁԼԽՄՁՆԽՋԾՇՊՌՀԽԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊ԰ԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅԱՁԽՊԞՋՋԹՑՋ
ԬԠ԰ԭԭԱԽՋՌԤՍՁԼԽՄՁՆԽ԰ԽՊՁԽՋԪԹՊԻՀԬԾԾՁԻԽՇԾԠՀԽՅՁԻԹՄ԰ԹԾԽՌՑԹՆԼԭՇՄՄՍՌՁՇՆԭՊԽՎԽՆՌՁՇՆԬԠ԰ԭԭԴԹՋՀՁՆԿՌՇՆԡԠՀՌՌՈՏՏՏԽՈԹԿՇՎԽՆԼՇՈՍԺՋԹՋՋԹՑՎԹՄՁԼԹՌՁՇՆԻՄԹՊՁԾՁԻԹՌՁՇՆԼՇԻՈԼԾ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

       !  "        #  $           ԴԽՊԽՈԹՊԽՆՌՋՅԹՁՆՌԹՁՆԽԼԹՋՁՆՀՇՍՋԽՋՌՇԻՃ!ԶԽՋԟՊԽԽԼՁՆԿՈԹՁՊՋՏԽՊԽՇՊՁԿՁՆԹՄՄՑՇԺՌԹՁՆԽԼԾՊՇՅ 

# ԵԽՆՇՈՍՋԢՐՈՊԽՋՋ
ԟՊՇՇՃՋՎՁՄՄԽԣՄՇՊՁԼԹ 

# ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՄԹՊՎԹԽ ՍՋԽԼ ՁՆ ՌՀԽ ԹՋՋԹՑԺԽԼԽՊՁՎԽԼԾՊՇՅՁՆ ՀՇՍՋԽԹԼՍՄՌՋԴԽՊԽՈԹՊԽՆՌԹՄԹԻԻՄՁՅԹՌՁՇՆԻՇՆԼՁՌՁՇՆՋՋԹՅԽԹՋԼԽԾՁՆՁՌՁՎԽՌԽՋՌ!ԥԹՌԻՀՁՆԿԹՉՍԹՊՁԹՏԽՊԽՅԹՁՆՌԹՁՆԽԼԹՌոԠԹՆԼՏԽՊԽՋՍԺՂԽԻՌՌՇՈԹՊՌՁԹՄՊԽՆԽՏԹՄՋՏՁՌՀՄԹԺՇՊԹՌՇՊՑԼՁՄՍՌՁՇՆՏԹՌԽՊԫՇԾՍՊՌՀԽՊԼԽՌԹՁՄՋՏԽՊԽՈՊՇՎՁԼԽԼԞԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼԾՇՊՈԹՊԽՆՌԹՄԾՊՇԿՋ
ՁԾԹՈՈՄՁԻԹԺՄԽԫՇՌՋՈԽԻՁԾՁԽԼԡԽՌԹՁՄՋՇՆՈԹՊԽՆՌԹՄԾԽԽԼՁՆԿԡԽՌԹՁՄՋՇՆՈԹՊԽՆՌԹՄԾԽԽԼՁՆԿՏԽՊԽՆՇՌՈՊՇՎՁԼԽԼԭՊՁՇՊՌՇՌԽՋՌՁՆՁՌՁԹՌՁՇՆՌԹԼՈՇՄԽՋԺԽԿՁՆՆՁՆԿԼԹՑՋՈՇՋՌՀԹՌԻՀԫԣՏԽՊԽԾԽԼ԰ԽՊԹԪՁԻՊՇՆՌՏՁԻԽԼԹՁՄՑԹՌԹՊԹՌԽՇԾԹՈՈՊՇՐՁՅԹՌԽՄՑՅԿԹՆՁՅԹՄԼԹՑ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌՌՀԽԺԽՋՌ

- ՁՆԼՁՎՁԼՍԹՄՋՈԹՏՆՋՏՁՌՀԹՅՁՆՁՅՍՅՇԾ ՄԹՊՎԹԽՋՈԹՏՆԺԽԽՎԹՄՍԹՌԽԼՌՇՁԼԽՆՌՁԾՑ ՌՀԽԺԽՋՌՋՁՆԿՄԽՋՈԹՏՆԹՆԼՌՀԹՌՌՀԽՄԹՊՎԹԽ ՋԽՄԽԻՌԽԼԾՇՊՌԽՋՌՁՆԿՇՊՁԿՁՆԹՌԽԾՊՇՅՌՀԽ ԺԽՋՌ ՋՁՆԿՄԽՋՈԹՏՆ
ՁԽ 

# ՌՀԽՋՈԹՏՆՋԹՊԽՆՇՌԻՇ

ՅՁՐԽԼԫՍՅԺԽՊՇԾՋՈԹՏՆՋԽՎԹՄՍԹՌԽԼ
ՁԾԹՈՈՄՁԻԹԺՄԽԫԞԫՍՅԺԽՊՇԾԽԿԿՋՋԹՅՈՄԽԼՈԽՊՋՈԹՏՆԫԞԫԣՋՌԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԫԣ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽԼԽԾՁՆՁՌՁՎԽՋՌՍԼՑ ԺԽՁՆՁՌՁԹՌԽԼՏՁՌՀՄԹՊՎԹԽԹՌ ԫՁԽՍՏՃՇՇՈ - ԣԹԺԽՊ
ԫԣԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽ , 

#  ԼԹՑՋՈՇՋՌԾԽՊՌՁՄՁՒԹՌՁՇՆԞԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԼԹՑՋՇՄԼ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

       !  "        #  $           ԪԽԹՆՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾՊԽՈՇՊՌԽԼԫՇՌՊԽՈՇՊՌԽԼԯԹՆԿԽՇԾՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾՊԽՈՇՊՌԽԼԫՇՌՊԽՈՇՊՌԽԼԴԹՋՌՀԽՇՈՌՁՇՆԹՄՋՁՒԽՋԽՄԽԻՌՁՇՆՅԽՌՀՇԼՍՋԽԼ!ԫՇԡԽՌԹՁՄՋՇՆՄԹՊՎԹՄՋԽՄԽԻՌՁՇՆԱԹԼՈՇՄԽՋՏԽՊԽՁՆԼՁՎՁԼՍԹՄՄՑՋՌԹԿԽԼՏՁՌՀՇՍՌԹՆԽՋՌՀԽՋՁԹԹՆԼԫԣՋՌԹԿԽՌԹԼՈՇՄԽՋՏԽՊԽՊԹՆԼՇՅՄՑԹՋՋՁԿՆԽԼՌՇՌԽՋՌՎԽՋՋԽՄՋ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

    !   "  #        $  %           ԩՇԹԼՁՆԿՊԹՌԽ
ՊԽԹՊՁՆԿԼԽՆՋՁՌՑԞՈՈՊՇՐՁՅԹՌԽՄՑՄԹՊՎԹԽԩԱՏԽՆՌՑՌԹԼՈՇՄԽՋՏԽՊԽԹԼԼԽԼՌՇԽԹԻՀՌԽՋՌՎԽՋՋԽՄԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼԽԹԻՀՌԽՋՌՎԽՋՋԽՄՏԹՋՅԹՁՆՌԹՁՆԽԼԹՌԩ 

# ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՊԽԹՊՁՆԿ ԼԽՆՋՁՌՑ ՄՇԹԼՁՆԿ ՊԹՌԽ ՆՇՌ ԽՐԻԽԽԼ ԹՈՈՊՇՐՁՅԹՌԽՄՑ  ՄԹՊՎԹԽԩ ԻՍՄՌՍՊՁՆԿ ՋՑՋՌԽՅ ԾՇՊ ԾՄՇՏ  ՌՀՊՇՍԿՀ ՋՑՋՌԽՅՋ ՇՊ   ՌԹԼՈՇՄԽՋԩ ՁՆ ՋՌԹՌՁԻ ՊԽՆԽՏԹՄ ԽՐՈՇՋՍՊԽՋՑՋՌԽՅՋԱՑՈԽՇԾԾՇՇԼ԰ԽՊԹԪՁԻՊՇՆն 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋ԰ԽՊԹԪՁԻՊՇՆ ն 

# ՌՀՊՇՍԿՀՇՍՌ

ՈՊԽ  ԽՐՈՇՋՍՊԽ
ԹԾՌԽՊԫԣՋՌԹԿԽԹՆԼ ԼՍՊՁՆԿՌՀԽԽՆՌՁՊԽ  ԼԼԽԾՁՆՁՌՁՎԽՋՌՍԼՑԦԾ ԹՆՇՌՀԽՊԼՁԽՌՁՋՍՋԽԼՌՀԽՋՌՍԼՑՊԽՈՇՊՌՋՀՇՍՄԼ ՈՊՇՎՁԼԽԹՆԹՄՑՋՁՋՇԾՁՇԼՁԼԽԻՇՆՌԽՆՌԹՆԼ ՈՇՌԽՆՌՁԹՄԻՇՆՌԹՅՁՆԹՆՌՋԹՆԼՌՀԽԼՁԽՌՋՀՇՍՄԼ ԼԽՅՇՆՋՌՊԹՌԽԽՉՍԹՄՈԽՊԾՇՊՅԹՆԻԽ ՌՇ԰ԽՊԹ

ԪՁԻՊՇՆ ն 

# ԰ՇՍՊԻԽՇԾԾՇՇԼ԰ԽՊԹԡՁՊԽԻՌԪԹՊՌՁՆԽՒԞՍԿՍՋՌԹԤԞԦՇԼՁԼԽԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆԼՁԽՌ
ՁԾՃՆՇՏՆՅԿՃԿԣՊԽՉՍԽՆԻՑՇԾԾԽԽԼՁՆԿՌՁՅԽՋԼԹՑ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾԽԽԼՁՆԿՇԻԻՍՊԹՌ ՄԽԹՋՌՌՏՁԻԽՈԽՊԼԹՑ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌԾՇՇԼՊԹՌՁՇՆՋԼՍՊՁՆԿ

ՌՀԽՈՊԽ  ԽՐՈՇՋՍՊԽՈԽՊՁՇԼԺԽՁՆԻՊԽԹՋԽԼԹՄՇՆԿ ՏՁՌՀՄԹՊՎԹՄԿՊՇՏՌՀՌՇԹՈՈՊՇՐՁՅԹՌԽՄՑ ՅԿՄԹՊՎԹԼԹՑԺՑՌԽՋՌՁՆՁՌՁԹՌՁՇՆԢԭԞԹՆԼ ԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌԾՇՇԼՊԹՌՁՇՆՋ ՁՆԻՊԽԹՋԽԾՊՇՅՅԿՄԹՊՎԹԼԹ ՑԹՌՌԽՋՌ ՁՆՁՌՁԹՌՁՇՆ
԰ՌՍԼՑԡԹՑ ՌՇ ՅԿՄԹՊՎԹԼԹՑՁՆՌՀԽՄԹՋՌՏԽԽՃՇԾՌՀԽՌԽՋՌ ԰ՌՍԼՑԡԹՑ

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# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԹԾՄՇՏ ՌՀՊՇՍԿՀՋՑՋՌԽՅ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ՁԾԹՈՈՄՁԻԹԺՄԽԠՇՆՌՁՆՍՇՍՋԾՄՇՏԼՁՄՍՌԽՊՋՑՋՌԽՅ 

# ԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊՋՇՊ ԻՇՆՌՁՆՍՇՍՋԾՄՇՏ ՋԽՊՁԹՄԼՁՄՍՌԽՊՋԹՊԽ ՊԽԻՇՅՅԽՆԼԽԼ ԣՄՇՏՌՀՊՇՍԿՀՊԹՌԽ
ՁԾԹՈՈՄՁԻԹԺՄԽչՅԩՅՁՆ 

# ԯԽԻՇՅՅԽՆԼԽԼԾՄՇՏՌՀՊՇՍԿՀՊԹՌԽՁՋ ՅԩՅՁՆ
ԻՇՅՈՄԽՌԽՎՇՄՍՅԽՊԽՈՄԹԻԽՅԽՆՌ ԻԹԽՎԽՊՑՀՊՋԡԽՌԹՁՄՋՇՆՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊԾՄՇՏՌՀՊՇՍԿՀՋՑՋՌԽՅՋ
ՁԾԹՈՈՄՁԻԹԺՄԽԞԻՇՆՌՁՆՍՇՍՋԾՄՇՏՋՑՊՁՆԿԽՈՍՅՈՍՋՁՆԿԿԹՋՌՁԿՀՌՋՑՊՁՆԿԽՋԻՇՆՌՁՆՍՇՍՋՄՑԼԽՄՁՎԽՊԽԼԹԻՇՆԻԽՆՌՊԹՌԽԼՌԽՋՌՋՍԺՋՌԹՆԻԽՋՌՇԻՃՋՇՄՍՌՁՇՆԹՌԹԼԽՋՁԿՆԹՌԽԼՊԹՌԽԾՊՇՅԹՋՌՇԻՃՎԽՋՋԽՄՌՇԹՋՌԹՁՆՄԽՋՋՋՌԽԽՄՅՁՐՁՆԿԻՀԹՅԺԽՊՏՀԽՊԽՁՌՏԹՋՅՁՐԽԼՏՁՌՀԼՁՄՍՌՁՇՆՏԹՌԽՊԱՀԽԾՄՇՏՊԹՌԽ
չՅԩՅՁՆՈՊՇՎՁԼԽԼԹԻՇՅՈՄԽՌԽՎՇՄՍՅԽՌՍՊՆՇՎԽՊԹՈՈՊՇՐՁՅԹՌԽՄՑԽՎԽՊՑՀՇՍՊՋԾՇՊԽԹԻՀՊԽՈՄՁԻԹՌԽՌԽՋՌՎԽՋՋԽՄԱՀԽԼՁՄՍՌԽՊՋՑՋՌԽՅՏԹՋԻԹՄՁԺՊԹՌԽԼՈՊՁՇՊՌՇՌԽՋՌՁՆՁՌՁԹՌՁՇՆԡՁՄՍՌԽՊՇՈԽՊԹՌՁՇՆՏԹՋՅՇՆՁՌՇՊԽԼԹՌՄԽԹՋՌՌՏՇՌՁՅԽՋԼԹՁՄՑՌՀՊՇՍԿՀՇՍՌՌՀԽՌԽՋՌԦԾՌՀԽՊԽՏԹՋԹՆՁՆԼՁԻԹՌՁՇՆՌՀԹՌՌՀԽԼՁՄՍՌԽՊԻԹՄՁԺՊԹՌՁՇՆՀԹԼԻՀԹՆԿԽԼ
ԽԿԼՁՄՍՌԽՊՅԹՄԾՍՆԻՌՁՇՆԻՀԹՆԿԽՁՆՏԹՌԽՊՉՍԹՄՁՌՑՇՊ 

# ԯԽԻՇՅՅԽՆԼԽԼՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊԾՄՇՏ ՌՀՊՇՍԿՀՋՑՋՌԽՅՋԪՁՐՁՆԿԻՀԹՅԺԽՊՁՋ ՊԽԻՇՅՅԽՆԼԽԼԺՍՌՆՇՌՊԽՉՍՁՊԽԼ ԞԽՊԹՌՁՇՆՁՋՆՇՌՊԽԻՇՅՅԽՆԼԽԼԾՇՊՅՁՐՁՆԿ ԞԼԽՅՇՆՋՌՊԹՌՁՇՆՌՀԹՌՌՀԽՌԽՋՌՋՇՄՍՌՁՇՆ ՁՋԻՇՅՈՄԽՌԽՄՑՅՁՐԽԼԺԽԾՇՊԽՁՆՌՊՇԼՍԻԽԼ ՁՆՌՇՌՀԽՌԽՋՌՋՑՋՌԽՅՁՋՊԽԻՇՅՅԽՆԼԽԼ ԱՀԽՊԽԻՇՅՅԽՆԼԽԼԾՄՇՏՋՈՄՁՌՌՁՆԿԹԻԻՍՊԹԻՑ ՁՋՏՁՌՀՁՆԞԼԼՁՌՁՇՆԹՄԿՍՁԼԹՆԻԽԾՇՊԹՉՍԹՌՁԻՌԽՋՌԼԽՋՁԿՆՁՋՄՇԻԹՌԽԼՁՆԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ԰ՈԽԻՁԹՄԠՇՆՋՁԼԽՊԹՌՁՇՆՋԾՇՊԠՇՆԼՍԻՌՁՆԿԞՉՍԹՌՁԻԩԹԺՇՊԹՌՇՊՑ԰ՌՍԼՁԽՋ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ՁԾԹՈՈՄՁԻԹԺՄԽԫԞ 

# ԦԾՋՌԹՌՁԻՊԽՆԽՏԹՄՁՋՍՋԽԼԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՀՊՊԽՆԽՏԹՄՊԽՆԽՏԹՄ ՈԽՊՁՇԼՁՋՊԽԻՇՅՅԽՆԼԽԼՆՇՌՌՇԽՐԻԽԽԼ

ՀՇՍՊՋԞԽՊԹՌՁՇՆ!ԫՇ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՅԹՁՆՌԹՁՆՁՆԿԼՁՋՋՇՄՎԽԼ ՇՐՑԿԽՆԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԹՁՊ ՋԹՌՍՊԹՌՁՇՆ ՅԿԩ ԞԽՊԹՌՁՇՆՅԹՑ ԺԽՅԹՁՆՌԹՁՆԽԼՌՀՊՇՍԿՀԺՍԺԺՄԽՊՋԦՌՁՋ ՊԽԻՇՅՅԽՆԼԽԼՌՇՋԽՌԺՍԺԺՄԽՊՋԹՌՄԽՎԽՄՋ ՌՀԹՌԼՇՆՇՌԻԹՍՋԽՋՌՊԽՋՋՇՆՌՀԽՌԹԼՈՇՄԽՋ԰ՇՍՊԻԽՇԾԼՁՄՍՌՁՇՆՏԹՌԽՊԩԹԺՇՊԹՌՇՊՑԼՁՄՍՌՁՇՆՏԹՌԽՊՏԹՋԩԹՃԽԥՍՊՇՆՏԹՌԽՊՋՍՈՈՄՁԽԼՌՇԱՀԽԡՇՏԠՀԽՅՁԻԹՄԠՇՅՈԹՆՑԺՑՌՀԽ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՆԹՌՍՊԹՄՇՊՊԽԻՇՆՋՌՁՌՍՌԽԼ ՏԹՌԽՊՁՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՆԹՌՍՊԹՄ ՏԹՌԽՊԺԽՋՌԽՊՁՄՁՒԽԼՏՁՌՀԲԳԹՆԼՌԽՋՌԽԼԾՇՊ ՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

       !  "        #  $           ԠՁՌՑՇԾԪՁԼՄԹՆԼԴԹՌԽՊԱՊԽԹՌՅԽՆՌԭՄԹՆՌԴԹՌԽՊՏԹՋՇԺՌԹՁՆԽԼԾՊՇՅՌՀԽՍՈՈԽՊ԰ԹԿՁՆԹՏԟԹՑՇԾԩԹՃԽԥՍՊՇՆՇԾԾԴՀՁՌԽՋՌՇՆԽԭՇՁՆՌԹՆԼՏԹՋՄՁՅԽԼԹՆԼԾՄՇԻԻՍՄԹՌԽԼՏՁՌՀԾԽՊՊՁԻԻՀՄՇՊՁԼԽԴԹՌԽՊՏԹՋՈՍՅՈԽԼՌՇՌՀԽՄԹԺՇՊԹՌՇՊՑՈՊՁՇՊՌՇՅՍՆՁԻՁՈԹՄՌՊԽԹՌՅԽՆՌԾՇՊՀՍՅԹՆԻՇՆՋՍՅՈՌՁՇՆԭՊՁՇՊՌՇՍՋԽՁՆՌՀԽՌԽՋՌՏԹՌԽՊՏԹՋՋԹՆԼԾՁՄՌԽՊԽԼՈԥԹԼՂՍՋՌԽԼՏՁՌՀԠԬԻԹՊԺՇՆԾՁՄՌԽՊԽԼԹՆԼԲԳՁՊՊԹԼՁԹՌԽԼ 

# ՈՇՋՋՁԺՄԽԻՇՆՌԹՅՁՆԹՆՌՋՁՆԻՄՍԼՁՆԿՃՆՇՏՆ ՋՍԺՋՌՊԹՌԽՋՇԾՌՀԽՁՇԼՁՆԽՌՊԹՆՋՈՇՊՌԽՊՇԾՌՀԽ ՌՀՑՊՇՁԼԿՄԹՆԼ
Խ Կ 

# ԾՄՍՇՊՁԼԽԻՀՄՇՊԹՌԽ ՈԽՊԻՀՄՇՊԹՌԽԬԢԠԡԹԻԻԽՈՌՋԹՆՑՏԹՌԽՊՁՆ ՏՀՁԻՀՌՀԽՌԽՋՌՋՈԽԻՁԽՋՋՀՇՏԻՇՆՌՊՇՄ ՋՍՊՎՁՎԹՄԹՌՄԽԹՋՌԹՋԿՇՇԼԹՋՁՆԼՁԻԹՌԽԼՁՆ ՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԴԹՋԼՁՄՍՌՁՇՆՏԹՌԽՊԹՆԹՄՑՒԽԼԾՇՊՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊԻՇՆՌԹՅՁՆԹՆՌՋ!ԶԽՋ԰ԽԽԯԽՎՁԽՏԽՊ0ՋԠՇՅՅԽՆՌՋ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԦԾՊԽԻՇՆՋՌՁՌՍՌԽԼՏԹՌԽՊՁՋՍՋԽԼՇՊՁԾ ԺԹԻՃԿՊՇՍՆԼՄԽՎԽՄՋՇԾՁՇԼՁԼԽՁՆՆԹՌՍՊԹՄ ՏԹՌԽՊԹՊԽՄԽՋՋՌՀԹՆսԿԩՁՇԼՁԼԽ ՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՋՊԽԻՇՅՅԽՆԼԽԼԱՀՁՋ ՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՋՁՆԹԼԼՁՌՁՇՆՌՇՌՀԽ ՊԽԻՇՅՅԽՆԼԽԼԼՁԽՌԹՊՑՋՇՍՊԻԽՇԾՁՇԼՁԼԽ ԽԿՁՆ԰ԽՊԹԪՁԻՊՇՆԱԽՋՌՎԽՋՋԽՄՌՑՈԽՅԹՌԽՊՁԹՄՋԱԽՋՌԹՉՍԹՊՁԹՏԽՊԽԻՇՆՋՌՊՍԻՌԽԼՇԾԿՄԹՋՋՋԽԹՄԽԼՌՇԿԽՌՀԽՊՏՁՌՀԻՄԽԹՊՋՁՄՁԻՇՆԽԹԼՀԽՋՁՎԽ 

# ԢԭԞ ԹՆԼ ԬԢԠԡ ՊԽԻՇՅՅԽՆԼ ՌՀԹՌ ՏԹՌԽՊ ԻՇՆՌԹԻՌ ՈՇՊՌՁՇՆՋ ՇԾ ՌՀԽ ՋՑՋՌԽՅ ՆՇՌ
ԻՇՅՈՊՇՅՁՋԽ ՌՀԽ ՋՌՍԼՑ 
ԽԿ 

# ԹՄՄ ԿՄԹՋՋ ՎԽՋՋԽՄՋ ՇՊ ԿՄԹՋՋ ՎԽՋՋԽՄՋ ՏՁՌՀ ՋՌԹՁՆՄԽՋՋ ՋՌԽԽՄԾՊԹՅԽՋԹՊԽԹԻԻԽՈՌԹԺՄԽԽՐԹՅՈՄԽՋԱԽՋՌՎԽՋՋԽՄՋՁՒԽԻՅՐԻՅՐԻՅԼԽԽՈԣՁՄՄՎՇՄՍՅԽԩԴԹՌԽՊԼԽՈՌՀՏԹՋՅԹՁՆՌԹՁՆԽԼԹՌԻՅԺՑԹՋԻՊԽԽՆԻՇՎԽՊԽԼՏՁՌՀԹԼՊԹՁՆ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԢԭԞՊԽԻՇՅՅԽՆԼՋՀԹՊԼՆԽՋՋՌՇ ՅԿԩԹՋԠԹԠԬ ՈԥԴԽԽՃՄՑՁՆԽԹԻՀՊԽՈՄՁԻԹՌԽՌԽՋՌՎԽՋՋԽՄ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՈԥչՁՆՌԽՊ ՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊ  ՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋ ՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼԡՁՋՋՇՄՎԽԼՇՐՑԿԽՆ
ՅԿԩԴԽԽՃՄՑՁՆԽԹԻՀՊԽՈՄՁԻԹՌԽՌԽՋՌՎԽՋՋԽՄ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆ ԡԬ ՅԿԩ
 ԹՁՊՋԹՌՍՊԹՌՁՇՆ ԬԢԠԡՊԽԻՇՅՅԽՆԼՋԡԬԻՇՆԻԽՆՌՊԹՌՁՇՆ
ՅԿԩ ԹՁՊՋԹՌՍՊԹՌՁՇՆ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

                      

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ոԠ ԴԽԽՃՄՑՁՆԽԹԻՀՊԽՈՄՁԻԹՌԽՌԽՋՌՎԽՋՋԽՄԹՆԼԻՇՆՌՁՆՍՇՍՋՄՑՁՆՇՆԽՊԽՈՄՁԻԹՌԽԾՊՇՅՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌԽՅՈԽՊԹՌՍՊԽչ Շ 

# Ԡ

ՁՆՌԽՊ ՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊՌՊԽԹՌՅԽՆՌ ԼՁԾԾԽՊԽՆՌՁԹՄՋՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼ Շ 

# ԠԦՇԼՁԼԽՅԿԩ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՉՍԹՌՁԻՁՇԼՁԼԽՊԹՆԿԽ

 - սԿԩ
ՋՍՈՈՄԽՅԽՆՌԹՄՁՇԼՁԼԽ ՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼսԿԩ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

                      

!     "           ԞՅՅՇՆՁԹՅԿԩ 

# ԤԽՆԽՊԹՄՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋԾՇՊԾՊԽՉՍԽՆԻՑ ՇԾՅԽԹՋՍՊԽՅԽՆՌՋԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌ ՏԹՌԽՊՉՍԹՄՁՌՑՈԹՊԹՅԽՌԽՊՋԺԽՅԽԹՋՍՊԽԼՁՆ ԹԻՇՆՌՊՇՄԹՆԼԹՌՇՆԽՌԽՋՌՁՌԽՅ ԻՇՆԻԽՆՌՊԹՌՁՇՆԹՌՄԽԹՋՌՏԽԽՃՄՑԦՆՋՌԹՌՁԻ ՊԽՆԽՏԹՄՋՑՋՌԽՅՋՏԹՌԽՊՉՍԹՄՁՌՑ ՈԹՊԹՅԽՌԽՊՋՁՆԻՄՍԼՁՆԿԹՅՅՇՆՁԹՋՀՇՍՄԼ ԺԽՅԽԹՋՍՊԽԼՂՍՋՌՈՊՁՇՊՌՇՊԽՆԽՏԹՄԦՆ ԹԼԼՁՌՁՇՆԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԡԬԺԽ ՅԽԹՋՍՊԽԼԹՌԽԹԻՀԻՇՆԻԽՆՌՊԹՌՁՇՆԹՌՄԽԹՋՌ ՏԽԽՃՄՑԹՆԼՌՀԹՌՌԽՅՈԽՊԹՌՍՊԽԺԽ ՅԽԹՋՍՊԽԼԻՇՆՌՁՆՍՇՍՋՄՑ ԬԢԠԡ ՊԽԻՇՅՅԽՆԼՋՌՀԹՌ ԡԬԹՆԼՌԽՅՈԽՊԹՌՍՊԽ ԺԽՅԽԹՋՍՊԽԼԹՌՄԽԹՋՌՏԽԽՃՄՑԹՆԼՌՀԹՌՈԥ ԹՆԼՀԹՊԼՆԽՋՋԺԽՅԽԹՋՍՊԽԼԹՌՄԽԹՋՌԹՌ ՌՀԽԺԽԿՁՆՆՁՆԿԹՆԼԽՆԼՇԾՌՀԽՌԽՋՌԣՄՍՇՊՁԼԽՅԿԩԭԽՊԻՀՄՇՊԹՌԽսԿԩԠՀՄՇՊԹՌԽսԿԩԞՄՃԹՄՁՆՁՌՑ
ՅԿԩԹՋԠԹԠԬԴԽԽՃՄՑՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈԠՇՆԼՍԻՌՁՎՁՌՑ
սՅՀՇՋԻՅ                                                  ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ՌՇՅԿԩ"ոԠ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾ ՌՀԽՌԽՋՌԻՇՅՈՇՍՆԼՅԿԩՇՊ ԼԽՅՇՆՋՌՊԹՌԽՋԹԼԽՉՍԹՌԽԽՎՁԼԽՆԻԽՇԾՌՇՐՁԻՁՌՑ

ԽԿ ՅՇՊՌԹՄՁՌՑՏՀՁԻՀԽՎԽՊ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌ԰ՈԽԻՁԽՋ 

# ԵԽՆՇՈՍՋՄԹԽՎՁՋԩՁԾԽՋՌԹԿԽԫՇՌՋՈԽԻՁԾՁԽԼԱԽՋՌԼՍՊԹՌՁՇՆԼԹՑՋ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ՆԽԿԹՌՁՎԽԹՆԼՋՇՄՎԽՆՌԻՇՆՌՊՇՄՋԹՆԼՅԿԹՁԩԹՆԼՌՀԽՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼՍՆԼԽՊԾՄՇՏՌՀՊՇՍԿՀԻՇՆԼՁՌՁՇՆՋԞԾՌԽՊԼԹՑՋՇԾԽՐՈՇՋՍՊԽՌՀԽՊԽՏԹՋԹՋՁՆԿՄԽՅՇՊՌԹՄՁՌՑՁՆՌՀԽՋՇՄՎԽՆՌԻՇՆՌՊՇՄՆՇՇՌՀԽՊՅՇՊՌԹՄՁՌՁԽՋՏԽՊԽՇԺՋԽՊՎԽԼԣՍՊՌՀԽՊՌԹԼՈՇՄԽՋԹՌՌՀԽՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՀԹԼՋՁԿՆՁԾՁԻԹՆՌՄՑՄՇՏԽՊԺՇԼՑՏԽՁԿՀՌՊԽՄԹՌՁՎԽՌՇՌՀԽՈՇՇՄԽԼԻՇՆՌՊՇՄ

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# ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՌՀԽ ԼՍՊԹՌՁՇՆ ՇԾ ՌՀԽ ԼԽԾՁՆՁՌՁՎԽՌԽՋՌԺԽԼԹՑՋ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾ ՌՀԽՌԽՋՌԻՇՅՈՇՍՆԼՅԿԩՇՊ ԼԽՅՇՆՋՌՊԹՌԽՋԹԼԽՉՍԹՌԽԽՎՁԼԽՆԻԽՇԾՌՇՐՁԻՁՌՑ

ԽԿ ՅՇՊՌԹՄՁՌՑՏՀՁԻՀԽՎԽՊ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌԯԽԾԽՊԽՆԻԽՋՌՍԼՑԻՁՌԹՌՁՇՆ
ՁԾԹՈՈՄՁԻԹԺՄԽԫԞ԰ԽՈԹՊԹՌՁՇՆՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ 

# ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՌՀԽ ՅԹՐՁՅՍՅ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՋԽՈԹՊԹՌՁՇՆ ԺԽ  ԹՆԼ ՌՀԽ ՅՁՆՁՅՍՅԺԽԫՍՅԺԽՊՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՇԾ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԹՆԼԹԻՇՆՌՊՇՄՈՄՍՋՋՇՄՎԽՆՌ ԻՇՆՌՊՇՄՁԾԹՈՈՊՇՈՊՁԹՌԽԞՊԽՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋԹԼՂՍՋՌԽԼԾՇՊՈՍՊՁՌՑ!ԶԽՋ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ԩԬԮՅԿԹՁԩ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՇՊԻՀԽՅՁԻԹՄՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽՄՇՏՌՀԽԩԬԮԹՆԹՄՑՋԽՋԺԽ ԻՇՆԼՍԻՌԽԼՇՆՌՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋԩԽՎԽՄՇԾԼԽՌԽԻՌՁՇՆ
ԩԬԡԫՇՌՊԽՈՇՊՌԽԼԣՊԽՉՍԽՆԻՑՇԾՅԽԹՋՍՊԽՅԽՆՌ԰ԹՅՈՄԽՋՏԽՊԽԹՆԹՄՑՒԽԼՇՆԡԹՑՋԹՆԼ 

# ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՌԽՋՌՁՌԽՅ ԻՇՆԻԽՆՌՊԹՌՁՇՆԺԽՅԽԹՋՍՊԽԼՁՆՇՆԽՌԹՆՃԹՌ ԽԹԻՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼ ԽՎԽՊՑՏԽԽՃՌՀԽՊԽԹԾՌԽՊԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆԻՇՆՌՊՇՄ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆՋՇՄՎԽՆՌԻՇՆՌՊՇՄ
ՁԾԹՈՈՄՁԻԹԺՄԽ 

# ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԽՍՋԽՇԾԹ ԻՇՆԻՍՊՊԽՆՌՋՇՄՎԽՆՌԻՇՆՌՊՇՄՏՀԽՆԹ ՋՇՄՍԺՁՄՁՒՁՆԿԹԿԽՆՌՁՋՍՋԽԼ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋԫՍՅԺԽՊՇԾՄԹՊՎԹԽՈԽՊՌՊԽԹՌՅԽՆՌԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԴԹՋԹՋՇՄՎԽՆՌՍՋԽԼ!ԶԽՋ԰ՇՄՎԽՆՌՌՑՈԽ
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# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՋՇՄՎԽՆՌՆՇՌ ԽՐԻԽԽԼՅՄԩ 

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# ԪԽԹՆՋԹՆԼՋՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆՋԾՇՊԥԩԩՏԽՊԽԺԹՋԽԼՇՆՊԽՎՁԽՏԽՊ0Ջ ԻԹՄԻՍՄԹՌՁՇՆՋՇԾՊԹՏԼԹՌԹ

 

#  ԰ՍՅՅԹՊՑՊԽՋՍՄՌՋԾՇՊՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ
԰ԳԩԹՊԽՈՊԽՋԽՆՌԽԼՁՆՌ ՀԽՆԽՐՌՌԹԺՄԽ  

 

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ԡԹՑ  ԺՇԼՑ ՏԽՁԿՀՌ ՊԹՆԿԽԼ ԾՊՇՅ  Կ ՁՆ ՌՀԽ ՀՁԿՀԽՋՌ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ՌՇ  ՁՆ ՌՀԽ ՅՁԼԼՄԽ ՌՊԽԹՌՅԽՆՌԿՊՇՍՈ  

 

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ԱԴԞՅԿԹՁ ԩՏԹՋ ՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼՊԽՄԹՌՁՎԽՌՇՌՀԽՈՇՇՄԽԼԻՇՆՌՊՇՄ
ԼԽԻՊԽ ԹՋԽ 

# Ո ԰ՁՅՁՄԹՊՄՑԡԹՑ԰Գԩ
ՊԹՆԿԽԼԾՊՇՅՅՅՁՆՌՀԽՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՌՇՅՅՁ ՆՌՀԽՅՁԼԼՄԽՌՊԽԹՌՅԽՆՌԿՊՇՍՈԞ ՋՁԿՆՁԾՁԻԹՆՌԼՁԾԾԽՊԽՆԻԽՁՆ԰ԳԩՏԹՋԼԽՌԽԻՌԽԼԺԽՌՏԽԽՆՌՀԽՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ
ՅՅԹՆԼՋՇՄՎԽՆՌԻՇՆՌՊՇՄ ՅՅՌՀԽՊԽԾՇՊԽՌՊԽԹՌՅԽՆՌԼԹՌԹՏԽՊԽԻՇՅՈԹՊԽԼՌՇՌՀԽՋՇՄՎԽ ՆՌԻՇՆՌՊՇՄՇՆՄՑ԰ԳԩԹՌՌՀԽՀՁԿՀԽՋՌ ՌՊԽԹՌՅԽՆՌԿՊՇՍՈՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼ
ԼԽԻՊԽԹՋԽ 

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# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

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# ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞ 

# ԫՇՌԹՈՈՄՁԻԹԺՄԽ ԫԡ 

# ԫՇՌԼԽՌԽՊՅՁՆԽԼ ԰ԡ 

# ԰ՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ



#  ԞՄՋՇՊԽԾԽՊՊԽԼՌՇԹՋ՟ ԺՇԼՑՏԽՁԿՀՌ ՠՁՆՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽ

ԞԾՌԽՊԼԹՑՋՇԾԽՐՈՇՋՍՊԽՆՇՌՀՑՊՇՁԼԹՌՊՇՈՀՑՇՊՀՑՈԽՊՌՊՇՈՀՑՏԹՋԽՐԹՅՁՆԽԼՁՆԹՆՑԻՇՆՌՊՇՄՇՊՌՊԽԹՌՅԽՆՌ ՌԹԼՈՇՄԽՋԱՀԽՊԽՏԹՋԹՋՁՆԿՄԽՁՆԻՁԼԽՆԻԽՇԾԾՇՄՄՁԻՍՄԹՊԻԽՄՄՀՑՈԽՊ ՈՄԹՋՁԹՁՆՌՀԽՋՇՄՎԽՆՌԻՇՆՌՊՇՄԹՆԼԱԴԞ ՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈԪՁՄԼԾՇՄՄՁԻՍՄԹՊԻԽՄՄՀՑՈԽՊՌՊՇ ՈՀՑՏԹՋՇԺՋԽՊՎԽԼՁՆԺՇՌՀԻՇՆՌՊՇՄՋԹՆԼ
ԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏՁՌՀԺԽՌՏԽԽՆԹՆԼՌԹԼՈՇՄԽՋԽՐՀՁԺՁՌՁՆԿՌ ՀՁՋԽԾԾԽԻՌՏՀՁԻՀՏԹՋՁՆՌԽՊՈՊԽՌԽԼՌՇԺԽ ՏՁՌՀՁՆՆՇՊՅԹՄՄՁՅՁՌՋԱՀԽՊԽՏԹՋՆՇԽՎՁԼԽՆԻԽՇԾԹՆՑՁՆԾՄԹՅՅԹՌՇՊՑ ՇՊԼԽԿԽՆԽՊԹՌՁՎԽԻՀԹՆԿԽՋՁՆՌՀԽՌՀՑՊՇՁԼ
ԿՄԹՆԼՋԽՐԹՅՁՆԽԼՁՆԺՇՌՀԻՇՆՌՊՇՄՋԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԞՄՄ ՇՌՀԽՊՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄԻՊՁՌԽՊՁԹՋՍԻՀԹՋ ՌՀԽՇՎԽՊԹՄՄՋՁՒԽՇԾՌՀԽԿՄԹՆԼՌՀԽԾՇՄՄՁԻՍՄԹՊՄՍՅԽՆԹՊԽԹԹՅՇՍՆՌԹՆԼՌՑՈԽՇԾԻՇՄՄՇՁԼԹՆԼՌՀԽԾՇՄՄՁԻՍՄԹՊ ԻԽՄՄՌՑՈԽԹՆԼԹՊՊԹՆԿԽՅԽՆՌՇԾԹՄՄՌԹԼՈՇՄԽՋՁՆՌՀԽՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԽՊԽԻՇՅՈԹՊԹԺՄԽՌՇՌՀՇՋԽՇԾՌՀԽ

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- ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑԫՇՌՊԽՅԹՊՃԹԺՄԽԪՁՄԼ
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# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

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ՋԹՅԽ ՎԽՋՋԽՄ ՇՆԽՌԹԼՈՇՄԽՏԹՋ ՇԺՋԽՊՎԽԼՏՁՌՀ ԹԺՆՇՊՅԹՄ ՋՍՋՈԽՆՋՁՇՆ ԹՆԼ ԹՈՈԽԹՊԽԼՌՇ ՀԹՎԽ ՁՆՌԽՊՆԹՄ ԺՄԽԽԼՁՆԿՇՆԡԹՑՋԹՆԼԹՆԼՏԹՋՇԺՋԽՊՎԽԼՊԽՋՌՁՆԿՇՆՌՀԽՋՍՊ ԾԹԻԽՇՆԡԹՑԱՀԽՋԽՇԺՋԽՊՎԹՌՁՇՆՋ
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# ԪԽԼՁԹՆ ՎԹՄՍԽՋ ՏԽՊԽ ՁՆՋՈԽԻՌԽԼ ԾՇՊ ԽԹԻՀ ՅԽԹՋՍՊԽԼ ԽՆԼՈՇՁՆՌ ԺՑ ՌՊԽԹ ՌՅԽՆՌ ՄԽՎԽՄ ՌՇ ԼԽՌԽՊՅՁՆԽ ՁԾ ՌՀԽ ՊԽՋՈՇՆՋԽՏԹՋՅՇՆՇՌՇՆՁԻՏՁՌՀ ՁՆԻՊԽԹՋՁՆԿ ԻՇՆԻԽՆՌՊԹՌՁՇՆ԰ՌԹՌՁՋՌՁԻ ԹՄ ԹՆԹՄՑՋԽՋ ՇԾ ԻՇՆՌՁՆՍՇՍՋ ԼԹՌԹ ՋԽՌ ՁՆԻՄՍԼՁՆԿ ՆՇՊՅԹՄՁՒԽԼ ԥԩԩ ԺՇԼՑ ՏԽՁԿՀՌ ԹՆԼ ԰Գԩ ՁԾ ՌՀԽ ԼԹՌԹ ՏԹՋ ԻՇՆՋՁՋՌԽՆՌ ՏՁՌՀ Թ ՅՇՆՇՌՇՆԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆՊԽՋՈՇՆՋԽ ԦԾ ՌՀԽՋԽ ԽՆԼՈՇՁՆՌՋ ՏԽՊԽ ՆՇՌ ԻՇՆՋՁՋՌԽՆՌ ՏՁՌՀ Թ ՅՇՆՇՌՇՆՁԻ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՊԽՋՈՇՆՋԽԼԹՌԹՏԽՊԽԹՋՋԽՋՋԽԼԾՇՊՆՇՊՅԹՄՁՌՑՍՋՁՆԿՌՀԽ԰ՀԹՈՁՊՇ ԴՁՄՃ0ՋՌԽՋՌԹՆԼՀՇՅՇԿԽՆԽՁՌՑՇԾՎԹՊՁԹՆԻԽ
ՍՋՁՆԿՌՀԽԩԽՎԽՆԽՌԽՋՌԴՀԽՊԽՆՇՆՆՇՊՅԹՄՁՌՑՇՊՀԽՌԽՊՇՋՃԽԼԹՋՌՁԻՁՌ ՑՏԽՊԽՇԺՋԽՊՎԽԼՆՇՊՅԹՄՁՒՁՆԿԹՆԼՇՊ ՎԹՊՁԹՆԻԽՋՌԹԺՁՄՁՒՁՆԿՌՊԹՆՋԾՇՊՅԹՌՁՇՆՋՏԽՊԽԹՈՈՄՁԽԼԦԾԼԹՌԹՏԽՊ ԽՆՇՊՅԹՄՄՑԼՁՋՌՊՁԺՍՌԽԼՏՁՌՀՀԽՌԽՊՇԿԽՆԽՇՍՋ ՎԹՊՁԹՆԻԽՌՀԽԪԹՆՆԴՀՁՌՆԽՑԴՁՄԻՇՐՇՆԲՌԽՋՌՏԹՋՍՋԽԼԦԾՆՇՆՇՊՅ ԹՄՁՒՁՆԿՌՊԹՆՋԾՇՊՅԹՌՁՇՆՏԹՋԾՇՍՆԼ
ՌՀԽԪԹՆՆԴՀՁՌՆԽՑԴՁՄԻՇՐՇՆԲՌԽՋՌ ՍՋՁՆԿԹԟՇՆԾԽՊՊՇՆՁԥՇՄԼԹԼՂՍՋՌ ՅԽՆՌՏԹՋՍՋԽԼԩԽՆԿՌՀԹՆԼՏԽՁԿՀՌ    

       



   



    

    







 





    





 

   



# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ 

 

# ՇԾ 



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ԼԹՌԹԾՇՊՌԹԼՈՇՄԽՋՊԽԹԻՀՁՆԿՋՌԹԿԽՋԿՊԽԹՌԽՊՌՀԹՆԫԣՏԽՊԽԽՐԻՄՍ ԼԽԼԾՊՇՅՋՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋԽՋԼՍԽՌՇ ԼՊԹՋՌՁԻՅՇՊՈՀՇՄՇԿՁԻԹՄԻՀԹՆԿԽՋԹՌՌՀՁՋՋՌԹԿԽԹՆԼԹԺՇՎԽԞՋՁԿՆՁԾ ՁԻԹՆՌՌՊԽԹՌՅԽՆՌԽԾԾԽԻՌԾՇՊԼԽՎԽՄՇՈՅԽՆՌԹՄ ՋՌԹԿԽՏԹՋԼԽՌԽՊՅՁՆԽԼԾՊՇՅՌՀԽՋՌԽՈԼՇՏՆԹՈՈՄՁԻԹՌՁՇՆՇԾՌՀԽՅՍՄՌ ՁՉՍԹՆՌԹՄԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌԽՋՌ

ԾՊՇՅՌՀԽ ՌՀ 

# ՌՇՌՀԽ ՌՀ 

# ՈԽՊԻԽՆՌՁՄԽ

ԡԹՑ ԡԹՑ ԺՇԼՑՏԽՁԿՀՌ ԹՆԼ԰ԳԩՏԽՊԽ ՆՇՌՅՇՆՇՌՇՆՁԻ ԺՍՌՏԽՊԽ ՆՇՊՅԹՄՄՑ ԼՁՋՌՊՁԺՍՌԽԼՏՁՌՀ ՀՇՅՇԿԽՆԽՇՍՋ

ՎԹՊՁԹՆ ԻԽՋԹՆԼՏԽՊԽՌՀԽՊԽԾՇՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԡՍՆՆԽՌՌ0ՋՌԽՋՌ ԟՇԼՑՏԽՁԿՀՌՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՁՆՀՁԺՁՌԽԼԹՌ
ՌՀԽԱԴԞՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՊԽՄԹՌՁՎԽՌՇՌՀԽՈՇՇՄԽԼ ԻՇՆՌՊՇՄՏՀԽՊԽԹՋ԰ԳԩՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑ
ՊԽԼՍԻԽԼԹՌՌՀԽՋԹՅԽՌՊԽԹՌՅԽՆՌԿՊՇՍՈՊԽՄԹՌՁՎԽՌՇՌՀԽՋՇՄՎԽՆՌԻՇՆ ՌՊՇՄԟՇՌՀԫԣՋՌԹԿԽԹՆԼՆՇՊՅԹՄՁՒԽԼ ԥԩԩՏԽՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌԽՋՌՆՇԼՁԾԾԽՊԽՆԻԽՋ ՏԽՊԽԾՇՍՆԼԺԽՌՏԽԽՆՌՀԽՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈՋԹՆԼՌՀԽՈՇՇՄԽԼԻՇՆՌՊՇՄԾՇՊԽՁՌՀԽՊԽՆԼՈՇՁՆՌ

ԡԹՑ ԡԹՑԺՇԼՑՏԽՁԿՀՌԹՆԼ԰ԳԩՏԽՊԽՆՇՌՅՇՆՇՌՇՆՁԻԹՆԼԺՇԼՑՏԽՁԿՀՌԾ ՊՇՅՌՀԽՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԹՋ ԻՇՅՈԹՊԽԼՌՇՌՀԽՋՇՄՎԽՆՌԻՇՆՌՊՇՄՍՋՁՆԿԡՍՆՆԽՌՌ0ՋՌԽՋՌ ԠՇՆՎԽՊՋԽՄՑ԰ԳԩՁՆՌՀԽՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԹՋ
ԻՇՅՈԹՊԽԼՌՇՌՀԽՈՇՇՄԽԼԻՇՆՌՊՇՄՍՋՁՆԿՌՀԽԪԹՆՆԴՀՁՌՆԽՑԴՁՄԻՇՐՇՆ ԲՌԽՋՌՏՁՌՀՌՀԽԟՇՆԾԽՊՊՇՆՁԥՇՄՅ ԹԼՂՍՋՌՅԽՆՌԟՇՌՀԿՊՇՏՌՀՈԹՊԹՅԽՌԽՊՋՏԽՊԽՋՁԿՆՁԾՁԻԹՆՌՄՑՁՆՀՁԺՁՌԽԼ ԹՌՌՀԽԱԴԞՅԿԹՁԩՌՊԽԹՌՅԽՆՌ

ԿՊՇՍՈ ԫՇՊՅԹՄՁՒԽԼԥԩԩԾՊՇՅՌՀԽՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԹՋԻՇՅՈԹՊԽԼՌՇՌՀԽՋՇ ՄՎԽՆՌԻՇՆՌՊՇՄՍՋՁՆԿԡՍՆՆԽՌՌ0Ջ
ՌԽՋՌՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՁՆՀՁԺՁՌԽԼԹՌՌՀԽԱԴԞՅԿԹՁԩՌՊԽ ԹՌՅԽՆՌԿՊՇՍՈԠՇՆՎԽՊՋԽՄՑԫԣՋՌԹԿԽ
ՏԹՋ ԻՇՅՈԹՊԹԺՄԽ ԺԽՌՏԽԽՆՌՀԽՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈՋ ԹՆԼ ՌՀԽ ՈՇՇՄԽԼ ԻՇՆՌՊՇՄ ԺԹՋԽԼ ՇՆ ՌՀԽՅՍՄՌՁՉՍԹՆՌԹՄ
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# ԰ՍՊՎՁՎԹՄՏԹՋՁՆՌՀԽԻՇՆՌՊՇՄՋԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՈՊԽ ԻՄՍԼՁՆԿՌՀԽԹՆԹՄՑՋՁՋ
ՇԾՅՇՊՌԹՄՁՌՑԼԹՌԹԯԽՈՄՁԻԹՌԽՅԽԼՁԹՆՎԹՄՍԽՋԾՇՊԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽ ՇՆԡԹՑԼՁԼՆՇՌԼԽՈԹՊՌԾՊՇՅ ՅՇՆՇՌՇՆՁԻՁՌՑ ԹՆԼ ՌՀԽՊԽԾՇՊԽ ՏԽՊԽ ԹՆԹՄՑՒԽԼ ՏՁՌՀ ՌՀԽ ՇՆԽՋՁԼԽԼ ԧՇՆ ԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ ՌԽՋՌ  ԡԹՑ  ՊԽՈՄՁԻԹՌԽՅԽԼՁԹՆՋԼՁԼՆՇՌԽՐՀՁԺՁՌԹՅՇՆՇՌՇՆՁԻՊԽՋՈՇՆՋԽՌՀԽՊԽԾՇՊԽՏԽՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽՌՏՇՋՁԼԽԼ    

       



   



    

    







 





    





 

   



# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ   

# ՇԾ 



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ԪԹՆՆԴՀՁՌՆԽՑԲՌԽՋՌԡԹՑ԰ԳԩՏԹՋԹԼԼՁՌՁՇՆԹՄՄՑՌԽՋՌԽԼՍՋՁՆԿՌ ՀՁՋՅԽՌՀՇԼԺԽԻԹՍՋԽՌՀԽԼԹՌԹԼՁԼՆՇՌ ՋԹՌՁՋԾՑՌՀԽԹՋՋՍՅՈՌՁՇՆՋՇԾՆՇՊՅԹՄՁՌՑԹՆԼՀՇՅՇԿԽՆԽՁՌՑՇԾՎԹՊՁԹՆԻ ԽՋԫՇՊՅԹՄՁՒԽԼԥԩԩՇՆԺՇՌՀԡԹՑՋ
ԹՆԼԼՁԼՆՇՌԽՐՀՁԺՁՌԹՅՇՆՇՌՇՆՁԻՊԽՋՈՇՆՋԽԺՍՌՌՀԽՅԽՌՌՀԽԹՋ ՋՍՅՈՌՁՇՆՋՇԾՈԹՊԹՅԽՌՊՁԻՋՌԹՌՁՋՌՁԻՋԹՆԼ ՏԹՋՌՀԽՊԽԾՇՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԞԫԬԳԞԾՇՄՄՇՏԽԼԺՑԹԡՍՆՆԽՌՌ0ՋՅՍՄՌ ՁՈՄԽԻՇՅՈԹՊՁՋՇՆՌԽՋՌ԰ՁՅՁՄԹՊՄՑԹՄՄ ՌՀՊԽԽ ԿՊՇՏՌՀ ՈԹՊԹՅԽՌԽՊՋ 
ՁԽ ԺՇԼՑ ՏԽՁԿՀՌ ԰Գԩ ԥԩԩ ՏԽՊԽ ՆՇՌ ՅՇՆՇՌՇՆՁԻ ԺՍՌ ՏԽՊԽ ՆՇՊՅԹՄՄՑ ԼՁՋՌՊՁԺՍՌԽԼՏՁՌՀՀՇՅՇԿԽՆՇՍՋՎԹՊՁԹՆԻԽՋԹՆԼՏԽՊԽՌՀԽՊԽԾՇՊԽԹՆԹՄՑՒ ԽԼՏՁՌՀԞԫԬԳԞԾՇՄՄՇՏԽԼԺՑԡՍՆՆԽՌՌ0 Ջ ՅՍՄՌՁՈՄԽԻՇՅՈԹՊՁՋՇՆՌԽՋՌԫԽԿԹՌՁՎԽԹՆԼՋՇՄՎԽՆՌԻՇՆՌՊՇՄԼԹՌԹՏԽՊ ԽԻՇՅՈԹՊԽԼՍՋՁՆԿԹՌՏՇ ՋՁԼԽԼ԰ՌՍԼԽՆՌ0Ջ
ՌՌԽՋՌԹՋՋՍՅՁՆԿՍՆԽՉՍԹՄՎԹՊՁԹՆԻԽՋՎՁԹԪՁԻՊՇՋՇԾՌԢՐԻԽՄԱԹԼՈՇՄԽՋ ԹՌԫԣՋՌԹԿԽ ՏԽՊԽԽՐԻՄՍԼԽԼԾՊՇՅ ՋՌԹՌՁՋՌՁԻԹՄ ԹՆԹՄՑՋՁՋ ՇԾ  ԼԹՑ ԿՊՇՏՌՀ ԹՆԼ ՆՇՊՅԹՄՁՒԽԼ ԥԩԩ ՈԹՊԹՅԽ ՌԽՊՋ ՆԹՅԽՄՑ ՇՆԽ ՌԹԼՈՇՄԽ ՁՆ ՌՀԽ ՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԾՇՍՊՁՆՌՀԽՋՇՄՎԽՆՌԻՇՆՌՊՇՄՋԽՎԽՆԹՆԼՆՁՆԽՁ ՆՌՀԽՄՇՏԹՆԼՅՁԼԼՄԽԻՇՆԻԽՆՌՊԹՌՁՇՆՋԹՆԼ
ՆՇՌԹԼՈՇՄԽՋԹՌՌՀԽՀՁԿՀԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆ

ԫՇԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌՏԹՋՇԺՋԽՊՎԽԼԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼ ՁԻԹՌԽԼԽԾԾԽԻՌՋՏԽՊԽԻՇՆՋՁԼԽՊԽԼ ՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌ 

# Ո 

!  



