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



# MEMORANDUM

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



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

# Assays

PC Code: 057701 

# Decision No.: 457627, 468179 

# Petition No.: NA 

# Risk Assessment Type: NA TXR 

# No.: 0052348 

# DP Barcode: D396178, D404274 

# Registration No.: NA 

# Regulatory Action: NA 

# CaseNo.: NA 

# CAS No.: 121-75-5 MRID 

# No.: See Table 40 

# CFR: NA



# FROM: Greg Akerman, Ph.D. 

# -ho .Ar----

Immediate Office Health Effects Division (7509P)



# THROUGH: Jess Rowland

# ~- c;2(LDeputy Director Health Effects Division



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



# I. ACTION REQUESTED Ver.Apr. 2010



# The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays submitted 

# in response to the agency's Test Order for malathion: Test Order# EDSP-057701-44.

Page 1 of2

# Page 2 of 2 

# II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for malathion.

III. MRID Table

Chemical: Malathion PC Code: 057701 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48617501 890.1150 Androgen Receptor Binding (Rat Prostate) 48617503 890.1200 Aromatase Assay (Human Recombinant) 48617504 890.1250 Estrogen Receptor Binding NA 890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) 48617505
890.1350 Fish Short-Term Reproduction 48617506 890.1400 Hershberger (Rat) 48617507 890.1450 Female Pubertal (Rat) 48685701 890.1500 Male Pubertal (Rat) 48685701 890.1550 Steroidogenesis (Human Cell Line – H295R) 48617509 890.1600 Uterotrophic (Rat) 48617510 Range finding study 48685702 Range finding study 48617508 Analytical method 48617512 NA= Not Applicable (requirement satisfied by ot her scientifically relevant information (OSRI))    

       



   



 

  







 



    





 

  


# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

# ՇԾ  



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ      

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#  ԭԹՄՅԽՊ ԰ԧ ԰Է ԰ԻՀՆԽՁԼԽՊ ԱԷ ԨԽՆԼԹՄՄ ԹՆԼ ԥԬ ԨՊՍԽԿԽՊ  ԪԹՄԹՌՀՁՇՆ ԞՅՈՀՁԺՁԹՆ ԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑԣՁՆԹՄԯԽՈՇՊՌԲՆՈՍԺՄՁՋՀԽԼՋՌՍԼՑՈԽՊԾՇՊՅԽԼԺ ՑԴՁՄԼՄՁԾԽԦՆՌԽՊՆԹՌՁՇՆԹՄԢԹՋՌՇՆԪԡ ԩԹԺՇՊԹՌՇՊՑԭՊՇՂԽԻՌԫՍՅԺԽՊԞ԰ՌՍԼՑՋՈՇՆՋՇՊԽԼԺՑԠՀԽՅՁՆՇՎ ԹԞ԰԰ՌՍԼՑՁՆՁՌՁԹՌԽԼԬԻՌՇԺԽՊ
ԹՆԼՋՍԺՅՁՌՌԽԼԬԻՌՇԺԽՊ

ԫՇՌԽԱՀԽԲ԰ԢԭԞԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊ԰ԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅ
Ԣԡ԰ԭԱ ՁԽՊՋԻՊԽԽՆՁՆԿԺԹՌՌԽՊՑ ՁՋԻՇՅՈՊՁՋԽԼՇԾ ԽՄԽՎԽՆՋԻՊԽԽՆՁՆԿԹՋՋԹՑՋՁՆՌԽՆԼԽԼՌՇՁԼԽՆՌՁԾՑԹԻՀԽՅՁԻԹՄ0ՋՄՁՃԽՄ Ց ԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՁԽՁՌՋՈՇՌԽՆՌՁԹՄՌՇ ՁՆՌԽՊԹԻՌՏՁՌՀՌՀԽԽՋՌՊՇԿԽՆԹՆԼՊՇԿԽՆՇՊՌՀՑՊՇՁԼ
ԢԞՇՊԱՈԹՌՀՏԹՑՋԱՀԽՊՇԺՍՋՌՆԽՋՋՇԾՌՀԽԱՁԽՊԺԹՌՌԽՊՑ
ՁՋԺԹՋԽԼՇՆՌՀԽՋՌՊԽՆԿՌՀՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՌՇՁԼԽՆՌՁԾՑՈՇՌԽՆՌՁԹՄ ԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՏՁՌՀ ԻՇՅՈՄԽՅԽՆՌԹՊՑԽՆԼՈՇՁՆՌՋՏՁՌՀՁՆՌՀԽԹՋՋԹՑՏՀԽՊԽԹՎԹՁՄԹԺՄԽԹՆԼՊԽԼՍՆԼԹՆԻՑԹԻՊՇՋՋՌՀԽԺԹՌՌԽՊՑ ԱՀՍՋՌՀԽ ՊԽՋՍՄՌՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՋՀՇՍՄԼՆՇՌԺԽԻՇՆՋՁԼԽՊԽԼՁՆՁՋ ՇՄԹՌՁՇՆԺՍՌՊԹՌՀԽՊՋՀՇՍՄԼԺԽԻՇՆՋՁԼԽՊԽԼՁՆՌՀԽ ԻՇՆՌԽՐՌՇԾՇՌՀԽՊԹՋՋԹՑՋՁՆՌՀԽԺԹՌՌԽՊՑԹՋՏԽՄՄԹՋԬՌՀԽՊ԰ԻՁԽՆՌՁ ԾՁԻԹՄՄՑԯԽՄԽՎԹՆՌԦՆԾՇՊՅԹՌՁՇՆ
Ԭ԰ԯԦԦՆՇՊԼԽՊ
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# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

# ՇԾ  



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ    !