 

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# ԡԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽՏԹՋԻՇՅՈԹՊԹԺՄԽԺԽՌՏԽԽՆԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԹՆԼՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՇՆԡԹՑՋ ԹՆԼ 

# Ո ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹԹՆԼԪԹՆՆԴՀՁՌՆԽՑԲՌԽՋՌՊԽՋՈԽԻՌՁՎԽՄ ՑԫՇՊՅԹՄՁՒԽԼԥԩԩ ՏԹՋ ՆՇՌ ԹԾԾԽԻՌԽԼ ՇՆ ԡԹՑ  
Ո  ՊԽՄԹՌՁՎԽ ՌՇ ՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆ ՌՊՇՄ ԺՍՌ ՏԹՋ ԹԾԾԽԻՌԽԼ ԹՌ ԽՎԽՊՑ ՌՊԽԹՌՅԽՆՌՄԽՎԽՄՇՆԡԹՑ
ՈԡՍՆՆԽՌՌ0ՋՌԽՋՌ ԱՀԽԼՁՊԽԻՌՁՇՆՇԾԽԾԾԽԻՌՋՏՁՌԻՀԽԼԺԽՌՏԽԽՆՌՀԽ ՅՁԼԼՄԽԹՆԼՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋԫՇՊՅԹՄՁՒԽԼԥԩԩՏԹՋՋՁԿՆՁԾ ՁԻԹՆՌՄՑՈՊՇՅՇՌԽԼԹՌՌՀԽՄՇՏ ԹՆԼՅՁԼԼՄԽԻՇՆԻԽՆՌՊԹՌՁՇՆԹՆԼՋՁԿՆՁԾՁԻԹՆՌՄՑ
ՈՊԽԼՍԻԽԼ ՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՌ ՌՀԽՀՁԿՀԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆԱՀԽԼՁՊԽԻՌՁՇՆՇԾԽԾԾԽԻՌՁՆՌՀԽՋՇՄՎԽ ՆՌԻՇՆՌՊՇՄՏԹՋՌՀԽՋԹՅԽԹՋՌՀԹՌԹՌՌՀԽ
ՄՇՏԽՋՌՌՏՇՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋԺՍՌՇՈՈՇՋՁՌԽՌՀԹՌԹՌՌՀԽՀՁԿՀԽՋՌՌ ՊԽԹՌՅԽՆՌՄԽՎԽՄ

ԬՆԡԹՑ  ՋՁԿՆՁԾՁԻԹՆՌ ՁՆՀՁԺՁՌՁՇՆՏԹՋ ԼԽՌԽԻՌԽԼԾՇՊ ԺՇԼՑՏԽՁԿՀՌ  ՊԽԼՍԻՌՁՇՆ 

# Ո  ԹՌՌՀԽ
ՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԡԹՑ ԰ԳԩՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՁՆԻՊԽԹՋԽԼԹՌՌՀԽ ՅՁԼԼՄԽԻՇՆԻԽՆՌՊԹՌՁՇՆ
ՁՆԻՊԽԹՋԽ 

# Ո ԹՆԼՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼԹՌՌՀԽՀՁԿՀԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇ Ն ՊԽԼՍԻՌՁՇՆ 

# Ո ԟՇԼՑՏԽՁԿՀՌԹՆԼ԰ԳԩՇՆԡԹՑՏԽՊԽԺՇՌՀՍՆԹԾԾԽԻՌԽԼՁ ՆՌՀԽՌՊԽԹՌՅԽՆՌ    

       



   



    

    







 





    





 

   



# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ   

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# Ո ԤՁՎԽՆՌՀԽՋՁԿՆՁԾՁԻԹՆՌԽԾԾԽԻՌ
ՈԹՌԹՄՄՌՊԽԹՌՅԽՆՌ
ՄԽՎԽՄՋԾՇՊԡԹՑՆՇՊՅԹՄՁՒԽԼԥԩԩՏՀՁԻՀԻԹՆՆՇՌԺԽԽՐՈՄԹՁՆԽԼԻՇՅ ՈՄԽՌԽՄՑԺՑՋՇՄՎԽՆՌՁՆՌԽՊԾԽՊԽՆԻԽՌՀԽՊԽ ՁՋԽՎՁԼԽՆԻԽՌՇՋՍԿԿԽՋՌՌՀԹՌՌՊՁԾՄՍՊԹՄՁՆՅԹՑԼԽՎԽՄՇՈՅԽՆՌԹՄԽՆԼՈՇ ՁՆՌՋՁՆՌԹԼՈՇՄԽՋԽՐՈՇՋԽԼՌՇՌՀԽՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄՋԹՆԼԻՇՆԼՁՌՁՇՆՋՇԾՌՀՁՋՋՌՍԼՑ

ԱՀԽՊԽՋՍՄՌՋՇԾՌՀՁՋԼԹՑՋՌՍԼՑԹՄՋՇՋՀՇՏՌՀԹՌԹՉՍԹՌՁԻԽՐՈՇՋՍՊ ԽՌՇՌՊՁԾՄՍՊԹՄՁՆԹՌՄԽՎԽՄՋԹՈՈՊՇԹԻՀՁՆԿՁՌՋ ՄՁՅՁՌՇԾՋՇՄՍԺՁՄՁՌՑՁՆՏԹՌԽՊՁՋԹՋՋՇԻՁԹՌԽԼՏՁՌՀՊԽԼՍԻԽԼԿՊՇՏՌՀՇԾ 

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# Ո ԥՁՆԼՄՁՅԺՄԽՆԿՌՀՁՋՆՇՊՅԹՄՁՒԽԼՌՇՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ
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# ՈՎԹՄՍԽԾՊՇՅԹՌՏՇՋՁԼԽԼՌՌԽՋՌԹՋՋՍՅՁՆԿՍՆԽՉՍԹՄՎԹՊՁԹՆԻԽ                                                 ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ԢԾԾԽԻՌՏԹՋՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌՏՀԽՆՌՊԽԹՌՅԽՆՌՋՏԽՊԽԻՇՅՈԹՊԽԼՌՇՌՀԽՋՇՄՎԽՆՌԻՇՆՌՊՇՄ
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# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

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# Ո ՎԹՄՍԽԾՊՇՅԹՌՏՇՋՁԼԽԼՌՌԽՋՌԹՋՋՍՅՁՆԿՍՆԽՉՍԹՄՎԹՊՁԹՆԻԽ

 

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ԻՊՁՌԽՊՁՇՆՎԹՄՍԽՇԾ 5 

# ՁՌԼՁԼՆՇՌՁՅՈԹԻՌՌՀԽՁՆՌԽՊՈՊԽՌԹՌՁՇՆՇԾՌՀՁՋՋՌՍԼՑ

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ՌՀԽԢՐԽԻՍՌՁՎԽ԰ՍՅՅԹՊՑԹՆԼԠՇՆԻՄՍՋՁՇՆՋՋԽԻՌՁՇՆՋՇԾՌՀՁՋԡԢԯ    

       



   



    

    







 





    





 

   



# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ   

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# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ԰ՆՇՍՌՎԽՆՌՄԽՆԿՌՀՇՆԡԹՑՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՁՆԻՊԽԹՋԽԼՁՆՌՀԽՋ ՇՄՎԽՆՌԻՇՆՌՊՇՄՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ
ՁՆԻՊԽԹՋԽ 

# Ո ՆՇՇՌՀԽՊԼՁԾԾԽՊԽՆԻԽՋՏԽՊԽԼԽՌԽԻՌԽԼԹՌ ԺԽՌՏԽԽՆՌՀԽՌՏՇ

ԻՇՆՌՊՇՄՋ

ԱՀԽ ՊԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼ ՅԽԹՆՋ ԼՁԾԾԽՊԽԼ ՋՄՁԿՀՌՄՑ ԾՊՇՅ ՌՀՇՋԽ ՊԽՈՇ ՊՌԽԼ ԺՑ ՌՀԽ ՋՌՍԼՑ ԹՍՌՀՇՊՋ ԱՀԽՋԽ ԼՁԾԾԽՊԽՆԻԽՋԹՊԽՄՁՃԽՄՑՌՀԽԻՇՅԺՁՆԽԼՊԽՋՍՄՌՇԾՊՇՍՆԼՁՆԿԽՊՊՇՊՋԹ ՆԼՌՀԽԽՐԻՄՍՋՁՇՆՇԾՌԹԼՈՇՄԽՋԹՌՋՌԹԿԽՋ ԫԣԾՊՇՅԹՆԹՄՑՋՁՋԱՀՇՍԿՀՌՀԽՊԽՏԽՊԽԼՁԾԾԽՊԽՆԻԽՋՌՀԽՑՏԽՊԽՅ ՁՆՇՊ

ԱՀԽՊԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼՌՀԽԱԴԞԻՇՆԻԽՆՌՊԹՌՁՇՆՋՍՋՁՆԿՌՀԽԾՇՄՄՇՏ ՁՆԿԽՉՍԹՌՁՇՆ

ՏՀԽՊԽ ԠԱԴԞՁՋՌՀԽՌՁՅԽՏԽՁԿՀՌԽԼԹՎԽՊԹԿԽԻՇՆԻԽՆՌՊԹՌՁՇՆ ԠՂՁՋՌՀԽԻՇՆԻԽՆՌՊԹՌՁՇՆՅԽԹՋՍՊԽԼԹՌՌՁՅԽՁՆՌԽՊՎԹՄՂ
Ղ Ն ՌՂՁՋՌՀԽՆՍՅԺԽՊՇԾՀՇՍՊՋ
ՇՊԼԹՑՋՇՊՏԽԽՃՋՍՆՁՌՋՍՋԽԼՂՍՋՌ ՆԽԽԼՌՇԺԽԻՇՆՋՁՋՌԽՆՌՁՆՌՀԽԽՉՍԹՌՁՇՆ
ՇԾՌՀԽՌԽՋՌԹՌՌՁՅԽՁՆՌԽՊՎԹՄՂ ԽԿՌՀՇՍՊՋ
ՌԽՋՌՁՆՁՌՁԹՌՁՇՆՌՀՇՍՊՋՌ ՀՇՍՊՋ

ԱՀԽ ՈԽՊՁՇԼՁԻ ՋԻՊԽԽՆՁՆԿ ԹՆԹՄՑՋՁՋ ՇԾՌՀԽ ԼՁՄՍՌՁՇՆՏԹՌԽՊԾՇՍՆԼՌՀԽ ԾՇՄՄՇՏՁՆԿ ԹՆԹՄՑՌԽՋ ԻՑԹՆՁԼԽ 
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ՅԿԩԾՄՍՇՊՁԼԽ
ՅԿԩԫԬ  

# ԫ
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ԢՎԹՄՍԹՌՁՇՆՇԾՌՀԽԹՅՈՀՁԺՁԹՆՅԽՌԹՅՇՊՈ ՀՇՋՁՋԹՋՋԹՑԢՐՈՇՋՍՊԽՌՇՌՀԽԿՇՁՌՊՇԿԽՆ
ՅԽՌՀՁՅԹՒՇՄԽԹՆԼՌՀԽԽՆԼՇԿԽՆՇՍՋՌՀՑՊՇՁԼՀՇՊՅՇՆԽԩՌՀՑՊՇՐՁՆԽ 

# ԢՆՎՁՊՇՆՅԽՆՌԹՄԱՇՐՁԻՇՄՇԿՑԹՆԼԠՀԽՅՁՋՌՊՑ

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ԤՊԽԽՆ԰ԩ
ԱՀԽԩԹԺՇՊԹՌՇՊՑԵԽՆՇՈՍՋՋՈԠԯԠԭՊԽՋՋԱԹՑՄՇ ՊԹՆԼԣՊԹՆԻՁՋԤՊՇՍՈԟՇԻԹԯԹՌՇՆ

ԣՄՇՊՁԼԹ

ԥՇՄՄԹՆԼԽՊԪ ԹՆԼԴՇՄԾԽԡԞ 
 

# ԫՇՆՈԹՊԹՅԽՌՊՁԻ ԰ՌԹՌՁՋՌՁԻԹՄԪԽՌՀՇԼՋ  ԧՇՀՆԴՁՄԽՑ ԹՆԼ԰ՇՆՋԫԽՏ

ԶՇՊՃ

ԨՆՇՏՄԽՋ ԰ԧ
ԡԽՌԽՊՅՁՆԹՌՁՇՆ ՇԾ ԭՀՑՋՁԻՇԠՀԽՅՁԻԹՄ ԭՊՇՈԽՊՌՁ ԽՋ ՇԾ ԱՊՁԾՄՍՊԹՄՁՆ 
ԭՍՊԽ ԞԤԯ
ԡԢԯԟԦԩԹԺԯԽՈՇՊՌԤԥԢԭԡՇՏԞԿՊՇ԰ԻՁԽՆԻԽՋԩԩԠԦՆ ԼՁԹՆԹՈՇՄՁՋԦՆԼՁԹՆԹ

ԨՇՇՊՋԟ
ԯԽԻԽՊՌՁԾՁԻԹՌՁՇՆՇԾՈՍՊՁՌՑԹՆԼՁԼԽՆՌՁՌՑՇԾԱ԰ԫ ԱՊՁԾՄՍՊԹՄՁՆԽՌԽԻՀՆՁԻԹՄԿՊԹԼԽԹԻՌՁՎԽ ՁՆԿՊԽԼՁԽՆՌՌՇՐԾՇՊՍՋԽՁՆԾՍՌՍՊԽՋՌՍԼՁԽՋԣԞԭԠԡՇՏ ԞԿՊՇ԰ԻՁԽՆԻԽՋԦՆԼՁԹՆԹՈՇՄՁՋԦՆԼՁԹՆԹ

ԫՁԽՍՏՃՇՇՈԭԡԹՆԼԣԹԺԽՊԧ
ԫՇՊՅԹՄԱԹԺՄԽՇԾ 

# ԵԽՆՇՈՍՋՄԹԽՎՁՋ 
ԡԹՍԼՁՆԤԹՊՄԹՆԼԭՍԺՄՁՋՀՁՆԿ

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# DATA EVALUATION RECORD

TRIFLURALIN

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

EPA Contract No. EP10H001452 Task Assignment No. 2-40-2012 (MRID 48619702)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Daniel R. Hunt, M.S. Date: 4/27/2012
Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/14/2012

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

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/17/2012

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

.

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

# TRIFLURALIN/036101 Androgen Receptor Binding (Rat 

# Prostate Cytosol) (2012) I 

# Page 1 of 13 OCSPP 890

. 1150/ OECD Nooe


# Primary Reviewer: Nancy McCarroll 

# Health Effects Division Secondary Reviewer: Gregory Akerman. Ph.D. 

# Health Effects Division Signature:

# ~i**the-~**

# Signa~:::~**--=~::::.s:J:dftS:=' :~~=ts~** **=** r<)")I**~** Date:

__ ,,..<2._,_/

.... 

# 1r

.....



# l+-J6':_,___ Template version 08/2011



# DATA EVALUATION RECORD

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



# PC CODE: 036101 

# DP BARCODE: D398779



# TXR#: 0055724 

# CAS No.: 1582-09-8



# TEST MATERIAL (PURITY): Trifluralin (97 .1 %)



# SYNONYMS: 2,6-Dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine; Trifluoralin



# CITATION: LeBaron
, M.J., Schisler, M.R.
, and Visconti, N.R. (2012). Evalua tion of trifluralin in an 

# in vitro androgen rece ptor binding assay. Toxicology 

# & Environmental Research and Consulting, The Dow Chemical Company, Midland, Ml. Laboratory Project ID: 111098
, January 11, 2012. MR.ID 48619702. Unpublished.



# SPONSOR: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN



# TEST ORDER#: CON -36101-26



# EXECUTIVE SYMMARY: In an androgen receptor (AR) binding assay (MRID 48619702), ventral prostate cytosol homogenate from Sprague Dawley rats 

# was used as the source of AR to perform a competitive binding experiment, which was conducted to measure the binding of a single concentration of [ 3 

# H]-Rl 8 81 ( 1 nM) 

# in the presence of increasing concentrations (logarithmic increase from 

# 10

- 10 

# to 

# 10- 3 

# M) of trifluralin (% purity: 97.1; Lot#: DKO-B916-33
, TSN100656). Ethanol 

# was used as solvent at a final assay concentration of <3%. A total of3

runs were performed
, and each run included dexamethasone as a weak positive control, and R 1881 as the ligand reference standard.

Although no 

# data from a saturation binding experiment were provided, a saturation binding experiment 

# was carried out by 

# the performing laboratory 

# with this batch of cytosol (MRID 48614301). A summary of these results indicate that acceptable responsiveness was determined for all parameters. In the saturation binding experiment, the maximwn binding capacity 

# (Bmax)

was 3 .245 fmoVl 00 μg protein and the dissociation constant 

# (K-0) was 

# 0.4641 

# nM. These val ue s

were below the range of values from the validation studies 

# (Bmax expected range = 7 to 

# 16 finol/100 μg protein and the 

# l<4 expected range = 

# 0.685 to 1.57 nM. Nonspecific binding as a percent of total binding was >20 % across the entire concentration range for the saturation

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 2 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None


# binding assay. Only a single run of the satu ration binding experiment was conducted on the batch of cytosol preparation used in the competitive binding experiment.

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

# s for R1881 (

− 8.996 M) and the weak positive control, dexamethasone (

− 4.522 M), were within the expected ranges. The mean RBA for the weak positive control was 0.0034% . Confidence in these numbers is high due to the small variation. The solv ent control responses indicated no drift in the study assay. All performance criteria were generally met.

Trifluralin had no effect on specific binding of the [ 3 

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

# and RBA could not be calculated.

Based on the results from the three runs, triflurali n 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 GLP Compliance, Data Confidentiality, and Quality Assurance statements were provided.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 3 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting Location: The Dow Chemical Company, Midland, MI Study Director: M.R. Schisler Other Personnel: M.J. LeBaron, Lead Scientist N.R. Visconti, Research Biologist B.B. Gollapudi, Technical Reviewer Study Period: June 15, 2011 to January 11, 2012



# 2. Test substance: Trifluralin
Description: Orange solid Source: Dow AgroSciences LLC, Indianapolis, IN Lot #: DKO-B916-33, TSN100656 Purity: 97.1% Solubility: Soluble in ethanol up to 30 mM; 0.3 ppm in water Volatility: 6.1 x 10 -3 

# Pa (physical state at 25°C) Stability: 4 years shelf life Storage conditions: Ambient CAS #: 1582-09-8 Molecular weight: 335.3 g/mol Structure:

N

CF 3 

# C H 2


# C H 2 

# CH 3 

# C H 2


# C H 2 

# CH

3



# O 2 

# N NO 2



# 3. Non-labeled ligand: R1881 Supplier: Perkin Elmer, Boston, MA Catalog #: Lot #: Not reported 614156 Purity: >97% CAS #: 965-93-5


# 4. Radioactive ligand: [ 3 

# H]-R1881 Supplier: Perkin-Elmer, Boston, MA Catalog (Lot #): NET590250UC (614814) Date of production: July 1, 2010 Date of use: June 16, 2011 to June 23, 2011 Radiochemical purity: >97% Specific activity: 85.1 Ci/mmol Concentration of stock: 1.0 mCi/mL

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 4 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None



# 5. Positive control: Dexamethasone Supplier: Sigma, St. Louis, MO Catalog # Lot #: Not reported BCBC9296 Purity: 98.9% CAS # : 50-02-2



# 6. Solvent/vehicle control: Ethanol Justification for choice of solvent: None provided

Final Concentration: <3%



# B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: The rat ventral prostate tissue was purchased from Charles River Laboratories (Wilm ington, MA). Male Sprague Dawley rats (number not reported) were castrated at a pproximately 90 days of age and euthanized approximately 24 hours later. The ventral pros tate tissues were collected and stored at approximately

− 80°C until use, and were processed as described in MRID 48614301 and used for multiple studies.

The cytosol was prepared by adding low-sa lt TEDG buffer [0.01 M Tris buffer, 10% glycerol, 1 mM sodium molybdate, 1.5 mM EDTA] and 1 mM phenylmethylsulfonyl fluoride (PMSF) with dithiothre itol (DTT)] at pH 7.4 to the ve ntral prostate tissues at 10 mL/g of tissue. The tissues were minced, hom ogenized on ice, and cen trifuged for 30 min at 30,000 × g at 4°C. The supernatant was collect ed, pooled from all tissues, aliquoted (amounts not reported) and stored at

− 80°C until used. Protein concentration of the cytosol prepared for this study was determined to be 6.566 mg/mL using th e Pierce BCA method (Thermo Scientific Pierce Research Lab, Rock ford, IL) compatible with DTT in the TEDG buffer.

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

# H]-R1881 was reportedly performed to demonstrate that the AR was present in reason able concentrations and had the appropriate affinity for the R1881 ligand (Table 1). Howeve r, the conditions for the saturation binding were reported separately and not include d with the AR binding report (Table 2).

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 5 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None



# TABLE 1. Summary of Conditions for Saturation Binding Experiment Source of receptor Rat prostate cytosol Concentration of radioligand (as serial dilutions) 0.25-10 nM Concentration of non-labeled ligand (100X [radioligand]) 25-1000 nM Optimization of receptor concentration Sufficient to bind 8.6-9.0% b 

# of radioligand at 0.25 nM Temperature ~2-8°C Incubation time ~16 hours Composition of assay buffer (TEDG) Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride 1.0 mM DTT 1.0mM a Data were not included in the study report but are reported in the study profile, submitted separately (MRID 48614301). b As indicated in the guideline for acceptable assay perf ormance, the receptor co ncentration bound <25 to 35% of the radiolabeled R1881.



# The specific activity of the stock solution [ 3 

# H]-R1881 was not adjusted for decay over time at the time of the experiment. Serial dilutions in TEDG buffer were pr epared to achieve thefinal concentrations of 0.25, 0.5, 0.7, 1.0, 1.5, 2.5, 5.0, and 10.0 nM. Solutions of nonlabeled R1881 were prepared in a similar manne r to achieve concentrations that were 100- fold greater than each respective radiolabeled concentration to re sult in final concentrations of 25, 50, 70, 100, 150, 250, 500 and 1000 nM. The optimal protein concentration was determined to be 1.97 mg protein/assay tube, which resulted in the binding of 8.6-9.0% of the total radioactivity added. The protein content was slightly greater than the recommended range in the guideline. Cytosolic protein used in this assay was thawed fresh for this experiment at ~4o C and maintained at ~4o C during the binding assay. Each run contained three concurrent replicates at each concentration, resu lting in the 72 samples depicted in Table 2.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 6 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None



# TABLE 2. Saturation Binding Experiment Run a, b 

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

# Tubes 25-48 d 

# Tubes 49-72 e 

# [ 3 

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

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

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

# H]-R1881 (μL) 0.25 0.25 25 10 7.5 0.50 0.50 50 10 15 0.70 0.70 70 10 21 1.00 1.00 100 10 30 1.50 1.50 150 10 45 2.50 2.50 250 100 7.5 5.00 5.00 500 100 15 10.00 10.00 1000 100 30 a Data were not included in the study report but are reported in the study profile, submitted separately (MRID 48614301). b Each concentration was run in triplicate for a total of 72 samples. c Tubes 1-24 contained 50 μL of triamcinolone 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 tria mcinolone acetonide and 7.5 - 45 μL [ 3 

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

# H]-R1881in 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 [ 3 

# H]-R1881 or 7.5, 15, or 30 μL of100 nM [ 3 

# H]- R1881without cytosol or other components to determine the total counts added.



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

TABLE 3. Summary of Conditions for Competitive Binding Experiment a 

# Source of receptor Rat ventral prostate cytosol Concentration of radioligand 1 nM Optimization of receptor concentration Sufficient to bind 5.3-5.8% of 1.0 nM radioligand b 

# Concentration of test substance (as serial dilutions) 10

− 10 

# to 10

− 3 

# M Incubation Temperature 4-8 oC Incubation time Overnight Composition of assay buffer Tris 0.01 M (pH 7.4) EDTA 1.5 mM Glycerol 10% (v/v) Phenylmethylsulfonyl fluoride with DTT 1 mM

Sodium molybdate 1 mM Triamcinolone acetonide 60 μM a Data were obtained from pages 16-17, 21 of the study report. b Data obtained from pages 39, 41 and 43 of the study report. Protein concentrations (μg/tube) were not reported.



# The competitive binding experiment was performed according to the protocol provided in the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 7 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None


# the binding of a singl e concentration of [ 3 

# H]-R1881 (specific activity of 85.1 Ci/mmol) to the AR in the presence of increasing concentrations of a test substance.

Ethanol was used as the solvent, and the solubi lity of the test material in the solvent and assay buffer was evaluated visua lly. No precipitation was noted.

Dilutions of the test subs tance, reference standard (R1881), weak positive control (dexamethasone), and solvent control (ethanol) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of th ree independent runs on three different days. Each run contained duplicate blanks, and three replicates at each concentration of the solvent blank, NSB, reference standard, weak positive control, and test chemical resulting in a total of 77 samples per run. In addition, duplicate blanks followed by six replicates [ 3 

# H]- R1881 only (for total binding cal culations) were run the day before each analysis run (the day of preparation of sample tubes).

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

# Solvent Control Reference standard We ak positive control Test Chemical None Ethanol R1881 Dexamethasone Trifluralin Tubes 3-5 and 72-74 Tubes 6-23 and 75-77 c 

# Tubes 24-47 Tubes 48-71 Tubes 1-2 -- 1×10

− 3 

# 1×10

− 3 

# -- 1×10

− 4 

# 1×10

− 4 

# -- 1×10

− 5 

# 1×10

− 5 

# 1×10

− 7 

# 1×10

− 6 

# 1×10

− 6 

# 1×10

− 8 

# 1×10

− 7 

# 1×10

− 7 

# 1×10

− 9 

# 1×10

− 8 

# 1×10

− 8 

# 1×10

− 10 

# 1×10

− 9 

# 1×10

− 9 

# 1×10

− 11 

# 1×10

− 10 

# 1×10

− 10 

# a Data were obtained from pages 18-20, and 39-44 of the study report. b Each concentration of each chemical was run in triplicate, plus duplicate blanks for a total of 77 tubes per run. Tubes 3-77 contained 50 μL of tr iamcinolone acetonide and 30 μL [ 3 

# H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 3-77 also contained 10 μL of the solvent control, reference standard (non-radiolabeled R1881), weak positive control, or test substance, with the exception of Tubes 6-8 and 75-77 that contained 30 μL of non-radiolabeled R1881 (used to evaluate non-specific binding). Six tubes analyzed the day prior to each run analysis contained only 30 μL of [ 3 

# H]-R1881 to determine ligand activity. c Tubes 6-8 and 75-77 were used to evaluate non-specific binding by adding 100x of cold (non-radiolabeled) R1881.



# Sample tubes were stored overn ight at 4-8°C in the dark to allow the reaction to reach equilibrium, bound R1881 was separated from free R1881 by washing with buffer and extraction with ethanol, followed by scintillation counting of bound [ 3 

# H]-R1881.

4. Data Analysis: The top and bottom of the curve, Hill Slope and inhibition concentration (IC 50 

# ) were calculated with nonlin ear regression analysis by using Graph Pad Prism v.5.0 (GraphPad, La Jolla , CA) and means were calculated for each run (mean and standard error were calculated for the composite three runs).

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 8 of 13 TRIFLURALIN/036101 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 follows:

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

− 0.6 to

− 1.4.

Equivocal: The average lowest portion of curves across runs is between 50% and 75% radioligand binding ( i.e. radioligand displacement is at le ast 25% but less than 50%), or the 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

II. RESULTS

A. SATURATION BINDING EXPERIMENT: The saturation binding experiment data with this batch of cytosol (Batch # 4) (see MRID 48614301) are shown below in Table 5.

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

# Parameter Run 1 b 

# Run 2 b 

# Run 3 b 

# Mean Runs 1-2 c 

# R 2 

# (unweighted) 0.9937 0.9871 ND 0.9871-0.9937 B max 

# (nM) 0.06194 0.06590 ND 0.06392 B max 

# (fmol/μg protein) 3.146 3.343 ND 3.245 K d 

# (nM) 0.4359 0.4922 ND 0.4641 a Data were not included in the study report but are reported in a separate study (MRID 48614301). b Two saturation runs were performed for this batch of cytosol, which was used for the competitive binding experiment. c The range of R 2 

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

# Goodness of fit for curve calculated for specific binding. ND Not determined.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 9 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None


# Specific, non-specific, and total binding curves for [ 3 

# H]-R1881 to the andr ogen receptor are presented below in Figure 1. The specific binding reached a plateau, and non-specific binding was generally less than 20 % of total binding at all concentr ations (range 6.2% - 19.8%), except the highest c oncentration in Run 1 (24.6%). All other values indicated acceptable performance of the assay. Figure 2 is a Scatchard plot that illustrates the binding of [ 3 

# H]-R1881 to the androgen receptor. The data fit results in a linear plot.

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

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 10 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None


# The estimated average log IC 50 

# was
− 8.996 M for R1881 and

− 4.522 M for the weak positive control (dexamethasone). Compared to R1881, the mean RBA for the positive control was 0.0034%. Confidence in these numbers is high due to the sm all variation. No precipitation of the test com pound was reported. The solvent c ontrol responses indicated no drift in the study assay.

Trifluralin had no effect on specific binding of the [ 3 

# H]-ligand to the AR at concentrations up to 10 -3 

# M. A log IC 50 

# and RBA for trifluralin could not be determined as exposure to trifluralin did not resu lt in 50% reduction in AR binding. Based on the results of the three valid assays, trifluralin is clas sified as a “non-binder” with the androgen receptor under the conditions of this assay.

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

# Parameter Run 1b Run 2b Run 3b Mean ± SEM c 

# R 2 

# (unweighted) R1881 1.0000 1.0000 0.9981 0.9981-1.000 Dexamethasone 0.9996 0.9997 0.9956 0.9956-0.9997 Trifluralin 0.9941 0.9582 0.8542 0.8542-0.9941 Log IC 50 

# (M) R1881 -9.009 -8.949 -9.051 -8.996 ± 0.030 Dexamethasone -4.576 -4.525 -4.467 -4.522 ± 0.031 Trifluralin NA NA NA NA IC 50 

# (M) R1881 9.803 × 10 -10 

# 1.124 × 10 -9 

# 8.902 × 10 -10 

# 1.008 × 10 -9 

# (± 0.681) Dexamethasone 2.655 × 10 -5 

# 2.988 × 10 -5 

# 3.410 × 10 -5 

# 3.004 × 10 -5 

# (± 0.219) Trifluralin NA NA NA NA Log %RBA c 

# Dexamethasone -2.43 -2.42 -2.59 -2.48 ± 0.05 Trifluralin NA NA NA NA RBA (%) Dexamethasone 0.0037 0.0038 0.0026 0.0034 ± 0.0004 Trifluralin NA NA NA NA a Data were obtained from pages 32-33 of the study report. b The mean is reported for the concu rrent replicates within each run. c The range of r 2 

# is reported and the mean and SE are reported for the other parameters. Log %RBA and SEM values are reviewer-calculated. Reviewer-calculated means differed slightly fro m study author-calculated means reported on p. 32 of the study report. For mean values reported in scientific notation, the SEM is reported in the same exponential. NA Not applicable R 2 

# Goodness of fit RBA Relative binding affinity

% RBA = IC 50 

# (R1881) X 100 IC 50 

# (Test Material)

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 11 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None


# FIGURE 3. Percentage R1881 Bound to th e Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and Trifluralin (Run 1).

FIGURE 4. Percentage R1881 Bound to th e Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and Trifluralin (Run 2).

FIGURE 5. Percentage R1881 Bound to th e Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and Trifluralin (Run 3).

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 12 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None


# FIGURE 6. Mean Percentage R1881 Bound to the Androgen Receptor in the Presence of Trifluralin in Three Competitive Binding Assays.

C. ANALYTICAL DETERMINATIONS: Nominal test concentrations of trifluralin used in this assay were found to contain ±1 1% of the target concentrations.

D. PERFORMANCE CRITERIA: To ensure that the competitive binding experiment was functioning properly, each run was evaluate d using the following criteria (Table 7):

TABLE 7. Criterion a 

# Tolerance Limit(s) b 

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

# H]-R1881 added per assay tube. ≤ 15% 5.3 to 5.8 X

Test chemical Top (% binding) 80 to 115 98.6 to 102.3 X R1881 fitted curve parameters Top (% binding) 82 to 114 96.7 to 100.9 X Bottom (% binding)

− 2.0 to 2.0 -3.6 to -0.6 X c 

# Hill Slope

− 1.2 to

− 0.8 -1.0 to -0.8 X Weak positive control (dexamethasone) fitted curve parameters Top (% binding) 87 to 106 99.3 to 105.0 X Bottom (% binding)

− 12 to 12 -1.8 to -1.1 X Hill Slope

− 1.4 to

− 0.6 -1.0 to -0.9 X Saturation Binding Experiment K d 

# (nM) 0.685 to 1.57 0.4641 X Non-specific binding (%) ≤ 20.0 6.65-6.88 d 

# X a Data were obtained from pages 32-33, 39, 41, and 43 of the study report. b These values represent ranges from the validation study. c The bottom % binding was low (-3.6) in Run 3, but within the acceptable range in the other two runs. d Values reported for the three NSB tubes at the beginning of the run; does not include the three NSB tubes at the end of the run. NR Not reported



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

# H]-R1881 in a manner consistent with one-site binding, as

# Androgen Receptor Binding (Rat Prostate Cytosol) (2012) / Page 13 of 13 TRIFLURALIN/036101 OCSPP 890.1150/ OECD None


# indicated by a Hill slope of -1.0. 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: Based on the combined responses in each of three independent androgen receptor binding assays, it was determined that trifluralin treatment did not result in significant alterations in the binding of the reference androgen (R1881) up to the assay limit concentration (1 mM, 10 -3 

# M). The results of the in vitro androgen receptor binding assay using rat prostate cyto sol indicate that, under th e conditions of this study, trifluralin was negative (non-bindi ng) for androgen receptor binding.

B. AGENCY COMMENTS: Although no data for the satura tion binding experiment were provided in the study report, a saturation binding experiment wa s carried out by the performing laboratory with the batch (Batch # 4) of cytosol (see MRID 48614301) used in the competitive binding experiment. A summary of the results stated that acceptable responsiveness was determined for the undiluted cytosol and the levels of bound 3 

# H-R1881 and non-specific binding were w ithin guideline recommendations.

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

# s for R1881 (

− 8.996 M) and the weak positive control, dexamethasone (

− 4.522 M), were within the expected ranges. The mean RBA for the weak positive control was 0.0034%. Confidence in these numbers is high due to the small variation. The solvent c ontrol responses indicated no drift in the study assay. All performance criter ia were generally met.

Based on the results of the three runs, triflurali n is classified as a non- binder in this assay.

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

 The B max 

# and the K d 

# values were below the range of recommended values for the validation studies. The study author state d, however, that the results were highly reproducible and all other performance cr iteria and the competitive binding data indicated acceptable performance of the assay.

 The amount of cytosol used in the co mpetitive binding experiment bound 5.3-5.8% of the radioactive ligand ([ 3 

# H]-R1881), which is lower than the guideline 10-15%.

# DATA EVALUATION RECORD

TRIFLURALIN

Study Type: OCSPP 890.1200, Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-40-2012 (MRID 48619703)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Joan Harlin, M.S. Date: 4/30/2012

Secondary Reviewer: Signature: David A. McEwen, B.S. Date: 5/17/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 6/04/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 6/04/2012

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

.

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

# TRIFLURALIN 

# I 

# 036101 Aromatase (Human Recombinant) Assay (2012) 

# I Page 1 of ll OCSPP 890.1200 I 

# OE.CD None


# Primary Reviewer: Nancy McCarroll 

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

# Health Effects Division Signature:

# J~ f1-.<..

# ~ Date: ' ().J°,;)..1-ilo

# ,~-- 

# Signature: 

# 4::**~** Date: 

# Gfu:b C Template version 08/2011



# DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200



# PC CODE: 036101 

# DP BARCODE: D398779

TXR#: 0055724 

# CAS No.: 1582-09-8



# TEST MATERIAL <PURITY): Trifluralin (97.1 % 

# ai.)



# SYNONYMS: 2,6-Dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine; T rifluoralin



# CITATION: Coady
, K.K. and L.K. Sosinski (2012). Trifluralin: Evaluation of Trifluralin in the Human Recombinant Aromatase Assay. Toxicology & 

# Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project Study ID: 111053, January 4, 2012. MRID 48619703. Unpublished.



# SPONSOR: Dow AgroSciences LLC
, 9330 Zionsville Rd, Indianapolis
, IN



# TEST ORDER#: CON-036101-26



# EXECUTIVE SUMMARY: In 

# an 

# in vitro aromatase (CYP19) assay (MRID 48619703), trifluralin (97.1% ai. Lot# DKO-B916-33, TSN100656) was incubated with human recombinant aromatase and tritiated androstenedione**([1~- H(N)]-androst-4-ene-3,**  

# 17-dione; [ 3 

# H]-ASDN) in 1 % dimethyl sulfoxide (DMSO) at log concentrations from 10- 10 

# to 10-4. 5 

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

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

# to 

# 10-s 

# M) using a known inhibitor ( 4-hydroxyandrostenedione; 4-0H ASDN), and the test chemical series c10

- 10 

# to 10-4.S M) 

# with three repetitions per concentration. At higher tested concentrations (10-4, l 

# o- 3

· 5

, 

# and 10- 3 

# M), trifluralin was visibly insoluble.

Aromatase activity in the full activity controls ranged from 0

. 133 to 0.225 nmol·mgprotein-1·min-1 for the three test runs, with a mean and standard deviation of 0.192 ± 

# 0.043 nmol·mg-protein- 1 ·min

- 1 • 

# The response of each full activity control within a run was 

# between 90
to 110% of the average full activity. Activity in the background controls was 0.23 to 0.31% of the full activity controls.

# Aromatase (Human Recombinant) A ssay (2012) / Page 2 of 12 TRIFLURALIN / 036101 OCSPP 890.1200 / OECD None


# For the positive control substance (4-OH ASDN), aromatase activity results were within the recommended ranges for the performance criteria. The estimated log IC 50 

# for 4-OH ASDN averaged

− 7.27 M and the Hill slope was

− 0.93.

For trifluralin, aromatas e activity averaged 0.198 ± 0.030 nmol·mg-protein

− 1 

# ·min

− 1 

# at the lowest tested concentration (10

− 10 

# M) and 0.191 ± 0.029 nmol·mg-protein

− 1 

# ·min

− 1 

# at the highest soluble concentration tested (10

− 4.5 

# M). As a percent of the control, ar omatase activity for trifluralin was ≥ 96.50% at all concentrations from 10 -10 

# to 10 -4.5 

# M.

Based on the data from the average response curve, trifluralin is classified as a Non-inhibitor of aromatase in this assay.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Aromatase assay (OCSPP 890.1200).

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

# Aromatase (Human Recombinant) A ssay (2012) / Page 3 of 12 TRIFLURALIN / 036101 OCSPP 890.1200 / OECD None



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Trifluralin Description: Orange solid (see MRID 48619702) Source: Dow AgroSciences LLC Lot # (expiration date): DKO-B916-33, TSN100656 (expiry not reported) Purity: 97.1% Volatility: Not reported Storage conditions: 5oC to ambient temperature Stability: Not reported Solvent: DMSO Solubility (in test solvent): 10 -4.5 

# M Highest Concentration Tested: 10

− 4.5 

# M Stock Solution Preparation: Serial dilution Molecular weight: 335.3 g/mol CAS #: 1582-09-8 Structure:

N

CF 3 

# C H 2


# C H 2 

# CH 3 

# C H 2


# C H 2 

# CH

3



# O 2 

# N NO 2



# 2. Non-Labeled Substrate: Androstenedione (ASDN)
CAS # : 63-05-8 Source: Steraloids, Inc. (Catalog # A6030-100) Batch # (expiration date): L1627 (expiry not reported) Purity: 98.36%



# 3. Radiolabeled Substrate: 1- β [ 3 

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

# H]ASDN)
Source: Perkin Elmer Life and Analytical Sciences (Boston, MA) Batch # (expiration date): 619344 (expiry not reported) Radiochemical Purity (Supplier): >97% Specific activity: 26.3 mCi/mmol Radiochemical Purity (In-lab determination): Not determined



# 4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN)
CAS # : 566-48-3 Source: Sigma (Catalog # F2552) Batch # (expiration date): 081K2133 (expiry not reported) Purity: 99.6%


# 5. Solvent (Vehicle Control): Dimethyl sulfoxide (DMSO)
Source: Batch # (expiration date): Fisher Scientific 06635A (not reported) Justification for choice of solvent Chosen for solubility; trifluralin more soluble in DMSO than in ethanol Concentration (% of total volume in assays): 1%

# Aromatase (Human Recombinant) A ssay (2012) / Page 4 of 12 TRIFLURALIN / 036101 OCSPP 890.1200 / OECD None



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

Source: BD Gentest TM 

# Products and Services, Woburn, MA (Catalog # 456260) Lot # (expiration date): 03897 (expiry not reported) Protein concentration: Not reported Cytochrome C reductase activity: Not reported Aromatase activity: 6.0 pmol/pmol P450/min



# B. METHODS

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

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

Test chemical stock solutions were prepared such that the total vol ume of DMSO used per assay was no more than 1% v/v of the total assay volume. DMSO was selected as the solvent because trifluralin is more soluble in it than in ethanol.

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 as the stock solution on the day of use. Dilutions were prepared such that the target concentrations of the positive control substance (0.1-10,000 nM; Table 4) were achieved by the addition of 20 μL of the dilution for a fina l assay volume of 2 mL.

Human recombinant microsomes were purchased from BD Gentest TM 

# , and stored at

− 80 ± 10oC (storage interval not repor ted). Microsomes we re portioned into i ndividual vials based on the protein concentration of the batch ( 0.004 mg/mL microsomal protein per tube) and stored at (storage in terval not reported).

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

TABLE 1. Assay Components and Conditions Assay Factor Values 0.1M sodium phosphate buffer (pH 7.4) Microsomal Protein 0.004 mg/mL NADPH 0.3 mM [ 3 

# H]ASDN 0.1μM Propylene Glycol 100 μL Temperature 37°C Incubation Time 15 min


# 2. Suitability Assessments: The protein concentration of the microsomes (Lot #03897) was not reported. Aromatase ac tivity of the microsomes was provided by the vendor as 6.0 pmol/min/pmol P450. The minimum aromatase ac tivity in the full activ ity control samples was determined to be 0.133 nmol·mg protein

− 1 

# ·min

− 1 

# , which was greater than the minimum acceptable aromatase activity of 0.1 nmol·mg protein

− 1 

# ·min

− 1



# .

# Aromatase (Human Recombinant) A ssay (2012) / Page 5 of 12 TRIFLURALIN / 036101 OCSPP 890.1200 / OECD None


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

The amount of 3 

# H 2 

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

− 1 

# ·min

− 1



# . 4. Demonstration of Proficiency: Proficiency testing of the CYP19 aromatase assay was not reported.

a. Positive Control

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

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

− 1 

# ·min

− 1



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

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

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

(2) Demonstration of Proficiency of Ne w Technician for Conducting Assay (when applicable): The positive control data for slope, t op and bottom percent met the criteria as listed in section (i) of OCSPP 890.1200.

TABLE 2. Performance Criteria for the Positive Control a 

# Parameter Lower Limit Criteria Upper Limit Crit eria Actual Lower Limit Actual Upper Limit Slope

− 1.2

− 0.8

− 1.0

− 0.86 Top (%) 90 110 101 107 Bottom (%)

− 5 +6

− 1.9 1.5 Log IC 50 

# (M)

− 7.3

− 7.0

− 7.3

− 7.2 a Data were obtained from pages 16 and 28 of the study report.



# b. Proficiency Chemicals: Assays to classify proficienc y chemicals (econazole, fenarimol, nitrofen, and atrazine) were not performed (Table 3).

# Aromatase (Human Recombinant) A ssay (2012) / Page 6 of 12 TRIFLURALIN / 036101 OCSPP 890.1200 / OECD None



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



# 5. Determination of Aromatase Ac tivity with Test Chemical(s): The response of aromatase activity to the presence of eight concentrations of triflurali n per run, in triplicate, was tested during three independent runs (T able 4). Solubility was assess ed (presence of cloudiness or a precipitate). Insolubility was obs erved at test concentrations of ≥ 10 -4 

# M; therefore, 10 -4.5 

# M was the highest test concentration used in the assay for al l three runs.

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


# Sample Type Repetitions (Tubes) Description Reference or Chemical (M) Full Activity Control 4 All test components b 

# plus solvent vehicle 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



# Trifluralin Conc 1 3 All test components plus Trifluralin 1×10 -4.5 

# Trifluralin Conc 2 3 All test components plus Trifluralin 1×10

− 5 

# Trifluralin Conc 3 3 All test components plus Trifluralin 1×10

− 5.5 

# Trifluralin Conc 4 3 All test components plus Trifluralin 1×10

− 6 

# Trifluralin Conc 5 3 All test components plus Trifluralin 1×10

− 7 

# Trifluralin Conc 6 3 All test components plus Trifluralin 1×10

− 8 

# Trifluralin Conc 7 3 All test components plus Trifluralin 1×10

− 9 

# Trifluralin Conc 10 3 All test components plus Trifluralin 1×10

− 10 

# a Data were obtained from pages 12-13 of the study report. b The complete assay contained buffer, pr opylene glycol, microsomal protein, [ 3 

# H]ASDN, and NADPH.



# C. DATA ANALYSIS

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

− 1 

# ·min

− 1 

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

# Aromatase (Human Recombinant) A ssay (2012) / Page 7 of 12 TRIFLURALIN / 036101 OCSPP 890.1200 / OECD None



# TABLE 5. Raw and Calculated Data Raw/Calculated Data Included (X) DPM/mL for each portion of extract ed aqueous incubation mixture 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 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: The response curve of the positive co ntrol and the test chemical was fitted by nonlinear regression analysis (Graph Pad Prism version) using a 4-parameter regression model program 4.0). Three independent concentrat ion response curves were generated (one for each run of the aromatase a ssay). For each run, the individual percent of control values were plotted versus logarithm of the test chemical concentration. The fitted concentration response curve was superimposed on the plot, with indivi dual plots prepared for each run. The average percent of control values versus logarithm of test chemical concentration for the individual runs for each te st chemical (with different symbols for each run) were included on the same graph with their respective fitted response curves. In addition, the average percent of control values for each run versus the logarithm of test chemical concentration were plotted on a separate graph along with the average concentration response.

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

TABLE 6. Interpret ation of Results Criteria Interpretation Data fit 4-parameter nonlinear regression model Average curve across runs crossed 50% a 

# Inhibitor Average lowest portion of curves across runs is between 50% and 75% activity b 

# Equivocal

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

# Non-inhibitor

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

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

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

− 6 

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



# II. RESULTS

A. CONTROL ACTIVITY: Aromatase activity in the full activity controls ranged from 0.133 to 0.225 nmol·mg-protein

− 1 

# ·min

− 1 

# for the 3 test runs, with a mean and standard deviation of 0.192 ± 0.043 nmol·mg-protein

− 1 

# ·min

− 1 

# . The response of each full activity control within a run was betw een 90 to 110% of the average full activity. Activity in the background controls was 0.23 to 0.31% of the full activity controls. The response of the full activity controls and background controls were acceptable.

# Aromatase (Human Recombinant) A ssay (2012) / Page 8 of 12 TRIFLURALIN / 036101 OCSPP 890.1200 / OECD None



# B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.197 ± 0.043 nmol·mg-protein

− 1 

# ·min

− 1 

# at the lowest tested concentration 10 − 10 

# M and 0.001 ± 0.000 nmol·mg-protein

− 1 

# ·min

− 1 

# at the highest test ed concentration 10 − 5 

# M. The mean aromatase activity of the positive control (expressed as % full control activity control) for each concen tration tested across all 3 runs is presented in Table 7, along with the overall standard deviation, SEM, a nd % CV. Inhibition re sponse curves for the positive control from each run and the average of all runs are shown in Figure 1. These results were generally within the recommende d ranges for the top of the curve, bottom curve, Hill Slope, log IC 50 

# , and coefficient of variation for re plicates of each concentration.

TABLE 7. Effect of Trifluralin on Aromatase Acti vity (as percent of contro l) from Independent Runs a



# Chemical Concen. (Log M) # Runs Overall Mean b 

# Overall SD b 

# Overall SEM b 

# Overall % CV b



# 4-OH ASDN (positive control) − 5 3 0.71 0.10 0.06 13.9

− 6 3 6.06 1.90 1.10 31.4

− 6.5 3 15.80 1.57 0.91 10.0

− 7 3 35.98 3.35 1.94 9.3

− 7.5 3 60.76 5.27 3.04 8.7

− 8 3 84.35 3.35 1.93 4.0

− 9 3 101.33 2.35 1.36 2.3

− 10 3 103.99 2.75 1.59 2.6 Trifluralin

− 4.5 3 104.37 6.46 3.73 6.2

− 5 3 100.47 4.11 2.37 4.1

− 5.5 3 102.47 6.44 3.72 6.3

− 6 3 100.60 1.64 0.95 1.6

− 7 3 96.50 2.40 1.39 2.5

− 8 3 101.47 3.56 2.05 3.5

− 9 3 103.67 4.73 2.73 4.6

− 10 3 100.47 5.32 3.07 5.3 a Data were obtained from Tables 2 and 3, pages 29 and 30; and Appendix C, pages 38-40 of the study report. b Calculated by the reviewers from data presented in this table. SD Standard Deviation SEM Standard error of the mean CV Coefficient of Variance

# Aromatase (Human Recombinant) Assay (2012) / Page 9 of 12 TRIFLURALIN / 036101 OCSPP 890.1200 / OECD None


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

C. TEST SUBSTANCE: For trifluralin, aromatase activity averaged 0.198 ± 0.030 nmol·mgprotein

− 1 

# ·min

− 1 

# at the lowest tested concentration 10

− 10 

# M and 0.191 ± 0.029 nmol·mgprotein

− 1 

# ·min

− 1 

# at the highest tested concentration 10

− 4.5 

# M. The mean aromatase activity of trifluralin (expressed as % full control activit y) for each concentration tested across all 3 runs is presented in Table 7 (presented above ), along with the overall standard deviation,

# Aromatase (Human Recombinant) A ssay (2012) / Page 10 of 12 TRIFLURALIN / 036101 OCSPP 890.1200 / OECD None


# SEM, and % CV. Inhibition response curves for trifluralin from each run are shown in Figure 2, and the average inhi bition response curve across all runs is shown in Figure 3.

FIGURE 2. Inhibition Response Curves for Trifluralin From Each Test Run.

# Aromatase (Human Recombinant) A ssay (2012) / Page 11 of 12 TRIFLURALIN / 036101 OCSPP 890.1200 / OECD None


# FIGURE 3. Mean Inhibition Response Curve for Trifluralin.

The effect of trifluralin on inhi bition of aromatase activity is presented in Table 8. The data for trifluralin could not be f it to the 4-parameter nonlinear re gression model; therefore, log IC 50 

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

# averaged

− 7.27 M and the Hill slope was

− 0.93 (Table 8).

TABLE 8. Effect of Trifluralin on Aromatase Acti vity (as Percent of Control) From Independent Runs a 

# Chemical Run 1 Run 2 Run 3 Mean b 

# SD b 

# %CV b 

# Log IC 50 

# (M) 

# Trifluralin NA NA NA NA NA NA 4-OH ASDN

− 7.3

− 7.2 -7.3 -7.27 0.06 -0.79 Hill Slope 

# Trifluralin NA NA NA NA NA NA 4-OH ASDN -0.92 -0.86 -1.00 -0.93 0.07 -7.58 a Data were obtained from Table 1, page 28 of the study report b Calculated by the reviewers from data presented in this table. SD Standard Deviation CV Coefficient of Variance NA Not applicable



# Based on the data from the average response curv e and the criteria listed above in Table 6, the results support the c onclusion that trifluralin does not in hibit aromatase in the aromatase assay.

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# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: Trifluralin was determined to be a non-inhibitor of aromatase activity in this assay, since there was no inhibition of aromatas e activity at any of the tested concentrations, including 10

− 4.5 

# M, which was also the limit of solubility of trifluralin in the test system.

B. AGENCY COMMENTS: Aromatase activity in the fu ll activity controls was 0.192 ± 0.043 nmol·mg-protein

− 1 

# ·min

− 1 

# . The response of each full activity control within a run was between 90 to 110% of the aver age full activity. Activity in the background controls was 0.23 to 0.31% of the full control activity.

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

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

# for 4-OH ASDN averaged

− 7.27 M and the Hill slope was

− 0.93.

It was stated that the trifluralin was vi sibly insoluble at c oncentrations of 10 -3 

# , 10 -3.5 

# and 10 -4 

# M. Aromatase activities at the lowest and highest tested concentrations, 10

− 10 

# and 10

− 4.5 

# M, were 100.47 ± 5.32% and 104.37 ± 6.46% of the control. The lowest portion of the response curve across runs was gr eater than 75% activity at all concentrations of trifluralin. The data could not be fit to the 4-parameter nonlinear regression model. Based on the data from the average response curve, trifluralin is classified as a non- inhibitor of aromatase activity up to the highest sol uble concentration tested (10 -4.5 

# M).

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

 The required proficiency da ta were not provided.

 Complete information on the Human recomb inant aromatase (CYP19) microsomes was not provided. The protein concentration a nd cytochrome C reductase activity were not reported, and the length of tim e that the microsomes were stored prior to be being portioned into individual vials and prior to us e in the aromatase a ssay was not reported.

 Some of the primary data presente d in Appendix C were not legible.

 The equation used to determine how the data presented in Table 7 were calculated was not provided.

# DATA EVALUATION RECORD

TRIFLURALIN

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-40-2012 (MRID 48619704)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Daniel R. Hunt, M.S. Date: 04/20/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 05/14/2012

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

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/17/2012

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

.

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

# TRJFLURALIN/036101


# Primary Reviewer: Deborah Smegal 

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

# Effects Division Sig.t;

# ~ :

# --A~~~~­ Date:**--~~.L,,C,.Qot--­** 

# Signature:**---JJ.~~-;.c=====­** 

# Date: 

# c I 

# u f.l.(11 

# r Temp

# lat~ version 08/lOl l



# DATA EVALUATION RECORD

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



# PC CODE: 036101 

# DP BARCODE: 0398779

TXR#: 0055724 

# CAS No.: 1582-09-8



# TEST MATERIAL (PURITY): Trifluralin (97.1 

# %)

SYNONYMS: 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine; Trifluoralin



# CITATION: LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2012). Evaluation of trifluralin in an 

# in vitro estrogen receptor binding assay. Toxicology 

# & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project ID: 111124, January 11, 2012. MRID 48619704. Unpublished.



# SPONSOR: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN



# TEST ORDER#: CON-36101-26



# EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MR.ID 48619704 ), uterine cytosol from Sprague Dawley rats was used as the source of ER for a competitive binding experiment, which was conducted to measure the binding of a single concentration of [ 3 

# HJ-1**7~­** 

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

# to 

# 10- 3 

# M) oftrifluralin (97.l % purity; Lot#DK0-8916-33, TSN100656). Ethanol was used as the solvent vehicle at a final concentration of <3%. A total of three runs were performed, and each

run included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17-P-estradiol as the natural ligand reference material.

A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radiolabeled reference estrogen prior to routinely conducting ER competitive binding experiments. Data from the saturation binding experiment were not included in the original report; however, the binding data were reported in a separate report for the batch of cytosol that

was utilized in the assay. In the saturation binding experiment, the maximum binding capacity (Bmax) was 59.28 fmol/100 μg protein and the dissociation constant (Kd) was 0.1032 nM. The Bmax was within the expected range of 10 to 150 fmol/l 00 μg protein, and the Kd was within the expected range of 0.03 to 1.5 

# nM. Nonspecific binding as a percent of total binding was 1.7% to 8.6% across the entire concentration range in the saturation binding assay. Only a single

# Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 2 of 13 TRIFLURALIN/036101 OCSPP 890.1250/ OECD None


# run of the saturation binding experiment was co nducted on the batch of cytosol preparation used for the competitive binding experiment.

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

# was
− 8.934 M for 17 β

estradiol and

− 5.442 M for the weak positive control. The mean Relative Binding Affinity (RBA) was 0.032% for the weak positive control.

Trifluralin had no effect on specific binding of the [ 3 

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

# and RBA could not be calculated for trifluralin. Specific binding was >75% at all concentrations of trifluralin tested.

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

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

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

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# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting
Location: The Dow Chemical Company, Midland, MI Study Director: M.R. Schisler Other Personnel: M.J. LeBaron, Lead Scientist N.R. Visconti, Research Biologist B.B. Gollapudi, Technical Reviewer Study Period: September 6, 2011 to January 11, 2012



# 2. Test substance: Trifluralin Description: Orange solid Source: Dow AgroSciences LLC, Indianapolis, IN Lot#: DKO-B916-33, TSN100656 Purity: 97.1% Solubility: Soluble in ethanol up to 30 mM; 0.3 ppm in water Volatility: 6.1 x 10 -3 

# Pa (physical state at 25°C) Stability: 4 years shelf life Storage conditions: Ambient CAS #: 1582-09-8 Molecular weight: 335.3 g/mol Structure:



# 3. Non-labeled ligand: 17 β -estradiol Supplier: Sigma, St. Louis, MO Catalog #: E8875 Lot #: 098K1372 Purity: 100% CAS #: 50-28-2


# 4. Radioactive ligand: [ 3 

# H]-17 β -estradiol Supplier: Perkin-Elmer, Boston, MA Catalog #: NET517001MC Lot#: 639068 Radiochemical purity: >97% Specific activity: 162.9 Ci/mmol Concentration of stock: 1.0 mCi/mL



# 5. Positive control: 19-Norethindrone Supplier: Sigma, St. Louis, MO Catalog #: N4128 Lot #: 030M1359

# Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 4 of 13 TRIFLURALIN/036101 OCSPP 890.1250/ OECD None

Purity: 99% CAS #: 68-22-4


# 6. Negative control: Octyltriethoxysilane Supplier: Sigma, St. Louis, MO Catalog #: 440213 Lot #: 72596AMV Purity: 98.58% CAS #: 2943-75-1



# 7. Solvent/vehicle control: Ethanol
Justification for choice of solvent: None provided

Final Concentration: <3%



# B. METHODS

1. Preparation of Rat Uterine Cytosol (RUC): Trimmed uterine tissue from 85- to 100-day old female Crl:CD(SD) rats, which were ovari ectomized approximately 7-10 days prior to being euthanized, was purchased from Charle s River (Wilmington, MA). The uteri were weighed, minced and homogenized in ice-cold TEDG (Tris, EDTA, DTT, glycerol) + PMSF (phenylmethylsulfonyl fluoride) buffer, then centrifuged for 10 min at 2500 × g at 4oC. Supernatant was transferred and centrifuged for 60 minutes at 105000 × g , discarding the resulting pellets. Protein c oncentration of the cytosol was determined to be 3.573 mg/mL using a protein kit compatible with DTT in the TEDG buffer (Pierce BCA method; Thermo Scientific Pierce Research Lab). Cytosol wa s divided into aliquots (volume not reported) that were stored at

− 80 oC for up to 90 days until use.

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

# H]-17 β -estradiol was performed to demonstrate that the ER was present in reason able concentrations and had the appropriate affinity for the native ligand (Table 1). Howe ver, the conditions for the saturation binding were reported separately in MRID 48843501 a nd are summarized below in Table 2.

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# TABLE 1. Summary of Conditions for Saturation Binding Experiment a 

# Source of receptor Rat uterine cytosol Concentration of radioligand (as serial dilutions) 0.03-3.0 nM Concentration of non-labeled ligand (100X [radioligand]) 3.0-300 nM Concentration of receptor Sufficient to bind 40.77% of radioligand at 0.03 nM b 

# Temperature 2-8 o 

# C Incubation time 16 hours Composition of assay buffer Tris 10 nM (pH 7.4) EDTA 1.5 nM Glycerol 10% Phenylmethylsulfonyl fluoride 1 nM DTT 1 nM a Data were not included in the study report, but were reported as a se parate validation report b This value was slightly higher than the suggested range in the guideline; however, all other values, including minimal ligand depletion, indicat ed acceptable performance in the assay.

TABLE 2. Saturation Binding Experiment Run a


# Total Binding b 

# Non-specific binding c 

# Radioligand alone d 

# Assay components

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

350 uL 300 uL --- TEDG + PMSF buffer 50 uL 50 uL 50 uL 3 

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



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

# 100 uL 100 uL --- Uterine cytosol (diluted to appropriate conc.) 500 uL 500 uL 50 uL Total volume in each assay tube a Data were not included in the study report, but are reported as a separate validation report MRID 48843501 b Total binding = 3 

# H]-17 β -estradiol bound to ER. 

# c Non-specific binding = 3 

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

# d Total 3 

# H]-17 β -estradiol alone for dpm determin ation at each concentration. 

# e. Final concentration of 3 

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

# f. Final concentration of non-labeled 17 β -estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.



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

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

# Source of receptor Rat Uterine Cytosol Concentration of radioligand 1.0 nM Concentration of receptor Sufficient to bind 5.66-7.15% of radioligand Concentration of test substance (as serial dilutions) 10

− 10 

# to 10

− 3 

# M Temperature 4 to 8  C Incubation time 16 to 20 hours Composition of assay buffer Tris 10 mM EDTA 1.5 mM Glycerol 10% Phenylmethylsulfonyl fluoride 1 mM DTT 1 mM a Data were obtained from pages 17, 22-23, and 41-46 of the study report.



# Solubility of trifluralin in ethanol and assay buffer wa s evaluated visually and no precipitation was noted. On the day of the assa y, the specific activity of the stock solution [ 3 

# H]-17 β -estradiol was not adjusted for decay over time, and was diluted in TEDG + PMSF buffer to achieve a final concentration of 1 nM. Uterine cytosol was used at a 1:2 dilution to achieve a protein concentr ation of 1.191 mg/mL that bound 5.66-7.15% of the total radioactivity added. Serial dilutions of the test substance, positive control (19- norethindrone), negative control (octyltriethoxysilane), and refe rence material (non-labeled 17 β -estradiol) were prepared to achieve the c oncentrations shown in Table 4. Each assay consisted of three independent r uns on three different days. E ach run included triplicate sets of 0.1 μ M estradiol (non-specific binding, NSB) at the beginning and end of each run, along with triplicate tubes of the reference standar d, the weak positive cont rol, negative control, and trifluralin at each concentr ation, resulting in a total of 104 samples per run. In addition, duplicate blanks followed by six replicates of [ 3 

# H]-17 β -estradiol only (for total binding calculations) were run the day before each analys is run (the day of preparation of sample tubes).

# Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 7 of 13 TRIFLURALIN/036101 OCSPP 890.1250/ OECD None



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



# Trifluralin Positive control Negative control Reference Chemical 19-norethindrone Octyltriethoxysilane Non-labeled 17 β -estradiol Tubes 75-98 c 

# Tubes 27-50 c 

# Tubes 51-74 c 

# Tubes 6-26 and 102-104 c 

# 10

− 10 

# 10

− 8.5 

# 10

− 10 

# Solvent control d 

# 10

− 9 

# 10

− 7.5 

# 10

− 9 

# 10

− 11 

# 10

− 8 

# 10

− 7 

# 10

− 8 

# 10

− 10 

# 10

− 7 

# 10

− 6.5 

# 10

− 7 

# 10

− 9.5 

# 10

− 6 

# 10

− 6 

# 10

− 6 

# 10

− 9 

# 10

− 5 

# 10

− 5.5 

# 10

− 5 

# 10

− 8.5 

# 10

− 4 

# 10

− 4.5 

# 10

− 4 

# 10

− 8 

# 10

− 3 

# 10

− 4 

# 10

− 3 

# 10

− 7 

# a Data were obtained from pages 34-35 and 41-46 of the study report. b Each tube contains: 10μL of either the test substance, positive control, negative control, solvent control, or non-labeled 17 β -estradiol; 390 μL of TEDG + PMSF buffer with [ 3 

# H]-17 β -estradiol; and 100 μL of uterine cytosol (with ER), for a total of 500 μL. c Each concentration of each chemical was run in triplicate, for a total of 104 tubes per run. d Solvent is ethanol in Tubes 3-5 and 99-101.



# Tubes were gently vortexed and incubated for 16-20 hours at 4-8  C. To separate bound from free estradiol, hydroxyapatite (HAP) slu rry was added to each tube and vortexed (4 times at 5 minute intervals). Subsequently, the contents of each tube were washed three times as follows: TEDG + PMSF buffer was added, vortexed, centrifuged for 10 min at 1000 × g , and the supernatant decanted and discarded. Ethanol was added to the HAP pellet remaining in each tube to extract the bound [ 3 

# H]-17 β -estradiol, followed by vortexing, and centrifugation for 10 min at 1000 × g . An aliquot of supernat ant was radioassayed by liquid scintillation counting. The temperature was main tained at 4-8 oC thr oughout the assay prior to extraction with ethanol.

C. DATA ANALYSIS: For the competitive binding assay, GraphPad Prism v. 5.0 (GraphPad, La Jolla, CA) was used to conduct non-linear re gression analyses and to fit a curve (for 17 β

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

# as a parameter to be estimated. For parameters reported from the 

# competitive binding experiment (log IC 50 

# and %RBA), m eans were calculated for each run and for the composite three runs.



# 1. Definitions

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

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

Not interactive = there are usable data points at or above 10 -6 

# M and either the lowest point on the fitted response curve within the range of the da ta is above 75% (i.e., <25% of the radiolabeled estradiol ha s been displaced from the ER) or a

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# binding curve cannot be fitted and the lowest average percent binding among concentration groups in the data is above 75%.

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

− 6 

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

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

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

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


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

Test Material Classifi cation Numerical Range Interactive average ≥ 1.5 Equivocal 0.5 ≥ average <1.5 Not interactive average <0.5 Equivocal up to the limit of concentrations tested “missing”


# b. Descriptors for receptor binding:

B max 

# : maximum specific binding number (fmol ER/ 100 μg cytosol protein) measures the concentration of active receptor sites K d 

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

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

# of 17 β -estradiol ÷ IC 50 

# of test substance) × 100

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Data pertaining to the saturation binding experiment were not included in this report (Table 5). A summary of the results was included that indicated that the batch of cytosol used for the ER binding analyses had acceptable responsiveness, including the determ ined percent ligand binding and non-specific binding.

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# TABLE 5. Saturation Binding Experiment of 17 β -estradiol with Estrogen Receptor from Rat Uterine Cytosol Parameter Run 1 Run 2 Run 3 Runs 1-3 R 2 

# (unweighted) NR NR NR NR B max 

# (nM) NR NR NR NR B max 

# (fmol/100 μ g protein) NR NR NR NR K d 

# (nM) NR NR NR NR NR Not reported



# B. COMPETITIVE BINDING EXPERIMENT: The results from the competitive binding experiments are presented in Table 6 and show n graphically in Figures 1 to 4. The three reference chemicals generally performed as expected. The estimated mean log IC 50 

# was

− 8.934 M for 17 β -estradiol and

− 5.442 M for the weak positive control, and the mean RBA was 0.032% for the weak positive control. The negative control, octyltriethoxysilane, had no effect on specific binding of the [ 3 

# H]-ligand (86.5 to 102.7%). C onfidence in these numbers is high due to the small variation. Repeat runs were unnecessary.

Trifluralin had no effect on speci fic binding (84.2-106.5%) of the [ 3 

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

# and RBA could not be calculated for trifluralin. Based on the results of the three valid runs, trifluralin is classified as not interactive with the estrogen receptor under the conditions of this assay (Table 7).

TABLE 6. Competitive Binding Assa y of Trifluralin with Estrogen Receptor from Rat Uterine Cytosol a 

# Parameter Run 1 b 

# Run 2 b 

# Run 3 b 

# Mean ± SEM c 

# R 2 

# (unweighted) 17 β -estradiol 0.9989 0.9990 0.9997 0.9989-0.9997 19-norethindrone 0.9983 0.9999 0.9997 0.9983-0.9999 Trifluralin 0.9195 0.9589 0.9315 NA Log IC 50 

# (M) 17 β -estradiol -8.913 -8.948 -8.942 -8.934 ± 0.011 19-norethindrone -5.431 -5.462 -5.435 -5.442 ± 0.010 Trifluralin NA NA NA NA IC 50 

# (M) 17 β -estradiol 1.223 × 10 -9 

# 1.127 × 10 -9 

# 1.142 × 10 -9 

# 1.165 × 10 -9 

# (± 0.298) 19-norethindrone 3.710 × 10 -6 

# 3.448 × 10 -6 

# 3.673 × 10 -6 

# 3.612 × 10 -6 

# (± 0.082) Trifluralin NA NA NA NA Log RBA 19-norethindrone -3.482 -3.486 -3.507 -3.491 ± 0.008 Trifluralin NA NA NA NA RBA (%) 19-norethindrone 0.033 0.033 0.031 0.032 ± 0.001 Trifluralin NA NA NA NA a Data were obtained from pages 34-35 of the study report. b The mean is reported for the concu rrent replicates within each run. c The range of R 2 

# is reported and the mean and SEM are reported for the other parameters. SEM values are reviewer-calculated. Reviewer-calculated means di ffered slightly from study author-calculated means reported on p. 34 of the study report. For mean valu es reported in scientific notation, the SEM is reported in the same exponential. R 2 

# Goodness of fit RBA (%) Relative binding affinity NA Not applicable

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# TABLE 7 Binding Classification of Trifluralin with Estrogen Receptor a 

# Run 1 2 3 Mean Binding Classification c 

# Classification category value b 

# 0 0 0 0 Not interactive a Data were obtained from page 35 of the study report. b Classification category value: Interac tive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of concentrations tested (“missing”, i.e., not included in calculation of mean). c Interactive = mean ≥ 1.5; Equivocal = 0.5 ≤ mean <1.5; Not inte ractive = mean <0.5



# FIGURE 1. Percentage E2 Bound to the Estrog en Receptor in the Presence of Unlabeled E2, 19-Norethindrone, Octyltriethoxysilane or Trifluralin, Run 1.

FIGURE 2. Percentage E2 Bound to the Estrog en Receptor in the Presence of Unlabeled E2, 19-Norethindrone, Octyltriethoxysilane or Trifluralin, Run 2.

# Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 11 of 13 TRIFLURALIN/036101 OCSPP 890.1250/ OECD None


# FIGURE 3. Percentage E2 Bound to the Estrog en Receptor in the Presence of Unlabeled E2, 19-Norethindrone, Octyltriethoxysilane or Trifluralin, Run 3.

FIGURE 4. Mean Percentage [ 3 

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

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

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# TABLE 8. Criterion a 

# Tolerance Limit(s) Value Yes No

17 β -estradiol fitted curve parameters Log e 

# residual SD ≤ 2.35 -0.1101 to 0.6136 X Top (% binding) 94 to 111 98.9 to 99.4 X Bottom (% binding)

− 4 to 1 -1.3 to -0.1 X Hill Slope (log 10 

# (M)

− 1 

# )

− 1.1 to

− 0.7 -1.033 to -0.9611 X Weak Positive control (19-norethindrone) fitted curve parameters b 

# Log e 

# residual SD ≤ 2.6 -0.5212 to 0.7816 X Top (% binding) 90 to 110 98.3 to 100.3 X Bottom (% binding) - 5 to +1 -1.5 to 0.6 X Hill Slope (log 10 

# (M)

− 1 

# ) -1.1 to – 0.7 -1.072 to -0.9885 X Solvent concentration Ethanol ≤ 3% <3% X Negative control (octyltriethoxysilane) does not displace more than 25% of [ 3 

# H]-17 β -estradiol from the ER on average across all concentrations ≤ 25% ≤ 13.5 c 

# X

a Data were obtained from pages 34-35 and 41-46 of the study report. b The EPA Guideline does not define a set of tolerance li mits for 19-norethindrone. A cceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable as they show 19-nor ethindrone to be an accepta ble weak positive control. c The minimum total binding of [ 3 

# H]-17 β -estradiol in the negative control assays was 86.50% (Run 3). NA Not applicable



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

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

− 0.9906.

Trifluralin was tested over a concentration range that fully defined the top of the curve. The percent binding at this top plateau 97.2-104.9% wa s within 25 percentage points of the value for solvent control 95.24-99.45% and the lowest concentration of the estradiol standard 98.9- 99.4%.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the combined responses in each of three independent estrogen receptor bi nding assays, it was determin ed that trifluralin had no appreciable effect on the binding of th e reference estrogen (radiolabeled 17 β -estradiol) at any concentration up to 10 -3 

# M. The results of the in vitro estrogen receptor binding assay using rat uterine cytosol indicate that, under the conditions of this study, trifluralin was determined to be negative (not inte ractive) for estrogen receptor binding.

B. AGENCY COMMENTS: No data from a saturation bind ing experiment were included in the report, but were included as a separate s ubmission. In the satura tion binding experiment, the maximum binding capacity (Bmax) was 59.28 fmol/100 μ g protein and the dissociation constant (Kd) was 0.1032 nM. The Bmax was within the expected range of 10 to 150 fmol/100 μ g protein, and the Kd was within the expected range of 0.03 to 1.5 nM. Nonspecific binding as a per cent of total binding was 1.7% to 8.6% across the entire

# Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 13 of 13 TRIFLURALIN/036101 OCSPP 890.1250/ OECD None


# concentration range in the sa turation binding assay. Only a single run of the saturation binding experiment was conducted on the batc h of cytosol prepar ation used for the competitive binding experiment.

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

# was
− 8.934 M for 17 β

estradiol and

− 5.442 M for the weak positive control. The mean RBA was 0.032% for the weak positive control. All pe rformance criteria were met.

The estimated mean IC 50 

# and RBA were not calculated for trifluralin as it did not achieve 50% competitive binding. Trifluralin is classified as not-interactive in this assay as the mean specific [ 3 

# H]-17 β -estradiol ER binding was >75% at trifluralin concentrations up to 10 –3 

# M in all three independent runs.

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

 The amount of cytosol used in the co mpetitive binding assay bound 5.66-7.15% of the radioactive estradiol, which is lower than the guideline 10-15%.

# DATA EVALUATION RECORD

TRIFLURALIN

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 2-40-2012 (MRID 48619705)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Michelle Sharpe-Kass, M.S. Date: 4/11/2012

Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/14/2012

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

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

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

.

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

# TRIFLURALIN/03610 I 

# Estrogen Receptor Transcriptional Activation (2012) I 

# Page 1 of 8 OCSPP 890.130-0/ OECD 455


# Primary 

# Reviewer: Deborah Smegal 

# Health Eff ect

. s Division Secondary Reviewer: Minerva Mercado, Ph.D. 

# Health Effects Division Date:
,,,.,.

# Si~~e~**Signature~** Date: 

# c-Zft- 

# IS

- Template version 0812011



# DATA EVALUATION RECORD

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



# PC CODE: 036101

TXR#: 0055724



# TEST MATERIAL (PURITY): Trifluralin (97.1 %) 

# DP BARCODE: D398779



# CAS No.: 1582-09-8



# SYNONYMS: 2,6-Dinitro-N ,N-di propy 1-4-( trifluoromethy l) benzenamine, Trifluoralin



# CITATION: LeBaron, M.J., Kan, H.L. (2012). Evaluation of Triflu ralin in an 

# In Vitro Estrogen Receptor Transcriptional Activation Assay i n Human Cell Line hERaHeLa-9903. Toxico logy & E nvironm e ntal Research and Consulting, Midland, MI. Laboratory Report No.: 101188, January 5, 2012. MRID 48619705. Unpublished.



# SPONSOR: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN



# TEST ORDER#: CON-036101-26



# EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID ( 48619705)
, hERa-HeLa-9903 cells cultured in vitro were exposed to trifluralin (97. l %, Lot #s DKO -B9 16-3 3, TSN100656) at logarithmically increasing concentrations from 10- 10 to 10 4 

# Min DMSO. Four independent runs were performed using 96-well plates; each concentration was tested in 3 wells/plate. Cells were exposed to the test agent for about 24 hours to induce reporter (luciferase) gene products. Luciferase expression in respons e to activation of the estrogen receptor was measured after addition of luciferase substrate and detection with a lwninometer.

Trifluralin was tested up to the limit of solubility, 10 4 

# M. Performance criteria were generally
met for all reference chemicals, except values for l 7a-methyltestosterone were consistently lower than the expected range, indica t ing increased s en s itivity of the assay to weak agonists. Tr ifluralin RPCMax was <0%
, 18%
, 16 .6% and 13.7% in runs 

# 1, 2, 3, and 4, respectivel y; associated PCMax was 10 4 

# M. The response was dose-dependent. Because the RPCMax 

# > PC10 in three out of four assay runs, trifluralin was considered positive for estrogen receptor tran sc riptional activation in this test system.
This assay 

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



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

# Estrogen Receptor Transcriptional Activation (2012) / Page 2 of 8 TRIFLURALIN/036101 OCSPP 890.1300/ OECD 455



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Trifluralin Description: Orange solid, MW 335.3 Source (cat #): Dow AgroSciences (not reported) Lot/Batch # (Exp date): DKO-B916-33, TSN100656 (not reported) Purity: 97.1% Solubility: DMSO 0.1M, 0.3 ppm in water Volatility: 6.1 × 10 -3 

# Pa at 25°C Stability: 4 year shelf life Storage conditions: Ambient CAS #: 1582-09-8 Structure:



# 2. Reference substances 17 β -estradiol (strong estrogen; positive control) Supplier: Sigma, St Louis, MO Catalog and Batch #: Cat# E-8875, Lot# 079K0131 Purity: 100% CAS # : 50-28-2



# 17 α -estradiol (weak estrogen) Supplier: Sigma, St Louis, MO Catalog and Batch #: Cat# E-8750, Lot# 029K4116 Purity: ≥ 99.5% CAS # : 57-91-0 

# Corticosterone (negative compound) Supplier: Sigma, St Louis, MO Catalog and Batch #: Cat# C-2505, Lot# 010M2010 Purity: 100% CAS # : 50-22-6



# 17 α -methyltestosterone (very weak agonist) Supplier: Sigma, St Louis, MO Catalog and Batch #: Cat# M-7252, Lot# 060M1543V Purity: 99% CAS # : 58-18-4


# 3. Vehicle(s)
Solvent: DMSO, Sigma-Aldrich, Cat # 276855
Solvent control (final concentration): 0.1%

# Estrogen Receptor Tran scriptional Activation (2012) / Page 3 of 8 TRIFLURALIN/036101 OCSPP 890.1300/ OECD 455



# B. METHODS

1. Cell Culture: Stably-transfected hER α -HeLa-9903 cells were obtained from the Japanese Collection of Research Bioresour ces Cell Bank, and were verified to be free of mycoplasma infection using RT PCR. Cells were mainta ined in Eagles Minimum Essential Medium without phenol red, supplemented with 60 mg/L kanamycin and 10% dextran-coated charcoal-treated fetal bovine serum (in-house prep; lot # DCC-FBS 040111), in an incubator under 5% CO 2 

# at 37oC. Data was not provided to verify that no hormones were present in the in-house prep of DCC-FBS. Cells were s ubcultured at least twi ce prior to exposure to the test material. The cells in this study were passage 12 and 14.

2. Transcriptional Activation Assays: For each test, cells were plated at a density of 1- 1.5x10 4 

# cells/100 μL medium/well in a 96 well plate and allowed to attach for three hours. Growth medium was replaced with medium contai ning serial log dilutions of trifluralin in DMSO (0.1% final concentration). Cells were incubated for approximately 24 hours at approximately 37°C. Cytotoxicity was determined using a CellTiter 96 Aq ueous 

# One Solution Cell Proliferation Assay kit (Prome ga, Madison, WI, Cat #G 3580). Transcriptional activation of the estrogen receptor was determined using a standard luciferase assay system (Promega, Cat #E1500). Chemiluminescence wa s detected with a Packard TopCount NXT counter with a detection limit of ~5 cells/100μL medium.

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

# to 10 -4 

# M was conducted to determine the appropriate concentration range and to determine concentrations resulting in insolubility a nd/or cytotoxicity. Concentrations of 10 -3 

# M and 10 -3.5 

# M had precipitation noted in the treatment me dium and was not tested for cytotoxicity.

b. Proficiency Chemicals: It was stated that a labora tory proficiency assay with 10 proficiency chemicals was performed to confirm the responsi veness of the ER transcriptional activation a ssay. These non-GLP, unpublished experimental results were reported to have demonstrated laboratory proficiency, but were not included.

c. Reference Chemicals: To ensure the stability of the response from the cell line, seven 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 (E2) 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. Graph Pad Prism v. 5.0 (GraphPad Software, Inc., La Jolla, CA) was used to calculate the EC 50 

# , PC 10 

# , PC 50 

# , RPC Max 

# , and PC Max 

# for trifluralin wh en applicable.

# Estrogen Receptor Tran scriptional Activation (2012) / Page 4 of 8 TRIFLURALIN/036101 OCSPP 890.1300/ OECD 455


# The test material was defined as positive for inducing estrogen receptor transcriptional activation if the RPC Max 

# ≥ PC 10 

# in at least 2 of 2 or 2 of 3 runs. Log EC 50 

# and Hill slope values are calculated only if a positive respons e is observed. Coefficien ts of variation (CV) were calculated for the luminescence data tr iplicates. Concentr ations showing >20% 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 which the response is 10% of the response induced by the positive control (E2 at 1 nM) in each plate

PC 50 

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

RPC Max 

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

PC Max 

# = concentration of a test chemical inducing the RPC Max



# II. RESULTS

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the transcriptional activation assays, preliminary cytotoxicity and precipitation assays were conducted. Due to precipitation in 10 -3 

# M trifluralin in medium, the highest concentration tested was 10 -4 

# M. The results of the cytotoxicity test are presented in Table 1. Based on these results, logarithmically in creasing concentrations from 10 -10 

# to 10 -4 

# M were selected for the assay.

TABLE 1. Preliminary Test for Solubility, Cytoto xicity, and Concentration-Selection for Trifluralin a 

# Concentration (M) % Viability Comments 10

− 4 

# 120.0 Solubility limit concentration 10

− 5 

# 120.4 10

− 6 

# 127.1 10

− 7 

# 124.3 10

− 8 

# 140.6 10

− 9 

# 132.5 10

− 10 

# 119.0 E2 1nM 130.8 VC b 

# 100.0 a Data were obtained from page 52 of the study report. b VC = Vehicle control



# B. POSITIVE AND NEGATIVE REFERENCE CHEMICALS

1. Proficiency Chemicals: The results from the proficiency test were not reported (Table 2).

# Estrogen Receptor Tran scriptional Activation (2012) / Page 5 of 8 TRIFLURALIN/036101 OCSPP 890.1300/ OECD 455



# TABLE 2. Proficiency Chemicals Compound Expected Response Lab Response Diethylstilbestrol Positive Not Reported 17 α -Ethynyl estradiol Positive Not Reported Hexestrol Positive Not Reported Genistein Positive Not Reported Estrone Positive Not Reported Butyl paraben Positive Not Reported 1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive Not Reported Dibutyl phthalate Negative Not Reported Atrazine Negativ e Not Reported Corticosterone Negative Not Reported



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

# log PC 10, 

# log EC 50, 

# Hill slope) for the four concurrently run reference materials are included in Table 3. Performance criteria were met for corticosterone, and all values but the Hill Slopes for 17 β -estradiol and 17 α -estradiol, the log EC 50 

# for 17 β -estradiol, and the Log PC 50 

# for 17 α -estradiol. The Hill slopes were consiste ntly lower than the expected values in at least half of the runs. Ho wever, all other parameters were within the acceptable range. The log PC 50 

# and log PC 10 

# for 17 α -methyltestosterone were consistently lower than the acceptable range, indicating an increased sensit ivity of the assay to weak agonists.

TABLE 3. Performance Criteria for Reference Chemicals a


# Reference Chemical Parameter Acceptable Range Values Acceptable Run 1 Run 2 Run 3 Run 4 Yes No 17 β -estradiol Log PC 50 

# − 11.4 to

− 10.1 -10.8 -11.1 -10.9 -10.7 X Log PC 10 

# <

− 11 -11.9 -12.6 -12.7 -12.5 X Log EC 50 

# − 11.3 to

− 10.1 -9.4 -10.4 -10.7 -10.6 Run 1 Hill Slope 0.7 to 1.5 0.3 0.3 0.7 0.8 X Test range 10

− 14 

# to 10

− 8 

# M 10

− 14 

# to 10

− 8 

# M X 17 α -estradiol Log PC 50 

# − 9.6 to

− 8.1 -9.3 -10.1 -9.0 -9.0 X Log PC 10 

# − 10.7 to

− 9.3 -10.6 -10.9 -10.6 -10.9 X Log EC 50 

# 9.6 to

− 8.4 -8.7 -9.6 -8.8 -8.6 X Hill Slope 0.9 to 2.0 0.5 1.1 0.7 0.6 X Test range 10

− 12 

# to 10

− 6 

# M 10

− 12 

# to 10

− 6 

# M X Corticosterone Test range 10

− 10 

# to 10

− 4 

# M 10

− 10 

# to 10

− 4 

# M X 17 α -methyltestosterone Log PC 50 

# − 6.0 to

− 5.1 -7.0 -8.8 -7.5 -6.5 X b 

# Log PC 10 

# − 8.0 to

− 6.2 -8.8 -9.8 -9.2 -9.2 X b 

# Test range 10

− 11 

# to 10

− 5 

# M 10

− 11 

# to 10

− 5 

# M X a Data were obtained from page 53 of the study report. b The Log PC 50 

# and Log PC 10 

# estimates were below the acceptable range, indicating increased sensitivity of the assay

system.



# C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls: Data for the vehicle and pos itive controls are included in Table 4. The overall mean TA value, expresse d as arbitrary light units, for the vehicle control was 1513-3035, and the overall mean TA value for the positiv e control was 1513- 3035. The induction for the positive control ra nged from 5.7- to 8.2-fold. The mean

# Estrogen Receptor Tran scriptional Activation (2012) / Page 6 of 8 TRIFLURALIN/036101 OCSPP 890.1300/ OECD 455


# normalized value for the positive control was 10912-16604. The PC 50 

# (50% of the maximum response) for E2 in this assay is 5456-8302 and the PC 10 

# (10% of the maximum response) is 1091-1660.

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

Sample Vehicle Control a 

# Positive Control a b 

# Normalized Positive Control bd


# Runs Mean SD Mean SD Fold Induction c 

# Mean SD
1 2743 248 15564 1604 5.7 12821 1605 2 1513 290 12425 1958 8.2 10912 1958 3 3035 271 19639 1116 6.5 16604 1116 4 2603 316 14761 860 5.7 12158 860 a Data were obtained from page 54 of the study report. b Positive control was 17 β -estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC) d Calculated by the reviewers from data on pages 38, 42, 46 and 50 of the study report.



# 2. Test Material: Relative (to the PC) transcriptional ac tivation at each concentration of the test chemical during the four a ssay runs is presented in Table 5. The concentration-response curves depicting fold induction of relative tran scriptional activation is presented in Figure 1 below. The mean RPC Max 

# was <0% for the first Run, but 13.7% - 18% for Runs 2, 3, and 4. The associated PC Max 

# was 10 -4 

# M. Because the RPC Max 

# ≥ PC 10 

# in three out of four runs, trifluralin was considered positive for estroge n receptor transcripti onal activation. The PC 50 

# was not calculable, but the PC 10 

# was 10 -5.5 

# to 10 -4.6 

# M.

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


# Parameter RTA (mean ± SD); % of Positive Control (PC) Run 1 Run 2 Run 3 Run 4 Conc. (M) Mean SD Mean SD Mean SD Mean SD 10

− 4 

# -0.6 1.4 18.0 1.8 16.6 4.5 13.7 2.7 10

− 5 

# -0.4 0.8 13.7 3.4 10.5 2.4 8.0 5.1 10

− 6 

# -2.3 2.6 6.1 5.0 6.3 1.4 5.6 4.5 10

− 7 

# -2.2 1.0 3.7 1.1 3.5 1.5 2.8 2.3 10

− 8 

# -5.4 1.5 2.5 2.0 0.9 1.5 2.6 1.8 10

− 9 

# -1.1 3.3 4.5 3.7 3.1 0.7 3.0 2.6 10

− 10 

# -1.1 4.5 2.9 2.1 2.1 3.5 4.1 0.6 Log EC 50 b 

# NA -5.3 -4.0 -5.1 Hill Slope b 

# NA 1.0 0.3 0.5 RPC Max 

# <0 18% 16.6% 13.7% PC Max 

# NA 10

− 4 

# 10

− 4 

# 10

− 4 

# PC 50 

# NA NA NA NA PC 10 

# NA 10

− 5.5 

# 10

− 5.1 

# 10

− 4.6 

# a Data were obtained from page 55 of the study report. b Report if appropriate NA = Not Applicable

# Estrogen Receptor Transcriptional Activation (2012) / Page 7 of 8 TRIFLURALIN/036101 OCSPP 890.1300/ OECD 455


# FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of Trifluralin Compared to the Positive Control.

VC= Vehicle Control PC= Positive Control (1 nM E2)



# 3. Performance Criteria: The laboratory proficiency assays were not reported. Performance criteria were met for corticosterone, and all values but the Hill Slopes for 17 β -estradiol and 17 α -estradiol, the log EC 50 

# for 17 β -estradiol, and the log PC 50 

# for 17 α -estradiol. The Hill slopes were consistently lower than the expect ed values in at leas t half of the runs. However, all other parameters were w ithin the acceptable range. The log PC 50 

# and log PC 10 

# for 17 α -methyltestosterone were consistently lowe r than the acceptable range, indicating an increased sensitivity of the assay to weak agonist s. Mean luciferase activity for the PC was >4-fold for all runs. The fold -induction corresponding to the PC 10 

# of the concurrent PC was greater than 1+2 standard devi ations of the vehicle control. Variability was minimal and results were reproducible among runs.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: Based on the combined responses in each of four independent estrogen receptor transactivation assays, it was determined that trifluralin treatment resulted in a weak ER-mediated transc riptional activation at two higher acceptable concentrations tested (10 -4 

# M and/or 10 -5 

# M).

# Estrogen Receptor Tran scriptional Activation (2012) / Page 8 of 8 TRIFLURALIN/036101 OCSPP 890.1300/ OECD 455



# B. AGENCY COMMENTS: The results of the laboratory pr oficiency tests were not reported. Performance criteria were met, and the slig ht deviations do not negatively impact the interpretation of the results. Trifluralin was tested up to the limit of solubility, 10 -4 

# M. The mean RPC Max 

# was <0% for the first Run, but 13.7% - 18% for Runs 2, 3, and 4. The associated PC Max 

# was 10 -4 

# M. Because the RPC Max 

# ≥ PC 10 

# in three out of four runs, trifluralin was considered positive for estroge n receptor transcripti onal activation. The PC 50 

# was not calculable but the PC 10 

# was 10 -5.5 

# to 10 -4.6 

# M. The response was dose-dependent and indicates that trifluralin is positive for estrogen receptor transcriptional activation in this test system.

C. STUDY DEFICIENCIES: The following deficiencies were noted:

 The results of the proficiency test were not provided.

 The status of hormone activity in the house-prepped DCC-FBS was not reported.

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

EPA MRID Number 48619706

Page 1 of 88 

# Data Requirement: EPA DP Barcode 427406

OECD Data Point

EPA MRID 48619706
EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test material: Trifluralin Purity: 97.1%

Common name

Chemical name: IUPAC CAS name: 2,6-Dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine.

CAS No.: 1582-09-8
Synonyms:

EPA PC Code: 036101

Primary Reviewer: Joan Gaidos Signature: Senior Scientist, Cambridge Environmental, Inc. Date: 04/27/2012

Secondary Reviewer: Teri S. Myers Signature:
Senior Scientist, Cambridge Environmental, Inc Date: 05/4/2012

Primary Reviewer: Amy Blankinship Signature:
Chemist, USEPA/OCSPP/OPP/EFED/ERB3 Date:

Additional Reviewer: Anita Ullagaddi Signature: No longer with EPA Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 06/13/2012

Final Additional Reviewer: Robin Sternberg Signature:
Wildlife Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 05/27/2015

Date Evaluation Completed: 05/27/2015 Digitally signed by ROBIN STERNBERG DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN STERNBERG, dnQualifier=0000039126 Date: 2015.06.02 13:11:49 -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 15:49:09 -04'00'

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

EPA MRID Number 48619706

Page 2 of 88 

# CITATION: Marino, T.A., K. Coady, E.L. McClymont, A.W. Perala, L.K. Sosinski, J. Thomas. 2011. Trifluralin:
A Fish Short-term Reproduction Assay with the Fathead Minnow, Pimephales promelas . Unpublished study performed by Toxicology & Environmental Research Consulting, The Dow Chemical Company, Midland,
Michigan. Lab Study No.: 101098. Study sponsored by Dow AgroSciences LLC, Indianapolis, Indiana.
Study completed September 8, 2011.

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

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

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

EPA MRID Number 48619706

Page 3 of 88 

# EXECUTIVE SUMMARY:

The 21-day short-term reproduction assay of trifluralin with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions. Adult fish (20 spawning groups; 2 males and 4 females in each group; 6 months old), were exposed to trifluralin (97.1% purity) at nominal concentrations of 0 (negative control), 0 (solvent control; 0.05 mL/L dimethylformamide; DMF), 0.000350, 0.00350, and 0.0350 mg a.i./L; time-weighted average (TWA), measured concentrations were <0.00003 (<LOQ, controls), 0.000195,
0.00198, and 0.0215 mg a.i./L. The test system was maintained at 24.5 to 25.6 o 

# C and a pH of 7.4 to 7.6.

Comparisons between the negative and solvent control groups revealed no significant differences (p>0.05) for
any endpoint. Unless otherwise indicated in this DER, all effects are reported based on comparison to the
negative control.