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# ԱՀԽԼԹՑԹՋՋԹՑՇԾՅԹՄԹՌՀՁՇՆՇՆԹՅՈՀՁԺՁԹՆՅԽՌԹՅՇՊՈՀՇՋՁՋՇԾ԰ՇՍ ՌՀԞԾՊՁԻԹՆԻՄԹՏԽԼԾՊՇԿՋ 

# ԵԽՆՇՈՍՋՄԹԽՎՁՋ 
ՏԹՋԻՇՆԼՍԻՌԽԼՍՆԼԽՊԾՄՇՏՌՀՊՇՍԿՀԻՇՆԼՁՌՁՇՆՋԞՅՈՀՁԺՁԹՆՄԹՊՎԹԽԹ ՌԫՁԽՍՏՃՇՇՈԣԹԺԽՊ
ԫԣ԰ՌԹԿԽ
ՈԽՊ ԻՇՆՌՊՇՄՇՊՌՊԽԹՌՅԽՆՌԿՊՇՍՈՊԽՈՄՁԻԹՌԽՏԽՊԽԽՐՈՇՋԽԼՌՇՅԹՄԹՌ ՀՁՇՆ
ՈՍՊՁՌՑԹՌՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ՇԾ  
ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ  ԹՆԼ ՅԿ ԹՁԩՅԽԹՆ ՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽ ԩԬԮԻՇՆՌՊՇՄԹՆԼՅԿԹՁԩԱՀԽՌԽՋՌՋՑՋՌ ԽՅՏԹՋՅԹՁՆՌԹՁՆԽԼԹՌՌՇոԠԹՆԼԹ
ՈԥՇԾՌՇ

ԱՀԽՊԽՏԽՊԽՆՇՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՇՆՋՍՊՎՁՎԹՄՇԾՌԹԼՈՇՄԽՋ ԼՍՊՁՆԿՌՀԽԼԹՑՌԽՋՌԣՊՇՅԡԹՑՋՌՀՊՇՍԿՀ ՋՍՊՎՁՎԹՄՏԹՋ  ԹՆԼ  ՁՆՌՀԽ ՆԽԿԹՌՁՎԽԻՇՆՌՊ ՇՄ ԹՆԼ ԹՆԼՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑԣՊՇՅԡԹՑՌՀՊՇՍԿՀՌԽՋՌՌԽՊՅՁՆԹ ՌՁՇՆՋՍՊՎՁՎԹՄՏԹՋԹՆԼ ՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑԞՌՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆ ՌԹՁՄԻՍՊՎԹՌՍՊԽՏԹՋՇԺՋԽՊՎԽԼՁՆՇԾՌԹԼՈՇՄԽՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՆԼՇԾՌԹԼՈՇՄԽՋՁՆ ՌՀԽ ՄՇՏ ՁՆՌԽՊՅԽԼՁԹՌԽ ԹՆԼ ՀՁԿՀ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԿՊՇՍՈՋ ՊԽՋՈԽԻՌՁՎ ԽՄՑ ԱՀԽ ՁՆԻՁԼԽՆԻԽ ՇԾ ՌԹՁՄ ԻՍՊՎԹՌՍՊԽ ԼՁԼ ՆՇՌ ԹՈՈԽԹՊՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼ

ԱՀԽՊԽՏԽՊԽ ՆՇ ՋՁԿՆՁԾՁԻԹՆՌ ԼՁԾԾԽՊԽՆԻԽՋ ՁՆ ԼԽՎԽՄՇՈՅԽՆՌԹՄ ԽՆԼՈՇՁՆՌ Ջ 

# ՁԽ ԫԣ ԼԽՎԽՄՇՈՅԽՆՌԹՄ ՋՌԹԿԽ ՀՁՆԼ ՄՁՅԺ ՄԽՆԿՌՀ ՆՇՊՅԹՄՁՒԽԼ ՀՁՆԼ ՄԽԿ ՄԽՆԿՌՀ ՁՆ ԹՆՑ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ՏՀԽՆ ԻՇՅՈԹՊԽԼ ՌՇ ՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ ԫՇ
ԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌՏԹՋՇԺՋԽՊՎԽԼԱՀԽՊԽՏԽՊԽՆՇԼՁԹԿՆՇՋՌՁԻԹԺՆՇՊՅԹՄՁՌՁԽՋՇԺՋԽՊՎԽԼԺՑՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄ ԽՐԹՅՁՆԹՌՁՇՆՇԾՌՀԽՌՀՑՊՇՁԼՋՇԾՌԹԼՈՇՄԽՋՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈ ՏՀԽՆԻՇՅՈԹՊԽԼՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՌՌՀԽ ՌԽՊՅՁՆԹՌՁՇՆՇԾՌՀԽԼԹՑՌԽՋՌ

ԡԹՑՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ
԰ԳԩԹՆԼԺՇԼՑՏԽՁԿՀՌՏԽՊԽՋՁԿՆՁԾՁԻԹՆՌՄՑՁՆԻՊԽԹՋԽԼ
ՈԺՑԹՆԼ
ՊԽՋՈԽԻՌՁՎԽՄՑՁՆՌՀԽՄՇՏԻՇՆԻԽՆՌՊԹՌՁՇՆԿՊՇՍՈՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽ ԿԹՌՁՎԽԻՇՆՌՊՇՄԥՇՏԽՎԽՊՌՀԽՊԽՏԽՊԽՆՇՋՁԿՆՁԾՁԻԹՆՌ ԽԾԾԽԻՌՋ ՇՆ ԿՊՇՏՌՀ ՈԹՊԹՅԽՌԽՊՋ ՁՆ ՌՀԽ ՁՆՌԽՊՅԽԼՁԹՌԽ ԹՆԼ ՀՁԿՀ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԿՊՇՍՈՋ ԻՇՅՈԹՊԽԼՌՇՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄԣՍՊՌՀԽՊՅՇՊԽ԰ԳԩԹՆԼԺՇԼՑՏԽՁԿՀՌՁՆՌՀԽՌՊԽԹՌԽԼԿՊՇՍՈՋ ՏԽՊԽԻՇՅՈԹՊԹԺՄԽՌՇՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՋՇՆԡԹՑ 
ԽԾԾԽԻՌՋՇՆԺՇԼՑՏԽՁԿՀՌՏԽՊԽՇԺՋԽՊՎԽԼԹՌԹՆԼՅԿԹՁԩՁՆՌՀԽՊԹՆԿԽԾՁՆԼԽՊՋՍԿԿԽՋՌՁՆԿՌՀԽՀՁԿՀ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀՁՋՋՌՍԼՑՏԹՋՋՍԾԾՁԻՁԽՆՌ    

       



   



 

  







 



    





 

  


# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

# ՇԾ  



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ԩԹՌԽՋՌԹԿԽ 
ԫԣ  ՌԹԼՈՇՄԽՋ ՏԽՊԽ ԽՐԻՄՍԼԽԼ ԾՊՇՅ ԹՆԹՄՑՋԽՋ ՏՁՌՀ ՊԽ ՋՈԽԻՌ ՌՇ ԺՇԼՑ ՏԽՁԿՀՌ ԰Գԩ ԹՆԼ ԥԩԩ ՁՆԻՄՍԼՁՆԿ ՆՇՊՅԹՄՁՒԽԼ ԥԩԩ ԺՑ ՌՀԽ ՊԽՎՁԽՏԽՊ ՌՀԽՋԽ ՁՆԻՄՍԼԽԼ  
   