There was no significant effect (p>0.05) on fish mortality. Clinical signs of toxicity were present at the high
treatment level, with five males showing partial loss of equilibrium, very thin bodies, partial paralysis, and/or
a bent spine. The same number of females appeared to be bloated and/or partially paralyzed with bent tails. These clinical effects were in contrast to no more than two fish from the control groups appearing extremely dark in color or aggressive. Female body weight was significantly increased (p<0.05; Dunnett’s) by 10% in
the high treatment group compared to the negative control. Male body weight was not significantly different (p>0.05) from the negative control. Total length for males and females was not significantly different (p>0.05)
from the negative control.

Average spawning frequency and fecundity in controls were at least every 4 days (except for one replicate in the solvent control) and 36 eggs/female/reproductive day (range: negative control, 24.3-50.6; solvent control, 17.2-41.5 egg/female/reproductive day), respectively; fertilization success in both controls was 99%. Fertility was significantly reduced (p<0.05; Jonckheere-Terpstra) by 0.5 and 4% at the low and high treatment levels, respectively, relative to the negative control. Fecundity was significantly decreased (p<0.05; Mann Whitney U)
by 87% at the high treatment level compared to the negative control.

Male nuptial tubercle scores were significantly reduced (p<0.05; Jonckheere-Terpstra) (p<0.05) at the high treatment level (median tubercle score: 20) relative to negative control (median tubercle score: 31); no

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# tubercles were noted for females. There was a significant monotonic decrease (p<0.05; Jonckheere-Terpstra) of the male gonado-somatic index (GSI) at all treatment levels, ranging from 29% (low treatment) to 39% (high treatment) relative to the negative control. Female GSI was not significantly different (p>0.05) between
the negative control and treatment groups. Observations of additional secondary sexual characteristics were
reported but results were not presented.

Plasma vitellogenin (VTG) was not significantly different (p>0.05) for males or females. There was a significant increase (p<0.05; Jonckheere-Terpstra) of 78% in male 17 ȕ -estradiol at the high treatment level relative to the negative control. Male plasma testosterone (T) was not significantly different (p>0.05). Plasma 17 ȕ

estradiol and testosterone were not measured in females.

Male gonadal development was altered at the high treatment level with a greater proportion of testes categorized
at the late spermatogenic stage compared to the mid-spermatogenic stage of testes in control fish and those
exposed to lower levels of trifluralin. For females, there was increased incidence of severe increased oocyte
atresia in the high treatment group. Finally, there was an increase in minimal subacute multifocal ovarian
inflammation in females exposed to the high treatment level.

All test validity and performance criteria were met.

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: Male 4.0 g; Female 1.8 g
Mean length at test initiation: Not reported
Test Type: Flow-throughData Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page5of88Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Trifluralin.Treatment(mg a.i./L)[TWA-measured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.000195NoYesNoNoYesNoNoNoNoNoNoNANoNA0.00198NoNoNoNoYesNoNoNoNoNoNoNANoNA0.0215YesYesYesNoYesNoYesYesNoNoNoNAYesNAAbbreviations.E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified. DMF solvent (0.05 mL/L) was used instudy.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may beheavily weighted by the expert opinion of a board-certified pathologist.

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# I. MATERIALS AND METHODS

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

1. The total organic carbon in the dilution water was reportedly measured periodically, but the value was not provided; to satisfy the OCSPP 890.1350 performance requirement, this value must not exceed 2
mg/L.
2. The dilution water reportedly had <0.10 mg/L unionized ammonia content and <0.02 mg/L residual chlorine content. To satisfy the OCSPP 890.1350 performance criteria, ammonia content must be
<0.001 mg/L and residual chlorine must be <0.01 mg/L.
3. There was no confirmation that the flow splitting accuracy was within 10%. The study authors reported
that the test solution was gravity fed from a mixing chamber and split equally among replicate vessels

at an initial flow rate of 45±5 mL/min.

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

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality claims
statements were provided. This study was conducted in accordance with GLP Standards as published by the U.S. EPA (40 CFR Parts 160), OECD Principles of GLP [ENV/MC/CHEM(98)17], and European Community (EC; 2004).

A. TEST MATERIAL: Trifluralin, CAS No. 1582-09-8

Description: The test material was an orange solid and water solubility was reported to
be 0.2 to 0.4 mg/L at 25 q C.

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# OECD recommends describing water so lubility, melting/boili ng point stability in water and light, pKa,
Pow or Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : DKO-B916-33, TSN100656 (Lot No.)

Purity: 97.1% (originally determined January 22, 2007; recertification verified purity on January 23, 2011)

Impurities: None reported

Stability of Compound: Analytical verification of diluter stock fortified with 7.0, 70 and 700 mg a.i./L
yielded recoveries ranging from 87.2 to 95.3% of nominal concentrations.
Analytical verification of the test solutions at 0, 1, 4, 7, 11, 14, 18 and 21
yielded recoveries ranging from 46.3% to 85% of nominal concentrations,
with a pattern of decline toward test termination. The %CV for some individual replicate vessels exceeded 20% (ranging from 20.3 to 24.3%); however the overall CVs for each test concentration were 20% or lower (using daily average at each sample point).

Storage Conditions of

Test Chemicals: The stock solution was stored at ambient temperature in the dark. Storage
stability was determined after 5, 8, 12, 15, and 22 days; recoveries ranged from 70.4% (Day 22) to 115% (Day 15).Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page8of88B. Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead Minnow 

# EPA recommends fathead minnow (Pimephales

promelas).Species scientific name: 

# Pimephales promelasSpecies strain (if stated):Not reportedWere fish obtained from a singlelaboratory stock?YesNew England bioassay, Manchester,Connecticut. 

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

# EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive test.Acclimation period:2 weeks 

# EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page9of88ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Mortalities <5% during 7 days prior to preexposure period.Fish did not receive any treatment fordisease during acclimation period.No behavioral abnormalities or clinical signswere noted. 

# EPA recommends that mortality during the 7 days prior to the pre -exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPA recommends that the fish be rejected. If mortality is between 5 - 10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur during this extended acclimation period, EPA recommends that the fish not be used.Type of food:Frozen brine shrimp 

# EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body condition.Source of food:Not reportedFrequency of feeding:Frozen shrimp: 2times/dayDetails on feeding:2.5-3.5 mL frozen (thawed) brineshrimp/vessel.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page10of88Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:4.5-6 months 

# EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at testinitiation (if determined):4.0 gBased on 64 males used to stock aquariafor pre-exposure phase. 

# EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for

each sex.Range of individual weights(males) at test initiation (ifdetermined):± 0.4 g (CV = 9.3%)Individual weights within ±20% of theestimated mean.Mean weight of females at testinitiation (if determined):1.8 gBased on 128 females used to stock aquariafor pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):± 0.2 (CV = 9.7%)Individual weights within ±20% 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 TrifluralinEPA MRID Number 48619706Page11of88ParameterValue(s)Details or RemarksGuideline RecommendationsWere pre-exposure conditionsidentical to the definitive test?Yes 

# EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:32 tanks 

# EPA recommends that additional tanks set up at the beginning of pre - exposure will ensure that sufficient replicates with the correct sex ratio are available for the definitive test.Number of males per tank:2 males/tankNumber of females per tank:4 females/tankPre-exposure fecundity:>15 eggs/female/reproductive day/replicate 

# EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Number of spawns during preexposure:>2 times in 7 daysSpawning occurred at least every 3 days. 

# EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Details on pre-exposure:None.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page12of88C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through 

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

# , the use of a static renewal system is not recommended for this

assay.Type of flow-through dilutionsystem:Continuous flowproportional diluter 

# Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:Exposure: 99 ±9.9 mL/minInitial flow: 45 ± 5 mL/minEquilibrium phase (prior to addition of fish): 90± 9 mL/min. 

# Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, D.C.(http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page13of88ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems:Solution from mixing chamber split equallyamong replicate test vessels.Treatment recoveries prior to test initiation werenot reported.The flow splitting accuracy was not reported.

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

10%.Aeration?No 

# EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (<60% saturation).Source of dilution water:Lake Huron waterDilution water was limed and flocculated andpumped to the laboratory prior to municipaltreatment.Prior to use, the water was land-filtered, pHadjusted (gaseous CO2), carbon-filtered andUV-irradiated. 

# EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated in the test

guideline.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page14of88ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzed forpesticides, heavy metals, andother contaminants?YesAll potential contaminants were confirmed to bebelow the detection limit.Test vessel type/materials:Glass aquaria with stainlesssteel screen drains

# 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:20 cm wide x 39 cm longx25 cm high; solutiondepth 13 cm. 

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

# EPA recommends 10 L solution per tank.Spawning substrate material:PVC pipe cut and sectionedlengthwise and inverted toform a semicircular archand positioned on stainlesssteel trays. 

# EPA recommends that each tank contain

three semi - circular spawning substrates, e.g., aged PVC pipe, 10 - 20 cm in length, split

lengthwise.Spawning substrate size:9 cm long and 10 cmdiameter positioned on 13cm long tray.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page15of88ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details on exposuresystem:None.Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsTemperature (°C)24.5 25.6 25.0 Continuous 

# EPA recommends temperature 25±1 o

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

# C.pH7.47.67.51Weekly 

# EPA recommends pH 6.5 to 9.0.Dissolvedoxygen (mg/L)6.88.57.81Weekly 

# EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3)28 42 351Weekly 

# EPA recommends total alkalinity >20 mg/L

as CaCO 3.Hardness (mg/L as CaCO3)4470621WeeklyTotalorganic carbon (mg/L)Not reportedNot reportedNot reportedNot reported 

# EPA recommends that total organic carbon in dilution water be <2 mg/L.Unionized ammonia (Pg/L)Not reportedNot reported<100 (as N)Once 

# 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 TrifluralinEPA MRID Number 48619706Page16of88ParameterMinimumMaximumMeanMeasurement IntervalGuideline RecommendationsResidual chlorine (Pg/L)Not reportedNot reported<20 Once 

# EPA recommends that residual chlorine in dilution water be <10 P g/L.Conductivity (Pmhos/cm) 132 238 182 Weekly 

# General recommendations for frequency of measurements: EPA recommends that temperature, pH, and dissolved oxygen be measured in all test tanks at least weekly and that hardness and alkalinity be measured in controls and in one tank at the highest test concentration at least weekly. In addition, continuous temperature monitoring of at least one tank is encouraged.1 Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levelsData Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page17of88D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?No 

# EPA recommends conducting a range-finder if

96 -hour LC

50 

# data for the fathead minnow are

unavailable.If yes, what was the method fordetermining the highest testconcentration in the range-finder?NA 

# EPA recommends that the highest test concentration be selected based on toxicity data

for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is

appropriate.Species:NALife stage:NA 

# EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in the test.Test duration:NA 

# EPA recommends a 96-hour exposure.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page18of88ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:NA 

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

50, 

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

test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page19of88Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days 

# EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in the definitivetest:The acute toxicity 96-hour LC50for trifluralinwith fathead minnow is0.105 mg/L.Selected based on 1/3 of the 96-hour LC50(0.105 mg a.i./L). 

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

hour LC 50 

# ), whichever concentration is lowest.Reference study citation (ifapplicable):Johnson and Finley, 1980Separation of test concentrations:Ten-fold 

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

# .Number of test concentrations:5 (3 treated, a negativeand a solvent control) 

# EPA recommends a minimum of 3 concentrations and a control, plus solvent control

if appropriate.Are nominal concentrationsadjusted for purity?YesData Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page20of88ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Time-weighted AverageLimit of quantification (LOQ):0.00003 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, 1, 4, 7, 11, 14 and 21days 

# It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two replicates per treatment level.Was the randomized completeblockdesign used?Yes 

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

# EPA recommends 4 replicates.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page21of88ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in solventcontrol (if applicable):4 

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

# EPA recommends 4 replicates.Number of male fish per replicateat test initiation:2 

# EPA recommends 2 males per replicate.Number of female fish per replicateat test initiation:4 

# EPA recommends 4 females per replicate.Was a solvent used?YesSolvent type (if applicable):N,N-dimethylformamide(DMF)Maximum solvent concentration (ifapplicable):0.05 mL/LThe maximum solvent concentrationexceeded the recommended maximum (0.02ml/L) 

# EPA recommends that the solvent not exceed

0.02 ml/L3 

# . OECD recommends that solvent have no effect on survival nor produce any other3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page22of88ParameterValue(s)Details or RemarksGuideline Recommendations 

# adverse effects and that concentration not be greater than 0.1 ml/L4 

# .Was a positive control used?NoPositive control (if applicable):NAPositive control concentration(s) (ifapplicable):NAPhotoperiod:16 hrs light:8 hrs dark 

# EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’s surface:589 to 866 lux 

# EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:Additional details about test solutionappearance during the study did not appearto be reported in study.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testingand Assessment. No. 23. Paris, France.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page23of88Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Trifluralin.Treatment IDNominalConcentration(mg a.i./L)Time-WeightedMeanMeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0<LOQNA 

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

# 20%.Solvent ControlNA<LOQNATreatment 10.000350.00019520.2Treatment 20.00350.0019816.8Treatment 30.0350.021515.2Abbreviations:CVCoefficient of variation.LOQ=0.000030 mg a.i./L.

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# E. Observations

Biological Endpoints: Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination (Day 21), secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal
staging and histopathology, plasma vitellogenin, and male plasma sex steroids (testosterone and 17 ȕ -estradiol) were evaluated.

Were raw (individual) data provided? Yes

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical signs occur at least daily. At test termination (Day 21), additional
observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma vitellogenin, and plasma sex steroids (testosterone and 17 ȕ -estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination.

Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding,

and other abnormal behavior.

I. RESULTS AND DISCUSSION

A. Results

No males died during the study. One female died in each of the negative control, 0.000195 and
0.00198 mg a.i./L treatment groups. Remaining groups exhibited 100% survival. Overall mean survival
values were 95.8, 100, 95.8, 95.8 and 100% at the negative control, solvent control, and TWAmeasured 0.000195, 0.00198, and 0.0215 mg a.i./L treatment levels, respectively.

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

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

n 1 

# # Surviving % Survival n # Surviving % Survival

Negative Control (<LOQ) 8 8 100 16 15 94

Solvent Control (<LOQ) 8 8 100 16 16 100

0.000195 8 8 100 16 15 94

0.00198 8 8 100 16 15 94

0.0215 8 8 100 16 16 100

1 

# Total number of fish at test initiation.
LOQ=0.000030 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page26of88Mean male body weight values were 4.19, 4.56, 4.14, 4.41 and 3.65 g and female body weight values were 1.80, 1.74, 1.67, 1.80 and 1.97 g inthe negative control, solvent control, and TWA-measured 0.000195, 0.00198, and 0.0215 mg a.i./L treatment levels, respectively.Mean male bodylength values were 54.8, 55.0, 53.5, 56.7, and 53.3 mm and female body length values were 44.5, 43.8, 43.9, 44.3 and 43.8 mm inthe negativecontrol, solvent control, and TWA-measured 0.000195, 0.00198, and 0.0215 mg a.i./L treatment levels, respectively.Table 10: Size at Test Termination in Fathead Minnow ( 

# Pimephales promelas).Treatment(mg a.i./L)[TWA-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDNegative Control(<LOQ)44.190.0704 1.800.072454.82.17444.50.12Solvent control(<LOQ)44.560.3664 1.740.161455.01.82443.80.690.00019544.140.38041.670.014453.53.06443.90.410.0019844.410.50641.800.045456.73.46444.31.460.021543.650.24041.9710.165453.31.23443.81.38LOQ=0.000030 mg a.i./L.

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# Mean fecundity values were 37.3, 33.8, 34.3, 38.8 and 4.85 and fertilization success was 99.0, 99.2,
98.5, 98.9 and 95.0% in the negative control, solvent control, and TWA-measured 0.000195, 0.00198,
and 0215 mg a.i./L treatment levels, respectively.

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

.

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

# Fertilization Success (%) 2



# Mean ± SD Mean ± SD

Negative Control (<LOQ) 37.3 12.9 99.0 0.54

Solvent Control (<LOQ) 33.8 11.4 99.2 0.05

0.000195 34.3 6.95 98.5 0.62

0.00198 38.8 5.97 98.9 0.38

0.0215 4.85 4.07 95.0 3.84

LOQ=0.000030 mg a.i./L.

1 

# Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate.

2 

# Fertilization success (%) is calculated as the number of embryos divided by the number of eggs, multiplied by 100.

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# Median male tubercle scores were 31, 32, 31, 31, and 20 in the negative control), solvent control, and
TWA-measured 0.000195, 0.00198, and 0.0215 mg a.i./L treatment levels, respectively. None of the
surviving females were found to have tubercles.

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

.

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

n Median Tubercle

Score n Median Tubercle

Score

Negative Control (<LOQ) 4 31 4 0

Solvent Control (<LOQ) 4 32 4 0

0.000195 4 31 4 0

0.00198 4 31 4 0

0.0215 4 20 4 0

LOQ=0.000030 mg a.i./L.

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# Mean male GSI was 1.10, 1.14, 1.42, 1.50 and 1.53 and mean female GSI was 12.3, 12.4, 11.6, 13.6
and 13.3 in the negative control, solvent control, and TWA-measured 0.000195, 0.00198, and 0.0215
mg a.i./L treatment levels, respectively.

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

.

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

n Mean GSI 1


# (%) ±SD n Mean GSI 1


# (%) ±SD

Negative Control (<LOQ) 4 1.10 0.131 4 12.3 1.35

Solvent Control (<LOQ) 4 1.14 0.084 4 12.4 1.99

0.000195 4 1.42 0.163 4 11.6 3.14

0.00198 4 1.50 0.240 4 13.6 1.97

0.0215 4 1.53 0.123 4 13.3 2.03

LOQ=0.000030 mg a.i./L.

1 

# Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100.

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# Median male gonadal stage was 2, 2, 2, 2, and 3 and median female gonadal stage was 3, 3, 3, 3, and
3 in the negative control, solvent control, and TWA-measured 0.000195, 0.00198, and 0.0215 mg a.i./L
treatment levels, respectively.

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

.

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

n Median Stage 1 

# n Median Stage 2



# Negative Control (<LOQ) 4 2.0 4 3.0

Solvent Control (<LOQ) 4 2.0 4 3.0

0.000195 4 2.0 4 3.0

0.00198 4 2.0 4 3.0

0.0215 4 3.0 4 3.0

LOQ=0.000030 mg a.i./L.

1 

# The guideline recommends the following gonadal staging scale for male fathead minnow: 0=undeveloped, 1=early
spermatogenic, 2=mid-spermatogenic, 3=late spermatogenic, 4=spent.

2 

# The guideline recommends the following gonadal staging scale for female fathead minnow: 0=undeveloped, 1=early
development, 2=mid-development, 3=late development, 4=late development/hydrated, 5=post-ovulatory.



# There was one finding of increased proportion of spermatogonia and one finding of granulomatous inflammation
in males exposed to the 0.0215 mg a.i./L level, compared to no incidents at any other level or in the control
groups. Multifocal duct mineralization did not appear to show any treatment-related pattern, with two instances
in the negative control, none in the solvent control, and only one instance at the 0.0215 mg a.i./L level. There
was one incident each of minimal to mild aggregation of histiocytic cells in the 0.0215 and 0.00198 mg a.i./L
levels, respectively, compared to no incidents in either control group. The study authors did not recognize any of these histopathological effects in males as being treatment related.

In females, there was one minimal finding of aggregation of macrophages and one incident of minimal inflammation of focal granulomatous, compared to no instances in either control or the lower treated conditions. The incidents of minimal inflammation of multifocal granulomatous were greater in the negative control (4/15)

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# than in the solvent control or trifluralin-treated groups (  2). The study authors recognized a higher incidence of minimal subacute multifocal inflammation (9/16) and oocyte atresia (mild to severe; 12/16) at the 0.0215
mg a.i./L level, compared to the controls indicating treatment-related effects.

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

.

Treatment (mg a.i./L)

[TWAmeasured] Diagnostic Observations 1



# Severity Increased Proportion of
Spermatogonia Presence of

Testis-Ova Increased

Testicular
Degeneration Interstitial Cell

Fibrosis Germinal
Epithelium,

Atrophy

n Incidence n Incidence n Incidence n Incidence n Incidence

Negative

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

1 8 0 NA NA 8 0 NA NA NA NA

2 8 0 NA NA 8 0 NA NA NA NA

3 8 0 NA NA 8 0 NA NA NA NA

4 8 0 NA NA 8 0 NA NA NA NA

Solvent

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

1 8 0 NA NA 8 1 NA NA NA NA

2 8 0 NA NA 8 0 NA NA NA NA

3 8 0 NA NA 8 0 NA NA NA NA

4 8 0 NA NA 8 0 NA NA NA NA

0.000195 0 8 8 NA NA 8 8 NA NA NA NA

1 8 0 NA NA 8 0 NA NA NA NA

2 8 0 NA NA 8 0 NA NA NA NA

3 8 0 NA NA 8 0 NA NA NA NA

4 8 0 NA NA 8 0 NA NA NA NA

0.00198 0 8 8 NA NA 8 7 NA NA NA NA

1 8 0 NA NA 8 1 NA NA NA NA

2 8 0 NA NA 8 0 NA NA NA NA

3 8 0 NA NA 8 0 NA NA NA NA

4 8 0 NA NA 8 0 NA NA NA NA

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[TWAmeasured] Diagnostic Observations 1



# Severity Increased
Proportion of Spermatogonia Presence of

Testis-Ova Increased

Testicular
Degeneration Interstitial Cell

Fibrosis Germinal
Epithelium,

Atrophy

n Incidence n Incidence n Incidence n Incidence n Incidence

0.0215 0 8 7 NA NA 8 8 NA NA NA NA

1 8 1 NA NA 8 0 NA NA NA NA

2 8 0 NA NA 8 0 NA NA NA NA

3 8 0 NA NA 8 0 NA NA NA NA

4 8 0 NA NA 8 0 NA NA NA NA

Abbreviations: NA 

# Not applicable.
LOQ=0.000030 mg a.i./L.

1 

# Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal,
2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.



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

.

Treatment (mg a.i./L)
[TWA-measured] Diagnostic Observations 1



# Severity Multifocal Duct

Mineralizaton Aggregates of Histiocytic Duct Cells

n Incidence n Incidence

Negative Control (<LOQ) 0 8 6 8 8

1 8 2 8 0

2 8 0 8 0

3 8 0 8 0

4 8 0 8 0

Solvent Control (<LOQ) 0 8 8 8 8

1 8 0 8 0

2 8 0 8 0

3 8 0 8 0

4 8 0 8 0

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[TWA-measured] Diagnostic Observations 1



# Severity Multifocal Duct

Mineralizaton Aggregates of
Histiocytic Duct Cells

n Incidence n Incidence

0.000195 0 8 5 8 8

1 8 3 8 0

2 8 0 8 0

3 8 0 8 0

4 8 0 8 0

0.00198 0 8 6 8 7

1 8 2 8 0

2 8 0 8 1

3 8 0 8 0

4 8 0 8 0

0.0215 0 8 7 8 7

1 8 1 8 1

2 8 0 8 0

3 8 0 8 0

4 8 0 8 0
LOQ=0.000030 mg a.i./L.

1 

# Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page35of88Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow ( 

# Pimephales promelas).Treatment(mg a.i./L)[TWA-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control(<LOQ)0NANANANANANANANA881NANANANANANANANA802NANANANANANANANA803NANANANANANANANA804NANANANANANANANA80Solvent Control(<LOQ)0NANANANANANANANA881NANANANANANANANA802NANANANANANANANA803NANANANANANANANA804NANANANANANANANA800.0001950NANANANANANANANA881NANANANANANANANA802NANANANANANANANA803NANANANANANANANA804NANANANANANANANA80Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page36of88Treatment(mg a.i./L)[TWA-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.001980NANANANANANANANA881NANANANANANANANA802NANANANANANANANA803NANANANANANANANA804NANANANANANANANA800.02150NANANANANANANANA871NANANANANANANANA812NANANANANANANANA803NANANANANANANANA804NANANANANANANANA80Abbreviations:NANot applicable.LOQ=0.000030 mg a.i./L.1Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe test guideline for reference.

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

.

Treatment (mg a.i./L)

[TWAmeasured] Additional Diagnostic Observations 1



# Severity Increased Oocyte

Atresia Perifollicular Cell
Hyperplasia/
Hypertrophy Decreased Yolk

Formation Infection,
Microsporidia

n Incidence n Incidence n Incidence n Incidence

Negative Control (<LOQ) 0 15 5 NA NA NA NA NA NA

1 15 6 NA NA NA NA NA NA

2 15 0 NA NA NA NA NA NA

3 15 2 NA NA NA NA NA NA

4 15 2 NA NA NA NA NA NA

Solvent Control (<LOQ) 0 16 5 NA NA NA NA NA NA

1 16 6 NA NA NA NA NA NA

2 16 1 NA NA NA NA NA NA

3 16 3 NA NA NA NA NA NA

4 16 1 NA NA NA NA NA NA

0.000195 0 15 6 NA NA NA NA NA NA

1 15 4 NA NA NA NA NA NA

2 15 2 NA NA NA NA NA NA

3 15 0 NA NA NA NA NA NA

4 15 3 NA NA NA NA NA NA

0.00198 0 15 8 NA NA NA NA NA NA

1 15 5 NA NA NA NA NA NA

2 15 0 NA NA NA NA NA NA

3 15 0 NA NA NA NA NA NA

4 15 2 NA NA NA NA NA NA

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[TWAmeasured] Additional Diagnostic Observations 1



# Severity Increased Oocyte

Atresia Perifollicular Cell
Hyperplasia/
Hypertrophy Decreased Yolk

Formation Infection,
Microsporidia

n Incidence n Incidence n Incidence n Incidence

0.0215 0 16 1 NA NA NA NA NA NA

1 16 3 NA NA NA NA NA NA

2 16 6 2 

# NA NA NA NA NA NA

3 16 0 NA NA NA NA NA NA

4 16 6 2 

# NA NA NA NA NA NA

Abbreviations: NA 

# Not applicable.
LOQ=0.000030 mg a.i./L.

1 

# Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

2 

# Effects interpreted by the study authors to be treatment-related.



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

.

Treatment (mg a.i./L)

[TWAmeasured] Diagnostic Observations 1



# Severity Subacute Multifocal

Inflammation Aggregates of
Macrophages

n Incidence n Incidence

Negative Control (<LOQ) 0 15 12 15 15

1 15 3 15 0

2 15 0 15 0

3 15 0 15 0

4 15 0 15 0

Solvent Control (<LOQ) 0 16 10 16 16

1 16 6 16 0

2 16 0 16 0

3 16 0 16 0

4 16 0 16 0

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[TWAmeasured] Diagnostic Observations 1



# Severity Subacute Multifocal

Inflammation Aggregates of
Macrophages

n Incidence n Incidence

0.000195 0 15 11 15 15

1 15 4 15 0

2 15 0 15 0

3 15 0 15 0

4 15 0 15 0

0.00198 0 15 12 15 15

1 15 3 15 0

2 15 0 15 0

3 15 0 15 0

4 15 0 15 0

0.0215 0 16 7 16 15

1 16 9 2 

# 16 1

2 16 0 16 0

3 16 0 16 0

4 16 0 16 0
LOQ=0.000030 mg a.i./L.

1 

# Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.

2 

# Effects interpreted by the study authors to be treatment-related.

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

.

Treatment (mg a.i./L)

[TWAmeasured] Additional Diagnostic Observations 1



# Severity Interstitial Fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

Negative

Control (<LOQ) 0 NA NA NA NA 15 11 NA NA

1 NA NA NA NA 15 4 NA NA

2 NA NA NA NA 15 0 NA NA

3 NA NA NA NA 15 0 NA NA

4 NA NA NA NA 15 0 NA NA

Solvent Control (<LOQ)) 0 NA NA NA NA 16 15 NA NA

1 NA NA NA NA 16 1 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

0.000195 0 NA NA NA NA 15 14 NA NA

1 NA NA NA NA 15 1 NA NA

2 NA NA NA NA 15 0 NA NA

3 NA NA NA NA 15 0 NA NA

4 NA NA NA NA 15 0 NA NA

0.00198 0 NA NA NA NA 15 14 NA NA

1 NA NA NA NA 15 1 NA NA

2 NA NA NA NA 15 0 NA NA

3 NA NA NA NA 15 0 NA NA

4 NA NA NA NA 15 0 NA NA

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[TWAmeasured] Additional Diagnostic Observations 1



# Severity Interstitial Fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence

0.0215 0 NA NA NA NA 16 13 NA NA

1 NA NA NA NA 16 3 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. LOQ=0.000030 mg a.i./L

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# Mean male VTG was 5.28x10 3 

# , 4.78x10 3 

# , 7.58x10 3 

# , 1.97x10 3 

# and 2.44x10 3 

# ng/mL and female VTG was

42.98x10 6 

# , 38.50x10 6 

# , 48.33x10 6 

# , 44.04x10 6 

# and 44.24x10 6 

# ng/mL in the negative control, solvent
control, and TWA-measured 0.000195, 0.00198, and 0.0215 mg a.i./L treatment levels, respectively.

Table 19: Plasma Vitellogenin in Fathead Minnow ( Pimephales promelas)

.

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

Males Females

n Mean (ng/mL plasma) ±SD n Mean (ng/mL plasma) ±SD

Negative Control (<LOQ) 4 5.28 x 10 3 

# 4.19 x 10 3 

# 4 42.98 x 10 6 

# 16.11 x 10 6



# Solvent Control (<LOQ) 4 4.78 x 10 3 

# 3.67 x 10 3 

# 4 38.50 x 10 6 

# 14.22 x 10 6



# 0.000195 4 7.58 x 10 3 

# 7.62 x 10 3 

# 4 48.33 x 10 6 

# 33.44 x 10 6



# 0.00198 4 1.97 x 10 3 

# 0.186 x 10 3 

# 4 44.04 x 10 6 

# 14.31 x 10 6



# 0.0215 4 2.44 x 10 3 

# 1.48 x 10 3 

# 4 44.24 x 10 6 

# 20.51 x 10 6



# LOQ=0.000030 mg a.i./L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page43of88Mean male plasma testosterone values were 9.86, 4.49, 5.88, 9.04 and 7.81 ng/L and mean male plasma 17ȕ-estradiolwas 0.165, 0.125, 0.178, 0.225and 0.293 ng/mL in the negative control, solvent control, and TWA-measured 0.000195, 0.00198, and 0215 mg a.i./L treatment levels, respectively. Femaleplasma testosterone and 17ȕ-estradiol were not reported in the study and no explanation was given for the absence of these measures, except that both maleand female sex steroid concentrations were an optional guideline parameter.Table 20: Plasma Sex Steroids in Fathead Minnow ( 

# Pimephales promelas).Treatment(mg a.i./L)[TWA-measured]Plasma Testosterone (T)Plasma 17ȕ-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDControl (<LOQ)49.869.85NANANA40.1650.0477NANANASolvent control (<LOQ)44.492.89NANANA40.1250.000NANANA0.00019545.884.616NANANA40.1780.0610NANANA0.0019849.045.060NANANA40.2250.0942NANANA0.021547.813.342NANANA40.2930.1034NANANALOQ=0.000030 mg a.i./L.Abbreviations:NANot applicable.SDStandard deviation.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page44of88There appeared to be an increase in clinical signs of overt toxicity at the 0.0215 mg a.i./L treatment level in both males andfemales. There was nomention in the study report about the dorsal nape pad of fish.Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow ( 

# Pimephales promelas).Treatment(mg a.i./L)[TWA-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceTypenIncidenceNegative Control(<LOQ)Extremely dark, dead82 Aggressive161Solvent Control(<LOQ)Extremely dark81Bent tail, aggressive1620.000195None80dead1610.00198None80Missing eye, dead1620.0215Partial loss of equilibrium and partialparalysis, extremely dark, bent spine, verythin851Bloated, partial paralysis with bent tail, benttail, bloated, bent tail1651LOQ=0.000030 mg a.i./L.1Effects interpreted by the study authors to be treatment-related.

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# B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, length, tubercle score, GSI, fertility (% eggs that were fertile), fecundity (#eggs/female/reproductive day), VTG and male testosterone and male 17 ȕ

estradiol. Data for growth, tubercle score, GSI, and VTG were analyzed separately by sex. For all parameters the negative and solvent control groups were compared using a Student’s t-test ( Į =0.05);
no difference was detected for any endpoint, so trifluralin-treatment conditions were compared to the
pooled control.

Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant effects were detected for p<0.05 unless otherwise noted (statistical program used was not reported). Effects on survival were determined using a Fisher’s exact test (with Bonferroni-Holm adjustment).
Effects on length, weight, female GSI, fecundity, VTG and male testosterone were determined byANOVA and a one-tailed Dunnett’s test. Effects on fertility were determined using a Mann-WhitneyWilcoxin U test. Effects on male GSI, tubercle score and male estradiol were determined using ANOVA
and a Jonckheere-Terpstra test, while effects on fertility were determined using ANOVA and Wilcoxon (with Bonferroni-Holm adjustment). Prior to Dunnett’s, data were analyzed by Shapiro-Wilk’s test and Levene’s to test for normality and homogeneity of variances, respectively. If normality and homogeneity tests passed (p>0.01), a parametric analysis was performed. If non-normality or unequal variance were indicated (p<0.01), a non-parametric analysis was performed on the ranks of the data. These
methods appear to be consistent with the methods recommended in the guideline.

Adult survival (range 94-100%) was not significantly different compared to the controls (97%). There
was a statistically significant increase in male GSI at all treatment levels compared to the pooled control (p<0.05; Jonckheere-Terpstra test). There was a statistically significant decrease in fertilization success at 0.000195 and 0.0215 mg a.i./L treatment levels compared to the pooled control (p<0.05; Mann-Whitney-Wilcoxin U test). There was also a statistically significant decrease in fecundity in the 0.0215 mg a.i./L treatment group compared to the pooled control (p<0.05; Dunnett’s test). There
was a statistically significant decrease in male weight and increase in female weight in the 0.0215 mg a.i./L treatment group compared to the pooled control (p<0.05; Dunnett’s test). There was also a
statistically significant increase in male 17 ȕ -estradiol in the 0.0215 mg a.i./L treatment group compared

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# to the pooled control (p<0.05; Dunnett’s test). Although not analyzed statistically, there were generalized overt signs of toxicity in both males and females (i.e., partial loss of equilibrium, very thin bodies, partial paralysis, bent spine, bloated, and bent tail), as well as, increased oocyte atresia and
subactute multifocal inflammation in ovaries in females and increased proportion of mature spermatozoa
in males in the 0.0215 mg a.i./L treatment group. The study authors also noted a delay in male gonadal stage development at the 0.0215 mg a.i./L concentration (late spermatogenic), relative to the control groups (mid-spermatogenic).

C. Reviewer’s Analysis and Conclusions

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via
Toxstat 3.5. Female and male weight, female and male length, female and male mean vitellogenin (VTG), female gonadal somatic index (GSI), male testosterone, and fecundity 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 (i.e., female and male body weight, male body length, female VTG, female GSI, and male testosterone were then analyzed using the parametric Dunnett’s test. Data that failed parametric assumptions (i.e., female body length, male VTG, and fecundity) were analyzed
using the non-parametric Mann-Whitney U test. Male GSI, fertility, and male 17 ȕ -estradiol data
exhibited monotonic trends and were analyzed using the non-parametric Jonckheere-Terpstra test, as recommended by the OCSPP 890.1350 guideline. While the response for fertility was not strictly
linear, the response pattern indicated a concentration-dependent reduction. The reviewer additionally compared the negative and solvent control groups using a 2-tailed Student’s t-test and detected no significant differences (p>0.05) for any parameter. The reviewer compared all trifluralin-treated groups
to the negative control group.

Male GSI exhibited a concentration-dependent monotonic increasing trend ranging from 29% to 39%, compared to the negative control, at all treatment levels (p<0.05; Jonckheere-Terpstra). Male tubercle
scores were significantly reduced 33% and fertility was significantly reduced 4% at the 0.0215 mg a.i./L treatment, compared to the negative control (p<0.05; Jonckheere-Terpstra). The reviewer’s analysis of fertility also indicated a slight (0.5%), but significant reduction (p<0.05), relative to the
negative control, at the lowest treatment level, 0.000195 mg a.i./L. While the response was not

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# strictly linear and the 0.00198 mg a.i./L level was not significantly different from the negative control (p>0.05), the reviewer relied on the Jonckheere-Terpstra test results for this endpoint because fertility at the two lower concentrations did not appreciably differ from one another (0.1 vs 0.5% difference from the negative control), making the overall response appear concentration-dependent. Male 17 ȕ

estradiol showed a significant increase of 78% in the 0.0215 mg a.i./L treatment, compared to the negative control (p<0.05; Jonckheere-Terpstra). Fecundity was significantly reduced 87% at the
highest treatment.

Histopathology, gonadal staging, and secondary sex characteristics were adversely indicated at the 0.0215 mg a.i./L level, but these effects were not statistically analyzed. Male fish at the 0.0215 mg a.i./L level appeared to be very thin, displaying partial loss of equilibrium, partial paralysis, and/or
bent spines. There was no mention of dorsal nape pad effects. Gonadal development was altered,
with testes appearing to be in the late spermatogenic stage, as opposed to the mid-spermatogenic
stage in the controls and lower treated levels. Male histopathology revealed individuals with increased proportion of spermatogonia, aggregation of histiocyctic duct cells, and granulomatous inflammation at
the 0.0215 mg a.i./L level, compared to no instances observed in the controls or lower treatment levels. Female fish at the 0.0215 mg a.i./L level appeared to be bloated with partial paralysis and/or
bent tails; no female fish displayed these signs in the control groups or lower treatment levels.
Examination of female histopathology revealed a greater number of individuals displaying subacute
multifocal inflammation, aggregation of macrophages, and severe oocyte atresia, relative to female fish from the controls and lower trifluralin-treated levels. Growth was unaffected in male fish (p>0.05),
while female fish body weights were increased an average of 10% at the 0.0215 mg a.i./L, relative to the negative control female body weights.

Conclusions:

Male GSI followed a concentration-dependent increase (p<0.05) of 29%-39% at all treatment levels,
compared to the negative control. Additionally, male tubercle scores were significantly reduced (p<0.05) by 33% and male 17 ȕ -estradiol was 78% higher (p<0.05) in the 0.0215 mg a.i./L
treatment group. Development of male testes in the 0.0215 mg a.i./L level was increased with a
higher proportion of mature spermatozoa. Males from the 0.0215 mg a.i./L level appeared very thin

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# with partial loss of equilibrium, partial paralysis, and/or bent spines. Fecundity was significantly reduced 87% in the 0.0215 mg a.i./L group. Fertility was slightly (0.5 and 4%), but significantly (p<0.05)
reduced at the 0.000195 and 0.0215 mg a.i./L levels and females from the 0.0215 mg a.i./L level
appeared bloated with partial paralysis and/or bent tails. There were clearly more females displaying severe oocyte atresia, subacute multifocal inflammation, at the 0.0215 mg a.i./L level, relative to the control groups. Finally, there was an effect on female growth, with a significant increased (10%; p<0.05) in female body weight at the 0.0215 mg a.i./L treatment level.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page49of88Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( 

# Pimephales promelas)in the FSTRA with Trifluralin.Treatment(mg a.i./L)[TWA-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.p% Diff.p%Diff.pNegative Control(<LOQ)31 NA 0 NANo 0 NA 0 NA 0 NASolvent Control(<LOQ)32 0.7144.540.586No -9.530.863-54.460.336-24.240.1450.000195310.50029.290.022No43.340.889-40.340.7017.580.2690.00198310.38536.330.009No-62.580.067-8.290.99536.520.1120.0215200.02539.060.006Yes-53.720.235-20.820.93677.580.009Statistical TestJonckheereJonckheere-TerpstraNAMann-Whitney UDunnett’sJonckheere-TerpstraAbbreviations:.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.LOQ=0.000030 mg a.i./L.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Effects are considered statistically significant at p<0.05.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page50of88Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( 

# Pimephales promelas)in the FSTRA with Trifluralin.Treatment(mg a.i./L)[TWAmeasured]FecundityFert. SuccessTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.pMedianp%Diff.pEffect?(Yes/No)% Diff.p%Diff.p% Diff.pNegative Control(<LOQ)0 NA 0 NA 0NA0NANo 0 NANANANANASolvent Control(<LOQ)-9.380.7050.200.4350NA0.810.920No-10.420.691NANANANA0.000195-7.920.886-0.530.0410NA-5.270.954No12.440.974NANANANA0.001984.030.857-0.080.2770NA11.020.726No2.451.000NANANANA0.0215-86.980.029-4.020.0120NA7.960.867Yes2.941.000NANANANAStatistical TestMann-Whitney UJonckheereTerpstraNA Dunnett’s NA Dunnett’s NA NAAbbreviations.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.LOQ=0.000030 mg a.i./L.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regarding histopathologymay be heavily weighted by the expert opinion of a board-certified pathologist.2Effects are considered statistically significant at p<0.05.

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# Table 24: Growth Endpoints 1,2 

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

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

Males Females Males Females

% Diff. p % Diff. p % Diff. p % Diff. p
Negative Control (<LOQ) 0 NA 0 NA 0 NA 0 NA

Solvent control (<LOQ) 8.83 0.095 -3.33 0.536 0.55 0.839 -1.79 0.069

0.000195 -1.19 0.993 -7.37 0.149 -2.33 0.832 -1.40 0.061

0.00198 5.37 0.681 0.00 1.000 3.61 0.595 -0.51 1.000

0.0215 -12.83 0.110 9.87 0.045 -2.56 0.793 -1.57 1.000

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

Abbreviations: Diff. 

# Difference. NA 

# Not applicable. LOQ=0.000030 mg a.i./L.

1 

# Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative (clean water)

control.

2 

# Effects are considered statistically significant at p<0.05.



# D. Study Deficiencies

There were deviations from the guideline as noted in Section I. Materials and Methods of the DER. The
reviewer noted that performance criteria for the OCSPP 890.1350 guideline were met for reported
parameters, but the TOC was not reported, and it was unclear if residual chlorine and unionized ammonia

levels fell below the recommended maxima because the maxima were lower than the detection levels for
the analyzed water samples. These deviations did not impact the interpretation of the study. All validity
and performance criteria were met.

E. Reviewer’s Comments

Despite the fact that the study authors based treated comparisons on the pooled control and the reviewer based comparisons on only the negative control, the reviewer’s and the study authors’ results were in

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# agreement regarding effects on female weight, fertility, male GSI, and male 17 ȕ -estradiol. The reviewer’s analysis did not detect reductions in fecundity (p>0.05), despite the rather large reduction from the negative control (i.e., 87%) at the 0.0215 mg a.i./L treatment level; the study authors’ analysis detected a significant reduction (p<0.05) at this level compared to the pooled control. The reviewer and study
authors agreed on the subjectively-determined effects on secondary sex characteristics, gonad
development, and histopathology. Conclusions regarding male weight and male tubercle scores differed, with the study authors’ analysis detecting a significant reduction in male body weight at the top concentration that the reviewer’s analysis did not, while the reviewer’s analysis detected a significant reduction (p<0.05) in median male tubercle score at the top concentration that the study authors’ analysis did not, relative to
the pooled control.

There were three endpoints exhibiting a monotonic concentration response which also satisfied parametric
assumptions. The OCSPP 890.1350 guidance recommends use of the non-parametric Jonckheere-Terpstra
test to detect treatment-related effects for any monotonic response, while the historical EFED method suggests use of William’s test when parametric assumptions are satisfied. For all cases, both tests resulted

in the same conclusions.

Some information was missing from the study report that did not allow the reviewer to confirm if all test
solution characteristics satisfied the OCSPP 890.1350 guideline requirements. The TOC was not reported.
There was no confirmation that the test solution was completely mixed prior to introduction to the test
system or that the flow splitting accuracy was within 10%. Residual chlorine in the dilution water was <20

P g/L; guideline 890.1350 recommends that residual chlorine in dilution water be <10 P g/L. Finally, ammonia (as N) in the dilution water was reported to be <100 P g/L, while guideline 890.1350 requires

that unionized ammonia in the dilution water be <1 P g/L.

While the overall test solution CVs were 20% or lower, some CVs were >20% when based on a per
replicate basis. The study authors addressed this, attributing the variation to the absorptive and lipophilic nature of the test substance, as well as the dynamic test system ( i.e. , fish addition, waste production and biofilm accumulation). They noted that attempts were made to improve the approximation of nominal target
concentrations by increasing the turn-over rate and cleaning the test vessels daily.

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# II. REFERENCES

U.S. Environmental Protection Agency. 2009. Endocrine Disruptor Screening Program Test Guidelines,
OCSPP 890.1350, Fish Short-Term Reproduction Assay, EPA 740-C-09-007, 93 pp.

Mount DI, Brungs WA. 1967. A Simplified Dosing Apparatus for Fish Toxicological Studies. Water Res

1:21-29.

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

Examination for Water and Wastewater. 20 th 

# ed. Washington, D.C. 2-27 to 2-29, 2-36 to 2-39.Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page54of88APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:Control Comparisons% Mort.(all)% Mort.(male)% Mort.(female)Fecundity% Fert.Wt (male)Length(male)Wt.(female)Length(female)Neg Control00045.899.34.0955.71.7644.616.702550.699.24.2151.81.7544.400028.398.24.2454.81.7844.600024.399.34.2256.81.9044.6Average4.20.06.25037.399.04.1954.81.8044.6SolventControl00017.299.24.5456.01.6343.200041.599.24.0553.31.7644.600041.299.24.8657.21.9644.100035.499.34.7853.81.6143.2Average0.00.00.00033.899.24.5655.11.7443.8t-test pvalue0.356NA0.3560.7050.4350.0950.8390.5360.069Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page55of88Control ComparisonsGSI(male)GSI(female)Tubercle(male)VTG(male)VTG(Female)Testosterone(male)17b-estradiol (male)Neg Control0.9710.831.5283036.985.140.1901.0912.128.0449022.913.240.1251.2812.130.0239056.166.560.1251.0614.039.01140055.8724.50.220Average1.1012.330.8527842.989.860.165SolventControl1.2312.624.5485027.091.720.1251.1410.932.5993033.244.650.1251.1815.132.0196034.388.440.1251.0310.934.5236059.283.140.125Average1.1512.432.3477538.504.490.125t-test pvalue0.5860.9200.7140.8630.6910.3360.145Data Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page56of88Reviewer’s statistical verification tableEndpointMonotonic?Parametric?890.1350EFEDCommentsSurvivalMales:Females:Overall:NoNAVisualVisualNo effect on male survival (100% all groups)No effect on female survival (control same as reduced trtgroups)No effect on overall combined survival (same as femaledata)Female body weightNoYesDunnett’s:Dose 3 p=0.045Dunnett’s:Dose 3 p=0.045890.1350 and EFED same conclusions, significant(p<0.05) promotion of female body weight of 10% at thehighest treatment level.Male body weightNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectFemale body lengthNoNoMann-Whitney:n.s. p>0.05Mann-Whitney:n.s. p>0.05890.1350 and EFED same conclusions, no effectMale body lengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectFemale VTGNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectMale VTGNoNoMann-Whitney:n.s. p>0.05Mann-Whitney:n.s. p>0.05890.1350 and EFED same conclusions, no effectFemale GSINoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectData Evaluation Record on the Fish Short-Term Reproduction Assay with TrifluralinEPA MRID Number 48619706Page57of88EndpointMonotonic?Parametric?890.1350EFEDCommentsMale GSIYesYesJonckheere:Dose 1 p=0.022Dose 2 p=0.009Dose 3 p=0.006William’s:Dose 1 p=0.011Dose 2 p=0.003Dose 3 p=0.002890.1350 and EFED same conclusions, a dose-dependentmonotonic increasing trend, ranging from 29 to 39% relativeto the negative control. Significant effects (p<0.05) detectedat all treated levels.Female tubercle scoreNANANANANo scoreMale tubercle scoreYesYesJonckheere:Dose 3 p=0.025William’s:Dose 3 p=0.009890.1350 and EFED same conclusions; a significant(p<0.05) 33% reduction from negative control at the0.0215 mg a.i./L treatment level.FecundityNoNoMann-Whitney:n.s. p>0.05Mann-Whitney:n.s. p>0.05890.1350 and EFED same conclusions, no effectFertilityYesNoJonckheere:Dose 1 p=0.041Dose 3 p=0.012Jonckheere:Dose 1 p=0.041Dose 3 p=0.012890.1350 and EFED same conclusions, significant(p<0.05) 0.5 and 4% reduction at the lowest and highestlevel; the middle dose average was only 0.1% lower thanthe control average.F testosteroneNANANANANAM testosteroneNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectF estradiolNANANANANAM estradiolYesYesJonckheere:Dose 3 p=0.009William’s:Dose 3 p=0.027890.1350 and EFED same conclusions; a significant(p<0.05) 78 % promotion from negative control at the0.0215 mg a.i./L treatment level.

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Page 58 of 88 Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.882 0.041 2.684 0.094 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.80 0.07 0.03 3.86 1.69, 1.91 Dose1 4 1.67 0.01 0.01 0.78 1.64, 1.69 Dose2 4 1.80 0.04 0.02 2.33 1.73, 1.86 Dose3 4 1.98 0.16 0.08 8.27 1.71, 2.24

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.77 1.75 1.90 . . Dose1 1.67 1.65 1.68 92.63 7.37 Dose2 1.81 1.74 1.84 100.00 0.00 Dose3 2.01 1.75 2.14 109.87 -9.87

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 7.74 0.004

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 1.80 . 1.81 . . . . . . Dose1 1.67 0.149 1.81 0.655 . . . . . Dose2 1.80 1.000 1.81 0.690 0.224 . . . . Dose3 1.98 0.045 1.81 0.709 0.002 0.073 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.99 0.019

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

Level Median MannWhit p-value Jonckheere p-value Ctrl 1.77 . . Dose1 1.67 0.067 0.010

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Page 59 of 88 Dose2 1.81 0.889 0.558 Dose3 2.01 0.283 0.958

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 7.74 0.004

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 -1.80 . -1.73 . . . . . . Dose1 -1.67 0.149 -1.73 0.902 . . . . . Dose2 -1.80 1.000 -1.80 0.618 0.224 . . . . Dose3 -1.98 0.045 -1.98 0.011 0.002 0.073 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 9.99 0.019

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

Level Median MannWhit p-value Jonckheere p-value Ctrl -1.77 . . Dose1 -1.67 0.067 0.990 Dose2 -1.81 0.889 0.442 Dose3 -2.01 0.283 0.042

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.932 0.267 2.641 0.097 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS

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

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Page 60 of 88 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 4.19 0.07 0.03 1.62 4.08, 4.30 Dose1 4 4.14 0.38 0.19 9.18 3.54, 4.74 Dose2 4 4.41 0.51 0.25 11.48 3.61, 5.22 Dose3 4 3.65 0.24 0.12 6.59 3.27, 4.04

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 4.22 4.09 4.24 . . Dose1 4.16 3.66 4.59 98.81 1.19 Dose2 4.50 3.77 4.89 105.37 -5.37 Dose3 3.56 3.49 4.01 87.17 12.83

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

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

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

Ctrl 4.19 . 4.25 . . . . . . Dose1 4.14 0.993 4.25 0.681 . . . . . Dose2 4.42 0.681 4.25 0.716 0.672 . . . . Dose3 3.65 0.110 3.65 0.028 0.233 0.036 . . .

******************************************************************************** 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 8.23 0.042

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 4.22 . . Dose1 4.16 0.678 0.282 Dose2 4.50 0.346 0.810 Dose3 3.56 0.067 0.068

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

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

Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing INCREASING trend

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Page 61 of 88 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 -4.19 . -4.10 . . . . . . Dose1 -4.14 0.993 -4.10 0.731 . . . . . Dose2 -4.42 0.681 -4.10 0.765 0.672 . . . . Dose3 -3.65 0.110 -4.10 0.783 0.233 0.036 . . .

******************************************************************************** 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 8.23 0.042

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 -4.22 . . Dose1 -4.16 0.678 0.718 Dose2 -4.50 0.346 0.190 Dose3 -3.56 0.067 0.932

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.925 0.205 5.471 0.013 USE NON-PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 44.55 0.10 0.05 0.22 44.39, 44.71 Dose1 4 43.93 0.38 0.19 0.86 43.32, 44.53 Dose2 4 44.33 1.48 0.74 3.35 41.96, 46.69 Dose3 4 43.85 1.39 0.70 3.17 41.64, 46.06

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 44.60 44.40 44.60 . . Dose1 44.00 43.40 44.30 98.60 1.40 Dose2 44.65 42.30 45.70 99.49 0.51 Dose3 44.05 42.20 45.10 98.43 1.57

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

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Page 62 of 88 Numerator df Denominator df F-stat P-value 3 12 0.41 0.748

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 44.55 . 44.55 . . . . . . Dose1 43.93 0.733 44.13 0.341 . . . . . Dose2 44.33 0.980 44.13 0.365 0.946 . . . . Dose3 43.85 0.667 43.85 0.236 1.000 0.914 . . .

******************************************************************************** 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 2.46 0.482

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 44.60 . . Dose1 44.00 0.061 0.009 Dose2 44.65 1.000 0.230 Dose3 44.05 1.000 0.241

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 0.41 0.748

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 -44.55 . -44.16 . . . . . . Dose1 -43.93 0.733 -44.16 0.782 . . . . . Dose2 -44.33 0.980 -44.16 0.814 0.946 . . . . Dose3 -43.85 0.667 -44.16 0.830 1.000 0.914 . . .

******************************************************************************** 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 2.46 0.482

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Page 63 of 88 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 -44.60 . . Dose1 -44.00 0.061 0.991 Dose2 -44.65 1.000 0.770 Dose3 -44.05 1.000 0.759

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.974 0.899 1.233 0.341 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 54.78 2.15 1.07 3.92 51.36, 58.19 Dose1 4 53.50 3.05 1.53 5.70 48.64, 58.36 Dose2 4 56.75 3.46 1.73 6.09 51.25, 62.25 Dose3 4 53.38 1.24 0.62 2.33 51.39, 55.36

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 55.25 51.80 56.80 . . Dose1 53.15 50.40 57.30 97.67 2.33 Dose2 57.70 52.00 59.60 103.61 -3.61 Dose3 53.40 52.20 54.50 97.44 2.56

******************************************************************************** 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 1.43 0.282

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

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

Ctrl 54.78 . 55.01 . . . . . . Dose1 53.50 0.832 55.01 0.635 . . . . . Dose2 56.75 0.595 55.01 0.670 0.340 . . . . Dose3 53.38 0.793 53.38 0.307 1.000 0.310 . . .

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

EPA MRID Number 48619706

Page 64 of 88 ******************************************************************************** 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 3.27 0.352

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 55.25 . . Dose1 53.15 0.580 0.234 Dose2 57.70 0.346 0.830 Dose3 53.40 0.346 0.463

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

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 1.43 0.282

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

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

Ctrl -54.78 . -54.14 . . . . . . Dose1 -53.50 0.832 -54.14 0.719 . . . . . Dose2 -56.75 0.595 -55.06 0.550 0.340 . . . . Dose3 -53.38 0.793 -55.06 0.568 1.000 0.310 . . .

******************************************************************************** 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 3.27 0.352

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 -55.25 . . Dose1 -53.15 0.580 0.766 Dose2 -57.70 0.346 0.170 Dose3 -53.40 0.346 0.537

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

Trifluralin 036101 48619706 890.1350

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

EPA MRID Number 48619706

Page 65 of 88 ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.918 0.158 1.165 0.363 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 42980000 16110756 8055378 37.48 17344192,68615808 Dose1 4 48325000 33442010 16721005 69.20 -4888701,1.0154E8 Dose2 4 44035000 14308931 7154465 32.49 21266298,66803702 Dose3 4 44242500 20506855 10253427 46.35 11611518,76873482

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 46425000 22910000 56160000 . . Dose1 36205000 23430000 97460000 112.44 -12.44 Dose2 46505000 24580000 58550000 102.45 -2.45 Dose3 38795000 25800000 73580000 102.94 -2.94

******************************************************************************** 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 0.04 0.987

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 42980000 . 45652500 . . . . . . Dose148325000 0.974 45652500 0.653 . . . . . Dose244035000 1.000 44138750 0.649 0.993 . . . . Dose344242500 1.000 44138750 0.668 0.994 1.000 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 0.26 0.967

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 46425000 . . Dose1 36205000 1.000 0.500 Dose2 46505000 0.889 0.670 Dose3 38795000 0.889 0.610

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

EPA MRID Number 48619706

Page 66 of 88 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 0.04 0.987

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 -4.298E7 . -4.298E7 . . . . . . Dose1-4.833E7 0.974 -4.553E7 0.515 . . . . . Dose2-4.404E7 1.000 -4.553E7 0.547 0.993 . . . . Dose3-4.424E7 1.000 -4.553E7 0.565 0.994 1.000 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 0.26 0.967

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 -4.643E7 . . Dose1 -3.621E7 1.000 0.500 Dose2 -4.651E7 0.889 0.330 Dose3 -3.88E7 0.889 0.390

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.826 0.006 4.183 0.030 USE NON-PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 5277.50 4180.63 2090.32 79.22 -1374.82,11929.82 Dose1 4 7565.00 7600.48 3800.24 100.47 -4529.06,19659.06

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

EPA MRID Number 48619706

Page 67 of 88 Dose2 4 1975.00 186.28 93.14 9.43 1678.59, 2271.41 Dose3 4 2442.50 1475.68 737.84 60.42 94.37, 4790.63

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 3660.00 2390.00 11400.00 . . Dose1 4450.00 2660.00 18700.00 143.34 -43.34 Dose2 1970.00 1760.00 2200.00 37.42 62.58 Dose3 1835.00 1460.00 4640.00 46.28 53.72

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 1.41 0.288

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 5277.50 . 6421.25 . . . . . . Dose1 7565.00 0.805 6421.25 0.728 . . . . . Dose2 1975.00 0.598 2208.75 0.219 0.321 . . . . Dose3 2442.50 0.696 2208.75 0.227 0.391 0.999 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 8.10 0.044

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 3660.00 . . Dose1 4450.00 0.889 0.614 Dose2 1970.00 0.067 0.014 Dose3 1835.00 0.235 0.004

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) 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 1.41 0.288

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 Short-Term Reproduction Assay with Trifluralin

EPA MRID Number 48619706

Page 68 of 88 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl -5277.50 . -4315.00 . . . . . . Dose1-7565.00 0.805 -4315.00 0.707 . . . . . Dose2-1975.00 0.598 -4315.00 0.741 0.321 . . . . Dose3-2442.50 0.696 -4315.00 0.760 0.391 0.999 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 8.10 0.044

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 -3660.00 . . Dose1 -4450.00 0.889 0.386 Dose2 -1970.00 0.067 0.986 Dose3 -1835.00 0.235 0.996

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.937 0.316 1.258 0.333 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 12.25 1.32 0.66 10.76 10.15, 14.35 Dose1 4 11.61 3.13 1.57 27.01 6.62, 16.59 Dose2 4 13.60 1.99 0.99 14.62 10.44, 16.76 Dose3 4 13.23 2.01 1.01 15.22 10.02, 16.43

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 12.10 10.80 14.00 . . Dose1 13.05 6.92 13.40 94.73 5.27 Dose2 12.95 12.00 16.50 111.02 -11.02 Dose3 13.45 10.90 15.10 107.96 -7.96

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.68 0.583

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

EPA MRID Number 48619706

Page 69 of 88 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC

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

Ctrl 12.25 . 12.67 . . . . . . Dose1 11.61 0.954 12.67 0.691 . . . . . Dose2 13.60 0.726 12.67 0.726 0.594 . . . . Dose3 13.23 0.867 12.67 0.745 0.732 0.995 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 1.23 0.745

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 12.10 . . Dose1 13.05 0.889 0.614 Dose2 12.95 0.491 0.746 Dose3 13.45 0.343 0.813

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

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.68 0.583

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

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

Ctrl -12.25 . -11.93 . . . . . . Dose1 -11.61 0.954 -11.93 0.667 . . . . . Dose2 -13.60 0.726 -13.41 0.301 0.594 . . . . Dose3 -13.23 0.867 -13.41 0.312 0.732 0.995 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 1.23 0.745

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

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

EPA MRID Number 48619706

Page 70 of 88 Level Median MannWhit p-value Jonckheere p-value Ctrl -12.10 . . Dose1 -13.05 0.889 0.386 Dose2 -12.95 0.491 0.254 Dose3 -13.45 0.343 0.187

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.984 0.987 0.353 0.788 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.10 0.13 0.07 11.82 0.89, 1.31 Dose1 4 1.42 0.17 0.08 11.64 1.16, 1.69 Dose2 4 1.50 0.24 0.12 15.86 1.12, 1.88 Dose3 4 1.53 0.12 0.06 7.86 1.34, 1.72

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.08 0.97 1.28 . . Dose1 1.44 1.21 1.60 129.29 -29.29 Dose2 1.52 1.19 1.77 136.33 -36.33 Dose3 1.55 1.38 1.65 139.06 -39.06

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 5.39 0.014

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

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

Ctrl 1.10 . 1.39 . . . . . . Dose1 1.42 0.050 1.39 0.994 . . . . . Dose2 1.50 0.016 1.39 0.996 0.915 . . . . Dose3 1.53 0.010 1.39 0.997 0.808 0.994 . . .

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

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

EPA MRID Number 48619706

Page 71 of 88 Degrees of Freedom TestStat P-value 3 7.64 0.054

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 1.08 . . Dose1 1.44 0.103 0.978 Dose2 1.52 0.101 0.991 Dose3 1.55 0.067 0.994

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 5.39 0.014

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

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

Ctrl -1.10 . -1.10 . . . . . . Dose1 -1.42 0.050 -1.42 0.011 . . . . . Dose2 -1.50 0.016 -1.50 0.003 0.915 . . . . Dose3 -1.53 0.010 -1.53 0.002 0.808 0.994 . . .

******************************************************************************** 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 7.64 0.054

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 -1.08 . . Dose1 -1.44 0.103 0.022 Dose2 -1.52 0.101 0.009 Dose3 -1.55 0.067 0.006

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS

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

EPA MRID Number 48619706

Page 72 of 88 Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.00 0.00 0.00 . . , . Dose1 4 0.00 0.00 0.00 . . , . Dose2 4 0.00 0.00 0.00 . . , . Dose3 4 0.00 0.00 0.00 . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.00 0.00 0.00 . . Dose1 0.00 0.00 0.00 . . Dose2 0.00 0.00 0.00 . . Dose3 0.00 0.00 0.00 . .

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

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

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

Ctrl 0.00 . . . . . . . . Dose1 0.00 . . . . . . . . Dose2 0.00 . . . . . . . . Dose3 0.00 . . . . . . . .

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

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

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.00 . . Dose1 0.00 1.000 . Dose2 0.00 1.000 . Dose3 0.00 1.000 .

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

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

EPA MRID Number 48619706

Page 73 of 88 Jonckheere Dose1

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

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

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

Ctrl 0.00 . . . . . . . . Dose1 0.00 . . . . . . . . Dose2 0.00 . . . . . . . . Dose3 0.00 . . . . . . . .

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

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

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.00 . . Dose1 0.00 1.000 . Dose2 0.00 1.000 . Dose3 0.00 1.000 .

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.902 0.086 0.478 0.703 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 32.13 4.80 2.40 14.95 24.48, 39.77 Dose1 4 32.38 5.91 2.95 18.25 22.98, 41.77 Dose2 4 30.50 3.72 1.86 12.19 24.58, 36.42

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

EPA MRID Number 48619706

Page 74 of 88 Dose3 4 21.63 6.28 3.14 29.02 11.64, 31.61

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 30.75 28.00 39.00 . . Dose1 31.25 27.00 40.00 100.78 -0.78 Dose2 31.00 25.50 34.50 94.94 5.06 Dose3 19.75 16.50 30.50 67.32 32.68

******************************************************************************** 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 3.73 0.042

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 32.13 . 32.25 . . . . . . Dose1 32.38 1.000 32.25 0.597 . . . . . Dose2 30.50 0.947 30.50 0.426 0.957 . . . . Dose3 21.63 0.039 21.63 0.009 0.058 0.134 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 5.35 0.148

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 30.75 . . Dose1 31.25 1.000 0.500 Dose2 31.00 0.889 0.385 Dose3 19.75 0.156 0.025

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

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 3.73 0.042

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 Short-Term Reproduction Assay with Trifluralin

EPA MRID Number 48619706

Page 75 of 88 Ctrl -32.13 . -29.16 . . . . . . Dose1 -32.38 1.000 -29.16 0.855 . . . . . Dose2 -30.50 0.947 -29.16 0.882 0.957 . . . . Dose3 -21.63 0.039 -29.16 0.895 0.058 0.134 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 5.35 0.148

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 -30.75 . . Dose1 -31.25 1.000 0.500 Dose2 -31.00 0.889 0.615 Dose3 -19.75 0.156 0.975

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.976 0.922 4.668 0.022 USE NON-PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 37.25 12.90 6.45 34.63 16.73, 57.77 Dose1 4 34.30 6.95 3.47 20.26 23.24, 45.36 Dose2 4 38.75 5.97 2.99 15.42 29.24, 48.26 Dose3 4 4.85 4.07 2.04 84.00 -1.63, 11.33

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 37.05 24.30 50.60 . . Dose1 33.10 27.20 43.80 92.08 7.92 Dose2 38.40 31.80 46.40 104.03 -4.03 Dose3 4.60 0.50 9.70 13.02 86.98

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 15.47 <.001

Dunnett - testing each trt mean signif. different than control

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

EPA MRID Number 48619706

Page 76 of 88 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 37.25 . 37.25 . . . . . . Dose1 34.30 0.919 36.53 0.530 . . . . . Dose2 38.75 0.987 36.53 0.563 0.866 . . . . Dose3 4.85 <.001 4.85 <.001 0.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 8.84 0.032

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

Level Median MannWhit p-value Jonckheere p-value Ctrl 37.05 . . Dose1 33.10 0.889 0.386 Dose2 38.40 0.889 0.670 Dose3 4.60 0.067 0.025

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

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 15.47 <.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 -37.25 . -28.79 . . . . . . Dose1 -34.30 0.919 -28.79 0.957 . . . . . Dose2 -38.75 0.987 -28.79 0.969 0.866 . . . . Dose3 -4.85 <.001 -28.79 0.974 0.001 <.001 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 8.84 0.032

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

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

EPA MRID Number 48619706

Page 77 of 88 Level Median MannWhit p-value Jonckheere p-value Ctrl -37.05 . . Dose1 -33.10 0.889 0.614 Dose2 -38.40 0.889 0.330 Dose3 -4.60 0.067 0.975

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.780 0.001 4.353 0.027 USE NON-PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 99.00 0.54 0.27 0.54 98.15, 99.85 Dose1 4 98.48 0.62 0.31 0.63 97.48, 99.47 Dose2 4 98.93 0.38 0.19 0.38 98.32, 99.53 Dose3 4 95.03 3.84 1.92 4.05 88.91, 101.14

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 99.25 98.20 99.30 . . Dose1 98.50 97.80 99.10 99.47 0.53 Dose2 99.00 98.40 99.30 99.92 0.08 Dose3 95.95 89.70 98.50 95.98 4.02

******************************************************************************** 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 3.71 0.043

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 99.00 . 99.00 . . . . . . Dose1 98.48 0.964 98.70 0.492 . . . . . Dose2 98.93 1.000 98.70 0.523 0.988 . . . . Dose3 95.03 0.037 95.03 0.009 0.116 0.068 . . .

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

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

EPA MRID Number 48619706

Page 78 of 88 3 8.02 0.046

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 99.25 . . Dose1 98.50 0.154 0.041 Dose2 99.00 0.669 0.277 Dose3 95.95 0.101 0.012

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 3.71 0.043

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 -99.00 . -97.86 . . . . . . Dose1 -98.48 0.964 -97.86 0.860 . . . . . Dose2 -98.93 1.000 -97.86 0.886 0.988 . . . . Dose3 -95.03 0.037 -97.86 0.900 0.116 0.068 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 8.02 0.046

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 -99.25 . . Dose1 -98.50 0.154 0.959 Dose2 -99.00 0.669 0.723 Dose3 -95.95 0.101 0.988

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR13 ( F testosterone (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01

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

EPA MRID Number 48619706

Page 79 of 88 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 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 3.71 0.043

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 3.71 0.043

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

Trifluralin 036101 48619706 890.1350 ANALYSIS RESULTS FOR VARIABLE VAR14 ( M testosterone (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01

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

EPA MRID Number 48619706

Page 80 of 88 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.916 0.145 1.831 0.195 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 9.86 9.85 4.93 99.94 -5.82, 25.54 Dose1 4 5.88 4.61 2.31 78.40 -1.46, 13.22 Dose2 4 9.04 5.06 2.53 55.96 0.99, 17.10 Dose3 4 7.81 3.33 1.67 42.71 2.50, 13.11

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 5.85 3.24 24.50 . . Dose1 4.91 1.92 11.80 59.66 40.34 Dose2 10.38 1.91 13.50 91.71 8.29 Dose3 7.99 3.55 11.70 79.18 20.82

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.31 0.819

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 9.86 . 9.86 . . . . . . Dose1 5.88 0.701 7.58 0.366 . . . . . Dose2 9.04 0.995 7.58 0.391 0.888 . . . . Dose3 7.81 0.936 7.58 0.404 0.971 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 0.93 0.819

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 5.85 . . Dose1 4.91 0.678 0.282 Dose2 10.38 0.889 0.558 Dose3 7.99 0.678 0.646

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

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

EPA MRID Number 48619706

Page 81 of 88 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.31 0.819

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 -9.86 . -7.87 . . . . . . Dose1 -5.88 0.701 -7.87 0.757 . . . . . Dose2 -9.04 0.995 -8.43 0.747 0.888 . . . . Dose3 -7.81 0.936 -8.43 0.766 0.971 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 0.93 0.819

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 -5.85 . . Dose1 -4.91 0.678 0.718 Dose2 -10.38 0.889 0.442 Dose3 -7.99 0.678 0.354

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

Trifluralin 036101 48619706 890.1350 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=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means)

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

EPA MRID Number 48619706

Page 82 of 88 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 0.31 0.819

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 0.31 0.819

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

Trifluralin 036101 48619706 890.1350 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=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.919 0.164 0.562 0.650 USE PARAMETRIC TESTS

******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.17 0.05 0.02 28.96 0.09, 0.24 Dose1 4 0.18 0.06 0.03 34.30 0.08, 0.27 Dose2 4 0.23 0.09 0.05 41.93 0.07, 0.38 Dose3 4 0.29 0.10 0.05 35.25 0.13, 0.46

Level Median Min Max %of Control(means) %Reduction(means)

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

EPA MRID Number 48619706

Page 83 of 88 Ctrl 0.16 0.13 0.22 . . Dose1 0.17 0.13 0.24 107.58 -7.58 Dose2 0.21 0.13 0.35 136.52 -36.52 Dose3 0.26 0.21 0.45 177.58 -77.58

******************************************************************************** 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 2.10 0.153

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

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

Ctrl 0.17 . 0.22 . . . . . . Dose1 0.18 0.992 0.22 0.875 . . . . . Dose2 0.23 0.595 0.22 0.900 0.832 . . . . Dose3 0.29 0.104 0.22 0.912 0.226 0.639 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 5.66 0.129

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

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.16 . . Dose1 0.17 0.659 0.731 Dose2 0.21 0.335 0.888 Dose3 0.26 0.101 0.991

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

******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 2.10 0.153

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

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

Ctrl -0.17 . -0.17 . . . . . . Dose1 -0.18 0.992 -0.18 0.489 . . . . .

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

EPA MRID Number 48619706

Page 84 of 88 Dose2 -0.23 0.595 -0.23 0.196 0.832 . . . . Dose3 -0.29 0.104 -0.29 0.027 0.226 0.639 . . .

******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 5.66 0.129

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

Level Median MannWhit p-value Jonckheere p-value Ctrl -0.16 . . Dose1 -0.17 0.659 0.269 Dose2 -0.21 0.335 0.112 Dose3 -0.26 0.101 0.009

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

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

EPA MRID Number 48619706

Page 85 of 88 

# Fecundity using the exact test for Mann-Whitney

**Obs con** 
**c endpoin**  **t 1**  0 45.8 **2** 0 50.6 **3** 0 28.3 **4** 0 24.3 **5** 1 32.3 **6** 1 27.2 **7** 1 43.8 **8** 1 33.9 **9** 2 31.8 **10** 2 46.4 **11** 2 38.4 **12** 2 38.4 **13** 3 2.7 **14** 3 9.7 **15** 3 6.5 **16** 3 0.5

The NPAR1WAY Procedure

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

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score**  **0** 4 42.0 34.0 8.240146 10.50 **1** 4 38.0 34.0 8.240146 9.50 **2** 4 46.0 34.0 8.240146 11.50 **3** 4 10.0 34.0 8.240146 2.50 **Average scores were used for ties.**

**Kruskal-Wallis Test Chi-Square**  8.8365 **DF** 3 **Asymptotic Pr > Chi-Square** 0.0315 **Exact Pr >= Chi-Square** 0.0147

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

EPA MRID Number 48619706

Page 86 of 88 

# The NPAR1WAY Procedure

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

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score**  **0** 4 19.0 18.0 3.464102 4.750 **1** 4 17.0 18.0 3.464102 4.250

**Wilcoxon Two-Sample Test Statistic (S)**  19.0000

**Normal Approximation Z**  0.1443 **One-Sided Pr > Z** 0.4426 **Two-Sided Pr > |Z|** 0.8852

**t Approximation One-Sided Pr > Z**  0.4447 **Two-Sided Pr > |Z|** 0.8893

**Exact Test One-Sided Pr >= S**  0.4429 **Two-Sided Pr >= |S - Mean|** 0.8857

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

**Kruskal-Wallis Test Chi-Square**  0.0833 **DF** 1 **Pr > Chi-Square** 0.7728

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

EPA MRID Number 48619706

Page 87 of 88 

# The NPAR1WAY Procedure

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

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score**  **0** 4 17.0 18.0 3.443420 4.250 **2** 4 19.0 18.0 3.443420 4.750 **Average scores were used for ties.**

**Wilcoxon Two-Sample Test Statistic (S)**  17.0000

**Normal Approximation Z**  -0.1452 **One-Sided Pr < Z** 0.4423 **Two-Sided Pr > |Z|** 0.8845

**t Approximation One-Sided Pr < Z**  0.4443 **Two-Sided Pr > |Z|** 0.8886

**Exact Test One-Sided Pr <= S**  0.4286 **Two-Sided Pr >= |S - Mean|** 0.8571

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

**Kruskal-Wallis Test Chi-Square**  0.0843 **DF** 1 **Pr > Chi-Square** 0.7715

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

EPA MRID Number 48619706

Page 88 of 88 

# The NPAR1WAY Procedure

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

**conc N**  **Sum of Scores**  **Expected Under H0**  **Std Dev Under H0**  **Mean Score**  **0** 4 26.0 18.0 3.464102 6.50 **3** 4 10.0 18.0 3.464102 2.50

**Wilcoxon Two-Sample Test Statistic (S)**  26.0000

**Normal Approximation Z**  2.1651 **One-Sided Pr > Z** 0.0152 **Two-Sided Pr > |Z|** 0.0304

**t Approximation One-Sided Pr > Z**  0.0336 **Two-Sided Pr > |Z|** 0.0671

**Exact Test One-Sided Pr >= S**  0.0143 **Two-Sided Pr >= |S - Mean|** 0.0286

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

**Kruskal-Wallis Test Chi-Square**  5.3333 **DF** 1 **Pr > Chi-Square** 0.0209

# DATA EVALUATION RECORD

TRIFLURALIN

Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-40-2012 (MRID 48619707)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 4/2/2012

Secondary Reviewer Signature: Kelly Luck, M.S. Date: 5/9/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/14/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/14/2012

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

.

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

# TRIFLURALIN I 

# 036101



# EPA Primary Reviewer: Yung G. Yang, Ph.D. Health Effects Division EPA Secondary Reviewer: Greg Akerman, Ph.D. Health Effects Division In vivo 

# Hershberger Assay (1011) 

# I Paget of 18 OCSPP 890.1400/ OECD 441



# Signature:

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STUDY TYPE: Jn Vivo 

# Hershberger Assay (Rat); OCSPP 890.1400; OECD 441

PC CODE: 03610 I DP BARCODE: D398779

TXR#: 0055724 CAS No.: 1582-09-8

TEST MATERIAL <PURITY): Trifluralin (97 .1 

# %)

SYNONYMS: 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine

CITATION:

SPONSOR: Marty, M.S., Marshall, V.A., and Jeong, YC. (2011). Trifluralin: Hershberger Assay in Castrated Adult Male Crl:CD(SD) Rats. Toxicology 

# & Enviromnental Research and Consulting, The Dow Chemical Company, Midland, ML Laboratory Study No.: 101133, December 20, 2011. MRID 48619707. Unpublished.

Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN

TEST ORDER#: CON-036101-26

EXECUTIVE SUMMARY: 

# In a Hershberger assay (MRID 48619707) screening for androgenic activity, trifluralin (97. l %, lot no. DKO-B916-33, TSNl 00656) in com oil was administered daily via oral gavage to groups of 55-day old castrated male Sprague Dawley rats (7/group) at dose levels of 0 (vehicle), 50, 150, or 450 mg/kg/day for 10 consecutive days. An 

# androgenic positive control group consisted of seven castrated male rats exposed to testosterone propionate (TP) at 0.4 mg/kg/day via subcutaneous (s.c.) injection in conjunction with com oil via oral gavage.

To screen for possible anti-androgenic activity, additional groups of 55-day old castrated male Sprague Dawley rats (7 /group) were dosed daily via oral gavage with trifluralin in com oil at 50, 150, or 450 mg/kg/day for 10 consecutive days in conjunction with a daily s.c. injection of TP at 0.4 mg/kg/day. The same seven animals used as positive controls in the androgenic portion of the study served as negative controls in the anti-androgenic portion of the study, and seven castrated male rats dosed orally with flutamide (FT) in com oil at 3 mg/kg/day for 10 days in conjugation with a daily s.c. dose ofTP (0.4 mg/kg/day) served as positive controls.

For both components of the assay, body weights and food consumption were determined daily. The animals were terminated approximately 24 hours after the final dose administration. At necropsy, the five androgen-dependent tissues (seminal vesicles with coagulating glands, ventral prostate, levator ani-bulbocavemosus (LABC), Cowper's glands, and glans penis), liver, adrenal

# In vivo Hershberger Assay (2011) / Page 2 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441


# glands, and kidneys were excised, examined macr oscopically, and weighed. Serum testosterone levels were measured in the anti-androgen assay.

No treatment-related effects were observed on mo rtality, clinical signs of toxicity, or gross pathology.

In the androgen agonist assay at 450 mg/kg/da y, body weights were decrea sed (not statistically significant [NS]) throughout the study ( ↓ 6–8%), compared to vehicle controls, and body weight gains were decreased (p ≤ 0.05) at each measured interval (Days 1–4, 1–7, and 1–11; ↓ 31–88%). In the anti-androgen assay at 450 mg/kg/day with TP, compared to negative controls, decreased (p ≤ 0.05) body weights were observed on Days 7 ( ↓ 8%) and 11 ( ↓ 10%), and body weight gains were decreased (p ≤ 0.05) at each measured interval ( ↓ 46–65%). Overall body weight gains (Days 1-11) were decreased (p ≤ 0.05) at 150 mg/kg/day with TP ( ↓ 22%). All other body weights and body weight gains in the trifluralin-treated groups were similar to controls in both the androgen agonist and anti -androgen assays.

In the androgen agonist assay at 450 mg /kg/day, food consumption was decreased (p ≤ 0.05) during the first 4-day interval (Days 1–4; ↓ 35%), compared to vehicle controls. In the antiandrogen assay at 450 mg/kg/day with TP, comp ared to negative controls, decreased food consumption was observed during each interval, Days 1–4 ( ↓ 31%; NS), 4–7 ( ↓ 12%; p ≤ 0.05), and 7–11 ( ↓ 14%; p ≤ 0.05). At 150 mg/kg/day with TP, food consumption was decreased (p ≤ 0.05) by 8% during Days 7–11, compared to negative controls . All other food consumption values in the trifluralin-treated groups were si milar to controls in both the androgen agonist and anti-androgen assays.
Liver weights were increased (p ≤ 0.05, except as noted) by triflu ralin treatment in both the androgenic and anti-androgenic portions of the study, by 16% at 150 mg/kg/day (NS without TP) and by 28 and 36% at 450 mg/kg/day (w ith and without TP, respectively).

For the androgenic portion of the study, there were no treatment-rel ated effects on the weights of any of the accessory sex tissues (seminal vesicl es, ventral prostate, LABC, Cowper's glands, or glans penis), adrenals, or kidney at any dose of trifluralin.

For the anti-androgenic portion of the study at 450 mg/kg/ day with TP, decreases (p ≤ 0.05) were noted in the weights of the seminal vesicles with coagulating glands ( ↓ 21%), ventral prostate ( ↓ 39%), and LABC ( ↑ 21%). Com pared to negative contro ls, serum testosterone was decreased by 27% (NS) at 150 mg/kg/day with TP and by 36% (p ≤ 0.05) at 450 mg/kg/day with TP. Body weights were decreased ≥ 10% at the dose (450 mg/kg/day) where the effects on the androgensensitive tissue weights were obser ved, indicating overt toxicity at this dose. Furthermore,

The increase in liver weight, si gnificant decrease in serum testos terone level, and results from previously performed studies, suggest that the anti-androgenic effects of trifluralin may be secondary to enhanced testoster one metabolism and clearance rather than direct interaction with the androgen receptor.

TP administration produced increases (p ≤ 0.05) of 20–48% in body weight gains at all intervals (Days 1–4, 1–7, and 1–11) and an increase in body wei ght compared to vehicle controls at Day 11 ( ↑ 8%). Food consumption was increased (p ≤ 0.05) by 16 and 18% in TP-treated animals

# In vivo Hershberger Assay (2011) / Page 3 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441


# relative to vehicle controls during Days 4–7 a nd 7–11, respectively. Only a minor increase (NS) in food consumption was noted in the TP-treated animals during th e first interval, Days 1–4. For the TP positive control group, there we re treatment-related increases (p ≤ 0.05) in the weights of the seminal vesicles with coagulating glands ( ↑ 1452%), ventral prostate ( ↑ 1328%), LABC ( ↑ 199%), Cowper’s glands ( ↑ 580%), and glans penis ( ↑ 66%), indicating that the test system was sensitive to an androgenic respon se. All these findings were e xpected androgenic responses of the positive control.

There was no effect of flutamide on body weights, body weight gains, or food consumption. For the flutamide positive control group, ther e were treatment-related decreases (p ≤ 0.05) in weights of the seminal vesicles with coagulating glands ( ↓ 70%), ventral prostate ( ↓ 66%), LABC ( ↓ 44%), Cowper’s glands ( ↓ 57%), and glans penis ( ↓ 14%) compared to weights in the negative control group. These data indicate that the test system was sensitive to an anti-androgenic response.

In the anti-androgen assay, all %CV levels were less than the guide line specified maximums. In the androgen agonist assay, only th e ventral prostate in the vehicl e control group did not meet the guideline criteria for maximum %CV (49.0% vs 45% maximum).

Trifluralin was negative for androgenic activity in the Hershberger assay as no statistically significant increases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist assay. Triflu ralin was positive for anti- androgenicity in the Hershberger assay based on statistically significan t decreases in organ weights seen in three of the five androgen sensitive tissues in the androg en antagonist assay. However, the target organ weight reductions were observe d at a dose (450 mg/kg/day) th at exhibited overt toxicity [decreased body weights (10%) and body we ight gains (46%)], which confounds the interpretation of the study.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for a Hershberger assay (OCSPP 890.1400).

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

# In vivo Hershberger Assay (2011) / Page 4 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting, The Dow Chemical Company
Location: Midland, MI Study Director: V. A. Marshall Other Personnel: M. S. Marty, Lead Scientist YC Jeong, Toxicologist Study Period: September 29, 2010 through December 20, 2011



# 2. Test Substance: Trifluralin Description: Molecular weight = 335.3 g/mol; a yellow-or ange crystal (not reported in study) Source: Dow AgroSciences LLC (Indianapolis, IN) Lot # (expiration): DKO-B916-33, TSN100656 (not reported) Purity: 97.1% Stability: Stable in corn oil at ambient temperature for up to 25 days CAS #: 1582-09-8 Structure:



# 3. Reference Androgen: Testosterone propionate (TP) Supplier Sigma-Aldrich (St. Louis, MO) Lot # (expiration): 048K1328 (not reported) Purity: ≥ 98% CAS # : 57-85-2


# 4. Reference Anti-androgen: Flutamide (FT) Supplier Sigma-Aldrich (St. Louis, MO) Lot # (expiration): 099K1112 (not reported) Purity: >99% CAS # : 13311-84-7



# 5. Solvent/Vehicle Control: Corn Oil Supplier: Sigma-Aldrich (St. Louis, MO) Lot #: Not provided Rationale (if other than water): Selected due to the solubility properties of trifluralin NO 2



# CF 3 

# O 2



# N N CH 3 

# CH

3

# In vivo Hershberger Assay (2011) / Page 5 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441



# 6. Test Animals: Species: Rat (castrated males only) Strain: Sprague Dawley (Crl:CD[SD]) Age/weight at dose initiation: Post-natal day (PND) 55/ 237.0 – 299.3 g Source: Charles River Laboratorie s, Inc. (Portage, MI) Housing: Rats were housed 2-3 per stainless steel cage with wire mesh floor suspended above absorbent paper Diet: LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO) , ad libitum (phytoestrogen concentration results not reported). Water: Tap water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 22±3oC 40–70% 12–15 changes/hour 12 hours light/12 hours dark Acclimation period: 5-6 days at facility prior to initiation of dosing dosing initiated 10 da ys post-castration



# B. STUDY DESIGN

1. In-Life Dates: Start: October 31, 2010 End: November 11, 2010

2. Study Design: In a Hershberger Assay conducted to screen for potentia l androgenic and anti-androgenic activity, th e test substance was administered daily at three dose levels via oral gavage to castrated male rats with or without a daily su bcutaneous injection of TP (0.4 mg/kg/day). Additionally, a si milar group of rats were treated with 0.4 mg/kg/day TP by injection and 3 mg/kg/day FT by daily oral gavage in order to compare the known antiandrogenic effect of FT with the test compound. Positive androge nic activity is defined as a significant increase in two or more organ we ights compared to the vehicle control. Anti-androgenic activity is indicated by a statis tically significant decrease in two or more target organ weights of the treated groups (t est substance + TP) comp ared to the TP-only (negative control) group. For both assays, the animals were treated once daily for 10 consecutive days. Animals were euthanized approximately 24 hours after the final dose administration.

3. Study Schedule: Rats were castrated at post-nata l day (PND) 45 at Charles River Laboratories, received from the animal s upplier on PND 49 or 50, randomized on PND 52, and the first dose was administered on PND 55 (10 days after castration, 5-6 days of acclimation). Doses were administered from PND 55-64 at about the same time each day. Rats were euthanized on PND 65, approximately 24 hours after the last dose, and subjected to necropsy and organ weight measurement.

4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1. Statistical anal ysis indicated that th ere were no significant differences in group means at study initiation. Th e same animals served as positive controls in the androgen agonist assay and negativ e controls in the anti-androgen assay.

# In vivo Hershberger Assay (2011) / Page 6 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441


# Table 1. Study Design a



# a Table 1 was copied from page 19 of the study report.



# 5. Dose-Selection Rationale: Dose-selection was based on a dose range finding study. 1 

# Crl:CD(SD) rats (4/sex/dose) were administered trifluralin in corn oil by gavage at dose levels of 0, 50, 200, 400 or 600 mg/kg/day for 15 days. Further discussion is restricted to the male rats for dose-selection for the Hershbe rger Assay. Male rats were dosed from postnatal day (PND) 30-44. Rats had 12%, 13% , and 20% decreases in body weight gains from Days 1-15 at 200, 400, and 600 mg/kg/ day, respectively. Similarly, respective terminal body weights were decreased by 6.3% , 6.5%, and 11.3%. At study termination, clear facial soiling was seen in one intermed iate and one high-dose rat. There were increases in absolute liver weights (9-21% at ≥ 200 mg/kg/day). Relative liver weights were increased in a dose-related manner from 200 mg/kg/day ( ↑ 18%) through 600 mg/kg/day ( ↑ 37%). Relative kidney weights were decreased by ≤ 10% in the high-dose group. Clinical chemistry parameters suggested a decrease in alkaline phosphatase and increased gamma glutamyl transpeptidase and cholesterol levels at 600 mg/kg/day. Creatinine was increased at 400 and 600 mg/kg/day. Terminal body wei ghts were decreased by more than 10% at 600 mg/kg/day. Therefore, 450 mg/kg/day was selected as the high dose for the Hershberger Assay. Administration of test material at 450 mg/kg/day was expected to produce some systemic toxicity without causing death, severe suffering or distress in the animals, or a greater than 10% reduction in terminal body weight relative to the control group.

1 

# Marty, M. S. and Marshall, V. A. (In progress). Triflu ralin: Hershberger, Uterotrophic, and Pubertal Assay Probe

Study in Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

# In vivo Hershberger Assay (2011) / Page 7 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441


# 6. (a) Dose Preparation: Appropriate amounts of the triflu ralin and positive controls were added to corn oil such that a dose volume of 4 mL/kg (0.5 mL/kg for testosterone propionate) yielded the targeted dose. Dose volumes were adjusted daily based on body weights. The study report st ated that trifluralin dosing solutions were prepared in accordance with established stability limits.

( b) Dose Analysis: The stability of trifluralin in corn oil at ambient temperature was evaluated for up to 25 days at concentrations of 0.25, 2.5, a nd 250 mg/mL prior to the start of dose administration. Concentr ation verification was determin ed for all trifluralin dose solutions from the first mix prior to admini stration to the animals. Homogeneity (top, middle, bottom) was determined concurrent with the initial concentration verification for the low and high doses.

Results

Stability (% of Time 0): 94.0-100.0%

Concentration (% of nominal): 94.9-97.2%

Homogeneity (%RSD): ≤ 3.7%

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

7. Dosage administration: Test formulations of triflurali n and FT were administered to the animals once daily via oral gavage (dose vol ume 4 mL/kg/day) for 10 consecutive days, beginning on Day 1. TP (0.4 mg/kg/day) wa s given via subcutaneous injection at 0.5 mL/kg/day. Dose volumes were adjusted daily based on the concurrent body weight measurement.

8. Statistics: For this study, the statistical comparisons of interest were as follows:

Androgenicity assay:

oral vehicle control admi nistration vs trifluralin- treated groups without TP oral vehicle control admi nistration vs TP group

Anti-androgenicity assay:

TP group vs trifluralin with TP-treated groups TP group vs TP with flutamide

Body weights, body weight gains, organ weight s (absolute) and feed consumption were evaluated by Bar tlett’s test ( α = 0.01) for equality of varian ces. Based on the outcome of Bartlett's test, a parametric or non-para metric analysis of variance (ANOVA) was performed. If the ANOVA was significant at α = 0.05, analysis by one-sided Dunnett's test ( α = 0.05) or the Wilcoxon Rank-Sum test ( α = 0.05) with Bonferroni's correction was performed, respectively. CVs were calculated for organ weights from the vehicle control

# In vivo Hershberger Assay (2011) / Page 8 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441


# and high-dose triflura lin groups and compared with perf ormance criteria provided in the OECD and OCSPP test guidelines. Preputial separation occurred in all animals in all test groups; therefore, the inciden ce of preputial separation was not statistically compared. Statistical outliers were identi fied using a sequential method ( α = 0.02), and excluded if justified by sound scientific reasons. The stat istical methods were considered appropriate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortal ity, moribundity, and clinical abnormalities were conducted twice daily. Appr oximately one hour after each dose, animals were given a hand-held examination for clinical signs of toxicity and alterations in behavior or demeanor.

2. Body Weight: Animals were weighed at randomi zation, daily during the dosing period, and on the day of euthanasia. Body weights were reported for Days 1, 4, 7, and 11. Mean body weight gains were calculated for Days 1-4, 1-7, and 1-11.

3. Food Consumption: Food consumption (g/rat/day) was determined for Days 1-4, 4-7, and 7-11.

4. Serum Hormone Measurements: Approximately 24 hours following each animal's last dose, blood samples were collected via cardiac puncture following anesthesia by isoflurane inhalation. Serum was obtained and stored at –80°C for anal ysis of serum testosterone levels. Samples were shipped (temperature not specified) to AniLytics Incorporated (Gaithersburg, MD) for analysis. Testosterone was measured from samples collected in the anti-androgen assay using a Coat-A-Count Total Testosterone RIA kit (Siemens Diagnostics, CA).

5. Dissection and Measurement of Tissue and Organ Weights: On PND 65, the rats were checked for preputial separation, euthanized by decapitation following anesthesia, and blood was collected as previously described. Th e following organs were dissected from all animals, trimmed of fat and connective tissue, and weighed: ventral prostate, seminal vesicles with coagulating gl ands and fluid, LABC, glans pe nis, Cowper’s (bulbourethral) glands, kidneys, and adrenals. Tissues were collected and preserved in neutral, phosphatebuffered 10% formalin. The liver also wa s dissected, trimmed, and weighed. Three sections of the liver were preserved in ne utral, phosphate-buffered 10% formalin for possible future histological evaluation, and a portion of the remainder was quick frozen in liquid nitrogen for possible future evaluati on of liver enzyme induction. Two animals (#1257 and 1279) that died spontaneously were submitted for a gross necropsy examination by a veterinary pathologist. Neither organs nor organ weights were collected from these animals at necropsy.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All rats survived the 10-day test pe riod with the exception of one control (animal #1257) and one TP (onl y) treated rat (animal #1279), which died spontaneously on

# In vivo Hershberger Assay (2011) / Page 9 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441


# Days 5 and 2, respectively. The cause of d eath was not identified during necropsy, and no further histopathological examination was perf ormed. These deaths were not related to treatment because trifluralin was no t administered to these animals.

2. Clinical signs of toxicity: Dark yellow-colored urine was seen in trifluralin-treated animals at all dose levels. There were no other obser vations related to treatment on any animal throughout the duration of the study. Trifluralin is a yellow-or ange crystal and the abnormal urine color may be related to the color of the test compound or its metabolites. In the absence of other evidence of toxicity, this clinical finding was not considered adverse.

B. BODY WEIGHT AND WEIGHT GAIN: Body weight and body weight gain data are presented in Table 2 for the androgen agonist assay and in Table 3 for the anti-androgen assay. In the androgen agonist assay at 450 mg/kg/day, body we ights were decreased (NS) throughout the study ( ↓ 6–8%), compared to vehicle cont rols, and body weight gains were decreased (p ≤ 0.05) at each measured interv al (Days 1-4, 1-7, and 1-11; ↓ 31–88%). In the anti-androgen assay at 450 mg/ kg/day with TP, compared to negative controls decreased (p ≤ 0.05) body weights were observed on Days 7 ( ↓ 8%) and 11 ( ↓ 10%), and body weight gains were decreased (p ≤ 0.05) at each measured interval ( ↓ 46-65%). Overall body weight gains (Days 1-11) were decreased (p ≤ 0.05) at 150 mg/kg/day with TP ( ↓ 22%). All other body weights and body weight gains in the triflura lin-treated groups were similar to controls in both the androgen agonist and anti-androgen assays.

There was no effect of flutamide on body weight s or body weight gains. TP administration produced increases (p ≤ 0.05) of 20–48% in body weight gains at all intervals (Days 1-4, 1-7, and 1-11) and an increase in body weight co mpared to vehicle controls at Day 11 ( ↑ 8%). These body weight gain increases were consiste nt with an androgenic response in the test animals.

# In vivo Hershberger Assay (2011) / Page 10 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441



# TABLE 2. Selected Group Mean (±SD ) Body Weights and Cumulative Body Weight Gains (g) in the Androgen Agonist Assay a



# Study Day # Dose (mg/kg/day)

Vehicle Control Positive Control Vehicle + TP (0.4) b 

# Trifluralin (50) Trifluralin (150) Trifluralin (450)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1 7 271.1 15.6 7 266.4 12.9 7 270. 4 13.0 7 267.5 20.4 7 265.7 17.2

4 7 291.7 16.0 6 287.7 11.4 7 290.1 14.9 7 284.3 20.8 7 268.2 ( ↓ 8) 19.1

7 6 305.6 11.2 6 319.4 14.5 7 310.0 17.5 7 300.3 19.9 7 287.7 ( ↓ 6) 20.8

11 6 330.6 11.1 6 356.7* ( ↑ 8) 18.3 7 335.2 20.1 7 326.6 23.3 7 309.1 ( ↓ 7) 23.0

BWG (Days 1-4) 7 20.5 1.6 6 24.6* ( ↑ 20) 3.4 7 19.7 3.0 7 16.8 3.3 7 2.5* ( ↓ 88) 6.7

BWG (Days 1-7) 6 38.3 5.7 6 56.3* ( ↑ 47) 7.3 7 39.5 5.1 7 32.8 7.4 7 22.0* ( ↓ 43) 7.5

BWG (Days 1-11) 6 63.3 8.8 6 93.5* ( ↑ 48) 11.1 7 64.7 8.4 7 59.1 9.2 7 43.4* ( ↓ 31) 10.7 a Data were obtained from Table 4 on pages 40-41 of the study report. b The positive control group for the androgen agonist assay is same as the negative control group for the anti-androgen assay. This did not affect the outc ome or conclusions of this study. * Significantly different from vehicle control at p ≤ 0.05. Percent differences from vehi cle control, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation BWG Body weight gain



# TABLE 3. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the AntiAndrogen Assay a



# Study Day # Dose (mg/kg/day) Negative Control Vehicle + TP (0.4) b 

# Positive Control FT/TP (3/0.4) Trifluralin/TP (50/0.4) Trifluralin/TP (150/0.4) Trifluralin/TP (450/0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1 7 266.4 12.9 7 266.1 17.9 7 271. 3 15.9 7 269.7 14.5 7 269.5 7.9 4 6 287.7 11.4 7 289.5 20.5 7 296. 6 18.4 7 289.3 13.0 7 278.3 11.1

7 6 319.4 14.5 7 318.2 23.3 7 327.0 20.0 7 313.8 14.9 7 295.0* ( ↓ 8) 17.6

11 6 356.7 18.3 7 351.2 28.2 7 361.1 19.7 7 342.3 19.8 7 320.3* ( ↓ 10) 16.1

BWG (Days 1-4) 6 24.6 3.4 7 23.3 4.9 7 25.3 4.3 7 19.6 3.0 7 8.7* ( ↓ 65) 5.9

BWG (Days 1-7) 6 56.3 7.3 7 52.1 8.8 7 55.7 6.6 7 44.2 4.1 7 25.5* ( ↓ 55) 16.3

BWG (Days 1-11) 6 93.5 11.1 7 85.1 13.4 7 89.8 7.7 7 72.6* ( ↓ 22) 9.7 7 50.8* ( ↓ 46) 16.1 a Data were obtained from Table 4 on pages 41-43 of the study report. b The negative control group for the anti-androgen assay is same as the positive control group for the androgen agonist assay. This did not affect the outc ome or conclusions of this study. * Significantly different from negative control at p ≤ 0.05. Percent differences from negati ve control, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation BWG Body weight gain

# In vivo Hershberger Assay (2011) / Page 11 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441



# C. FOOD CONSUMPTION: Food consumption data are presented in Table 4 for the androgen agonist assay and in Table 5 for the anti-androgen assay. In the androgen agonist assay at 450 mg/kg/day, food consumption was decreased (p ≤ 0.05) during the first 4-day interval (Days 1-4; ↓ 35%), compared to vehicle controls. In the anti-androgen assay at 450 mg/kg/day with TP, compared to negative controls decreased food consumption was observed during each interval, Days 1-4 ( ↓ 31%; NS), 4-7 ( ↓ 12%; p ≤ 0.05), and 7-11 ( ↓ 14%; p ≤ 0.05). At 150 mg/kg/day with TP, food consumption was decreased (p ≤ 0.05) by 8% during Days 7-11, compared to negative co ntrols. Food consumption at 50 and 150 mg/kg/day was similar to vehicle controls in the androgen ag onist assay, and food consumption at 50 mg/kg/day was similar to nega tive controls in the an ti-androgen assays.