  
 ԹՆԼ  

ՌԹԼՈՇՄԽՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՄՇՏՁՆՌԽՊՅԽԼՁԹՌԽԹՆԼՀՁԿ ՀՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ

ԱՀԽՋՌՍԼՑՅԽՌԹՄՄՇԾՌՀԽՈԽՊԾՇՊՅԹՆԻԽԹՆԼՎԹՄՁԼՁՌՑԻՊՁՌԽՊՁԹՏՁՌՀ ՌՀԽԾՇՄՄՇՏՁՆԿԽՐԻԽՈՌՁՇՆՋ

x ԱՀԽԠԳԾՇՊՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՇԾՌՀԽ ՄՇՏՌՊԽԹՌՅԽՆՌ  ԿՊՇՍՈ 
 ՅԿ ԹՁԩ ՏԹՋ
 ԽՐԻԽԽԼՁՆԿ ՌՀԽ ՈԽՊԾՇՊՅԹՆԻԽ ԻՊՁՌԽՊՁՇՆ ՇԾ ՅԹՁՆՌԹՁՆՁՆԿ Թ ԠԳ ՇԾ  

#  ՇՎԽՊ ՌՀԽ ԼԹՑ ՌԽՋՌ ԥՇՏԽՎԽՊՁՌՋՀՇՍՄԼԺԽՆՇՌԽԼՌՀԹՌՌՀՁՋԠԳՏԹՋԻԹՄԻՍՄԹՌԽԼԺՑՌՀԽ ՊԽՎՁԽՏԽՊՍՋՁՆԿԼԹՑՋԹՅՈՄԽՋՏՀՁԻՀ ՏԽՊԽՄՇՏԹՆԼՊԽՈՇՊՌԽԼՌՇԺԽԼՍԽՌՇՋԹՅՈՄՁՆԿՇՊԹՆԹՄՑՌՁԻԹՄԽՊՊՇՊԦԾՌՀԽԹԼԼՁՌՁՇՆԹՄՄՑԻՇՄՄԽԻՌԽԼԡԹՑ ՋԹՅՈՄԽՋ
ՌԽՋՌՋՑՋՌԽՅՅԹՁՆՌԹՁՆԽԼՁՆԹԺՋԽՆԻԽՇԾՌԹԼՈՇՄԽՋԹՊԽՍՋԽ ԼՁՆՄՁԽՍՇԾԡԹՑՋԹՅՈՄԽՋՌՀԽԠԳ ՇԾԼՇԽՋՆՇՌԽՐԻԽԽԼՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆ

x ԴԹՌԽՊՌԽՅՈԽՊԹՌՍՊԽՏԹՋՅԹՁՆՌԹՁՆԽԼՏՁՌՀՁՆչւԠՏՁՌՀՌՀԽԽՐ ԻԽՈՌՁՇՆՇԾԡԹՑՏՀԽՆՌԽՅՈԽՊԹՌՍՊԽՋ ՁՆՋՇՅԽՌԽՋՌՎԽՋՋԽՄՋՏԽՊԽԹՋՄՇՏԹՋւԠԬՆԡԹՑՋԹՆԼՌ ՀԽԼՁԾԾԽՊԽՆԻԽՁՆՌԽՅՈԽՊԹՌՍՊԽԺԽՌՏԽԽՆ ՌԽՋՌՎԽՋՋԽՄՋՏԹՋԹՆԼւԠՊԽՋՈԽԻՌՁՎԽՄՑԱՀԽՋԽՄՁՅՁՌՋԽՐԻԽ ԽԼՌՀԽՌԽՅՈԽՊԹՌՍՊԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆ ՇԾչւԠՏՁՌՀՁՆՌԽՊՊԽՈՄՁԻԹՌԽՁՆՌԽՊՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋՆՇՌԽՐԻԽԽԼՁՆԿւԠ

ԴՀՁՄԽ ՌՀԽՋԽ ԼԽՈԹՊՌՍՊԽՋ ԾՊՇՅ ԿՍՁԼԽՄՁՆԽ ՈԽՊԾՇՊՅԹՆԻԽ ԻՊՁՌԽՊՁԹ ԹՆԼ ԼԽՎՁԹՌՁՇՆՋ ԾՊՇՅ ԿՍՁԼԽՄՁՆԽ ՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ՏԽՊԽ ՇԺՋԽՊՎԽԼ ՌՀԽՑ ԼՁԼ ՆՇՌ ՋՍԾԾՁԻՁԽՆՌՄՑ ՁՅՈԹԻՌ ՌՀԽ ԹԺՁՄՁՌՑ ՌՇ ՁՆՌԽՊՈՊԽՌ ՌՀԽ ՊԽՋՍՄՌՋ ՇԾ ՌՀԽ ԹՋՋԹՑ ԱՀԽ ԹՋՋԹՑ

    



 

# ՌՀԽԢԡ԰ԭԱՁԽՊԱԽՋՌԬՊԼԽՊՊԽՉՍՁՊԽՅԽՆՌՋԾՇՊԹՆԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ 

 

     

 





# ԱԽՋՌՇՊԿԹՆՁՋՅԫԣՋՌԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ԱԽՋՌՇՊԿԹՆՁՋՅՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՇՈՌՁՇՆԹՄԫՇՌՊ ԽՈՇՊՌԽԼ ԱԽՋՌՌՑՈԽԾՄՇՏՌՀՊՇՍԿՀ    

       



   



 

  







 



    





 

  


# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

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# ԡՁԾԾԽՊԽՆԻԽ ԫԞ 

# ԫՇՌԹՈՈՄՁԻԹԺՄԽ

 

# Ԟ՟ՑԽՋՠՁՆԼՁԻԹՌԽՋԹՋՁԿՆՁԾՁԻԹՆՌԼՁԾԾԽՊԽՆԻԽԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆ ՌՇՌՀԽՆԽ ԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄՍՆՄԽՋՋ
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ԭՊՇՌԽԻՌՁՇՆԞԿԽՆԻՑ0ՋԬԠ԰ԭԭԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋ
ԣՊ ՇԿ ԹՆԼ ԬԢԠԡԱԽՋՌԤՍՁԼԽՄՁՆԽԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԡԽՎՁԹՌՁՇՆՋԾՊ ՇՅ
ԹՊԽ