There was no effect of flutamide on food c onsumption. Food consumption was increased (p ≤ 0.05) by 16 and 18% in TP-treated animals rela tive to vehicle cont rols during Days 4-7 and 7-11, respectively, corresponding to th e increased body weights and supporting an anabolic response to androgen treatment. Only a minor increase (NS) in food consumption was noted in the TP-treated animals during the first interval, Days 1-4.

TABLE 4. Selected Group Mean (±SD) Food Consum ption (g/rat/day) in the Androgen Agonist Assay a



# Study Day # Dose (mg/kg/day)

Vehicle Control Positive Control Vehicle + TP (0.4) b 

# Trifluralin (50) Trifluralin (150) Trifluralin (450)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD
Days 1–4 7 21.3 1.0 4 c 

# 21.8 0.1 7 20.4 1.7 7 19.0 1.6 7 13.8* ( ↓ 35) 2.9

Days 4–7 5 c 

# 19.9 0.5 6 23.1* ( ↑ 16) 1.0 7 21.1 2.4 7 19.4 2.1 7 19.5 1.3

Days 7–11 5 c 

# 20.6 0.4 6 24.4* ( ↑ 18) 1.3 7 21.1 2.1 7 20.2 1.4 7 19.7 1.8 a Data were obtained from Table 5 on pages 44-45 of the study report. b The positive control group for the androgen agonist assay is same as the negative control group for the anti-androgen assay. This did not affect the outcome or conclusions of this study. c Values for one or two animals were eliminated due to "scratched feed" or statistical outlier; in addition, one animal died. * Significantly different from vehicle control at p ≤ 0.05. Percent differences from vehicle c ontrol, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation

# In vivo Hershberger Assay (2011) / Page 12 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441



# TABLE 5. Selected Group Mean (±SD) Food Cons umption (g/rat/day) in the Anti-Androgen Assay a



# Study Day # Dose (mg/kg/day) Negative Control Vehicle + TP (0.4) b 

# Positive Control FT/TP (3/0.4) Trifluralin/TP (50/0.4) Trifluralin/TP (150/0.4) Trifluralin/TP (450/0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD Days 1–4 4 c 

# 21.8 0.1 7 21.2 1.5 7 22.0 1.6 7 19.2 0.8 7 15.1 ( ↓ 31) 1.5
Days 4–7 6 23.1 1.0 7 22.8 1.9 7 24.1 1.6 7 22.0 0.5 4 d 

# 20.3* ( ↓ 12) 2.4
Days 7– 11 6 24.4 1.3 7 23.7 1.9 7 24.9 1.4 7 22.5* ( ↓ 8) 0.6 7 21.0* ( ↓ 14) 0.6 a Data were obtained from Table 5 on pages 45-47 of the study report. b The negative control group for the anti-androgen assay is same as the positive control group for the androgen agonist assay. This did not affect the outcome or conclusions of this study. c Values for one or two animals were eliminated due to "scratched feed" or statistical outlier; in addition, one animal died. d Values for three animals were eliminated due to "scratched feed." * Significantly different from negative control at p ≤ 0.05. Percent differences from nega tive control, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation



# D. SERUM HORMONE CONCENTRATIONS: Serum testosterone (T) measurements in the anti-androgen assay are presented in Tabl e 6. Compared to the negative control group, serum testosterone was decreased by 27% (N S) at 150 mg/kg/day with TP and by 36% (p ≤ 0.05) at 450 mg/kg/day with TP.

TABLE 6. Hormone Measurements in the Anti-Androgen Assay a



# Hormone Dose (mg/kg/day) Negative Control Vehicle + TP (0.4) Trifluralin/TP (50/0.4) Trifluralin/TP (150/0.4) Trifluralin/TP (450/0.4)

N Mean SD CV (%) b 

# N Mean SD CV (%) b 

# N Mean SD CV (%) b 

# N Mean SD CV (%) b


# Serum T (ng/mL) 6 0.70 0.13 18.6 7 0.69 0.21 30.4 7 0.51 ( ↓ 27) 0.17 33.3 7 0.45* ( ↓ 36) 0.19 42.2
a Data were obtained from Table 9 on page 54 of the study report. b Calculated by the reviewers from data presented in this table. N Number of animals in the group SD Standard Deviation CV= Coefficient of Variation * Significantly different from negative control at p<0.05. Per cent differences from negative control, calculated by the reviewers, are included in parentheses.



# E. ORGAN WEIGHTS: Accessory sex organ weights are presented in Tables 7 (androgen agonist assay) and 8 (a nti-androgen assay).

For the androgenic portion of the study, ther e were no treatment-related effects on the weights of any of the accessory sex tissues (seminal vesicles, ventral prostate, LABC, Cowper's glands, or glans penis) , adrenals, or kidney at any dos e of trifluralin. For the TP positive control group, there were treatment-related increases (p ≤ 0.05) in the weights of the seminal vesicles with coagulating glands ( ↑ 1452%), ventral prostate ( ↑ 1328%), LABC

# In vivo Hershberger Assay (2011) / Page 13 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD 441


# ( ↑ 199%), Cowper’s glands ( ↑ 580%), and glans penis ( ↑ 66%), indicating that the test system was sensitive to an androgenic response.

For the anti-androgenic portion of the study at 450 mg/kg/ day with TP, decreases (p ≤ 0.05) were noted in the weights of the semina l vesicles with co agulating glands ( ↓ 21%), ventral prostate ( ↓ 39%), and LABC ( ↓ 21%), indicating an anti-androgenic response. For the flutamide positive control group, there were treatment-related decreases (p ≤ 0.05) in weights of the seminal vesicles with coagulating glands ( ↓ 70%), ventral prostate ( ↓ 66%), LABC ( ↓ 44%), Cowper’s glands ( ↓ 57%), and glans penis ( ↓ 14%) compared to weights in the negative control group. These data indicate that the test system was sensitive to an antiandrogenic response.

Liver weights were increased (p ≤ 0.05, except as noted) by triflu ralin treatment in both the androgenic and anti-androge nic portions of the study, by 16% at 150 mg/kg/day (NS without TP) and by 28 and 36% at 450 mg/kg/da y (with and without TP, respectively).

In the anti-androgen assay, all %CV levels were less than the guideline specified maximums. In the androgen agonist assay, only the ventral prostate in the vehicle control group failed to meet the guideline criteria for maximum %CV (49.0% vs 45% maximum).In vivoHershberger Assay (2011) / Page 14 of 18TRIFLURALIN/ 036101OCSPP 890.1400/ OECD NoneTABLE 7. Selected Accessory Sex Organs, Adrenals, Kidneys and Liver Weights (g) fromAndrogen Agonist Assay in Sprague-Dawley RatsaOrganDose (mg/kg/day)Vehicle controlTrifluralin (50)Trifluralin (150)Trifluralin (450)Positive ControlVehicle + TP (0.4)bN Mean SDCV(%)dN Mean SDCV(%)dN Mean SDCV(%)dN Mean SDCV(%)dN Mean SDCV(%)dSeminalvesiclesc6 0.0434 0.0088 20.3 7 0.0497 0.0172 34.6 7 0.0482 0.0098 20.3 7 0.0472 0.0051 10.8 60.6737*(↑1452)0.0802 11.9Ventralprostate6 0.0147 0.0072 49.0 7 0.0143 0.0053 37.1 7 0.0159 0.0048 30.2 7 0.0169 0.0025 14.8 60.2099*(↑1328)0.0435 20.7LABC 6 0.1783 0.0296 16.6 7 0.1757 0.01448.2 7 0.1871 0.0217 11.6 7 0.1662 0.0308 18.5 60.5325*(↑199)0.0631 11.8Cowper’sglands6 0.0074 0.0020 27.0 7 0.0069 0.0018 26.1 7 0.0094 0.0019 20.2 7 0.0073 0.0028 38.4 60.0503*(↑580)0.0151 30.0Glans penis 6 0.0542 0.0074 13.7 7 0.0598 0.0046 7.7 7 0.0559 0.0083 14.8 7 0.0599 0.0058 9.7 60.0902*(↑66)0.0107 11.9Liver 6 13.0 0.8 6.2 7 14.5 1.4 9.7 715.1(↑16)1.5 9.9 717.7*(↑36)3.1 17.5 6 14.1 1.2 8.5Adrenalglands6 0.0580 0.0080 13.8 7 0.0600 0.0099 16.5 7 0.05450.0079 14.5 7 0.0554 0.006912.5 6 0.0546 0.0046 8.4Kidney 6 2.0999 0.1460 7.0 7 2.1558 0.1514 7.0 72.0402 0.1647 8.1 7 2.0214 0.1776 8.8 6 2.2304 0.1450 6.5a Data were obtained from Table 6 onpages 48-49 of the study report.b The positive control group for the androgenagonist assay is same as the negative control group for the anti-androgen assay.This did not affect the outcomeor conclusions of this study.c Seminal vesicles and coagulating glandsd Calculated by the reviewers from data presented in this table.N Number of animals in the groupSD Standard DeviationCV Coefficient of Variation (%).* Significantly different from vehicle control at p≤0.05. Percent differences of the treated groups from the vehicle control were calculated by the reviewers and included in parentheses.In vivoHershberger Assay (2011) / Page 15 of 18TRIFLURALIN/ 036101OCSPP 890.1400/ OECD NoneTABLE 8. Selected Accessory Sex Organs, Adrenals, Kidneys and Liver Weights (g) from Anti-Androgen Assay in Sprague-Dawley RatsaOrganDose (mg/kg/day)Negative controlVehicle + TP (0.4)bTrifluralin/TP (50/0.4)Trifluralin/TP (150/0.4) Trifluralin/TP (450/0.4)Positive ControlFT+TP (3/0.4)N Mean SDCV(%)cN Mean SDCV(%)cN Mean SDCV(%)cN Mean SDCV(%)cN Mean SDCV(%)cSeminalvesiclesc6 0.6737 0.0802 11.9 7 0.7060 0.109015.46 0.6097 0.126820.870.5350*(↓21)0.096618.170.1991*(↓70)0.033016.6Ventralprostate6 0.2099 0.0435 20.7 7 0.1636 0.025115.37 0.1792 0.058632.770.1283*(↓39)0.022917.870.0721*(↓66)0.011315.7LABC 6 0.5325 0.0631 11.8 7 0.5314 0.067812.87 0.4641 0.053611.570.4184*(↓21)0.056413.570.3003*(↓44)0.039413.1Cowper’sglands6 0.0503 0.0151 30.0 7 0.0436 0.011827.17 0.0413 0.007518.27 0.0378 0.009023.870.0216*(↓57)0.003315.3Glans penis 6 0.0902 0.0107 11.9 7 0.0964 0.00707.37 0.0935 0.00384.17 0.0915 0.00596.470.0773*(↓14)0.00749.6Liver 6 14.1 1.2 8.5 7 16.0 1.5 9.4 716.4*(↑16)1.8 11.0 718.1*(↑28)1.9 10.5 7 14.9 2.1 14.1Adrenalglands6 0.0546 0.0046 8.4 7 0.0504 0.008717.37 0.0485 0.005812.07 0.0505 0.006312.57 0.0617 0.008213.3Kidneys 6 2.2304 0.1450 6.5 7 2.4399 0.22329.17 2.2963 0.19198.47 2.0908 0.11845.77 2.1523 0.15317.1a Data were obtained from Table 6 on pages 50-51 of the study report.b The negative control group for the anti-androgen assay is same as the positive control group for the androgen agonist assay.This did not affect the outcomeor conclusions of this study.c Calculated by the reviewers from data presented in this table.dSeminal vesicles and coagulating glandsN Number of animals in the groupSD Standard DeviationCV Coefficient of Variation (%)* Significantly different fromthe negative control at p≤0.05. Percent differences of the treated groups from the negative control were calculated by the reviewers and included in parentheses.

# In vivo Hershberger Assay (2011) / Page 16 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD None



# F. GROSS PATHOLOGY: No treatment-related effects were observed during gross pathology.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the lack of statistically significant, treatment-related increases in two accessory sex tissue (AST) wei ghts in the androgenic portion of the assay, trifluralin at doses ≤ 450 mg/kg/day was deemed negative for androgenic activity in the Hers hberger assay. Based on signifi cant decreases in three AST weights in the anti-androgenic por tion of the assay, the highest dose level of trifluralin was deemed positive for anti-androgenic potential; however, the significant increase in liver weight, significant decrease in serum testosterone level, and pr evious toxicity data suggest that the anti-androgenic effects of trifluralin are likely second ary to enhanced testosterone metabolism and clearance, resulting in decr eased AST weights, and not due to direct interaction with the androgen receptor.

B. AGENCY COMMENTS: No treatment-related effect s were observed on mortality, clinical signs of toxi city, or gross pathology.

In the androgen agonist assay at 450 mg /kg/day, body weights were decreased (NS) throughout the study ( ↓ 6–8%), compared to vehicle cont rols, and body weight gains were decreased (p ≤ 0.05) at each measured interv al (Days 1-4, 1-7, and 1-11; ↓ 31–88%). In the anti-androgen assay at 450 mg/ kg/day with TP, compared to negative controls decreased (p ≤ 0.05) body weights were observed on Days 7 ( ↓ 8%) and 11 ( ↓ 10%), and body weight gains were decreased (p ≤ 0.05) at each measured interval ( ↓ 46-65%). Overall body weight gains (Days 1-11) were decreased (p ≤ 0.05) at 150 mg/kg/day with TP ( ↓ 22%). All other body weights and body weight gains in the triflura lin-treated groups were similar to controls in both the androgen agonist and anti-androge n assays. The effect on body weights at 450 mg/kg/day was sufficient to indicate that this dose was adequate.

In the androgen agonist assay at 450 mg /kg/day, food consumption was decreased (p ≤ 0.05) during the first 4-day interval (Days 1-4; ↓ 35%), compared to vehicle controls. In the antiandrogen assay at 450 mg/kg/day with TP, comp ared to negative controls decreased food consumption was observed during each interval, Days 1-4 ( ↓ 31%; NS), 4-7 ( ↓ 12%; p ≤ 0.05), and 7-11 ( ↓ 14%; p ≤ 0.05). At 150 mg/kg/day with TP , food consumption was decreased (p ≤ 0.05) by 8% during Days 7-11, compared to negative controls. All other food consumption values in the trifluralin-treated gr oups were similar to controls in both the androgen agonist and anti -androgen assays.

For the androgenic portion of the study, ther e were no treatment-related effects on the weights of any of the accessory sex tissues (seminal vesicles, ventral prostate, LABC, Cowper's glands, or glans penis), adrenals , or kidney at any dos e of trifluralin.

For the anti-androgenic portion of the study at 450 mg/kg/ day with TP, decreases (p ≤ 0.05) were noted in the weights of the semina l vesicles with co agulating glands ( ↓ 21%), ventral prostate ( ↓ 39%), and LABC ( ↑ 21%), indicating an anti-androge nic response. In the antiandrogen assay, compared to negative contro ls, serum testosterone was decreased by 27% (NS) at 150 mg/kg/day with TP and by 36% (p ≤ 0.05) at 450 mg/kg/day with TP. The report

# In vivo Hershberger Assay (2011) / Page 17 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD None



# stated that available data for trifluralin support a mode-of-action related to enhanced testosterone metabolism/clearance. Trifluralin was negative in a concurrently submitted in vitro androgen receptor binding assay (MRID 48619702) using rat ventral prostate cytosol. In a previous mode-of-action study 2 

# with trifluralin, male rats given diet containing 6500 ppm trifluralin (441 ± 77 mg/ kg/day) for 2 weeks had a 2.4-fold increase in hepatic uridinediphosphate glucuronyltransferases, key enzymes for testosterone glucuronide conjugation and subseque nt urinary excretion. 3 

# These trifluralin-tr eated animals had a 14% decrease in terminal body weight and a 28% in crease in liver weights similar to the highdose animals in the current study. Moody et al., (1991) 4 

# also reported similar findings wherein three successive days of intraperitoneal injections of trifluralin at 250 mg/kg /day resulted in significant increases in cytochrome P450, gluthathione-s-tra nsferase, and epoxide hydrolase in rat livers. The report stated that these resu lts support the co nclusion that treatment with trifluralin induced metabolic enzymes responsible for testosterone metabolism and/or clearance, which subsequen tly resulted in decreased AST weights in high-dose animals. The reviewers agree that this conclusi on is plausible.

Liver weights were increased (p ≤ 0.05, except as noted) by triflu ralin treatment in both the androgenic and anti-androge nic portions of the study, by 16% at 150 mg/kg/day (NS without TP) and by 28 and 36% at 450 mg/kg/ day (with and without TP, respectively).

TP administration produced increases (p ≤ 0.05) of 20–48% in body weight gains at all intervals (Days 1–4, 1–7, and 1–11) and an increa se in body weight compared to vehicle controls at Day 11 ( ↑ 8%). Food consumption was increased (p ≤ 0.05) by 16 and 18% in TPtreated animals re lative to vehicle contro ls during Days 4–7 and 7–11, respectively. Only a minor increase (NS) in food consumption was noted in the TP-treated animals during the first interval, Days 1–4. For the TP positive control group, there were treatment-related increases (p ≤ 0.05) in the weights of the seminal ve sicles with coagulating glands ( ↑ 1452%), ventral prostate ( ↑ 1328%), LABC ( ↑ 199%), Cowper’s glands ( ↑ 580%), and glans penis ( ↑ 66%), indicating that the test system was sensitive to an an drogenic response. All these findings were expected androgenic re sponses of the positive control.

There was no effect of flutamide on body weight s, body weight gains, or food consumption. For the flutamide positive control group, there were treatment-related decreases (p ≤ 0.05) in weights of the seminal vesicles with coagulating glands ( ↓ 70%), ventral prostate ( ↓ 66%), LABC ( ↓ 44%), Cowper’s glands ( ↓ 57%), and glans penis ( ↓ 14%) compared to weights in the negative control group. These data indicate that the test system was sensitive to an antiandrogenic response.

In the anti-androgen assay, all %CV levels were less than the guideline specified maximums. In the androgen agonist assay, only the ventral prostate in the vehicle control

2 

# Saghir, S. A., Charles, G. D., Bartels, M. J., Kan, L. H., Dryzga, M. D., Brzak, K. A., and Clark, A. J. (2008).

Mechanism of trifluralin-induced thyroid tumors in rats. Toxicol Lett. 180, 38-45.
3 

# Schulze, J. J., Lundmark, J., Garle, M., Skilving, I., Ekström, L., and Rane, A. (2008). Doping test results

dependent on genotype of UGT2B17, the major enzyme for testosterone glucuronidation. J Clin Endocrinol Metab 93 , 2500–2506.
4 

# Moody, D. E., Narloch, B. A., Shull, L. R., Hammock, B. D. (1991). The effect of structurally divergent

herbicides on mouse liver xenobiotic-metabolizing enzymes (P-450-dependent mono-oxygenases, epoxide hydrolases and glutathione S-transfer ases) and carnitine acetyltransferase. Toxicol Lett

. 59 , 175-85.

# In vivo Hershberger Assay (2011) / Page 18 of 18 TRIFLURALIN / 036101 OCSPP 890.1400/ OECD None


# group failed to meet the guideline criteria for maximum %CV (49.0% vs 45% maximum).

Trifluralin was negative for androgenic activity in the Hershberger assay as no statistically significant increases in organ weights were seen in two or more of the five androgen sensitive t issues in the androgen agonist assay. Trifluralin was positive for antiandrogenicity in the Hershberger assay based on statistically significant decreases in organ weights seen in three of the five androgen sensitive tissues in the andr ogen antagonist assay. However, the target organ weight reductions were observed at a dose (450 mg/kg/day) that exhibited overt toxicity [d ecreased body weights (10%) a nd body weight gains (46%)], which confounds the interp retation of the study.

C. STUDY DEFICIENCIES: The following deficiency wa s noted but did not have an adverse effect on the results, interpreta tion or conclusions of this study:

 In the androgen agonist assay, the %CV for ve ntral prostate in the vehicle control group exceeded the maximum recommended value (49.0% vs 45% maximum).

# DATA EVALUATION RECORD

TRIFLURALIN

Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-76-2012 (MRID 48674001)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Joseph Rieth, Ph.D., DABT Date: 6/11/2012

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 7/13/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/19/2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/18/2012

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

.

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

# TRIFLURALIN I 

# 036101


# Primary Reviewer: Nancy McCarroll 

# Health Effects Division Secondary Reviewer: Elizabeth Mendez, Ph.D. 

# Health Effects Division Female Pubertal Assay (2012)/ Page 1 of 16 OCSPP 890.14501 OECD None


# Signature:**~~~.!..!.!J!~~=-­** 

# Date:**--:"7"'"~~~a~** 

# Signature

. °"'·**~-+rtW._.illii6IJIM~** 

# Date :

___**__,,~+..r..::."'--**



# DATA EVALUATION RECORD

STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None.



# PC CODE: 036101 

# DP BARCODE: D401753

TXR#: 0055724 CAS No: 1582-09-8



# TEST MATERIAL (PURITY): Trifluralin (97.l %)



# SYNONYMS: 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine



# CITATION: Marty, M.S., C.L. Zablotny, 

# J. Thomas. (2012). Trifluralin: Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female Crl:CD(SD) Rats. Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Report No. 111078, April 9, 2012. "MRID 48674001. Unpublished.



# SPONSOR: Dow AgroSciences, LLC
, 9330 Zionsville Road
, Indianapolis, IN.



# TEST ORDER#: CON 036101-26



# EXECUTIVE SUMMARY: In 

# a Female Pubertal Assay (MRID 48674001), 16 Crl:CD(SD) rats/dose group were treated daily via oral gavage with trifluralin (97.1% a.i., Lot# DKO-B916- 33, TSN 100656) in com oil at doses ofO, 25, 75 and 250 mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day of attainment were recorded. After sacrifice on PND 42, blood was collected for clinical chemistry analyses, including analysis of total thyroxine (T4) 

# and thyro id stimulat ing hormone (TSH) using immunoassays. Liver, pituitary, adrenals and urogenital organs were collected and weighed, and microscopic examinations were performed on the ovaries, uterus, thyroid
, and kidneys.

No treatment-related effects were observed on survival, clinical signs, age of attainment of VO, body weight at VO
, fmal body weight, or gross or histological pathology. No treatment-related effects were noted on cycle status at necropsy, mean age at first estrus, mean cycle**len~** 

# percent cycling, or percent regularly cycling.

At 250 mg/kg/day, body weights were decreased (p<0.05) by 7% on PND 28 and 8% on PND 32. The mean terminal body weight on PND 42 was decreased (not statistically significant; NS) by 5%. Body weight gains were decreased (p<0.05) over the intervals from PND 22-25 (30%), PND 22-28 (21%), and PND 22-32 (17%). After this initial decrease, the body weights of recovered slightly such that the decrease in body weight gains over the entire dosing period

# Female Pubertal Assay (2012)/ Page 2 of 16 TRIFLURALIN / 036101 OCSPP 890.1450/ OECD None


# (PND 22-42) was 8% (NS). This effect on body weight was transient and not considered adverse; there was no evidence that the delayed growth impacted sexual development. There were no effects on body weights or body weight gains at the lo w or intermediate dose.

Absolute, adjusted, and relative liver weights were increased (p <0.05) at the intermediate dose by 11-13% and at the high dose by 17-24%. There was no gross or clinical chemistry evidence to support hepatotoxicity. There were no ot her effects of treatm ent on organ weights.

Serum T 4 

# was decreased (p<0.05) in the mid- and high-dose groups by 33% and 45%, respectively. TSH was increased (p<0.05) by 73 % at the high dose. No effect was noted on thyroid weight. In the thyroid at the high dose, increased (p<0.05) incidences of higher follicular cells (Grade 3; 8/16 treated vs 1/16 controls) with correspondingl y smaller colloid areas (Grade 3; 8/16 treated vs 1/16 controls) were observed. No differences in these parameters were observed in the low- and mid-dose groups.

The high dose was considered to be an adequa te dose based on increased serum creatinine, increased liver weights, and changes in serum T 4 

# and TSH levels.

The assay satisfies the EDSP Tier 1 Test Order requireme nts for a Female Pubertal Assay (OCSPP 890.1450).

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

# Female Pubertal Assay (2012)/ Page 3 of 16 TRIFLURALIN / 036101 OCSPP 890.1450/ OECD None



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting, The Dow Chemical Co.
Location: Midland, MI Study Director: C.L. Zablotny Other Personnel: M.S. Marty, Lead Scientist J. Thomas, Pathologist Study Period: August 5, 2011 to April 9, 2012



# 2. Test Substance: Trifluralin Description: Technical, yellow crystals Source: Dow AgroSciences LLC, Indianapolis, IN Lot/Batch #: Lot no. DKO-B916-33, TSN100656. Exp. date not provided Purity: 97.1 % a.i. Stability: Not provided CAS #: 1582-09-8 Structure:

N

CF 3 

# C H 2


# C H 2 

# CH 3 

# C H 2


# C H 2 

# CH

3



# O 2 

# N NO 2



# 3. Vehicle: Corn oil

4. Test Animals: Species: Rat Strain: Sprague Dawley Crl:CD(SD) Age/Weight at Study Initiation: PND 22, 36.7-59.0 g females only

Source: Charles River Laboratorie s, Inc., Portage, MI Housing: Two per cage in plastic solid bottom cages c ontaining heat-treated aspen wood shavings. Diet: Teklad Diet #2016, Harlan Labora tories, Inc., Indianapolis, IN, ad libitum. Phytoestrogen content <325 μ g of genistein equivalents/gram diet Water: Deionized water, ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 22±3 °C 40-70% 12-15/hr 12 hrs light/ 12 hrs dark



# B. STUDY DESIGN

1. In-Life Dates: Start: August 12, 2011 End: September 2, 2011

2. Mating: Time-mated nulliparous pregnant dams were received on gestation days (GD) 7-10. The dams were ordered from the supplie r to reach GD 21 on the same day; however, litters born on either GD 21 or 22 were used in the assay. Litters with more than 10 pups were standardized by culling (r andom selection) on PND 4 to te n pups per litter with 5/sex when possible.

# Female Pubertal Assay (2012)/ Page 4 of 16 TRIFLURALIN / 036101 OCSPP 890.1450/ OECD None


# 3. Animal Assignment: Offspring were weaned on PND 21 and weighed. Animals were assigned randomly with a computer program designed to provide uniform group mean weights and standard deviations to the test groups noted in Table 1. Littermates were not assigned to the same treatment group.

TABLE 1. Study Design a 

# Test group Dose (mg/kg/day) # of Females Control 0 16 Low 25 16 Mid 75 16 High 250 16 a Data were obtained from page 22 of the study report.



# 4. Dose Selection Rationale: Several studies were considered in the selection of the doses for this study and are described below. In partic ular, the high dose level was selected to avoid the excessive increases in serum creatinin e levels observed in the probe study at 400 mg/kg/day.

In a probe study 1 

# , groups of four male and four female Crl:CD(SD) rats were administered trifluralin by gavage at dose levels of 0, 50, 200, 400, or 600 mg/kg/day in corn oil for 15 days. Male rats were dose d from postnatal day (PND) 30- 44 and female rats from PND 22-36. Over the entire treatment period (Days 1-15), there were no treatment-related effects on body weights or body weight gains in female rats. Male rats had 12%, 13%, and 20% decreases in body weight gains during the Da y 1-15 interval at 200, 400, and 600 mg/kg/day, respectively. Similarly, respective termin al body weights were decreased by 6.3%, 6.5%, and 11.3% in males. At study termination, peri anal (urine) soiling was observed in three high-dose females and clear facial soiling was seen in one intermediate and one high dose male and one high-dose female. There were incr eases in absolute liver weights in both males (9-21% at >200 mg/kg/day) and females (16-52% at >50 mg/kg/da y). In females, relative liver weights were increased in a dose-rela ted manner from 50 mg/kg/day (13%) through 600 mg/kg/day (48%). In males, relative weight s were increased in a dose-related manner from 200 mg/kg/day (18%) through 600 mg/kg/day (3 7%). Relative kidney weights were decreased by <10% in both males and females in the high-dose group. Clinical chemistry parameters suggested a decrease in alka line phosphatase (ALP) and increased gamma glutamyl transpeptidase (GGT) and choleste rol levels at 600 mg/kg/day in males and females. Albumin, globulin, a nd albumin/globulin ratio were altered in high-dose females only. Creatinine was increased at 400 and 600 mg/kg/day in both males and females.

In a dominant lethal study 2 

# , mature male Wistar rats (15/ dose) were given trifluralin by gavage at doses of 0, 100 or 1000 mg/kg/day in 10% aqueous acacia for 5 days. The high dose animals had a 50% reduction in body weight ga in at the end of the exposure period.

1 

# Marty, M. S., Marshall, V. A. and Je ong, Y-C. (2011). Trifluralin: Hershbe rger Assay in Castrated Adult Male Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. MRID 48619707. 2 

# Markham, J. K. and Hoyt, J. A. (1983). A Dominant Lethal Study with Technical Trifluralin (Compound 36352) in the Wistar Rat. Report of The Dow Chemi cal Company performed by Lilly Research Laboratories, Greenfield, Indiana.

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# In a developmental toxicity study 3 

# , groups of 25 mated female Crl:CD(SD) rats were administered trifluralin at dose levels of 0, 100, 225, 475 or 1000 mg/kg/day in 10% aqueous acacia from GD 6-15. Body weight gains for dams given trifluralin on GD 6-10 were decreased 66% at 100 mg/kg/day, 50% at 225 mg /kg/day, and 95% at 475 mg/kg/day; dams given 1000 mg/kg/day lost wei ght during this interval (

− 2.8 g vs. 18.7 g gain in controls). On GD 11-15, dams in the 475 and 1000 mg/kg/ day groups had a 23 and 28% decrease in body weight gain, respectively, with corres ponding decreases in food consumption.

In a two-week mode-of-action study 4 

# , trifluralin was administered to F344 rats in the diet at 6500 ppm (441 mg/kg/day), a con centration that produced thyr oid tumors in the chronic study. Trifluralin caused a 20% reduction in body weight gain and a 14% decrease in terminal body weight compared to controls on Day 15. Relative liver and thyroid weights were increased by 49% and 17%, respectively.

In a uterotrophic study 5 

# , groups of six immature female Cr l:CD(SD) rats were exposed to 0 (vehicle control), 25, 75, or 225 mg/kg/day trif luralin by oral gavage from PND 19-21 with necropsy on PND 22. There we re no significant effects on body weights at any dose of trifluralin. At 225 mg/kg/day trifluralin, body weight gains we re decreased significantly by 34% on Days 1-4. Relative liver weights we re significantly increased by 13% and 17% in the 75 and 225 mg/kg/day triflu ralin groups, respectively. Ther e were no treatment-related increases in mean uterine weights (wet or bl otted) in immature animals treated with <225 mg/kg/day.

In a Hershberger assay 6 

# , trifluralin was administered by oral gavage at dose levels of 0, 50, 150 or 450 mg/kg/day to castrated male CD rats from 55-64 days of age in the presence or absence of 0.4 mg/kg/day testos terone propionate (TP; administ ered subcutaneously). At 450 mg/kg/day trifluralin with and without TP, animals had signifi cantly decreased body weight gains. High-dose trifluralin rats co -administered TP also had a 10% decrease in terminal body weights compared to TP only rats. There were significant increases in absolute liver weights at 450 mg/kg/day trif luralin without TP a nd at >150 mg/kg/day trifluralin with TP. In the anti-androgen ic portion of the assay, high-dose animals had significant decreases in weights of the levator ani-bulbocavernosus muscles, ventral prostate, and seminal vesicles with coagulating glands . Serum testosterone concentrations were significantly decreased in these animal s compared to the TP only rats.

3 

# Markham, J. K. and Byrd, R. A. (1984). A Teratology Study of Trifluralin (EL-152, Compound 36352) Administered Orally to Charles River CD Rats. Re port of The Dow Chemical Company performed by Lilly Research Laboratories, Greenfield, Indiana. 4 

# Saghir, S. A., Charles, G. D., Kan, L. H., Dryzga, M. D ., Brzak, K. A., and Clark, A. J. (2005). Trifluralin: Mechanism of Thyroid Tumors in Rats. Report of Toxi cology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. 5 

# Marty, M. S. and Brooks, K. J. (2011). Trifluralin : Uterotrophic assay in the immature Crl:CD(SD) Rat. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. MRID 48619709.
6 

# Marty, M. S., Marshall, V. A., and Jeong, Y-C. (20 11a). Trifluralin: Hershberger Assay in Castrated Adult Male Crl:CD(SD) Rats. Report of Toxicology & Environm ental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

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# Based on these data, dose levels were select ed to be 0, 25, 75 and 250 mg/kg/day for this female pubertal assay.

5. Dose Preparation and Analysis: Dose formulations were prepared by adding appropriate amounts of test substance with co rn oil to yield the target co ncentration. The report stated that dose solutions were prepared periodi cally throughout the study based upon stability data. Stability in corn oil at ambient temperature was ev aluated for up to 25 days at concentrations of 0.25, 2.5, and 250 mg/mL in a previous study (MRID 48619707). During the study, samples of dose formulations from the first mix of the study were analyzed for achieved concentration; homogeneity was test ed concurrently with the concentration analyses.

Results of Dose Analysis

Homogeneity (%RSD): 2.3-5.7%

Stability (% of Time 0): 94.0–100.0%

Concentration (% nominal): 95.6-98.6%

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: All doses were administered once daily by gavage, from PND 22 through PND 42, in a volume of 4 mL/kg of bo dy weight. Dose volumes were adjusted using the most current body weight.

7. Statistics: For continuous variab les, a Bartlett’s test was conducted to check for homogeneity of variance. Based on the outco me of the Bartlett’s test (alpha = 0.01), variables may have been transformed prior to statistical analysis. Initial body weight, body weight gain, clinical chemistries, mean cycl e length, age at vagina l opening, body weight at vaginal opening, age at first estrus, organ weights, organ- to-body weight ratios (liver, kidney, adrenal, and pituitary weights), and serum hormones were analyzed with a two-way analysis of variance (ANOVA) with block and treat ment as main effects. When significant dose effects were determined in the two-wa y ANOVA at alpha = 0.05, th en individual dose groups were compared to controls using D unnett's test at alpha = 0.05, experiment-wise error. Age and body weight at vaginal ope ning and organ weights (thyroid, ovaries and uterus) were analyzed with analysis of covariance (ANCOVA) with body weight on PND 22 as the covariate. When the dose eff ect was significant at al pha = 0.05, least square means with a Dunnett’s correction was used to determine differences (experiment-wise alpha = 0.05). The report includes values that were unadjusted and adjusted for body weight on PND 22. When the ANCOVA was not statistically significant, a Dunnett’s test also was performed. Globulin and albumin/globulin ratio , which were calculated values, were not statistically analyzed. A chi-square analysis was used to analyze cycling status and percent cycling regularly. When the ch i-square statistic was significan t, it was followed by pairwise comparisons to the control group via a Fish er’s exact test with a Bonferroni-Holm adjustment. Coefficients of variation (CVs) were calculated where appropriate. Control means and CVs were compared with performan ce criteria outlined in the test guideline.

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# Statistical outliers were identified by a sequential test (alpha = 0.02). Outliers and questionable data points ( i.e ., values due to technical e rrors) were excluded only for documented scientifically sound reasons. Hi stopathology results were not subjected to outlier tests. The incidence of specific hist opathological observations were analyzed using the pairwise Chi-square test with Yates’ continuity correc tion (alpha = 0.0 5; two-sided).

C. METHODS

1. Mortality and Clin ical Examinations: Cageside examinations on all animals were performed twice daily for general condition, mo rtality and moribundity. Detailed clinical examinations were conducted daily at about the same time each day following dosing.

2. Body Weight: Animals were weighed on PND 21 and da ily prior to dosing. Body weights were reported and statistica lly analyzed on PND 21, 22, 25, 28, 32, 35 and 42. Body weight gains were reported and statistically analyzed for all reported wei ght intervals beginning with PND 22-25 and including the entire dosing period (PND 22-42).

3. Vaginal Opening (VO): Beginning on PND 22, all animals were examined daily for onset of VO. The appearance of a small “pin hol e”, a vaginal thread, and complete VO were recorded for all days they were observed. Age and weight on the da y of completion of VO were recorded. If an animal had not co mpleted VO by PND 42, PND 43 was used as the day of vaginal opening when determining th e mean age at puberty onset for the group.

4. Estrous Cyclicity: Beginning on the day of VO, up to and including the day of necropsy, daily vaginal lavage smears were obtained at approximately the same time each day and evaluated for day of first estrus and estrous cycl e pattern. The age at fi rst vaginal estrus was recorded.

5. Sacrifice and Pathology: Groups were counter-balanced during necropsy, and necropsies were completed before 1300 hours in accordance with the test guidelin e. On the day of termination, approximately 2 hours post-dos e, rats were anesthetized by whole body isoflurane exposure (c a. 2 min), the abdomen was opene d and blood was collected via the inferior vena cava or cardiac puncture. Blood sa mples were transferred into three separate tubes for clinical chemistry and hormone an alyses. Blood samples for hormone analyses were kept refrigerated or on ice durin g necropsy. Exsanguination was followed by decapitation. A complete necr opsy was conducted on all animals.

a. Hormone Analysis: Blood samples were stored at

− 80°C until shipment on dry ice to WIL Research Laboratories, LLC (Ashland, OH). Hormone levels were analyzed using a chemiluminescent immunoassay (T 4 

# ) or a radioimmunoassay (TSH).
b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER
X Calcium X Albumin X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol X Potassium X Globulins (calculated)

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ELECTROLYTES OTHER X Sodium Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (L DH) X A/G ratio (calculated) X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase (SDH) X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in fe male rats based on guideline 890.1450.



# c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological examination.

UROGENITAL OTHER XX Ovaries (paired, without oviducts)*+ XX Thyroid*+ XX Uterus*+ (with oviducts and cervix) X Liver* XX Kidneys (paired)*+ X Adrenals (paired)* X Pituitary* * Weights required base d on guideline 890.1450 + Histopathological examination re quired based on guideline 890.1450



# All organs except the thyroid/tr achea and pituitary were weighe d prior to fixation. Paired organs (kidneys, adrenals, and ovaries without oviducts) were weighed together. The uterus was weighed again following removal of the fluid in the lumen (blotted weight). The ratios of organ weight to terminal body weight were calculated but only re ported for liver, kidney, adrenal, and pituitary weights.

All collected tissue samples were fixed in ne utral, phosphate-buffered 10% formalin. The kidneys, right ovary, and uterus were fixed in formalin for at least 24 hours and stored in graded ethanol prior to embedding. The thyroi d (with parathyroid) a nd kidneys were fixed in formalin for at least 24 hrs. Followi ng fixation, the thyroid was dissected from the trachea.

All collected tissues were routinely processe d into paraffin blocks, sectioned, and stained with hematoxylin and eosin. Tissues from th e high dose and control groups were examined microscopically, and the other groups were ex amined when treatment-related differences were observed.

Thyroid sections were subjectively evaluated for follicular cell height and colloid area using a five point grading scale (1 = shortes t/smallest; 5 = tallest/largest), and any abnormalities/lesions were noted. At least two sections from each of the two lobes of the thyroid were examined. The right ovar y histology included evaluations of any

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# abnormalities/lesions and follicular development, including: presence/absence of primary, atretic, tertiary/antral follicles, presence/abse nce of corpora lutea, and changes in corpus luteum development. Five random sections were evaluated. Uter ine histology documented cases of uterine hypertrophy or atrophy as charac terized by changes in uterine horn diameter and myometrial, stromal or endometrial gland de velopment. The stage of the estrous cycle of the female at the time of necropsy was documented.

Selected histopathologic findings were graded very slight, slight, moderate, or severe.

II. RESULTS

A. MORTALITY: All animals survived until scheduled termination.

B. CLINICAL SIGNS OF TOXICITY: There were no treatment-related clinical observations in the study.

C. GENERAL GROWTH AND VAGINAL OPENING (VO): Body weights, body weight gains, and age and weight at attainment of VO are presented in Table 2. There were no significant differences in initial (PND 22) or final (PND 42) body weights at any dose level. There were no significant differe nces in age or body weight at VO in any of the treatment groups.
At 250 mg/kg/day, body weights were decreased (p<0.05) by 7% on PND 28 and 8% on PND 32. The mean terminal body weight on PND 42 was decreased by 5% (NS). Body weight gains were decreased (p<0.05) over the intervals from PND 22-25 (30%), PND 22- 28 (21%), and PND 22-32 (17%). Following this initial decrease, the body weights recovered slightly such that the decrease in body weight gains over th e entire dosing period (PND 22-42) was 8% (NS). There were no si gnificant, treatment- related effects on body weight or body weight ga ins at 25 or 75 mg/kg/day.

Guideline criteria in the cont rols for final body weight was met; however, the mean age at VO (36.4 days) was above the maximum acceptable value (35.62 days), and the CV for weight at VO (15.58%) was slightly greater than the maximum acceptable value (13.97%).

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# TABLE 2. General Growth and Vaginal Opening (VO) a



# Parameter Evaluated Vehicle Control Trifluralin 25 mg/kg/day Trifluralin 75 mg/kg/day Trifluralin 250 mg/kg/day

N Mean SD/ SE b 

# CV (%) N Mean SD/ SE b 

# CV (%) N Mean SD/ SE b 

# CV (%) N Mean SD/ SE b 

# CV (%) Initial body weight (PND 22; g) U 16 47.98 4.95 10.32 16 48.21 4.21 8.74 16 48.19 4.51 9.35 16 48.18 3.15 6.54

Body weight at vaginal opening (g) U 16 120.49 

# 18.77 15.58 16 113.84 

# 16.36 14.37 16 123.73 

# 17.47 14.12 16 121.68 

# 17.20 14.14

A 16 120.75 

# 4.044 NA 16 113.72 

# 4.044 NA 16 123.65 

# 4.044 NA 16 121.62 

# 4.044 NA

Final body weight (g) U 16 154.44 

# 11.72 7.59 16 153.75 

# 15.02 9.77 16 156.95 

# 12.80 8.15 16 146.13 

# 13.19 9.02

A 16 154.64 

# 3.049 NA 16 153.66 

# 3.049 NA 16 156.89 

# 3.049 NA 16 146.09 

# 3.048 NA

Final body weight (% of control ) U NA NA NA NA 16 99.6 9.7 9.8 16 101.6 8.3 8.2 16 94.6 8.5 9.0

A NA NA NA NA NR NR NR NR NR NR NR NR NR NR NR NR

Body weight gain (final – initial; g) U 16 106.46 

# 10.87 10.21 16 105.54 

# 14.03 13.29 16 108.76 

# 11.12 10.22 16 97.96 

# 12.30 12.56

A NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR

Age at vaginal opening (PND) U 16 36.4 1.8 4.9 16 35.1 2.0 5.8 16 36.8 2.4 6.6 16 37.6 2.5 6.6

A 16 36.4 0.55 NA 16 35.1 0.55 NA 16 36.7 0.55 NA 16 37.6 0.55 NA

Proportion unopened (#/N) 0/15 0/15 0/15 0/14

a Data were obtained from page 52 of the study report. b Standard deviation (SD) reported for unadjusted values and st andard error of the mean (SE) reported for adjusted values. U Unadjusted for body weight on PND 22 A Adjusted for body weight on PND 22 N Number of animals examined CV Coefficient of Variation NA Not applicable NR Not required



# D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 3. Absolute, adjusted, and relative liver weights were increased (p< 0.05) at 75 mg/kg/day by 11-13% and at 250 mg/kg/day by 17-24%. There were no othe r effects of treatmen t on organ weights.

Guideline criteria were met for organ weight s in the controls, except the mean adrenal weight (32.8 mg) was slightly outside the acceptable range of 38.34-48.84 mg.

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# TABLE 3. Organ Weights at Necropsy a



# Organ Vehicle Control Trifluralin 25 mg/kg/day Trifluralin 75 mg/kg/day Trifluralin 250 mg/kg/day

N Mea

n SD/ SE b 

# CV (%) N Mean SD/ SE b 

# CV (%) N Mean SD/ SE b 

# CV (%) N Mea

n SD/ SE b 

# CV (%)

Liver (g) U 16 7.04 0.60 8.53 16 7.38 1.16 15.71 16 7.92* (13) 0.98 12.42 16 8.24* (17) 1.08 13.10

A 16 7.05 0.232 NA 16 7. 37 0.232 NA 16 7.91* (12) 0.232 NA 16 8.24* (17) 0.232 NA

R 16 4.55 0.13 2.86 16 4.78 0.36 7.53 16 5.03* (11) 0.33 6.60 16 5.63* (24) 0.37 6.51

Kidneys (g) U 16 1.20 0.07 5.97 16 1.21 0.11 8.93 16 1.24 0.11 9.25 16 1.16 0.11 9.31
A 16 1.20 0.022 NA 16 1.20 0.022 NA 16 1.23 0.022 NA 16 1.16 0.022 NA
R 16 0.78 0.07 8.43 16 0.79 0.03 4.24 16 0.79 0.06 7.28 16 0.80 0.06 7.14

Pituitary (mg) U 16 7.8 0.8 9.9 16 8.0 1.3 15.7 16 7.7 0.8 10.2 15 7.2 1.3 17.9
A 16 7.8 0.26 NA 16 8.0 0.26 NA 16 7.7 0.26 NA 15 7.2 0.27 NA
R 16 5.1 0.5 10.1 16 5.2 1.0 18.4 16 4.9 0.5 10.8 15 4.9 1.2 23.4

Adrenals (mg) U 16 32.8 4.5 13.7 16 34.0 3.8 11.3 16 33.6 4.2 12.5 16 32.1 4.8 15.1
A 16 32.9 1.07 NA 16 34.0 1.07 NA 16 33.6 1.07 NA 16 32.1 1.07 NA
R 16 21.3 2.6 12.3 16 22.3 3.1 14.0 16 21.4 2.2 10.4 16 22.2 4.5 20.4
Ovaries (mg) U 16 51.8 8.7 16.8 16 54.3 9.9 18.2 16 51.1 6.0 11.7 16 46.2 8.6 18.6
A 16 51.8 2.09 NA 16 54.2 2.09 NA 16 51.1 2.09 NA 16 46.2 2.09 NA Uterus, wet (mg) U 16 294.6 118.6 40.3 16 318.8 134.4 42.2 16 269.2 151.1 56.1 16 248.3 76.8 30.9
A 16 294.9 31.03 NA 16 318.7 31.03 NA 16 269.1 31.03 NA 16 248.3 31.03 NA Uterus, blotted (mg) U 16 258.3 66.0 25.5 16 278.0 74.2 26.7 16 233.2 80.3 34.4 16 232.0 58.1 25.0
A 16 258.6 17.62 NA 16 277.9 17.62 NA 16 233.1 17.61 NA 16 231.9 17.61 NA Thyroid, fixed (mg) U 16 8.4 2.3 27.1 16 7.8 1.1 14.7 16 8.6 1.7 19.5 16 8.4 1.8 21.8
A 16 8.4 0.44 NA 16 7.8 0.44 NA 16 8.6 0.44 NA 16 8.4 0.44 NA a Data were obtained from page 55 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. b Standard deviation (SD) reported for unadjusted values and st andard error of the mean (SE) reported for adjusted values. U Unadjusted for body weight on PND 22 A Adjusted for body weight on PND 22 N Number of animals examined SD Standard Deviation CV Coefficient of Variation R Organ-to-body weight ratio (relative to body weight) * Significantly different fro m controls at p<0.05.



# E. ESTROUS CYCLICITY: Estrous cycle data are provi ded in Table 4. No treatmentrelated effect was noted on cycle status at ne cropsy, mean age at first estrus, mean cycle length, percent cycling, or percent regularly cycling.

TABLE 4. Estrous Cyclicity a



# Treatment Groups N Mean Age at First Vaginal Estrus (PND) Mean Cycle Length (days) b 

# Cycling (%) b 

# Regularly Cycling

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not Cycling Vehicle 15 37.0 4.8 (6) 93.8 (16) 87.5 14 1 1 0 Trifluralin: 25 mg/kg/day 16 35.8 4.5 (10) 87.5 (16) 75.0 10 0 6 0

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# TABLE 4. Estrous Cyclicity a



# Treatment Groups N Mean Age at First Vaginal Estrus (PND) Mean Cycle Length (days) b 

# Cycling (%) b 

# Regularly Cycling

(%) Cycle Status at Necropsy (# Females)

Diestrus Proestrus Estrus Not Cycling 75 mg/kg/day 15 37.7 4.4 (4) 87.5 (16) 87.5 15 0 1 0 250 mg/kg/day 14 38.3 4.9 (4) 87.5 (16) 87.5 11 0 5 0 a Data were obtained from page 53 of the study report. b Number in parenthesis is N for that parameter N Number of animals



# F. HORMONE LEVELS AND CLINICAL CHEMISTRY: Hormone levels and clinical chemistry values are presented in Table 5. Serum T 4 

# was decreased (p<0.05) at 75 and 250 mg/kg/day by 33 and 45%, respectively. TSH was increased (p<0.05) by 73% at 250 mg/kg/day.

At 250 mg/kg/day, values for the following parameters in the treated group differed (p<0.05) from control: (i) in creased creatinine (1 00%); (ii) decreased alkaline phosphatase (26%); (iii) decreased total b ilirubin (40%); (iv) decrea sed potassium (10%); and (v) increased calcium (3%). At 75 mg/kg/day, cr eatinine was also increased (p<0.05) by 100%, and calcium was increased (p<0.05) by 4%. In addition, the study report provided normal ranges for selected clinic al chemistry measures.

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# TABLE 5. Hormone Levels and Clinical Chemistry a



# Parameter Evaluated Vehicle Control Trifluralin 25 mg/kg/day Trifluralin 75 mg/kg/day Trifluralin 250 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Hormones Serum T 4 

# , Total (μg/dL) 16 3.57 0.72 20.29 16 3.14 0.53 16.95 16 2.40* ( ↓ 33) 0.53 21.92 16 1.95* ( ↓ 45) 0.27 13.73

Serum TSH (ng/mL) 16 3.25 1.32 40.71 16 4.64 1.64 35.33 16 3.56 2.02 56.57 16 5.62* ( ↑ 73) 2.41 42.87

Clinical Chemistry Creatinine (μmol/L) 16 0.1 0.0 36.5 16 0.2 0.1 38.1 16 0.2* ( ↑ 100) 0.0 18.2 16 0.2* ( ↑ 100) 0.00 18.4

Serum urea nitrogen (mg/dL) 16 13 3 22 16 14 2 18 16 13 2 18 16 15 4 26

Alkaline phosphatase (U/L) 16 333 73 22 16 325 54 17 16 290 71 25 16 248* ( ↓ 26) 47 19

Alanine aminotransferase (U/L) 16 57 6 10 16 61 13 21 16 57 6 11 16 55 6 11

Aspartate aminotransferase (U/L) 16 112 18 16 16 122 17 14 16 128 34 26 16 127 30 24

Gamma glutamyl transferase (U/L) 16 1.5 0.0 0.0 16 1.5 0.0 0.0 16 1.5 0.0 0.0 16 1.5 0.0 0.0

Albumin (g/dL) 16 4.0 0.2 5.7 16 4.0 0.3 6.3 16 4.0 0.2 5.8 16 3.9 0.3 6.5 A/G ratio 16 3.0 0.3 11.3 16 2.9 0.3 11.4 16 2.9 0.3 12.0 16 2.7 0.3 12.4 Cholesterol (mg/dL) 16 91 7 8 16 92 9 10 16 101 17 17 16 101 12 12 Globulin (g/dL) 16 1.3 0.1 8.1 16 1.4 0.1 8.3 16 1.4 0.2 10.9 16 1.5 0.1 8.4 Total bilirubin (mg/dL) 16 0.05 0.01 23.53 16 0.05 0.01 23.53 16 0.05 0.02 27.91 16 0.07* ( ↓ 40) 0.03 45.18

Total protein (g/dL) 16 5.3 0.2 4.2 16 5. 4 0.3 4.8 16 5.4 0.3 5.7 16 5.4 0.3 4.7 Sodium (mmol/L) 16 143 2 1 16 143 1 1 16 143 1 1 16 143 2 1 Potassium (mmol/L) 16 4.9 0.5 9.2 16 4.7 0.3 7. 2 16 4.9 0.3 6.3 16 4.4* ( ↓ 10) 0.2 4.6

Chloride (mmol/L) 16 100 1 1 16 100 1 1 16 100 2 2 16 99 2 2 Calcium (mg/dL) 16 11.1 0.3 2.4 16 11.1 0.2 2.1 16 11.5* ( ↑ 4) 0.4 3.3 16 11.4* ( ↑ 3) 0.3 3.0

Phosphorus (mg/dL) 16 9.1 0.8 9.0 16 8.8 0.9 10.0 16 9.3 0.8 8.3 16 9.1 0.5 5.3 a Data were obtained from pages 36 and 54 of the study report. Percent differences from control, calculated by reviewers, are presented in parentheses. N Number of animals examined SD Standard deviation SE Standard error CV Coefficient of variation * Significantly different fro m controls at p<0.05.



# G. GROSS PATHOLOGY: No treatment-related gross lesions were observed. All rats given 250 mg/kg/day had yellow discol oration of the subcutaneous and intra-abdominal adipose tissue which was interpreted to be due to staini ng by the test material (yellow) and/or its metabolites.

H. Histopathology: The incidences of histopathological findings of the thyroid gland are presented in Table 6. In the thyroid at 250 mg/kg/day, increased (p<0.05) incidences of higher follicular cells (Grade 3; 8/16 treated vs 1/16 controls) with correspondingly smaller colloid areas (Grade 3; 8/16 treated vs 1/16 controls) were observed. No differences in these parameters were observed at 25 or 75 mg/kg/day.

# Female Pubertal Assay (2012)/ Page 14 of 16 TRIFLURALIN / 036101 OCSPP 890.1450/ OECD None



# TABLE 6. Incidence of Histopatho logical Lesions of the Thyroid Gland a



# Treatment Groups Parameter Evaluated Colloid Area Follicular Cell Height

Grade b 

# Incidence Grade b 

# Incidence O E O E

Vehicle Control 1 0 16 1 7 16 2 0 16 2 8 16 3 1 16 3 1 16 4 8 16 4 0 16 5 7 16 5 0 16

Trifluralin 25 mg/kg/day 

# 1 0 16 1 6 16 2 0 16 2 8 16 3 2 16 3 2 16 4 8 16 4 0 16 5 6 16 5 0 16

Trifluralin 75 mg/kg/day 

# 1 0 16 1 7 16 2 0 16 2 8 16 3 1 16 3 1 16 4 8 16 4 0 16 5 7 16 5 0 16

Trifluralin 250 mg/kg/day 

# 1 0 16 1 5 16 2 0 16 2 3 16 3 8* 16 3 8* 16 4 3 16 4 0 16 5 5 16 5 0 16 a Data were obtained from page 61 of the study report. b Thyroid histopathology is graded on a 1 – 5 scale: Follicular cell height,1 = lowest, 5 = highest; and Colloid area, 1 = most colloid, 5 = least colloid. O Number observed E Number examined * Significantly different fro m controls at p<0.05.



# No histological lesions were not ed in the ovaries or uterus. No treatment-related findings were observed in the kidney (Table 7). Th e following renal lesions were observed in the high dose group, but occurred at a similar incide nce in the control group (# affected/16 in treated vs # affected/16 controls): unilateral tu bular cyst (1 treated vs 0 controls), tubule degeneration (8 treated vs 9 c ontrols), and tubule dilatation (1 treate d vs 2 controls).

TABLE 7. Incidence (# affected/16) of Histological Lesions in the Kidney a 

# Finding Control 250 mg/kg/day Trifluralin Focal unilateral tubular cyst 0 1 Very slight multifocal tubule degeneration 9 8 Slight unilateral pelvis dilatation 1 0 Very slight focal tubule dilatation 2 1 Very slight focal interstitium fibrosis 1 0 Very slight focal subacute to chronic inflammation 3 0 Primary benign tubular adenoma 1 0 a Data were obtained from Table 13 on page 62 of the study report.

# Female Pubertal Assay (2012)/ Page 15 of 16 TRIFLURALIN / 036101 OCSPP 890.1450/ OECD None



# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: Based on the lack of treatment-related effects on puberty onset, estrous cycle, reproductive organ weights, and uterine and ovarian histopathology, there was no evidence of alteratio ns in the estrogenic pathway at any dose of trifluralin. There was some evidence for alte rations in the thyroid pathway at 250 mg/kg/day trifluralin, which incl uded decreased serum T 4 

# , increased TSH, and altered thyroid histopathology; these effects re quire consideration in the trifluralin weight-of-evidence (WoE) assessment for the thyroid pathway. The WoE also should include previous trifluralin mode-of-action studies, which show hepa tic enzyme induction and enhanced T 4 

# and steroid hormone clearance.

B. AGENCY COMMENTS: No treatment-related effect was observed on survival, clinical signs, age of attainment of VO, body wei ght at VO, final body weight, or gross or histological pathology. No treatment-related effect was not ed on cycle status at necropsy, mean age at first estrus, mean cycle length, pe rcent cycling, or percent regularly cycling.

At 250 mg/kg/day, body weights were decreased (p<0.05) by 7% on PND 28 and 8% on PND 32. The mean terminal body weights on PND 42 were decreased by 5% (NS) at this dose level. Body weight gains were decrea sed (p<0.05) over the intervals from PND 22-25, PND 22-28, and PND 22-32. After this initial decrease, the b ody weights of animals in the 250 mg/kg/day group recovered slightly such that the decrease in body weight gains over the entire dosing period (PND 22-42) was 8% (NS). This effect on body weight was transient and not considered adverse; ther e was no evidence that the delayed growth impacted sexual development. There were no effects on body weights or body weight gains at 25 or 75 mg/kg/day.

Absolute, adjusted, and relative liver weights were increased (p<0.05) at 75 and at 250 mg/kg/day. There was no gross or clinical ch emistry evidence to support hepatotoxicity. Histology was not performed, but is required to distinguish hepatotoxicity from an adaptive response. There were no other effe cts of treatment on organ weights.

Serum T 4 

# was decreased (p<0.05) at 75 and 250 mg /kg/day by 33 and 45%, respectively, and TSH was increased (p<0.05) by 73% at 250 mg/kg/day. No effect was noted on thyroid weight. In the thyroid at 250 mg/kg/day, increased (p<0.05) in cidences of higher follicular cells (Grade 3; 8/16 treated vs 1/16 controls) with correspondingly smaller colloid areas (Grade 3; 8/16 treated vs 1/16 controls) were observed. No differences in these parameters were observed at 25 or 75 mg/ kg/day. It has been reported 7 

# that trifluralin causes the induction of hepatic enzymes resulting in the increased conjugation and excretion of T 3 

# and T 4 

# and an increase in serum TSH.

At 250 mg/kg/day, values for the following parameters in the treated group differed (p<0.05) from control: (i) incr eased creatinine; (ii) decreased alkaline phosphatase; (iii) decreased total bilirubin; (i v) decreased potassium; and (v ) increased calcium. At 75 mg/kg/day, creatinine and calcium were increas ed. The toxicological significance of a decrease in alkaline phosphatase is unclear. The decrease in bilirubin, minor increases in

7 

# Saghir SA, Charles GD, Bartels MJ, et al. (2008). Mechanism of trifluralin-induced thyroid tumors in rats. Toxicol Lett. 2008 Jul 30;180(1):38-45. Epub 2008 Jun 6.

# Female Pubertal Assay (2012)/ Page 16 of 16 TRIFLURALIN / 036101 OCSPP 890.1450/ OECD None


# serum calcium levels, and minor decrease in potassium levels were not considered adverse.

There was no evidence of endocrine-related e ffects at any dose of trifluralin. Thyroidrelated effects include d decreased serum T 4 

# , increased TSH, and altered thyroid histopathology at 250 mg/kg/ day trifluralin.

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

 Guideline specifies a diet with ≤ 300 μg/g genistein-equivale nt content; however, the diet in this study was report ed to have <325 μg/g.

 The control mean age at VO (36.4 days) wa s above the performance criteria maximum of 35.62 days

 The control %CV for weight at VO (15.58%) wa s slightly greater th an the performance criteria maximum of 13.97%

 The control mean adrenal we ight (32.8 mg) was slightly outside the performance criteria acceptable range of 38.34-48.84 mg

# DATA EVALUATION RECORD

TRIFLURALIN

Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-76-2012 (MRID 48674002)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Joseph Rieth, Ph.D., DABT Date: 6/15/2012

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 7/12/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/19/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/19/2012

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

.

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

# TRIFLURALIN I 

# 036101 Male Puberbl Assay (2012) I 

# Page 1 of 19 OCSPP 890.1500 I 

# OECD Nooe


# Primary Reviewer:

# ~N~an ..............

# cy~M'""'c""""C ..... arro=-=ll

________ 

# Signature:

# ~j lit c,(

# ~ Health Effects Division

.

# Date~:J'**~~'** - Secondary Reviewer: John Liccione. Ph.D. 

# Signature· 

# = Health Effects Division Date•
•

# ~I( 08/2011



# DATA EVALUATION RECORD

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500



# PC CODE: 036101 

# DP BARCODE: D401753 ·



# TXR#: 0055724 

# CAS No.: 1582-09-8



# TEST MATERIAL (PURITY): Trifluralin (97. I% a.i.)



# SYNONYMS: 2,6-Dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine



# CITATION: Marty, M.S., A.K. Andrus, J. Thomas. (2012). Trifluralin: Pubertal Development and Thyroid Function 

# in Intact Juvenile/Peripubertal Male Crl:CD(SD) Rats. Toxicology and Environmental Research and Consulting, The Dow Chemical Company,**Midlan~** 

# MI. Laboratory Report No.: 111079, April 9
, 2012. MRID 48674002. Unpublished.



# SPONSOR: Dow AgroSciences, LLC
, 9330 Zionsvill e Road, Indianapolis, IN.



# TEST ORDER#: CON 036101-26



# EXECUTIVE SUMMARY: In a Male Pubertal Assay 

# (MRID 48674002), 16 Sprague Dawley Crl:CD(SD) rats/dose group were treated daily via oral gavage with trifluralin (97.1% a.i., Lot# DKO-B916-33, TSN100656) 

# in corn oil at doses of 0, 25, 75 or 250 mg/kg/day from post-natal day (PND) 23 

# to 53. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and weight at day of attainment was recorded. Following sacrifice on PND 53, blood was collected for analysis of clinical chemistry parameters, including total serum testosterone, thyroxine 

# (T4), and thyroid stimulating hormone (TSH}, which were analyzed using immunoassays. Liver, pituitary, adrenals, thyroid, and urogenital organ weights were recorded and microscopic examinations were performed on the testes, epididymides
, thyroid, and kidneys.

No treatment-related effects were observed on mortality, clinical signs, or gross pathology. Age and body weight at PPS 

# in the treated groups were similar to controls. In the thyroid, colloid

area and follicular cell height scores were similar in the treated groups to the control group. No treatment-related findings were noted in the epididymis and testis.

Body weights and body weight gains at 250 mg/kg/day were not significantly (NS) different from controls on PND 53; however, body weights at this dose were decreased on PND 30 and 37 by 11 and 9%, respectively (p<0.05), and body weight gains were decreased (p<0.05) for the periods PND 23-30, 23 -37
, and 23-42 by 24%, 14%, and 11 %, respectively. Although this effect

on body weight was transient and

. not considered adverse, the delayed growth could affect sexual

# Male Pubertal Assay (2012) / Page 2 of 18 TRIFLURALIN / 036101 OCSPP 890.1500 / OECD None


# development. There were no effects on body weights or body weight gains at 25 or 75 mg/kg/day.

Absolute, adjusted, and relative liver wei ghts were increased (p <0.05) by 13-16% at 75 mg/kg/day and by 29-37% at 250 mg/kg/day. At 25 mg/kg/day, relative liver weights were increased (p<0.05) by 6%. There was no gross or clinical chemistry evidence to support hepatotoxicity, and histology was not performed. The slight effect at 75 mg/kg/day and the substantial effect at 250 mg/kg/day are considered equivocal findings, becau se this effect could be an adaptive response.

At 250 mg/kg/day, decreased (p< 0.05) adjusted organ weights were noted in the following androgen-sensitive tissues: dorsolateral prostate (12%), levator ani-bulbocavernosus muscle complex (LABC, 13%), left epididymis (14%), and right epididymis (11%). Similar effects were noted on the unadjusted weights of these organs . Additionally, a minor decrease (8%; p<0.05) was noted in the right epididymis at 75 mg/kg/day. These effects were slight and may have been related to the delayed growth observed at 250 mg/kg/day.

Serum T 4 

# levels were decreased (p<0.05) dose-de pendently by 25, 42, and 58% at 25, 75 and 250 mg/kg/day, respectively. At 250 mg /kg/day there also appeared to be an effect on serum TSH (increased by 74%) and testos terone (decreased by 30%); ho wever, neither difference was statistically significant, nor were there any effects observed on t hyroid weight or histopathology.

At 250 mg/kg/day, creatinine was increased (p<0 .05) by 50%, which was consistent with the probe study. Alkaline phosphatase (ALP) was s lightly decreased (p<0.0 5) by 17%; however, the toxicological significance of a s light decrease is unclear. Calcium and sodium were also increased (p<0.05) by 4% and 1%, respectively, at 250 mg/kg/day, but these minor effects were not considered adverse.

Increased hyaline droplet formation within th e kidney proximal convoluted tubule (multifocal) was observed in the 25 (13/16, very slight severity), 75 (13/15, ve ry slight), and 250 (15/15, very slight to slight) mg/kg/day groups compared to the cont rol (1/16, very slight ). However, this finding was not associated with degenerative or cytotoxic changes in the kidneys. This was the only treatment-related histological lesion, and there was no other ev idence of kidney toxicity. There were no treatment-related histopatholog ical changes in the thyroid, testes, or epididymides.

The high dose was considered to be an adequa te dose based on increased serum creatinine, increased liver weights, and decreased serum T 4 

# levels.

The assay satisfies the EDSP Tier 1 Test Or der requirements for a Male Pubertal Assay (OCSPP 890.1500).

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

# Male Pubertal Assay (2012) / Page 3 of 18 TRIFLURALIN / 036101 OCSPP 890.1500 / OECD None



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting, Dow Chemical Co.
Location: Midland, MI Study Director: A.K, Andrus Other Personnel: M.S. Marty, Lead Scientist J. Thomas, Pathologist Study Period: August 5, 2011 to April 9, 2012



# 2. Test Substance: Trifluralin Description: Technical, yellow crystals Source: Dow AgroSciences LLC, Indianapolis, IN Lot/Batch #: Lot no. DKO-B916-33, TSN100656. Exp. date not provided Purity: 97.1 % a.i. Stability: Not provided CAS #: 1582-09-8 Structure:

N

CF 3 

# C H 2


# C H 2 

# CH 3 

# C H 2


# C H 2 

# CH

3



# O 2 

# N NO 2



# 3. Vehicle: Corn oil

4. Test Animals: Species: Rat Strain: Sprague Dawley Crl:CD(SD) Age/Weight at Study Initiation: PND 23-53, 36.8 – 59.3 g, males only Source: Charles River Laboratorie s, Inc., Portage, MI Housing: Two per cage in plastic solid bottom cages c ontaining heat-treated, aspen wood shavings. Diet: Teklad Diet #2016, Harlan Labora tories, Inc., Indianapolis, IN, ad libitum. Phytoestrogen content <325 μ g of genistein equivalents/gram diet Water: Deionized water, ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 22±3 °C 40-70% 12-15/hr 12 hrs light/ 12 hrs dark

# Male Pubertal Assay (2012) / Page 4 of 18 TRIFLURALIN / 036101 OCSPP 890.1500 / OECD None



# B. STUDY DESIGN

1. In-Life Dates: Start: August 13, 2011 End: September 13, 2011

2. Mating: Time-mated nulliparous pregnant dams were received on gestation days (GD) 7-10. The dams were ordered from the suppl ier to reach GD 21 on the same day; however, litters born on either GD 21 or 22 were used in the assay. Litters with more than 10 pups were standardized by culling (random selecti on) on PND 4 to 10 pups per litter with 5 males and 5 females per litter where possible.

3. Animal Assignment: Offspring were weaned on P ND 21 and weighed. Animals were assigned randomly with a computer program designed to provide uniform group mean weights and standard deviations to the test groups noted in Table 1. Littermates were not assigned to the same treatment group.

TABLE 1. Study Design a 

# Test group Dose (mg/kg/day) # of Males Control 0 16 Low 25 16 Mid 75 16 High 250 16 a Data were obtained from page 19 of the study report.



# 4. Dose Selection Rationale: Several studies were considered in the selection of the doses for this study and are described below. In partic ular, the high dose level was selected to avoid the excessive increases in serum creatinin e levels observed in the probe study at 400 mg/kg/day.

In a probe study 1 

# , groups of four male and four female Crl:CD(SD) rats were administered trifluralin by gavage at dose levels of 0, 50, 200, 400, or 600 mg/kg/day in corn oil for 15 days. Male rats were dose d from postnatal day (PND) 30- 44 and female rats from PND 22-36. Over the entire treatment period (Days 1-15), there were no treatment-related effects on body weights or body weight gains in female rats. Male rats had 12%, 13%, and 20% decreases in body weight gains during the Day 1-15 interval at 200, 400, and 600 mg/kg/day, respectively. Similarly, respectiv e terminal body weights were decreased by 6.3%, 6.5%, and 11.3% in males. At study te rmination, perianal (urine) soiling was observed in three high-dose fema les and clear facial soiling was seen in one intermediate and one high dose male and one high-dose female . There were increases in absolute liver weights in both males (9-21% at > 200 mg/kg/day) and females (16-52% at > 50 mg/kg/day). In females, rela tive liver weights were increased in a dose-related manner from 50 mg/kg/day (13%) through 600 mg/kg/day (48 %). In males, relative weights were increased in a dose-related manner from 200 mg/kg/day (18%) through 600 mg/kg/day (37%). Relative kidney weights were decrease d by < 10% in both males and females in the high-dose group. Clinical chemistry parameters suggested a decrease in ALP and increased

1 

# Marty, M. S., Marshall, V. A. and Je ong, Y-C. (2011). Trifluralin: Hershbe rger Assay in Castrated Adult Male Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. MRID 48619707.

# Male Pubertal Assay (2012) / Page 5 of 18 TRIFLURALIN / 036101 OCSPP 890.1500 / OECD None


# gamma glutamyl transpeptidase (GGT) and chol esterol levels at 600 mg/kg/day in males and females. Albumin, globulin, and albumin /globulin ratio were altered in high-dose females only. Creatinine was increased at 400 and 600 mg/kg/day in both males and females.

In a dominant lethal study 2 

# , mature male Wistar rats (15/ dose) were given trifluralin by gavage at doses of 0, 100, or 1000 mg/kg/day in 10% aqueous acacia for 5 days. The high dose animals had a 50% reduction in body weight ga in at the end of the exposure period.

In a developmental toxicity study 3 

# , groups of 25 mated female Crl:CD(SD) rats were administered trifluralin at dose levels of 0, 100, 225, 475 or 1000 mg/kg/day in 10% aqueous acacia from GD 6-15. Body weight ga ins for dams given trifluralin on GD 6-10 were decreased 66% at 100 mg/kg/day, 50% at 225 mg/kg/day, and 95% at 475 mg/kg/day; dams given 1000 mg/kg/day lost weight during this interval (-2.8 g vs. 18.7 g gain in controls). On GD 11-15, dams in the 475 and 1000 mg/kg/day groups had a 23 and 28% decrease in body weight gain, respectivel y, with corresponding decreases in food consumption.

In a two-week mode-of-action study 4 

# , trifluralin was administered to F344 rats in the diet at 6500 ppm (441 mg/kg/day), a con centration that produced thyr oid tumors in the chronic study. Trifluralin caused a 20% reduction in body weight gain and a 14% decrease in terminal body weight compared to controls on Day 15. Relative liver and thyroid weights were increased by 49% and 17%, respectively.

In a uterotrophic study 5 

# , groups of six immature female Cr l:CD(SD) rats were exposed to 0 (vehicle control), 25, 75, or 225 mg/kg/day trif luralin by oral gavage from PND 19-21 with necropsy on PND 22. There we re no significant effects on body weights at any dose of trifluralin. At 225 mg/kg/day trifluralin, body weight gains we re decreased significantly by 34% on Days 1-4. Relative liver weights were significantly increased by 13% and 17% in the 75 and 225 mg/kg/day triflu ralin groups, respectively. Ther e were no treatment-related increases in mean uterine weights (wet or bl otted) in immature animals treated with <225 mg/kg/day.

In a Hershberger assay 6 

# , trifluralin was administered by oral gavage at dose levels of 0, 50, 150, or 450 mg/kg/day to castrated male CD rats from 55-64 days of age in the presence or

2 

# Markham, J. K. and Hoyt, J. A. (1983) . A Dominant Lethal Study with Technical Trifluralin (Compound 36352) in the Wistar Rat. Report of The Dow Chemical Company performed by Lilly Research Laboratories, Greenfield, Indiana. 3 

# Markham, J. K. and Byrd, R. A. ( 1984). A Teratology Study of Trifluralin (EL-152, Compound 36352) Administered Orally to Charles River CD Rats. Report of The Dow Chemical Company performed by Lilly Research Laboratories, Greenfield, Indiana. 4 

# Saghir, S. A., Charles, G. D., Kan, L. H. , Dryzga, M. D., Brzak, K. A., and Clark, A. J. (2005). Trifluralin: Mechanism of Thyroid Tumors in Rats. Report of Toxicology & Environmen tal Research and Consulting, The Dow Chemical Company, Midland, Michigan. 5 

# Marty, M. S. and Brooks, K. J. (2011). Trifluralin: Uterotrophic assay in th e immature Crl:CD(SD) Rat. Report of Toxicology & Environmental Research and Consulting, Th e Dow Chemical Company, Midland, Michigan. MRID 48619709.
6 

# Marty, M. S., Marshall, V. A., and Jeong, Y-C. (2011a). Trifluralin: He rshberger Assay in Ca strated Adult Male Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

# Male Pubertal Assay (2012) / Page 6 of 18 TRIFLURALIN / 036101 OCSPP 890.1500 / OECD None


# absence of 0.4 mg/kg/day testos terone propionate (TP; administ ered subcutaneously). At 450 mg/kg/day trifluralin with and without TP, animals had signifi cantly decreased body weight gains. High-dose trifluralin rats co -administered TP also had a 10% decrease in terminal body weights compared to TP only rats. There were significant increases in absolute liver weights at 450 mg/kg/day trif luralin without TP a nd at >150 mg/kg/day trifluralin with TP. In the anti-androgen ic portion of the assay, high-dose animals had significant decreases in weights of the le vator ani-bulbocavernosus muscles, ventral prostate, and seminal vesicles with coagulati ng glands. Serum testosterone concentrations were significantly decreased in these an imals compared to the TP only rats.

Based on these data, dose levels of 0, 25, 75, and 250 mg/kg/day were selected for this male pubertal assay.

5. Dose Preparation and Analysis: Dose formulations were prepared by adding appropriate amounts of test substance to corn oil to yield the target concen tration. The report stated that dose solutions were prepared periodically throug hout the study based upon stability data. Stability in corn oil at ambient temper ature was evaluated for up to 25 days at concentrations of 0.25, 2.5, and 250 mg/mL in a previous study (MRID 48619707). Prior to dosing, samples of dose formulations from th e first mix of the study were analyzed for achieved concentration, and homogeneity was te sted for the low and high dose solutions.

Results of Dose Analysis

Homogeneity (%RSD): 2.3 – 5.7%.

Stability (% of Time 0): 94.0–100.0%

Concentration (% nominal): 95.6

− 98.6%.

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: All doses were administered once daily by gavage, from PND 23 through PND 53, in a volume of 4 mL/kg of bo dy weight. Dose volumes were adjusted using the most current body weight.

7. Statistics: For continuous variab les, a Bartlett’s test was conducted to check for homogeneity of variance. Based on the outco me of the Bartlett’s test (alpha = 0.01), variables may have been transformed prior to statistical analysis. Body weight, body weight gain, clinical chemistries, age at PPS, body we ight at PPS, organ we ights, organ-to-body weight ratios (liver, kidney, adrenal, and pituita ry weights) and serum hormones were analyzed with a two-way analysis of varian ce (ANOVA) with block and treatment as main effects. When significant dose effects were determined in the two-way ANOVA at alpha = 0.05, then individual dose groups were compared to controls using Dunnett's test at alpha = 0.05, experiment-wise error. Age and body we ight at PPS and organ weights (thyroid, testes, epididymides, seminal ve sicle, prostate and LABC) were analyzed with analysis of covariance (ANCOVA) with body weight on P ND 23 as the covariate. When the dose effect was significant at alpha = 0.05, least square means w ith a Dunnett’s correction was

# Male Pubertal Assay (2012) / Page 7 of 18 TRIFLURALIN / 036101 OCSPP 890.1500 / OECD None


# used to determine differences (experiment-wi se alpha = 0.05). The report includes values that were unadjusted and adjusted for body weight on PND 23. When the ANCOVA was not statistically significan t, a Dunnett’s test also was performed. Globulin and albumin/globulin ratio, which were calculated valu es, were not statistically analyzed. When the chi-square statistic was significant, it was followed by pairwise comparisons to the control group via a Fisher’s exac t test with a Bonferroni-Holm adjustment. When Bartlett's test was significant ev en after data transformation, nonpa rametric analyses was conducted: Kruskal-Wallis test followed by the Wilcoxon rank-sum test was performed with a Bonferroni-Holm adjustment for multiple compar isons to the control. Coefficients of variation (CVs) were calculated where appropriate. Statistical outliers were identified by a sequential test (alpha = 0.02). Outliers and que stionable data points ( i.e ., values due to technical errors) were ex cluded only for documented sc ientifically sound reasons. Histopathology results were not subjected to outlier tests. The incidence of specific histopathological observations we re analyzed using the pairwise Chi-square test with Yates’ continuity correction (alpha = 0.05; two-sided).

C. METHODS

1. Mortality and Clinic al Examinations: Cageside examinations on all animals were performed twice daily for general condition, mo rtality, and moribundity. Detailed clinical examinations were conducted daily at about the same time each day following dosing.

2. Body Weight: Animals were weighed on PND 21 and da ily prior to dosing. Body weights were reported and statisti cally analyzed on PND 21, 23, 26, 30, 37, 42, 45, and 53. Body weight gains were reported and statistically analyzed for all repor ted weight intervals beginning with PND 23-26 and including the entire dosing period (PND 22-53).

3. Preputial Separation (PPS): Beginning on PND 30, all animals were examined daily for onset of PPS. Age and weight at on the day of completion of PPS were recorded. When only a thread of tissue remained between the gl ans and prepuce, this was considered to be the onset of puberty. If the thread persisted, this was recorded. Examinations continued until complete PPS occurred or until PND 53; in the latter case, PND 54 was used for the animal in the calculati on of the group mean.

4. Sacrifice and Pathology: Groups were counter-balanced during necropsy, and necropsies were completed before 1300 hours in accordance w ith the test guideline. On the afternoon of the day prior to necropsy, the rats were transf erred to a holding room to avoid cage transport on the day of necropsy. All scheduled activ ities were performed in the holding room between approximately 0600 and 0800, and the body weight at the time of dosing served as the terminal body weight. On the day of termination, approximately 2 hours post-dose, rats were anesthetized by whole body isoflurane e xposure (ca. 2 min), the abdomen was opened and blood was collected via cardiac puncture. Blood samples were tr ansferred into three separate tubes for clinical chemistry and hormone analyses. Blood samples for hormone analyses were kept refrigerated or on i ce during necropsy. Exsanguination was followed by decapitation. A complete necr opsy was conducted on all animals.

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# a. Hormone Analysis: Blood samples were stored at

− 80°C until shipment on dry ice to WIL Research Laboratories, LLC (Ashland, OH). Hormone levels were analyzed using a chemiluminescent immunoassay (T 4 

# and testosterone) or a radioimmunoassay (TSH).

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

ELECTROLYTES OTHER
X Calcium X Albumin X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total cholesterol X Potassium X Globulins (calculated) X Sodium Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (L DH) X A/G ratio (calculated) X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase (SDH) X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended based on Guideline 890.1500.



# c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological examination.

UROGENITAL OTHER XX Testes (left and right separately)* + 

# XX Thyroid* + 

# XX Epididymides (left and right separately)* + 

# X Liver* X Seminal vesicle plus coagulating glands (w ith and without fluid)* X Adrenals (paired)* X Ventral prostate* X Pituitary* X Dorsolateral prostate* X Levator ani-bulbocavernosus (LABC) muscle complex* XX Kidneys (paired)* + 

# * Weights required based on Guideline 890.1500 + Histopathological examination re quired based on Guideline 890.1500



# The kidneys and adrenals were weighed as pair s. Organs were wei ghed prior to fixation except the thyroid and pituitary (weighed afte r fixation). After a seminal vesicle weight with fluid was collected, each side of the semi nal vesicles was cut sagittally. The seminal vesicles were placed between saline-moistened absorbent papers, and blotted by gently placing a 500 g weight over the paper-covere d seminal vesicles for approximately 10 seconds to remove seminal fluid, then re-wei ghed to determine the weight post-blotting.

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# The ratios of organ weight to terminal body weight were calculated but only reported for liver, kidney, adrenal, and pituitary weights.

Representative samples of all weighed tissues were preserved in neutral, phosphate-buffered 10% formalin, with the exception of testes and epididymides, which were preserved in Bouin’s fixative for 24-72 hours. Following fixa tion, the tissues were ri nsed three times in 50% ethanol and stored in 70% ethanol. Typically the right testis and epididymis were evaluated histologically although both right and le ft were preserved.

The thyroid was fixed fo r at least 24 h and then dissected with parathyroids and weighed. The thyroid follicular cell height and colloid ar ea was evaluated on a five point graded scale (1 = shortest/smallest and 5 = tallest/largest). At least two sections from each of the two lobes of the thyroid were examined.

The histopathological examinati on of the testis included a qua litative assessment of the stages of spermatogenesis. A cross section thr ough the approximate cente r of the right testis from control and high-dose males was embedded in paraffin, sectioned at 5 um and stained with modified periodic acid-Sc hiffs (PAS)/hematoxylin. The presence and integrity of the stages of spermatogenesis were qualitatively evaluated. Microscopic evaluation included a qualitative assessment of the relationships between spermatagonia, spermatocytes, spermatids, and spermatozoa seen in cross se ctions of the seminiferous tubules. The progression of these cellular associations define s the cycle of spermatogenesis. In addition, the testis was examined for degenerative changes ( e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). Examination of the intact epididymis included the caput, corpus, and cauda by evaluation of a longitudi nal section. This examination identified histopathological lesi ons, which could have included sperm granulomas, leukocytic infiltration (inflammation), aberrant cell types wi thin the lumen, or the absence of clear cells in the cauda epididymal epithelium.

Tissues from the high dose and control groups were examined microscopically, and the other groups were examined when treatment-re lated differences were observed. Selected histopathologic findings were graded very slight, sli ght, moderate, or severe.

II. RESULTS

A. MORTALITY: All animals survived the 31-day treatment period.

B. CLINICAL SIGNS OF TOXICITY: No treatment-related clini cal signs of toxicity were observed in animals for any dose group. Two animals (#7828 and #7855 in the 75 and 250 mg/kg/day dose groups, respectively) had pa lpable masses that we re noted beginning on PNDs 32 and 35, respectively. Each male wa s found with a unilatera l undescended testis not related to treatment because testicular des cent occurs prior to PND 23 (the first day of dosing). These animals were excluded from the study.

C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Selected body weight and body weight gain data are presented in Tabl e 2. Body weights and body weight gains at 250 mg/kg/day were not signifi cantly different from contro ls on PND 53; however, body

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# weights at this dose were decreased on PND 30 and 37 by 11 and 9%, respectively (p<0.05), and body weight gains were decreased (p <0.05) for the periods PND 23-30, 23-37, and 23-42 by 24%, 14%, and 11%, respectively. There were no effects on body weights or body weight gains at 25 or 75 mg/kg/day.

TABLE 2. Body weights in Control and High Dose Trifluralin-Treated Male Rats a



# BW PND

23 BW PND

26 BWG PND 23-26 BW PND

30 BWG PND 23-30 BW PND

37 BWG PND 23-37 BW PND

42 BWG PND 23-42 BW PND

53 BWG PND 23-53

Control 55.0 68.6 13.6 92.9 37.8 147. 5 92.4 192.9 137.8 289.1 234.0

250 mg/kg/day 53.9 63.2 9.3† ( ↓ 32) 82.6* ( ↓ 11) 28.7* ( ↓ 24) 133.7* ( ↓ 9) 79.8* ( ↓ 14) 176.5 ( ↓ 9) 122.6* ( ↓ 11) 273.2 ( ↓ 5) 219.3 ( ↓ 6) a Data were obtained from page 32 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. BW Body weight (g) BWG Body weight gain (g) * Significantly different from controls at p<0.05. † Significantly different from control by Bonf erroni-Holm adjusted W ilcoxon test at p<0.05.


# Body weights, body weight gains, age and wei ght at attainment of PPS are presented in Table 3. There were no significan t differences in age or body weight at PPS in any of the treatment groups.

TABLE 3. General Growth and Preputial Separation (PPS) a



# Parameter Evaluated Vehicle Control Trifluralin 25 mg/kg/day Trifluralin 75 mg/kg/day Trifluralin 250 mg/kg/day

N Mean SD/ SE b 

# CV (%) N Mean SD/ SE b 

# CV (%) N Mean SD/ SE b 

# CV (%) N Mean SD/ SE b 

# CV (%) Initial body weigh t (PND 22; g) U 16 55.04 5.86 10.64 16 54.83 5.06 9.23 15 54.55 4.08 7.49 15 53.91 4.97 9.21
Body weight at PPS (g) U 16 211.69 

# 22.42 10.59 16 212.20 

# 13.03 6.14 15 208.41 

# 20.08 9.64 15 205.52 

# 23.86 11.61 A 16 210.72 

# 4.302 NA 16 211.69 

# 4.299 NA 15 208.50 

# 4.438 NA 15 207.00 

# 4.449 NA Final body weight (g) U 16 289.09 

# 26.67 9.23 16 289.66 

# 21.54 7.44 15 281.13 

# 24.65 8.77 15 273.23 

# 32.47 11.88 A 16 287.44 

# 4.793 NA 16 288.79 

# 4.789 NA 15 281.29 

# 4.945 NA 15 275.77 

# 4.957 NA Final body weight (% of control) U NA NA NA NA 16 100.2 7.5 7. 4 15 97.2 8.5 8.8 15 94.5 11.2 11.9 A NA NA NA NA -- -- -- -- -- -- -- -- -- -- -- -- Body weight gain (final – initial; g) U 16 234.04 

# 22.29 9.52 16 234.83 

# 19.09 8.13 15 226.58 

# 22.28 9.83 15 219.33 

# 28.83 13.15 A -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
Age at PPS (PND) U 16 44.2 1.2 2.8 16 44.2 1.8 4.1 1 5 44.5 1.2 2.7 15 45.3 1.8 4.1 A 16 44.2 0.36 NA 16 44.2 0.36 NA 15 44.5 0.37 NA 15 45.3 0.37 NA Proportion unseparated (#/N) 0/16 0/16 0/15 0/15 a Data were obtained from page 54 of the study report. b Standard deviation (SD) reported for unadjusted values and st andard error of the mean (SE) reported for adjusted values. U Unadjusted for body weight on PND 23 A Adjusted for body weight on PND 23 N Number of animals examined CV Coefficient of Variation NA Not applicable -- Not required by test guideline

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# D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 4. Absolute, adjusted, and relative liv er weights were increased (p<0.05) by 13-16% at 75 mg/kg/day and by 29-37% at 250 mg/kg/day. At 25 mg/kg/day, relative liver weights were increased (p<0.05) by 6%. Left and right, absolute and adjusted, epididymis weights were decreased (p<0.05) by 11-15% at 250 mg/kg/day. At 75 mg/kg/day, the adjusted right epididymis weight was decreased (p<0.05) by 8%. At 250 mg/kg/day, absolute and adjusted LABC weights were decreased (p< 0.05) by 14 and 13%, respectively, and dorsolateral prostate weights were decreased (p< 0.05) by 12%. No other effect s on organ weights were observed.

The mean kidney weight in the control (1.91 g) was below the minimum acceptable value in the performance criteria (2.242 g). The mean thyroid weight in the control (12.5 mg) was below the minimum acceptable value in the perf ormance criteria (14 mg). The CVs for the seminal vesicles weights in the control group (25.6%) was slightly greater than the maximum acceptable values in the performance criteria (21.06%). Other guideline criteria were met for organ weights in the controls.

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# TABLE 4. Organ Weights at Necropsy a



# Organ Vehicle Control Trifluralin 25 mg/kg/day Trifluralin 75 mg/kg/day Trifluralin 250 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 16 12.97 1.51 11.62 16 13.76 1.39 10.1 15 14.63* ( ↑ 13) 1.63 11.13 15 16.78* ( ↑ 29) 1.93 11.51

A 16 12.90 0.345 NA 16 13.72 0.345 NA 15 14.64* ( ↑ 13) 0.356 NA 15 16.90* ( ↑ 31) 0.357 NA

R 16 4.48 0.23 5.21 16 4.75* ( ↑ 6) 0.27 5.76 15 5.20* ( ↑ 16) 0.21 4.1 15 6.15* ( ↑ 37) 0.23 3.73

Kidneys (g) U 16 1.91 0.23 11.85 16 1.96 0.19 9.58 15 1.95 0.19 9.94 15 1.87 0.22 11.69 A 16 1.90 0.037 NA 16 1.95 0.037 NA 15 1.96 0.038 NA 15 1.89 0.038 NA R 16 0.66 0.03 4.91 16 0.68 0.05 7.22 15 0.7 0.03 4.99 15 0.68 0.04 5.45
Pituitary (mg) U 16 9.9 1.5 15.7 16 9.9 1.0 9.6 15 9.4 1.7 17.7 15 9.0 1.2 13.1 A 16 9.9 0.33 NA 16 9.9 0.33 NA 15 9.4 0.34 NA 15 9.0 0.35 NA R 16 3.4 0.4 12.3 16 3.4 0.3 9.8 15 3.3 0.6 16.9 15 3.3 0.3 8.1

Adrenals (mg) U 16 41.6 6.2 14.9 16 40.6 3.9 9.7 15 42.1 6.6 15.8 15 39.0 7.3 18.6 A 16 41.4 1.37 NA 16 40.4 1.37 NA 15 42.1 1.42 NA 15 39.4 1.42 NA R 16 14.4 1.8 12.7 16 14 1.3 9. 4 15 15 2 13.3 15 14.2 1.6 11.4 Seminal vesicle + coag. gland, with fluid (mg) U 16 453.9 116.2 25.6 16 424.1 108.3 25. 5 15 435 74.5 17.1 15 369.8 167.3 45.2 A 16 448.9 27.10 NA 16 421.5 27.08 NA 15 435.5 27.96 NA 15 377.4 28.02 NA Seminal vesicle + coag. gland, without fluid (mg) U 16 221.8 43 19.4 16 212.1 35.4 16.7 1 5 221.2 37.3 16.8 15 188.0 61.5 32.7 A 16 220.0 10.16 NA 16 211.2 10.15 NA 15 221.4 10.48 NA 15 190.8 10.51 NA Ventral prostate (mg) U 16 208.2 42.9 20.6 16 199.9 37.9 18.9 1 5 192.2 25.2 13.1 15 181.4 47.7 26.3 A 16 206.5 8.66 NA 16 199.0 8.65 NA 15 192.4 8.93 NA 15 184.0 8.95 NA Dorsolateral prostate (mg) U 16 194.6 27.1 13.9 16 181 21.2 11.7 1 5 189.1 29.4 15.6 15 167.8 39.3 23.5

A 16 193.2 6.43 NA 16 180.2 6.4 2 NA 15 189.2 6.63 NA 15 169.9* ( ↓ 12) 6.65 NA

LABC (mg) U 16 510.2 59.7 11.7 16 486.6 64. 2 13.2 15 470.9 57.2 12.1 15 436.9* ( ↓ 14) 92.4 21.1

A 16 506.8 14.81 NA 16 484.9 14. 80 NA 15 471.2 15.28 NA 15 441.9* ( ↓ 13) 15.32 NA

Epididymis, left (mg) U 16 225.5 33.3 14.8 16 208.7 16.8 8 15 207.1 19.1 9.2 15 191.1* ( ↓ 15) 25 13.1

A 16 224.4 5.27 NA 16 208.1 5.2 7 NA 15 207.2 5.44 NA 15 192.8* ( ↓ 14) 5.45 NA

Epididymis, right (mg) U 16 226.9 18.3 8.1 16 212.9 19. 7 9.2 15 208.2 17.3 8.3 15 198.7* ( ↓ 12 30.6 15.4

A 16 226.0 4.94 NA 16 212.4 4.94 NA 15 208.3* ( ↓ 8) 5.10 NA 15 200.1* ( ↓ 11) 5.11 NA
Testis, left (mg) U 16 1396 96.9 6.9 16 1393 82.7 5.9 15 1453 114.3 7.9 15 1356 120.8 8.9 A 16 1391 21.9 NA 16 1390 21.9 NA 15 1454 22.6 NA 15 1364 22.6 NA Testis, right (mg) U 16 1418 94.5 6.7 16 1412 99.6 7.1 15 1482 152.6 10.3 15 1394 128.6 9.2 A 16 1412 25.2 NA 16 1409 25.2 NA 15 1483 26.0 NA 15 1403 26.1 NA Thyroid, fixed (mg) U 16 12.5 1.3 10.5 16 12.1 1.6 13. 4 15 12.1 1.3 11 15 12.6 1.6 12.6 A 16 12.4 0.33 NA 16 12.0 0.33 NA 15 12.1 0.35 NA 15 12.7 0.35 NA a Data were obtained from pages 57-59 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. U Unadjusted for body weight on PND 23 A Adjusted for body weight on PND 23 R Organ-to-body weight ratio (relative to body weight) N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different fro m controls at p<0.05.

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# E. CLINICAL CHEMISTRY AND HORMONE LEVELS: Mean hormone and clinical chemistry levels are presented in Table 5. Serum T 4 

# levels were dose-dependently decreased (p<0.05) by 25-58% at all dose levels . It was noted that the mean control T 4 

# level (5.28 μg/dL) was above the laboratory’s hi storical control range of 4.61-5.02 μg/dL; however, the values for the treated groups were below the provided historical control range. At 250 mg/kg/day, serum TSH was increased ( ↑ 74%) and testosterone was decreased ( ↓ 30%), but neither was statistically significant. The lack of significance for these values was likely associated with the high SDs for both parameters.

The CV for serum TSH in the control gr oup (70.02%) was greater than the maximum acceptable value in the performance criteria (58.29%). Guideline criteria for serum T 4 

# and testosterone levels were met in the control.

At 250 mg/kg/day, ALP was decreased (p< 0.05) by 17%, and creatinine was increased (p<0.05) by 50%. Calcium and sodium we re increased (p<0.05) by 4% and 1%, respectively, each at 250 mg/kg/day. Mean serum sodium concentrations at 25 and 250 mg/kg/day were marginally higher than that of the controls and statistically significant. These were interpreted to be unr elated to treatment due to lack of a dose response relationship and the values were within the historical control range.

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# TABLE 5. Hormone Levels and Clinical Chemistry a



# Parameter Evaluated Vehicle Control Trifluralin 25 mg/kg/day Trifluralin 75 mg/kg/day Trifluralin 250 mg/kg/day

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Hormones Serum T 4 

# , Total (μg/dL) 16 5.28 0.493 9.4 16 3.98* ( ↓ 25) 0.416 10.5 15 3.08* ( ↓ 42) 0.288 9.4 15 2.22* ( ↓ 58) 0.366 16.50



# Serum TSH (ng/mL) 16 7.20 5.041 70.0 16 7.63 2.816 36.9 15 8.85 8.159 92.2 15 12.55 ( ↑ 74) 6.134 

# 50.30

Serum testosterone (ng/mL) 16 2.735 1.341 

# 49.0 16 3.294 1.9924 

# 60.5 15 3.483 2.6584 

# 76.3 15 1.902 ( ↓ 30) 1.9695 

# 103.6 Clinical Chemistry Creatinine (μmol/L) 16 0.2 0.1 32.8 16 0.2 0 28.4 15 0.2 0.1 27.7 15 0.3* ( ↑ 50) 0 16.7 Serum urea nitrogen (mg/dL) 16 14 2 15 16 15 2 12 15 14 2 15 15 16 3 20 Alkaline phosphatase (U/L) c 

# 16 389 72 18 16 341 44 13 15 364 66 18 15 321* ( ↓ 17) 60 19

Alanine aminotransferase (U/L) 16 54 6 11 16 54 8 16 15 56 15 27 15 55 12 23Aspartate aminotransferase (U/L) 16 114 26 22 16 121 23 19 15 119 36 31 15 115 22 19 Gamma glutamyl transferase (U/L) 16 1.5 0 0 16 1.5 0 0 15 1.5 0 0 15 1.6 0.5 33.1 Albumin (g/dL) 16 3.8 0.2 4.5 16 3.9 0.2 5.6 15 3.8 0.2 6.3 15 3.8 0.1 3.7 A/G ratio 16 2 0.3 12.9 16 2.1 0.2 11.7 15 1.9 0.3 15.8 15 1.9 0.2 12.2 Cholesterol (mg/dL) 16 103 12 11 16 110 16 15 15 112 16 15 15 105 12 11 Globulin (g/dL) 16 1.9 0.2 10.8 16 1.9 0.2 8.7 15 2.1 0.3 12.6 15 2 0.2 12.1 Total bilirubin (mg/dL) 16 0.07 0.03 41.66 16 0.06 0.02 33.19 15 0.08 0.03 39.1 9 15 0.09 0.04 39.62 Total protein (g/dL) 16 5.7 0.2 3.6 16 5.7 0.2 4 15 5.9 0.3 4.3 15 5.8 0.3 5.2 Sodium (mmol/L) 16 143 1 1 16 144* 1 1 15 143 3 2 15 144* ( ↑ 1) 1 1 Potassium (mmol/L) 16 5.3 0.3 5.8 16 5. 2 0.3 5.3 15 5.2 0.4 7.7 15 5 0.3 6.4 Chloride (mmol/L) 16 98 1 1 16 99 1 1 15 98 2 2 15 97 1 1 Calcium (mg/dL) 16 11.1 0.3 2.7 16 11.2 0. 3 2.8 15 11.3 0.3 2.5 15 11.5* ( ↑ 4) 0.2 2.1 Phosphorus (mg/dL) 16 9.4 0.7 7.6 16 9. 3 1 10.3 15 9.1 0.6 6.7 15 9.3 0.9 9.3 a Data were obtained from pages 35, 55, and 56 of the study report. Percent differences from control, calculated by reviewers
, are presented in parentheses. N Number of animals examined SD Standard deviation SE Standard error CV Coefficient of variation * Significantly different fro m controls at p<0.05.