 ԦՆԾՇՊՅԹՌՁՇՆՇՆՌՀԽՈԹՊԽՆՌԹՄԻԹՊԽԹՆԼՋՈԹՏՆՁՆԿՏԹՋՆՇՌՊԽՈՇՊՌ ԽԼ  ԱՀԽ ԾՄՇՏՌՀՊՇՍԿՀ ՊԹՌԽ ՇԾ  ՅԩՅՁՆ ԹՋ ԻԹՄԻՍՄԹՌԽԼ ԺՑ ՌՀԽ ՊԽ ՎՁԽՏԽՊ ԽՐԻԽԽԼՋ ՌՀԽ ԿՍՁԼԽՄՁՆԽ
ՊԽԻՇՅՅԽՆԼԹՌՁՇՆՇԾՅԩՅՁՆ

 ԱՀԽԠԳԾՇՊՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՇԾՌՀԽՄՇՏՌՊԽԹՌՅ ԽՆՌԿՊՇՍՈ 

# ՁԽ ՅԿԹՁԩ
ՏԹՋԽՐԻԽԽԼՁՆԿՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆՇԾՅԹՁՆՌԹՁՆՁՆԿԹ ԠԳՇԾ ? 

# ՇՎԽՊՌՀԽԼԹՑ
ՌԽՋՌ ԥՇՏԽՎԽՊ ՁՌ ՋՀՇՍՄԼ ԺԽ ՆՇՌԽԼՌՀԹՌ ՌՀՁՋԠԳՏԹՋ ԻԹՄԻՍՄԹՌԽԼ ԺՑ ՌՀԽ ՊԽՎՁԽՏԽՊ ՍՋՁՆԿ ԼԹՑ  ՋԹՅՈՄԽՋՏՀՁԻՀՏԽՊԽՄՇՏԹՆԼՊԽՈՇՊՌԽԼՌՇԺԽԼՍԽՌՇՋԹՅՈՄՁՆԿՇՊԹՆ ԹՄՑՌՁԻԹՄԽՊՊՇՊԦԾՌՀԽԹԼԼՁՌՁՇՆԹՄՄՑ ԻՇՄՄԽԻՌԽԼ ԡԹՑ  ՋԹՅՈՄԽՋ 
ՌԽՋՌ ՋՑՋՌԽՅՅԹՁՆՌԹՁՆԽԼ ՁՆ ԹԺՋԽՆԻԽ ՇԾ ՌԹԼՈՇՄԽՋ ՁՆ ՄՁԽՍ ՇԾ ԡԹՑ 
ՋԹՅՈՄԽՋՌՀԽԠԳՇԾԼՇԽՋՆՇՌԽՐԻԽԽԼՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊ ՁՇՆ    

       



   



 

  







 



    





 

  


# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

# ՇԾ  



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ  ԴԹՌԽՊ ՌԽՅՈԽՊԹՌՍՊԽ ՏԹՋ ՅԹՁՆՌԹՁՆԽԼ ՏՁՌՀՁՆ  չ ւԠ ՏՁՌՀ ՌՀԽ  ԽՐԻԽՈՌՁՇՆ ՇԾ ԡԹՑ  ՏՀԽՆ ՌԽՅՈԽՊԹՌՍՊԽՋՁՆՋՇՅԽՌԽՋՌՎԽՋՋԽՄՋՏԽՊԽԹՋՄՇՏԹՋւԠԬՆԡԹ ՑՋԹՆԼՌՀԽԼՁԾԾԽՊԽՆԻԽՁՆ ՌԽՅՈԽՊԹՌՍՊԽ ԺԽՌՏԽԽՆ ՌԽՋՌ ՎԽՋՋԽՄՋ ՏԹՋ  ԹՆԼ ւԠ ՊԽՋՈԽԻՌՁՎԽՄ Ց  ԱՀԽՋԽ ՄՁՅՁՌՋ ԽՐԻԽԽԼ ՌՀԽ ՌԽՅՈԽՊԹՌՍՊԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆՇԾչւԠՏՁՌՀՁՆՌԽՊՊԽՈՄՁԻ ԹՌԽՁՆՌԽՊՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋՆՇՌ ԽՐԻԽԽԼՁՆԿւԠ

ԴՀՁՄԽՌՀԽՋԽԼԽՈԹՊՌՍՊԽՋԾՊՇՅԿՍՁԼԽՄՁՆԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹԹՆԼԼԽՎՁԹՌՁՇՆՋԾՊՇՅԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ՏԽՊԽՇԺՋԽՊՎԽԼՌՀԽՑԼՁԼՆՇՌՋՍԾԾՁԻՁԽՆՌՄՑՁՅՈԹԻՌՌՀԽԹԺՁՄՁՌՑՌՇ ՁՆՌԽՊՈՊԽՌՌՀԽՊԽՋՍՄՌՋՇԾՌՀԽԹՋՋԹՑԱՀԽԹՋՋԹՑ

    



 

# ՌՀԽԢԡ԰ԭԱՁԽՊԱԽՋՌԬՊԼԽՊՊԽՉՍՁՊԽՅԽՆՌՋԾՇՊԹՆԞՅՈՀՁԺՁԹՆԪԽՌԹՅ ՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԬԠ԰ԭԭ ԤՍՁԼԽՄՁՆԽ

 



  