# F. GROSS PATHOLOGY: No treatment-related effect was observed on gross findings. At 250 mg/kg/day, all rats had yellow discoloration of intra-abdomi nal adipose tissue attributed to staining by the test material. One 75 mg /kg/day rat had a hiatal hernia and one had alopecia at the neck. One 75 mg/kg/day rat and one 250 mg/kg/day rat had a small left testicle. No other gross findings were reported.

G. HISTOPATHOLOGY: There were no treatment-relate d histopathological changes in the thyroid, testes or epididymides. The incidences of histopathological findings of the thyroid gland are presented below in Table 6.

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# The incidences of histopathological findings of the kidney are presented in Table 7. Increased hyaline droplet formation with in the kidney proximal convoluted tubule (multifocal) was observed in the 25 (13/16, very slight severity), 75 (13/15, very slight), and 250 (14/15, very slight to sli ght) mg/kg/day groups compared to the control (1/16, very slight). However, this finding was not associat ed with degenerative or cytotoxic changes in the kidneys.

TABLE 6. Incidence of Histopatho logical Lesions of the Thyroid Gland a



# Treatment Groups Parameter Evaluated Colloid Area Follicular Cell Height

Grade b 

# Incidence Grade b 

# Incidence O E O E

Vehicle Control 1 0 16 1 3 16 2 4 16 2 6 16 3 3 16 3 3 16 4 6 16 4 4 16 5 3 16 5 0 16

Trifluralin 25 mg/kg/day 

# 1 NA 0 1 NA 0 2 NA 0 2 NA 0 3 NA 0 3 NA 0 4 NA 0 4 NA 0 5 NA 0 5 NA 0

Trifluralin 75 mg/kg/day 

# 1 NA 0 1 NA 0 2 NA 0 2 NA 0 3 NA 0 3 NA 0 4 NA 0 4 NA 0 5 NA 0 5 NA 0

Trifluralin 250 mg/kg/day 

# 1 0 15 1 3 15 2 3 15 2 4 15 3 5 15 3 5 15 4 4 15 4 3 15 5 3 15 5 0 15 a Data were obtained from page 65 of the study report. b Thyroid histopathology is graded on a 1 – 5 scale: Follicular cell height,1 = lowest, 5 = highest; and Colloid area, 1 = most colloid, 5 = least colloid. O Number observed E Number examined NA Not applicable

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# TABLE 7. Incidence of Histopatho logical Lesions of the Kidney a



# Findings Vehicle Control Trifluralin 25 mg/kg/day Trifluralin 75 

# mg/kg/day Trifluralin 250 

# mg/kg/day # Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined 

# Within normal limits 4 16 2 16 2 15 1 15 Casts, proteinaceous tubule, focal, very slight 0 16 1 16 0 15 0 15 Casts, proteinaceous tubule, focal, very slight 0 16 0 16 1 15 0 15 Cyst, tubular, cortex, unilateral 1 16 0 16 0 15 0 15 Degeneration, tubule, multifocal, very slight 11 16 5 16 6 15 8 15 Dilatation, pelvis, unilateral, slight 2 16 0 16 0 15 0 15
Dilatation, t ubule, focal, slight 0 16 0 16 1 15 1 15
Dilatation, tubul e, multifocal, very slight 0 16 1 16 0 15 0 15
Fibrosis, interstitium, focal, very slight 0 16 1 16 0 15 0 15 Hyaline droplet formation increased, proximal convoluted tubule, multifocal, very slight 1 16 13 16 13 15 4 15

Hyaline droplet formation increased, proximal convoluted tubule, multifocal, slight 0 16 0 16 0 15 10 15

Inflammation, subacute to chronic, interstitium, focal, very slight 0 16 2 16 1 15 0 15

Inflammation, subacute to chronic, interstitium, multifocal, very slight 0 16 0 16 1 15 1 15

a Data were obtained from page 67 of the study report.



# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Despite a significant decrease in T 4 

# at all doses, there was no evidence of sustained alterations in thyroid function (i.e., decreased weight or histopathology) at any dose of trifluralin. De creases in androgen-sensi tive organ weights at 75 and 250 mg/kg/day require cons ideration in the weight-of-ev idence (WoE) assessment of the androgen pathway. The WoE should include previous trifluralin mode-of-action studies, which show hepatic enzyme induction and enhanced thyr oxine clearance.

B. AGENCY COMMENTS: No treatment-related effect s were observed on mortality, clinical signs, or gross pathology. Age and body weight at PPS in the treated groups were similar to controls. In the thyroid, colloid ar ea and follicular cell height scores were similar in the treated groups to the c ontrol group. No treatment-relate d findings were noted in the epididymis and testis.

Body weights and body weight gain s at 250 mg/kg/day were not si gnificantly different from controls on PND 53; however, body weights at this dose we re decreased on PND 30 and 37

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# by 11 and 9%, respectively (p<0.05), and body we ight gains were decreased (p<0.05) for the periods PND 23-30, 23-37, and 23-42 by 24%, 14%, a nd 11%, respectively. Although this effect on body weight was transient and not cons idered adverse, the delayed growth could affect sexual development. There were no e ffects on body weights or body weight gains at 25 or 75 mg/kg/day.

Absolute, adjusted, and relative liver wei ghts were increased (p <0.05) by 13-16% at 75 mg/kg/day and by 29-37% at 250 mg/kg/day. At 25 mg/kg/day, relative liver weights were increased (p<0.05) by 6%. There was no gross or clinical chemistry evidence to support hepatotoxicity, and histology was not performed. The slight effect at 75 mg/kg/day and the substantial effect at 250 mg/kg/day are consid ered equivocal findings, because this effect could be an adaptive response.

At 250 mg/kg/day, decreased (p< 0.05) adjusted organ weights were noted in the following androgen-sensitive tissues: dorsolateral prostate, LABC, left epididymis, and right epididymis. Similar effects were noted on the unadjusted weights of these organs. Additionally, a minor decrease (p<0 .05) was noted in the right epididymis at 75 mg/kg/day. These effects were slight and may have been related to the delayed growth observed at 250 mg/kg/day. Serum testosterone was decr eased by 30% (NS) at 250 mg/kg/day.

Serum T 4 

# levels were decreased (p<0.05) by 25, 42, and 58% at 25, 75 and 250 mg/kg/day, respectively. It was noted that the mean control T 4 

# level (5.28 μg/dL) was above the laboratory’s historical contro l range of 4.61-5.02 μg/dL; however, the values for the treated groups were below the provided historical control range. At 250 mg/kg/day, serum TSH was increased by 74% (NS); however, no eff ect was observed on t hyroid weight or histopathology.

At 250 mg/kg/day, creatinine was increased (p<0 .05) by 50%, which is consistent with the probe study. ALP was slightly decreased (p<0.05) by 17%; however , the toxicological significance of a slight decrease is unclear. Calcium and sodium were also increased (p<0.05) by 4% and 1%, respectively, at 250 mg /kg/day, and these minor effects were not considered adverse.

In the kidney, increased hyaline droplet form ation within proximal convoluted tubule (multifocal) was observed in the 25 (13/16, very slight severity), 75 (13/15, very slight), and 250 (14/15, very slight to sli ght) mg/kg/day groups compared to the control (1/16, very slight). However, this finding was not associat ed with degenerative or cytotoxic changes in the kidneys. As this was the only treatment -related histological le sion and there was no other evidence of kidney toxicity, this finding was not considered adverse. There were no treatment-related histopathological changes in the thyroid, testes or epididymides.

The highest dose tested was determined to be an adequate dose based on increased serum creatinine, increased liver we ights, and decreased serum T 4 

# levels.

# Male Pubertal Assay (2012) / Page 18 of 18 TRIFLURALIN / 036101 OCSPP 890.1500 / OECD None



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

 The CV for mean seminal vesicle weight was 25.6% (PC top of range: 21.06%).

 The mean thyroid weight was 12.5 mg (PC lower boundary: 14 mg).

 The CV for mean TSH level wa s 70.02% (PC top of range 58.29).

 The CV for weight at PPS wa s 10.59% (PC top of range: 7.47%).

 The CV for final body weight was 9.23% (PC top of range: 7.47%).

 The mean kidney weight was 1.91 g (PC lower boundary: 2.242 g).

 The CV for mean weaning weight was 10.4% (PC top of range 10.25%).

# DATA EVALUATION RECORD

TRIFLURALIN

Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-40-2012 (MRID 48619708)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 3/28/2012

Secondary Reviewer Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 5/15/2012

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

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/17/2012

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

.

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

# TRIFLURALIN 

# I 036101 Steroidogenesis Assay (2011) 

# I Page 1 of 13 OCSPP 890.1550/ OECD None



# DATA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550



# PC CODE: 036101 

# DP BARCODE: D398779

TXR#: 0055724 

# CAS No.: 1582-09-8

TEST MATERIAL 

# {PURITY): Trifluralin (97.1 %)



# SYNONYMS: Trifluoralin, 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine



# CITATION: LeBaron, M.J.; 

# Kan, H.L; Perala, A.W. (2011) Evaluation oftrifluralin in the 

# in vitro steroidogenesis assay. Toxicology & 

# Environmental Research and Consulting, The Dow Chemical Company, Midland, ML Laboratory Study No.: 101187, December 21, 2011. MRID 48619708. Unpublished.



# SPONSOR: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN

TEST 

# ORDER#: CON-036101-26

EXECUTIVE 

# SUMMARY: 

# In a steroidogenesis assay (MR.ID 48619708), H295R cells cultured 

# in vitro in 24-well plates were incubated with trifluralin (97. l % purity; LOT# DK0- 8916-33, TSN100656) at log concentrations from 0.0001 to 100 μM for 48 hours in triplicate in three independent experiments. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.1 %.

Testosterone and estradiol levels were measured using LC-APPi-MS/MS. A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels. The positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol and testosterone production.

Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls. The exceptions were the minimum basal hormone production level of 500 pg/mL testosterone was not met in the solvent control (SC) in the testosterone assay for 2 runs (491and454 pg/mL), and cells with a passage number of 11.5 instead of 10.5 were used in a single run.

No effects of trifluralin were observed on testosterone levels. Decreased**(p~0.05)** 

# estradiol levels were observed at 0.0001 μM trifluralin 

# in Runs I and 2 (0.8-fold the SC) and at 0.001 μM

# Steroidogenesis Assay (2011) / Page 2 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None 

# in Run 2 (0.9-fold). These changes were minimal, unrelated to dose, and not reproducible across all three runs; therefore, the a pparent effect on decreased estr adiol level was not considered treatment related. However, statistically significant increases (p ≤ 0.05) of estradiol levels were noted at 10 and 100 μM in all runs (1.2–1.4-fold ). Although the magnitude of increase was slight, these effects of increas ed estradiol levels were repr oducible and were considered treatment related.

Based on hormone responses in each of the inde pendent runs, triflurali n treatment resulted in statistically significant and re producible increases in estrad iol production, but trifluralin treatment did not result in statis tically significant and reproducibl e alterations in testosterone production.

The assay satisfies the EDSP Tier 1 Test Order requi rements for a Steroidogenesis assay (OCSPP 890.1550).

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

# Steroidogenesis Assay (2011) / Page 3 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Toxicology & Environmental Research and Consulting, The Dow Chemical Company
Location: Midland, MI Study Director: H. L. Kan Other Personnel: M. J. LeBaron, lead scientist A. W. Perala, analytical chemist B. B. Gollapudi, te chnical reviewer Study Period: February 17, 2011 – December 21, 2011



# 2. Test Substance: Trifluralin Description: Orange solid Lot # (expiration date): DKO-B916-33, TSN100656 (not reported) Purity: 97.1% Solubility: Soluble in DMSO up to 0.1 M; up to 0.3 ppm in water Vapor pressure: 6.1× 10

− 3 

# Pa at 25°C Stability: 4 year shelf life; stability und er test conditions not provided Storage conditions: Ambient CAS #: 1582-09-8 Molecular weight: 335.3 Structure:

N

CF 3 

# C H 2


# C H 2 

# CH 3 

# C H 2


# C H 2 

# CH

3



# O 2 

# N NO 2



# 3. Positive Control (Inducer): Forskolin Description (molecular weight): White powder (410.50) Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): 097K50653 (not provided) Purity: 99% Solubility (in solvent): Soluble in DMSO up to 0.01 M Storage conditions: Ambient CAS #: 66575-29-9


# 4. Positive Control (Inhibitor): Prochloraz Description (molecular weight): Off-white powder (376.67) Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): SZE6220X (not provided) Purity: 99.1% Solubility (in solvent): Soluble in DMSO up to 0.01 M Storage conditions: Ambient CAS #: 67747-09-5



# 5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) Description (molecular weight): Clear liquid (78.13) Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): 68996LMV (not provided)

# Steroidogenesis Assay (2011) / Page 4 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None

Purity: ≥ 99.9% Storage conditions: Room temperature CAS #: 67-68-5 Justification for choice of solvent: Not provided. DMSO is a Guideline reco mmended solvent in which trifluralin is reasonably soluble. Final concentration in assay: 0.1% v/v



# 6. Stock Medium: Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture
Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): RNBB 2720 (not provided) Sodium bicarbonate: Component of stock medium Nu-Serum: 2.5%, BD-Biosciences; Lot # 81515; tested for background hormone concentrations by performing laboratory ITS+premix: 1%, BD-Biosciences; Lot # 84337 Other components: 2.5 mM L-glutamine (Life Technologi es, GIBCO, Grand Island, NY), and penicillin/streptomycin (25 IU/mL/25 μ g/mL; GIBCO).



# 7. Test Cells: H295R human adrenocortical carcinom a cells (ATCC CLR-2128; Batch # not provided) at passage 10.5 – 11.5 were incubated in the stock medium. Incubation conditions were at approximately 37°C with 5% CO 2 

# for approximately 24 hours prior to exposure.

The following performance criteria were met (indicated by an “x”):

x Cell passage identifier. Cell Passage #: 10.5 – 11.5 x Cells frozen down at passage 5 x Frozen cells cultured for 4 additional passages x Total number of passages does not exceed 10



# B. METHODS

1. Pre-Test Information

a. Hormone Assay Interference Test: The Sponsor stated that a separate chemical interference assay was not performed, as LC/A PPI-MS/MS was used along with internal spike-in controls to measure extraction and qua ntitation efficiency. No interference would be anticipated using this analytical methodology.

b. Hormone Extraction: Briefly, standards and samples were vortexed with methylene chloride containing both testoste rone and estradiol internal st andards, and the organic phase was transferred to clean vials. The aliquot s were evaporated to dryness, and the final residue in each vial was deri vatized with dansyl chloride in sodium bicarbonate buffer (100 mM, pH 10.5). Samples were subject ed to LC/APPI-MS/MS analysis.

c. Laboratory Proficiency Test: The Sponsor stated that labor atory proficiency assays were performed to optimize and validate the H295R steroidogenesis assay. These non-GLP, unpublished experimental results with positive control chemicals demonstrated laboratory proficiency and validation, and were reported se parately (data not presented and report not cited).

2. Test Solutions: Trifluralin, forskolin, and prochloraz we re dissolved in DMSO and diluted 1:1000 in the final treatment medi um. When added to the cell cu lture plates, these dilutions

# Steroidogenesis Assay (2011) / Page 5 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None 

# yielded final concentrations of 1 μM a nd 10 μM for forskolin, 0.1 μM and 1 μM for prochloraz, and 0.0001 to 100 μM fo r trifluralin. The selected concentrations of the test material in the dosing solutions used for the treatment in a definitive assay were verified analytically. The final concentration of DMSO in the medium was held constant at 0.1% (v/v).

3. Cell Plating and Preincubation: Cells were maintained in the stock medium described above. The hormone concentrations in undiluted Nu-Serum were 974 pg/mL for testosterone and 681 pg/mL for estradiol. H295R cells were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for ten to eleven additional passages prior to use in the assay. The cells were plated into wells of a 24-well cell culture plate at a density of approximately 200,000 – 300,000 cells/mL/well (50 – 60% confluency). The cells were then placed into an incubator (37°C/5% CO 2 

# ) for approximately 24 hours prior to chemical exposure. After a 24-hour pre-incubation, th e cells were checked microscopically for good attachment and proper morphology.

4. Exposure: The medium was removed from the cells and replaced with medium containing concentrations of trifluralin (or solvent) in tr iplicate, according to the schematic presented in Table 1.

TABLE 1. Dosing Schematic for the Exposure of H295R Cells to Trifluralin (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 Text Tables 2 an d 3 on page 18 of the study report.



# A concurrent QC plate was included with each of the three independent runs of the test chemical plates to demonstrate the assay’s res ponse to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testoste rone and estradiol production). The QC plate was prepared and dosed in the same manner w ith either forskolin or prochloraz, according to the schematic presented in Table 2.

TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM). a 

# 1 2 3 4 5 6 A Blank b 

# Blank Blank Blank + methanol c 

# Blank + methanol Blank + methanol B DMSO DMSO DMSO DMSO + methanol DMSO + methanol DMSO + methanol C Forskolin 1 μM Forskolin 1 μM Forskolin 1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM Prochloraz 0.1 μM D Forskolin 10 μM Forskolin 10 μM Forskolin 10 μM Prochloraz 1 μM Prochloraz 1 μM Prochloraz 1 μM a Data were obtained from Text Table 1 on page 17 of the study report. b Blank wells received medium only. c Methanol (MeOH) was added after the exposure was terminated and the medium was removed.

# Steroidogenesis Assay (2011) / Page 6 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None 

# Following dosing, the plates were incuba ted for approximately 48 hours under the conditions previously described. After the incu bation period, two equal portions of medium were collected and frozen at

− 80°C until further processing.

5. Cell Viability/Cytotoxicity Assay: The cell viability/cytotoxic ity testing was conducted in the QC plate and in the test chemical exposur e plate immediately afte r termination of the exposure experiments. In addition to viability /cytotoxicity testing, cells were checked for the degree of confluence, homogeneity from well- to-well, and any signs of cytotoxicity or altered morphology. A subjective parallel visu al assessment of each well was conducted. The CellTiter 96 Aqueous One Solution Cell Pr oliferation Assay kit (Madison, WI, Cat# G3580) was used for assessment of cell viability testing, as per manufact urer’s instructions. This cell proliferation assay kit is a colorime tric modified MTT cell viability assay. The Aqueous One Solution contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo lium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate). Th e quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of viable cells in culture. The minimum cell vi ability required per well was ≥ 80%; wells with lower viability were excluded in the final data analysis.

6. Hormone Measurement System: The concentration of eac h hormone (testosterone and estradiol) was evaluated in triplicate in the culture medium. Hormone s were extracted from the samples with methylene chloride, derivatiz ed with dansyl chloride, and analyzed by LC/APPI-MS/MS. The lower limit of quantification (LLQ) for this measurement system was 25 pg/mL for testosterone and 10 pg/mL for estradiol.

The following performance criteria were met (indicated by an “x”):

x Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol) x Spiked sample recovery acceptable for two concentrations of testosterone and estrad iol (mean measured amount from triplicate samples ≤ 30% of nominal concentration) NA Hormone cross-reactivity (antibody-based assays only; ≤ 30% of basal production of the respective hormone)

x Solvent control within 75% range be low maximum response on standard curve NA Test compound tested for interf erence with meas urement system



# C. DATA ANALYSIS: To evaluate the relative increas e or decrease of hormone production after test chemical exposure, the results were normalized to the mean SC value for each assay and results were expressed as fold-change relative to the SC in each exposure plate. All data were expressed as mean ± standa rd deviation (SD). Relative changes were calculated as follows:

Relative Change = Hormone concentration in each well ÷ Hormone c oncentration of mean solvent control

Prior to conducting statistical analyses, the assumptions of normality and variance homogeneity were evaluated. Homogeneity of va riance was evaluated by Bartlett’s test and normality by Shapiro-Wilk’s test at alpha = 0.01. If the data were not homogeneous or normally distributed, then the data were tr ansformed to approximate homogeneity or a normal distribution. If the da ta were homogeneous and appr oximately normally distributed, differences between chemical treatments and SC were analyzed using a parametric analysis of variance followed by Dunnett’s te st, if significant. If the da ta were not homogeneous or

# Steroidogenesis Assay (2011) / Page 7 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None 

# normally distributed, a non-parametric test was used (Kruskal-Wallis) and if significant, was followed by the Wilcoxon rank sum test with a Bonferroni-Holm correction. Differences were considered significant at p ≤ 0.05. These statistical analyses were considered appropriate.

II. RESULTS

A. TEST COMPOUND: Precipitation of the trifluralin was not reported at any concentration tested. Cytotoxicity was not observed at any tested trifluralin concentration. Inter- and intraplate variability (CV) were calculated by the reviewers from the raw data, and differed from the range of 0.6 to 22.0% CV provided in the report on page 27. Variability (CV) between the runs for the solvent controls wa s 11–12% for the testos terone and estradiol assays, and the variability (CV) within each run for the solvent controls in the testosterone and estradiol assays was less than 10%. The inter- and intraplate variability was within the guideline recommendation of ≤ 30%.

The effects of trifluralin on te stosterone and estradiol production are summarized in Table 3 and shown in Figures 1 and 2. Test osterone levels were decreased (p ≤ 0.05) to 0.9-fold the solvent control values in R un 2 at both 0.001 μM and 0.01 μM trifluralin. These changes were minimal, unrelated to dose, and not repr oducible across runs; ther efore, the apparent effect on testosterone was not considered treatm ent-related. No other effects were noted.

Statistically significant decreases (p ≤ 0.05) of estradiol levels were observed at 0.0001 μM trifluralin in Runs 1 and 2 (0.8-fold the solven t control) and at 0.001 μM in Run 2 (0.9-fold). These changes were minimal, unrelated to dose, and not reproducible across all three runs; therefore, the apparent effect on estradiol was not considered treatment-related. Statistically significant increases (p ≤ 0.05) of estradiol levels were noted at 10 and 100 μM in all runs (1.2–1.4-fold). These effects were reproducible and were considered related to treatment.

# Steroidogenesis Assay (2011) / Page 8 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None

TABLE 3. Mean (±SD) Hormone Concentrations Follo wing Treatment with Trifluralin for 48 Hours. a


# Nominal Concentration (μM) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean ± SD Statistical Significance

Testosterone (pg/mL) Fold Difference
DMSO 553.3 652.0 528.3 — — — — — — 0.0001 538.7 589.0 496.3 1.0 0.9 0.9 0.9 0.0 — 0.001 510.7 577.7 516.7 0.9 0.9 1.0 0.9 0.0 Run 2 0.01 510.0 572.7 490.3 0.9 0.9 0.9 0.9 0.0 Run 2 0.1 560.7 609.3 479.7 1.0 0.9 0.9 1.0 0.1 — 1 540.0 591.3 495.3 1.0 0.9 0.9 0.9 0.0 — 10 628.7 635.3 496.0 1.1 1.0 0.9 1.0 0.1 — 100 569.3 709.7 490.3 1.0 1.1 0.9 1.0 0.1 — Estradiol (pg/mL) Fold Difference
DMSO 57.4 60.5 71.8 — — — — — — 0.0001 45.7 50.8 65.6 0.8 0.8 0.9 0.8 0.1 Runs 1 and 2 0.001 48.6 52.4 65.2 0.8 0.9 0.9 0.9 0.0 Run 2 0.01 52.5 50.8 62.4 0.9 0.8 0.9 0.9 0.0 — 0.1 50.4 58.0 62.9 0.9 1.0 0.9 0.9 0.0 — 1 55.7 54.7 71.0 1.0 0.9 1.0 1.0 0.0 — 10 79.6 72.3 92.0 1.4 1.2 1.3 1.3 0.1 All runs 100 79.2 80.0 90.9 1.4 1.3 1.3 1.3 0.1 All runs a Data were obtained from Table 2 on page 31 of the study re port. The lower limit of quantification (LLQ) was 25 pg/mL for testosterone and 10 pg/mL for estradiol.

# Steroidogenesis Assay (2011) / Page 9 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None 

# FIGURE 1. Change in Testosterone Produc tion Relative to Trifluralin Concentration.

# Steroidogenesis Assay (2011) / Page 10 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None 

# FIGURE 2. Change in Estradiol Producti on Relative to Trifluralin Concentration.

# Steroidogenesis Assay (2011) / Page 11 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None



# B. CYTOTOXICITY: Trifluralin was not cytotoxic to the H295R cells (Table 4). Viability in each run of the test plate ranged from 106–116% relative to solv ent controls. Viability in each run of the QC plate was 96–124% relative to solvent controls, except for the methanoltreated wells which had viabilities of 30–36%.

TABLE 4. Mean (±SD) MTT Cell Viab ility Results after Treatment with Forskolin, Prochloraz, or Trifluralin for 48 Hours. a


# Compound Concentration (μM) Cell Viability (% of DMSO Controls) Mean SD Media NA 98.90 2.52 Media + Methanol NA 31.20 2.08 DMSO + Methanol NA 34.13 2.56 Forskolin 1 119.80 4.17 Forskolin 10 118.97 1.69 Prochloraz 0.1 103.97 5.68 Prochloraz 1 102.87 3.85 Trifluralin 0.0001 108.80 1.91 Trifluralin 0.001 111.37 4.08 Trifluralin 0.01 114.27 0.32 Trifluralin 0.1 108.67 1.07 Trifluralin 1 109.53 1.37 Trifluralin 10 109.87 3.72 Trifluralin 100 109.03 2.89 a Values were calculated by the reviewers from data obt ained from Appendix B on page 43 of the study report.



# C. QC PLATE: The hormone concentrations after expos ure to the reference chemicals, SC, and blank samples, as well as the fold difference change relative to SC (individual and mean ± SD) for the three assay runs are presente d in Table 5. The minimum basal hormone production levels (500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both blank and SC wells for testosterone, with the ex ceptions of Runs 1 and 3 for the SC in the testosterone assay (491 and 454 pg/mL, resp ectively). Forskolin at 10 μM induced testosterone an average of 2.5-fo ld and estradiol 13.3-fold over the SC. Prochloraz at 1 μM inhibited concentrations of test osterone and estradiol by 0.2- to 0.4- fold compared to SC. Criteria for the QC plate were me t for induction and inhibition.

The variability (%CV) between the runs (calculated by the re viewer) based on the absolute hormone concentrations in the SC were 5.3% fo r testosterone and 5.9% for estradiol, which were within the guideline criteria of ≤ 30% for the assays. The %CVs within each run for the QC plates were 6.0-8.3% for testosterone and 5.7-8.3% for estradiol, which were within the guideline criteria ( ≤ 30%).

# Steroidogenesis Assay (2011) / Page 12 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None



# TABLE 5. Mean (±SD) Hormone Concentrations Followi ng Treatment with Forskolin or Prochloraz for 48 Hours. a



# Concentration (μM) Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Mean ± SD

Testosterone (pg/mL) Fold Difference (Relative to DMSO)
Blank 589 667 554 — — — — — DMSO 491 503 454 — — — — — 1 μM Forskolin 1009 1008 866 2.1 2.0 1.9 2.0 0.1 10 μM Forskolin 1243 1433 934 2.5 2.8 2.1 2.5 0.4 0.1 μM Prochloraz 211 251 258 0.4 0.5 0.6 0.5 0.1 1 μM Prochloraz 91.7 123 133 0.2 0.2 0.3 0.2 0.1 Estradiol (pg/mL) Fold Difference (Relative to DMSO)
Blank 55.2 56 67.3 — — — — — DMSO 51.7 55.6 58.2 — — — — — 1 μM Forskolin 442 454 416 8.5 8.2 7.2 8.0 0.7 10 μM Forskolin 700 809 686 13.5 14.5 11.8 13.3 1.4 0.1 μM Prochloraz 32.9 30.7 38.7 0.6 0.6 0.7 0.6 0.1 1 μM Prochloraz 22.2 23.1 26.2 0.4 0.4 0.5 0.4 0.0 a Data were obtained from Table 3 on page 32 of the study re port. The lower limit of quantification (LLQ) was 25 pg/mL for testosterone and 10 pg/mL for estradiol.



# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Based on the combined hormone responses in each of three independent H295R steroidogenesis assays, it was determined that trifluralin treatment resulted in a slight, statistically significant incr ease in estradiol production at highest two concentrations includ ing the limit-concentration of 100 μ M (10 -4 

# M), while no effects on estradiol production we re seen at lower concentrati ons and there were no effects on T production at any concentration of triflurali n. The slight 1.4-fold or less increase in estradiol production did not meet the 1.5-fold cut-off criteria establis hed in the validation program for this assay and hence was not interpreted to be bi ologically relevant.

B. AGENCY COMMENTS: Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxi city, adequate production of testosterone and estradiol, acceptable reproducib ility (low %CV), and appropria te induction and inhibition with positive controls. The exception was the minimum basal hormone production level of 500 pg/mL testosterone was not met in the SC in the testosterone assay for 2 runs (491 and 454 pg/mL).

No effects of trifluralin were observe d on testosterone levels. Decreased (p ≤ 0.05) estradiol levels were observed at 0.0001 μM trifluralin in Runs 1 and 2 ( 0.8-fold the solvent control) and at 0.001 μM in Run 2 (0.9-fold). These ch anges were minimal, unrelated to dose, and not reproducible across all three runs; therefore, the apparent effect on decreased estradiol level was not considered treatment-related. Statistically significant increases (p ≤ 0.05) of estradiol levels were noted at 10 and 100 μM in all runs (1.2–1.4- fold). Although the magnitude of change was slight, these effects of increased estradiol level were reproducible and were considered treatment related.

# Steroidogenesis Assay (2011) / Page 13 of 13 TRIFLURALIN / 036101 OCSPP 890.1550/ OECD None 

# Based on hormone responses in each of the inde pendent runs, triflurali n treatment resulted in statistically significant and re producible increases in estrad iol production, but did not result in statistically significant a nd reproducible alterations in testosterone 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:

 Stability of trifluralin in the test system was not reported.

 The minimum basal hormone production level of 500 pg/mL testosterone was not met in the SC in the testosterone a ssay for 2 runs (491 and 454 pg/mL).

# DATA EVALUATION RECORD

TRIFLURALIN

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-40-2012 (MRID 48619709)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Rebecca L. Bryan, B.S. Date: 4/24/2012

Secondary Reviewer: Signature: Kelly Luck, M.S. Date: 5/9/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 5/11/2012

Quality Assurance: Signature: Steven Brecher, Ph.D., D.A.B.T. Date: 5/11/2012

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

.

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

# TRIFLURALIN/ 036101 Io vivo Uterotrophic Assay (2012) 

# I Page 1 of9 

# OCSPJ> 90.1600/ OECD 440 

# Primary Reviewer:

# -=N~an=c.,._y"""M=c:;..;:C=arr=cr..ol=l------- Signature: 

# b/he.,

# ~ Health Effects Division Date: 

# J**"S~.s-** Secondary Reviewer:

# ~J~e=ss'-'R~o"'"""w""""l=an=d.._ _______ 

# Signature: 

# deaa**~** Health Effects Division Date: ---=&"f"'-".t...;;..;7'-"/;+/,.,.S-.____ 

# I DATAEVALUATIONRECORD 

# I Template version 09/2011



# STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440



# PC CODE: 036101 

# DP BARCODE: 0398779

TXR#: 0055724 

# CA.S#: 1582-09-8



# TEST MATERIAL <PURITY): Trifluralin (97.1% a.i.)



# SYNONYMS: 2,6-Dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine



# CITATION: Marty, M.S.; Brooks, K.J. (2012). Trifluralin: Uterotrophic Assay in the Inunature Female Crl:CD(SD) Rat. The Dow Chemical Company, Toxicology 

# & Environmental Research and Consulting, Midland, Ml. Laboratory Project Study ID.: 101144, Jwie 10, 2011; revised date: April 4, 2012. 

# MRlD 48619709. Unpublished.



# SPONSOR: Dow AgroSciences, LLC, 9330 Zionsville Road, Indianapolis, IN



# TEST ORDER#: CON-036101-26



# EXECUTIVE SUMMARY: In an uterotrophic assay (MR.ID 

# 48619709) conducted to screen for potential estrogenic activity, trifluralin (97.1 % a.i., Lot# DK0-8916-33, TSNl 00656) in

com oil was administered daily for 3 consecutive days via oral gavage to groups of six inunature female Sprague Dawley rats at dose levels of 0 (vehicle), 25, 75, or 225 mg/kg/day on post-natal days (PND) 19-21. A positive control group was treated with a daily dose of l 7a-ethynyl estradiol (EE) at 10 μg/kg/day by oral gavage. Body weights were detennined daily. All animals were examined for precocious vaginal opening, and then terminated and necropsied on PND 22 approximately 24 hours after the final dose to determine wet and blotted uterine weights.

All animals survived until scheduled termination. No clinical signs of toxicity were observed, and no precocious vaginal opening was noted in the trifluralin-treated females. At 225 mg/kg/day, overall body weight gains were decreased (p<0.05) by 34%. Body weights in the low- and mid-dose groups, and the positive control group were comparable to the vehicle controls throughout the duration of the assay. Uterine weights in the trifluralin treated groups

were comparable to the vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p<0.05) by 707% and 396%, respectively, as expected.

# In vivo Uterotrophic Assay (2012) / Page 2 of 9 TRIFLURALIN/ 036101 OCSPP 890.1600/ OECD 440



# The doses tested were determined to be ad equate based on a 34% decrease in overall body weight gain in the high-dose group.

No statistically significant changes were seen in the uterine weight in this assay. Trifluralin was negative in the uterotrophic assay

The assay satisfies the EDSP Tier 1 Test Order require ments for a uterotrophic assay (OCSPP 890.1600).

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

# In vivo Uterotrophic Assay (2012) / Page 3 of 9 TRIFLURALIN/ 036101 OCSPP 890.1600/ OECD 440



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: The Dow Chemical Company, Toxicology & Environmental Research and Consulting
Location: Midland, MI Study Director: K.J. Brooks Other Personnel: M.S. Marty (Lead Scientist); J.L. Fairchild (Animal Technician) Study Period: November 3, 2010 to June 10, 2011



# 2. Test Substance: Trifluralin Description: Molecular weight = 335.3 g/mol Source: Dow AgroSciences, LLC (Indianapolis, IN) Lot #: DKO-B916-33, TSN100656 (expiry date: not reported) Purity: 97.1% a.i. Stability: Stable in corn oil at ambient temperature for up to 25 days CAS #: 1582-09-8 Structure:



# 3. Reference Estrogen: 17 α -ethynyl estradiol (EE) Supplier: Sigma Aldrich Lot/Batch #: 028K1411 (expiry date: not reported) Purity: Not reported CAS #: 57-63-6



# 4. Solvent/Vehicle Control: Corn oil Supplier: Sigma-Aldrich (St Louis, MO) Lot/Batch #: Not reported Rationale (if other than water): Selected as the vehicle due to solubility properties of the test material and because it has been shown in previous studies that corn oil does not exert any estrogenicity or toxicity in immature female rats. Dosing volume : 4 mL/kg



# 5. Test Animals: Species: Rat (females only) Strain: Sprague Dawley [Crl:CD(SD)] Age/weight at dose initiation: 19 days old/ 42.1-50.5 g Source: Charles River Laboratories, Inc. (Portage, MI); immature rats (PND 11) were received with lactating dams. Pups were weaned on PND 18 (1 day prior to dosing). Housing: Pups were group-housed (6/cage) in solid bottom cages with paper pulp bedding. Diet: Teklad Diet #2016 in meal form (Harlan Laboratories, Inc., Indianapolis, IN)

, ad libitum (total genistein equivalents <325 μ g/g). Water: Tap water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 22±3 oC 40-70% 12-15/hr 12 hrs light/ 12 hrs dark Acclimation period: 8 days (7 days housed with thei r dam plus 1 day after weaning ) NO 2



# CF 3 

# O 2



# N N CH 3 

# CH

3

# In vivo Uterotrophic Assay (2012) / Page 4 of 9 TRIFLURALIN/ 036101 OCSPP 890.1600/ OECD 440



# B. STUDY DESIGN

1. In Life Dates: Start: November 5, 2010 End: November 8, 2010

2. Study Design: Following an initial 8-day acclimation period, immature, intact female rats were administered the test substance from PND 19-21. Rats were euthanized (PND 22) approximately 24 hours after the last dose and ne cropsied for uterine weight measurements. The pups were housed with their dam fo r 7 days prior to weaning on PND 18.

3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1 using a computer program designed to increase the probability of uniform group mean weights and standard devi ations at the start of the study. Statistical analysis indicated that there were no significan t differences between group mean weights at study initiation.

TABLE 1. Study Design a 

# Test Group Dose (mg/kg/day) # of Females Estrogen Agonist Assay Vehicle Control (corn oil) 0 6 Trifluralin Low 25 6 Trifluralin Mid 75 6 Trifluralin High 225 6 17 α -ethynyl estradiol (EE), Reference Estrogen 10 μg/kg/day 6 a Data were obtained from Text Table 1 on page 15 of the study report.



# 4. Dose Selection Rationale: The dose levels were selected based on the results from a probe study 1 

# in which four female Sprague Dawley rats /dose group were administered trifluralin in corn oil via oral gavage at doses of 0, 50, 200, 400, or 600 mg/kg/day for 15 days. Clinical observations included urine soiling of the peri neum for one 600 mg/kg/day rat, and clear perioral soiling for one 600 mg/kg/day rat. Body weight gains (D ays 1-3) at 200, 400, and 600 mg/kg/day were decreased by 17, 68, and 80% , respectively. Absolute body weights at 200, 400, and 600 mg/kg/day were decreased on Day 3 by 3, 11, and 11%, respectively. Feed consumption had correspond ing decreases of 15-45% at ≥ 200 mg/kg/day doses. No effects on body weights were observed on Day 15 for any trifluralin dose group. Female rats at 400 and 600 mg/kg/day doses had increas ed levels of blood urea nitrogen, creatinine, gamma glutamyl transpeptidase, and globulin, a nd decreased levels of alkaline phosphatase and albumin. There were dose-related increa ses in relative liver weights, ranging from 12.5% at 50 mg/kg/day to 32% at 600 mg/kg/day. The high dose level in the uterotrophic study was expected to cause a significant d ecrease in body weight gain over the 3-day dosing period. The lower dose levels were sel ected to provide dose response data for any estrogenic effects that may be obs erved among the high-dose group rats.

1 

# Marty, M. S. and Marshall, V. A. (In progress A). Tr ifluralin: Hershberger, Uter otrophic, and Pubertal Assay Probe Study in Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

# In vivo Uterotrophic Assay (2012) / Page 5 of 9 TRIFLURALIN/ 036101 OCSPP 890.1600/ OECD 440



# 5. Dose Preparation and Analysis: Dose formulations in corn oil were prepared once prior to initiation of treatment in concentrations su ch that a dose volume of 4 mL/kg yielded the targeted doses

. Concentration verification (all dose s) and homogeneity (low and high doses) were tested prior to initiation of dosing. Stability of trifluralin in corn oil was determined in a concurrently submitted Hershberger study (MRID 48619707; concurrently reviewed) 2



# . Results of Dose Analysis

Homogeneity (%RSD): 0.7 and 1.3%

Stability (% of Day 1): 94.0–100% at concentrations of 0.25-250 mg/mL in corn oil for up to 25 days at ambient temperature3

Concentration (% of nominal): 99.0-102.5%

The analytical data indicated that the mixing procedure was ad equate and that the variation between nominal and actual dosage to the animals was acceptable.

6. Dosage Administration: Animals were administered the test formulations, positive control (EE) or vehicle control daily via oral gavage for three consecu tive days in a dose volume of 4 mL/kg body weight. Dose volumes were ad justed daily based on the concurrent body weight measurement.

7. Statistics: Body weights and gains were analyzed by a forced parametric test. For uterine weights (blotted and wet), a Bartlett’s test was conducted to check for homogeneity of variance. Based on the outcome of the Bartlett ’s test (p<0.01), the wet and blotted uterine weights for vehicle control versus positive control were subjected to a log transformation. An analysis of covariance ( ANCOVA) was performed with dos e in the model and terminal body weight as the covariate. Data from the vehicle controls versus trifluralin treated groups and vehicle controls vers us EE-treated group were statistic ally compared. If the dose effect was significant (p<0.05), one-sided leas t squared means with Dunnett’s correction were used to determine differences since the least square means were adjusted for the covariant. Liver weights were evaluated usi ng a parametric analysis of variance (ANOVA); if the ANOVA was significant (p<0.05), a Dunne tt's test was performed. Incidence of vaginal opening was reported but not statistica lly analyzed. The statistical analyses were considered adequate.

2 

# Marty, M.S., Marshall, V.A., and Jeong, YC. Trifluralin : Hershberger Assay in Castrated Adult Male Crl:CD(SD) Rats. Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Study No.: 101133, December 20 , 2011. MRID 4861 9707. Unpublished 3 Ibid.

# In vivo Uterotrophic Assay (2012) / Page 6 of 9 TRIFLURALIN/ 036101 OCSPP 890.1600/ OECD 440



# C. METHODS

1. Clinical Examinations: Cage-side checks for mortality, morbidity and clinical signs of toxicity were conducted twice daily. Detailed physical examin ations were performed prior to initiation of dosing.

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing period and at termination.

3. Food Consumption (Optional): Food consumption was not measured.

4. Necropsy and Measurement of Uterine and Liver Weight: On PND 22 (approximately 24 hours after final administration of the test substance), all animals were checked for precocious vaginal opening, anesthetized by CO 2 

# inhalation, euthanized by cervical dislocation, and subjected to a gross necropsy. Dissection of the uterus was performed according to the Guidelines (OCSPP 890.1600). Briefly, the vagina was removed just below the cervix in order to retain the lumina l fluid in the uterus. The “wet” uterus (i.e., containing the luminal fluid) was weighed. S ubsequently, each uterine horn was “nicked” three times, covered with saline-moistened abso rbent paper, and blotted by gently placing a 500 g weight on the paper-covered uterus to remove the luminal fluid while preventing desiccation. The blotted uter us was then weighed. Add itionally, the liver weight was determined.

5. Microscopic Examination (Optional): Microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination.

2. Clinical Signs of Toxicity: No clinical signs of toxicity were observed in animals for any dose groups. No precocious vaginal opening was observed in the vehicle control and trifluralin treated animals. Precocious va ginal opening was observed in one of the EE positive control animals, an effect consistent with an estrogenic response.

B. BODY WEIGHT AND WEIGHT GAIN: Body weight and body weight gain data are presented in Table 2. Overall body weight ga ins were decreased (p<0.05) by 34% at 225 mg/kg/day. Body weights in the low- and mid-dose groups, and the positive control group were comparable to the vehicle contro ls throughout the duration of the assay.

# In vivo Uterotrophic Assay (2012) / Page 7 of 9 TRIFLURALIN/ 036101 OCSPP 890.1600/ OECD 440



# TABLE 2. Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a



# Study Day Dose (mg/kg/day)

Vehicle Control Trifluralin (25) Trifluralin (75) Trifluralin (225) Reference Estrogen EE (10 μ g/kg/day) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1 6 46.5 2.6 6 45.2 2.3 6 45.8 1.7 6 45.7 2.8 6 46.0 2.0 2 6 51.4 2.9 6 48.1 3.3 6 48.8 1.9 6 47.2 3.4 6 49.3 3.6 3 6 55.2 3.1 6 52.1 3.1 6 53.2 2.6 6 51.2 2.9 6 52.0 3.1 4 (termination) 6 59.6 3.8 6 56.0 3.3 6 57.3 3.5 6 54.3 3.5 6 56.9 3.5 Body Weight Gain (Days 1-4) 6 13.0 1.6 6 10.9 1.6 6 11.5 2.3 6 8.6* ( ↓ 34) 2.1 6 10.9 1.9 a Data were obtained from Table 3 on page 27 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05


# C. FOOD CONSUMPTION (Optional): Food consumption was not measured.

D. PATHOLOGY

1. Uterine and Liver Weights: Uterine (wet and blotted) we ight and liver weight data are presented in Table 3. Uterine weights in the trifluralin treated groups were comparable to the vehicle controls. Absolute wet and blotted uterus weight s for the positive control (EE) group were increased (p<0.05) by 707% a nd 396%, respectively, as expected.

The mean blotted uterine weight for the ve hicle control group was 0.043% of terminal body weight, which was deemed acceptable to yield su fficient assay sensitivity and was consistent with the requirement (<0.09%) of th e test guideline (OCSPP 890.1600).

Absolute liver weights in the trifluralin trea ted groups and positive control were comparable to the vehicle controls. Rela tive liver weights (not reported in Table 3) in the 75 and 225 mg/kg/day group were increased (p< 0.05) by 13% and 17%, respectively.

# In vivo Uterotrophic Assay (2012) / Page 8 of 9 TRIFLURALIN/ 036101 OCSPP 890.1600/ OECD 440



# TABLE 3. Uterine and Liver Weights from Estrogen Agonist Assay in SD Rats a



# Parameter Dose (mg/kg/day)

Vehicle Control Trifluralin (25) Trifluralin (75) Trifluralin (225) Reference Estrogen EE (10 μ g/kg/day) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD Terminal BW (g) 6 59.6 3.8 6 56.0 3.3 6 57.3 3.5 6 54.3 3.5 6 56.9 3.5 Wet, absolute (mg) 6 27.1 2.8 5 b 

# 27.7 1.6 4 b 

# 26.3 1.3 6 27.8 4.1 6 218.7* ( ↑ 707) 41.9 Wet, relative (%) c 

# 6 0.046 0.006 5 0.050 0.002 4 0.046 0.003 6 0.051 0.007 6 0.384 0.067 Blotted, absolute (mg) 6 25.4 2.6 6 24.0 3.3 6 24.5 1.0 6 26.1 3.6 6 126.0* ( ↑ 396) 11.0
Blotted, relative (%) c 

# 6 0.043 0.006 6 0.043 0.007 6 0.043 0.004 6 0.048 0.007 6 0.222 0.021 Liver, absolute (g) 6 2.745 0.0381 6 2.638 0.159 6 2.964 0.331 6 2.904 0.193 6 2.445 0.287 a Data were obtained from Tables 4 and 5 on pages 28 and 29 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. b Wet uterine weights for one animal in the 25 mg/kg/day group and two animals in the 75 mg/kg/day group were excluded from analysis. c Relative wet and blotted uterine weights were calculated by the reviewer from the individual data (Appendix Table 3, pages 36 and 37 ). BW Body weight N Number of animals in the group SD Standard Deviation * Significantly different from controls at p<0.05



# 2. Microscopic Examination (Optional): Microscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: There was no animal mortality or treatmentrelated clinical observations in this st udy. There were no significant effects on body weights at any dose of trifluralin. At 225 mg/kg/day trifluralin, body weight gains were decreased significantly by 69%, 37% and 34% on Days 1-2, 1-3, and 1-4, respectively. Body weights/body weight gains were not affected at ≤ 75 mg/kg/day trifluralin. There were no significant increases in absolute liver weights with trifluralin treatment; however relative liver weights were significantly increased by 13% and 17% in the 75 and 225 mg/kg/day trifluralin groups, respectively. Relative liver weights were not significantly affected in the 25 mg/kg/day trifluralin or EE-treated groups. There were no treatment-related increases in mean uterine weights (wet or blotted) in immature animals treated with ≤ 225 mg/kg/day trifluralin. Neither the vehicle control anim als nor the trifluralin-treated animals had precocious vaginal opening. The positive control compound, EE (10 μ g/kg/day), had no effect on body weight; however, there was a significant (32%) d ecrease in body weight gains at one interval (Days 1-3 only) . Immature rats treated with 10 μ g/kg/day EE showed significant (p<0.05) increases in mean imbibe d and blotted uterine weights (707 and 396%, respectively). One of the EE positive control animals exhibited precocious vaginal opening, an effect consistent with an estrogenic res ponse. Vehicle-treated control uterine weights met the performance criteria outlined in the ap plicable test guidelines, indicating acceptable assay sensitivity. Based on these uterotrophi c assay results, there was no indication of

# In vivo Uterotrophic Assay (2012) / Page 9 of 9 TRIFLURALIN/ 036101 OCSPP 890.1600/ OECD 440 

# estrogenicity in immature female rats at doses <225 mg/kg bw/day trifluralin when compared to the vehicle controls.

B. AGENCY COMMENTS: All animals survived until scheduled termination. No clinical signs of toxicity were observed in anim als for any trifluralin treated groups, and no precocious vaginal opening was observed in th e treated immature females. Overall body weight gains were decreased (p<0.05) by 34% at 225 mg/kg/day. Body weights in the lowand mid-dose groups, and the positive control gro up were comparable to the vehicle controls throughout the duration of the assa y. Uterine weights in the tr ifluralin treated groups were comparable to the vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) group were signifi cantly increased as expected.

The doses tested were determined to be ad equate based on a 34% decrease in overall body weight gain for the high-dose group.

No statistically sign ificant changes were seen in the uter ine weight in this study. Trifluralin was negative in the uterotrophic assay.

C. STUDY DEFICIENCIES: None