#  ԰ՁԿՆԽԼԹՆԼԼԹՌԽԼԫՇԡԹՌԹԠՇՆԾՁԼԽՆՌՁԹՄՁՌՑԤԩԭԹՆԼԮՍԹՄՁՌՑԞՋՋ ՍՊԹՆԻԽ ՋՌԹՌԽՅԽՆՌՋՏԽՊԽՈՊՇՎՁԼԽԼԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼՁՆԻՇՅՈՄՁԹՆԻ ԽՏՁՌՀՌՀԽ ԤՇՇԼԩԹԺՇՊԹՌՇՊՑԭՊԹԻՌՁԻԽ԰ՌԹՆԼԹՊԼՋԹՋՋՈԽԻՁԾՁԽԼՁՆԠԣԯՏ ՁՌՀՌՀԽ ԾՇՄՄՇՏՁՆԿԽՐԻԽՈՌՁՇՆՋՈԽՊՁՇԼՁԻԹՆԹՄՑՋԽՋՇԾՏԹՌԽՊԾՇՊՈՇՌԽՆՌՁԹՄ ԻՇՆՌԹՅՁՆԹՆՌՋՏԽՊԽ ՆՇՌՈԽՊԾՇՊՅԽԼԹԻԻՇՊԼՁՆԿՌՇԤԩԭՋՌԹՆԼԹՊԼՋԺՍՌՏԽՊԽՈԽՊԾՇՊՅԽԼՍՋ ՁՆԿԹԻԽՊՌՁԾՁԽԼ ՄԹԺՇՊԹՌՇՊՑԹՆԼՋՌԹՆԼԹՊԼԲ԰ԢԭԞԹՆԹՄՑՌՁԻԹՄՅԽՌՀՇԼՋՈՊԽՄՁՅՁՆԹՊՑ ԹՆԹՄՑՋԽՋՇԾ ՏԹՌԽՊՁՇԼՁԼԽԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽՆՇՌՈԽՊԾՇՊՅԽԼԹԻԻՇՊԼՁՆԿՌՇԤԩԭ



 

   



  

#    ԪԹՄԹՌՀՁՇՆԱԽԻՀՆՁԻԹՄ

    

  

# ԩՁՉՍՁԼ

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ՎՁԹՊԽՎԽՊՋԽ ՇՋՅՇՋՁՋԽՎԽՊՑՌՇԼԹՑՋԼՍՊՁՆԿՌՀԽՌԽՋՌ    

       



   



 

  







 



    





 

  


# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

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#    ԱԽԻՀՆՁԻԹՄՅԹՄԹՌՀՁՇՆՏԹՋՋՌՇՊԽԼՁՆԹՊԽԾՊՁԿԽՊԹՌՇՊԹՌ+ւԠԱՀԽՋՌՇԻՃ
ՋՇՄՍՌՁՇՆՏԹՋՀԽՄԼՍՆԼԽՊԹՅԺՁԽՆՌԻՇՆԼՁՌՁՇՆՋՈՊՇՌԽԻՌԽԼԾՊՇՅՄՁԿՀ ՌԹՆԼ ՅՁՐԽԼԻՇՆՌՁՆՍՇՍՋՄՑՍՋՁՆԿԹՌՇՈԼՇՏՆԽՄԽԻՌՊՁԻՅՁՐԽՊ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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

# ՀՇՏԽՎԽՊԹ ՄՁՋՌՇԾԹՄՄՇԾՌՀԽՆԽԻԽՋՋԹՊՑՈՊՇՌՇԻՇՄ ԼԽՎՁԹՌՁՇՆՋՌՇԹԻԻՇՅՅՇԼԹՌԽՌՀՁՋՋՈԽԻՁԽՋՁՋ ՊԽԻՇՅՅԽՆԼԽԼԾՇՊՁՆԻՄՍՋՁՇՆՁՆՌՀԽՋՌՍԼՑ ՊԽՈՇՊՌԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽ

ՈԽՊԾ ՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹՍՋԽԼՌՇՋՍՈՈՇՊՌՌՀԽ ՊԽՄՁԹԺՁՄՁՌՑՇԾՌՀԽՌԽՋՌԺԽՁԼԽՆՌՁԾՁԽԼ԰ՈԽԻՁԽՋՋԻՁԽՆՌՁԾՁԻՆԹՅԽ 

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

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

         !        "  #           ԴԽՊԽՈԹՊԽՆՌՋՅԹՁՆՌԹՁՆԽԼԹՋՁՆՀՇՍՋԽՋՌՇԻՃ!ԶԽՋԫՇՁՆԾՇՊՅԹՌՁՇՆՁՋՈՊՇՎՁԼԽԼՇՆՈԹՊԽՆՌՋԱՀԽՋՌՍԼՑԹՍՌՀՇՊՋՊԽՈՇՊՌՌՀԹՌՄԹՊՎԹԽՏԽՊԽՇԺՌԹՁՆԽԼԾՊՇՅԻՍՄՌՍՊԽՋՅԹՁՆՌԹՁՆԽԼԺՑԴՁՄԼՄՁԾԽԦՆՌԽՊՆԹՌՁՇՆԹՄԩՌԼԹՆԼՌՀԹՌԹՄՄՌԹԼՈՇՄԽՋՏԽՊԽԼԽՊՁՎԽԼԾՊՇՅԽԿԿՋՋՈԹՏՆԽԼՇՆՌՀԽՋԹՅԽԼԹՑ 

# ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՄԹՊՎԹԽ ՍՋԽԼ ՁՆ ՌՀԽ ԹՋՋԹՑԺԽԼԽՊՁՎԽԼԾՊՇՅՁՆ ՀՇՍՋԽԹԼՍՄՌՋԴԽՊԽՈԹՊԽՆՌԹՄԹԻԻՄՁՅԹՌՁՇՆԻՇՆԼՁՌՁՇՆՋՋԹՅԽԹՋԼԽԾՁՆՁՌՁՎԽՌԽՋՌ!ԫՇՌՊԽՈՇՊՌԽԼԞԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼԾՇՊՈԹՊԽՆՌԹՄԾՊՇԿՋ
ՁԾԹՈՈՄՁԻԹԺՄԽԫՇՌՊԽՈՇՊՌԽԼԡԽՌԹՁՄՋՇՆՈԹՊԽՆՌԹՄԾԽԽԼՁՆԿԫՇՌՊԽՈՇՊՌԽԼԡԽՌԹՁՄՋՇՆՈԹՊԽՆՌԹՄՀԽԹՄՌՀԫՇՌՊԽՈՇՊՌԽԼ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌՌՀԽԺԽՋՌ

- ՁՆԼՁՎՁԼՍԹՄՋՈԹՏՆՋՏՁՌՀԹՅՁՆՁՅՍՅՇԾ ՄԹՊՎԹԽՋՈԹՏՆԺԽԽՎԹՄՍԹՌԽԼՌՇՁԼԽՆՌՁԾՑ ՌՀԽԺԽՋՌՋՁՆԿՄԽՋՈԹՏՆԹՆԼՌՀԹՌՌՀԽՄԹՊՎԹԽ ՋԽՄԽԻՌԽԼԾՇՊՌԽՋՌՁՆԿՇՊՁԿՁՆԹՌԽԾՊՇՅՌՀԽԺԽՋՌ ՋՁՆԿՄԽՋՈԹՏՆ
ՁԽ 

# ՌՀԽՋՈԹՏՆՋԹՊԽՆՇՌԻՇ

ՅՁՐԽԼԫՍՅԺԽՊՇԾՋՈԹՏՆՋԽՎԹՄՍԹՌԽԼ
ՁԾԹՈՈՄՁԻԹԺՄԽԫՇՌՊԽՈՇՊՌԽԼԫՍՅԺԽՊՇԾԽԿԿՋՋԹՅՈՄԽԼՈԽՊՋՈԹՏՆԫՇՌՊԽՈՇՊՌԽԼԫԣՋՌԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽԼԽԾՁՆՁՌՁՎԽՋՌՍԼՑ ԺԽՁՆՁՌՁԹՌԽԼՏՁՌՀՄԹՊՎԹԽԹՌԫՁԽՍՏՃՇՇՈ - ԣԹԺԽՊ
ԫԣԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽ + 

#  ԼԹՑՋՈՇՋՌԾԽՊՌՁՄՁՒԹՌՁՇՆԞԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԼԹՑՋՈՇՋՌԾԽՊՌՁՄՁՒԹՌՁՇՆ
ԼՈԾԪԽԹՆՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾՊԽՈՇՊՌԽԼԫՇՌՊԽՈՇՊՌԽԼԯԹՆԿԽՇԾՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾՊԽՈՇՊՌԽԼԫՇՌՊԽՈՇՊՌԽԼԴԹՋՌՀԽՇՈՌՁՇՆԹՄՋՁՒԽՋԽՄԽԻՌՁՇՆՅԽՌՀՇԼՍՋԽԼ!ԫՇ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

         !        "  #           ԡԽՌԹՁՄՋՇՆՄԹՊՎԹՄՋԽՄԽԻՌՁՇՆԩԹՊՎԹՄՋԽՄԽԻՌՁՇՆՏԹՋՋՁՅՁՄԹՊՌՇԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋԡԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽՇԾԽԹԻՀՌԹԼՈՇՄԽՍՋԽԼՁՆՌՀԽՌԽՋՌՏԹՋԼԽՌԽՊՅՁՆԽԼԹՋԹԻԻՍՊԹՌԽՄՑԹՋՈՇՋՋՁԺՄԽՍՋՁՆԿՌՀԽԼՁՋՋԽԻՌՁՇՆՅՁԻՊՇՋԻՇՈԽԱՀԽՈՊՁՅԹՊՑԼԽՎԽՄՇՈՅԽՆՌԹՄԾԽԹՌՍՊԽԾՇՊՋԽՄԽԻՌՁՆԿՋՌԹԿԽՌԹԼՈՇՄԽՋՏԹՋՀՁՆԼՄՁՅԺՅՇՊՈՀՇՄՇԿՑԱԹԼՈՇՄԽՋՌՀԹՌՅԽՌՌՀԽՋՌԹԿԽԻՊՁՌԽՊՁԹԾՇՊՌՀԽՌԽՋՌՏԽՊԽՁՆԼՁՋԻՊՁՅՁՆԹՌԽՄՑՋԽՄԽԻՌԽԼԹՆԼՈՄԹԻԽԼՇՆԽՇՊՌՏՇԹՌԹՌՁՅԽՁՆՌՇՌՊԹՆՋԾԽՊՎԽՋՋԽՄՋՍՆՌՁՄԽԹԻՀՎԽՋՋԽՄԻՇՆՌԹՁՆԽԼՌԹԼՈՇՄԽՋԬՆԽՌՊԹՆՋԾԽՊՎԽՋՋԽՄՏԹՋՌՀԽՆՊԹՆԼՇՅՄՑԹՋՋՁԿՆԽԼՌՇԽԹԻՀՌԽՋՌԻՀԹՅԺԽՊԹՆԼՌՀԽՌԹԼՈՇՄԽՋՏԽՊԽԿԽՆՌՄՑՌՊԹՆՋԾԽՊՊԽԼՌՇՌՀԽՌԽՋՌԻՀԹՅԺԽՊՋԩՇԹԼՁՆԿՊԹՌԽ
ՊԽԹՊՁՆԿԼԽՆՋՁՌՑԫՇՌՋՈԽԻՁԾՁԽԼ 

# ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՊԽԹՊՁՆԿ ԼԽՆՋՁՌՑ ՄՇԹԼՁՆԿ ՊԹՌԽ ՆՇՌ ԽՐԻԽԽԼ ԹՈՈՊՇՐՁՅԹՌԽՄՑ  ՄԹՊՎԹԽԩ ԻՍՄՌՍՊՁՆԿ ՋՑՋՌԽՅ ԾՇՊ ԾՄՇՏ  ՌՀՊՇՍԿՀ ՋՑՋՌԽՅՋ ՇՊ  ՌԹԼՈՇՄԽՋԩ ՁՆ ՋՌԹՌՁԻ  ՊԽՆԽՏԹՄ ԽՐՈՇՋՍՊԽՋՑՋՌԽՅՋ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

       !  "        #  $           ԱՑՈԽՇԾԾՇՇԼ԰ԽՊԹԪՁԻՊՇՆն 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋ԰ԽՊԹԪՁԻՊՇՆ ն 

# ՌՀՊՇՍԿՀՇՍՌ

ՈՊԽ  ԽՐՈՇՋՍՊԽ
ԹԾՌԽՊԫԣՋՌԹԿԽԹՆԼ ԼՍՊՁՆԿՌՀԽԽՆՌՁՊԽ  ԼԼԽԾՁՆՁՌՁՎԽՋՌՍԼՑԦԾ ԹՆՇՌՀԽՊԼՁԽՌՁՋՍՋԽԼՌՀԽՋՌՍԼՑՊԽՈՇՊՌՋՀՇՍՄԼ ՈՊՇՎՁԼԽԹՆԹՄՑՋՁՋՇԾՁՇԼՁԼԽԻՇՆՌԽՆՌԹՆԼ ՈՇՌԽՆՌՁԹՄԻՇՆՌԹՅՁՆԹՆՌՋԹՆԼՌՀԽԼՁԽՌՋՀՇՍՄԼ ԼԽՅՇՆՋՌՊԹՌԽԽՉՍԹՄՈԽՊԾՇՊՅԹՆԻԽՌՇ԰ԽՊԹ

ԪՁԻՊՇՆ ն 

# ԰ՇՍՊԻԽՇԾԾՇՇԼ԰ԽՊԹԫՇՊՌՀԞՅԽՊՁԻԹԪՇՆՌԿՇՅԽՊՑՎՁՄՄԽԭԞԦՇԼՁԼԽԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆԼՁԽՌ
ՁԾՃՆՇՏՆԫՇՌՊԽՈՇՊՌԽԼԣՊԽՉՍԽՆԻՑՇԾԾԽԽԼՁՆԿՌՁՅԽՋԼԹՑԱԹԼՈՇՄԽՋՏԽՊԽԾԽԼՌՏՁԻԽՇՆՌՀԽԾՁՊՋՌԼԹՑԹՆԼՇՆԻԽՇՆՌՀԽՄԹՋՌԼԹՑՇԾՌՀԽՌԽՋՌ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾԽԽԼՁՆԿՇԻԻՍՊԹՌ ՄԽԹՋՌՌՏՁԻԽՈԽՊԼԹՑԡԽՌԹՁՄՋՇՆԾԽԽԼՁՆԿՊԽԿՁՅԽԣԽԽԼՁՆԿՊԽԿՁՅԽՏԹՋՌՀԽՋԹՅԽԹՋԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋԣՇՇԼՊԹՌՁՇՆՋԼՍՊՁՆԿՌՀԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼՋՌԹՊՌԽԼԹՌՅԿՄԹՊՎԹԼԹՑԹՆԼՁՆԻՊԽԹՋԽԼԺՑՁՆԻՊԽՅԽՆՌՋՇԾՅԿՍՆՌՁՄՊԽԹԻՀՁՆԿՅԿՄԹՊՎԹԼԹՑԼՍՊՁՆԿՌՀԽԾՁՆԹՄՏԽԽՃՇԾՌԽՋՌՁՆԿ 

# ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌԾՇՇԼՊԹՌՁՇՆՋԼՍՊՁՆԿ

ՌՀԽ ՈՊԽ ԽՐՈՇՋՍՊԽՈԽՊՁՇԼԺԽՁՆԻՊԽԹՋԽԼԹՄՇՆԿ ՏՁՌՀՄԹՊՎԹՄԿՊՇՏՌՀՌՇԹՈՈՊՇՐՁՅԹՌԽՄՑ ՅԿՄԹՊՎԹԼԹՑԺՑՌԽՋՌՁՆՁՌՁԹՌՁՇՆԢԭԞԹՆԼ ԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌԾՇՇԼՊԹՌՁՇՆՋ ՁՆԻՊԽԹՋԽԾՊՇՅՅԿՄԹՊՎԹԼԹՑԹՌՌԽՋՌ ՁՆՁՌՁԹՌՁՇՆ
԰ՌՍԼՑԡԹՑ ՌՇ ՅԿՄԹՊՎԹԼԹՑՁՆՌՀԽՄԹՋՌՏԽԽՃՇԾ ՌՀԽՌԽՋՌ ԰ՌՍԼՑԡԹՑ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ՁԾԹՈՈՄՁԻԹԺՄԽԠՇՆՌՁՆՍՇՍՋԾՄՇՏԼՁՄՍՌԽՊ 

# ԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊՋՇՊ ԻՇՆՌՁՆՍՇՍՋԾՄՇՏՋԽՊՁԹՄԼՁՄՍՌԽՊՋԹՊԽ ՊԽԻՇՅՅԽՆԼԽԼ ԞԼԼՁՌՁՇՆԹՄԿՍՁԼԹՆԻԽԾՇՊԹՉՍԹՌՁԻՌԽՋՌԼԽՋՁԿՆՁՋՄՇԻԹՌԽԼՁՆԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ԰ՈԽԻՁԹՄԠՇՆՋՁԼԽՊԹՌՁՇՆՋԾՇՊԠՇՆԼՍԻՌՁՆԿԞՉՍԹՌՁԻԩԹԺՇՊԹՌՇՊՑ԰ՌՍԼՁԽՋ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

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# ԯԽԻՇՅՅԽՆԼԽԼԾՄՇՏՌՀՊՇՍԿՀՊԹՌԽՁՋ ՅԩՅՁՆ
ԻՇՅՈՄԽՌԽՎՇՄՍՅԽՊԽՈՄԹԻԽՅԽՆՌ ԻԹԽՎԽՊՑՀՊՋ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

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ՁԾԹՈՈՄՁԻԹԺՄԽԣՄՍՁԼՅԽՌԽՊՁՆԿՈՍՅՈՋ
ԣՄՍՁԼԪԽՌԽՊՁՆԿԦՆԻ԰ՑՇՋՋԽՌԫԶՏԽՊԽՍՋԽԼՌՇԼԽՄՁՎԽՊՎՇՄՍՅԽՋՇԾԹՋՁՆԿՄԽՌԽՋՌՋՍԺՋՌԹՆԻԽՋՌՇԻՃՋՇՄՍՌՁՇՆՌՇՅՁՐՁՆԿԻՀԹՅԺԽՊՋՁՆԼՁՋԻՊՁՅՁՆԹՌԽՄՑԹՋՋՁԿՆԽԼՌՇԽԹԻՀՌՊԽԹՌՅԽՆՌԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՏԹՋԼՁՄՍՌԽԼՏՁՌՀՏԽՄՄՏԹՌԽՊՁՆՌՀԽՅՁՐՁՆԿԻՀԹՅԺԽՊՋՁՆՇՊԼԽՊՌՇՇԺՌԹՁՆՌՀԽԼԽՋՁՊԽԼՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՈՊՁՇՊՌՇԼԽՄՁՎԽՊՑՌՇՌՀԽՌԽՋՌԻՀԹՅԺԽՊԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՏԹՋԼՁՄՍՌԽԼՏՁՌՀՏԽՄՄՏԹՌԽՊՁՆՌՀԽՅՁՐՁՆԿԻՀԹՅԺԽՊՋՁՆՇՊԼԽՊՌՇՇԺՌԹՁՆՌՀԽԼԽՋՁՊԽԼՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՈՊՁՇՊՌՇԼԽՄՁՎԽՊՑՌՇՌՀԽՌԽՋՌԻՀԹՅԺԽՊՋԞԾՌԽՊՅՁՐՁՆԿՌՀԽՌԽՋՌՏԹՌԽՊՁՆԽԹԻՀՅՁՐՁՆԿԻՀԹՅԺԽՊՏԹՋՈՍՅՈԽԼՁՆՌՇՌՀԽԹՈՈՊՇՈՊՁԹՌԽՊԽՈՄՁԻԹՌԽՌԽՋՌԻՀԹՅԺԽՊՍՋՁՆԿԹՈԽՊՁՋՌԹՄՌՁԻՈՍՅՈ
ԠՇՄԽԭԹՊՅԽՊԦՆՋՌՊՍՅԽՆՌԠՇՅՈԹՆՑԠՀՁԻԹՇԦԩԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆԹՆԼՌՀԽՌԽՋՌՋՇՄՍՌՁՇՆՋՏԽՊԽՋՌՁՊՊԽԼԻՇՆՌՁՆՍՇՍՋՄՑԹՆԼՏԽՊԽՈՊՇՌԽԻՌԽԼԾՊՇՅՄՁԿՀՌԱՀԽՈՊՇՈՇՊՌՁՇՆՇԾՌԽՋՌՏԹՌԽՊՌՀԹՌՏԹՋՈՍՅՈԽԼՁՆՌՇԽԹԻՀՊԽՈՄՁԻԹՌԽՌԽՋՌԻՀԹՅԺԽՊՏԹՋԻՀԽԻՃԽԼՈՊՁՇՊՌՇՌՀԽՌԽՋՌԹՆԼԹՈՈՊՇՐՁՅԹՌԽՄՑՏԽԽՃՄՑԼՍՊՁՆԿՌՀԽՌԽՋՌՌՇԽՆՋՍՊԽՌՀԹՌԾՄՇՏՊԹՌԽՋՎԹՊՁԽԼԺՑՆՇՅՇՊԽՌՀԹՆչՇԾՌՀԽՅԽԹՆԾՄՇՏՊԹՌԽԾՇՊՌՀԽՊԽՈՄՁԻԹՌԽՋ 

# ԯԽԻՇՅՅԽՆԼԽԼՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊԾՄՇՏ ՌՀՊՇՍԿՀՋՑՋՌԽՅՋԪՁՐՁՆԿԻՀԹՅԺԽՊՁՋ ՊԽԻՇՅՅԽՆԼԽԼԺՍՌՆՇՌՊԽՉՍՁՊԽԼ

ԞԽՊԹՌՁՇՆՁ ՋՆՇՌՊԽԻՇՅՅԽՆԼԽԼԾՇՊ
ՅՁՐՁՆԿ ԞԼԽՅՇՆՋՌՊԹՌՁՇՆՌՀԹՌՌՀԽՌԽՋՌ

ՋՇՄՍՌ ՁՇՆՁՋԻՇՅՈՄԽՌԽՄՑՅՁՐԽԼԺԽԾՇՊԽ ՁՆՌՊՇԼՍԻԽԼՁՆՌՇՌՀԽՌԽՋՌՋՑՋՌԽՅՁՋ ՊԽԻՇՅՅԽՆԼԽԼ ԱՀԽՊԽԻՇՅՅԽՆԼԽԼԾՄՇՏ ՋՈՄՁՌՌՁՆԿԹԻԻՍՊԹԻՑՁՋՏՁՌՀՁՆ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ՁԾԹՈՈՄՁԻԹԺՄԽԫԞ 

# ԦԾՋՌԹՌՁԻՊԽՆԽՏԹՄՁՋՍՋԽԼԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՀՊՊԽՆԽՏԹՄՊԽՆԽՏԹՄ ՈԽՊՁՇԼՁՋՊԽԻՇՅՅԽՆԼԽԼՆՇՌՌՇԽՐԻԽԽԼ

ՀՇՍՊՋԞԽՊԹՌՁՇՆ!ԫՇ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՅԹՁՆՌԹՁՆՁՆԿԼՁՋՋՇՄՎԽԼ ՇՐՑԿԽՆԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԹՁՊ ՋԹՌՍՊԹՌՁՇՆ ՅԿԩ ԞԽՊԹՌՁՇՆՅԹՑԺԽ ՅԹՁՆՌԹՁՆԽԼՌՀՊՇՍԿՀԺՍԺԺՄԽՊՋԦՌՁՋ ՊԽԻՇՅՅԽՆԼԽԼՌՇՋԽՌԺՍԺԺՄԽՊՋԹՌՄԽՎԽՄՋ ՌՀԹՌԼՇՆՇՌԻԹՍՋԽՋՌՊԽՋՋՇՆՌՀԽՌԹԼՈՇՄԽՋ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

       !  "        #  $           ԰ՇՍՊԻԽՇԾԼՁՄՍՌՁՇՆՏԹՌԽՊԴԽՄՄՏԹՌԽՊԱՀԽՏԽՄՄՏԹՌԽՊՈԹՋՋԽԼՌՀՊՇՍԿՀԹՋԹՆԼԾՁՄՌԽՊՌՇՊԽՅՇՎԽՈԹՊՌՁԻՄԽՋԿՊԽԹՌԽՊՌՀԹՆԹՈՈՊՇՐՁՅԹՌԽՄՑսՅԹՆԼՈՍՅՈԽԼՁՆՌՇԹԩՋՌՇՊԹԿԽՌԹՆՃՏՀԽՊԽՌՀԽՏԹՌԽՊՏԹՋԹԽՊԹՌԽԼՏՁՌՀՋՈՊԹՑՆՇՒՒՄԽՋԭՊՁՇՊՌՇՍՋԽՌՀԽՏԹՌԽՊՏԹՋԾՁՄՌԽՊԽԼՌՇսՅՌՇՊԽՅՇՎԽԾՁՆԽՈԹՊՌՁԻՄԽՋԱՀԽՏԽՄՄՏԹՌԽՊՁՋԻՀԹՊԹԻՌԽՊՁՒԽԼԹՋՅՇԼԽՊԹՌԽՄՑՀԹՊԼՏԹՌԽՊ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՆԹՌՍՊԹՄՇՊՊԽԻՇՆՋՌՁՌՍՌԽԼ ՏԹՌԽՊՁՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՆԹՌՍՊԹՄ ՏԹՌԽՊԺԽՋՌԽՊՁՄՁՒԽԼՏՁՌՀԲԳԹՆԼՌԽՋՌԽԼԾՇՊ ՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊ ՈՇՋՋՁԺՄԽԻՇՆՌԹՅՁՆԹՆՌՋՁՆԻՄՍԼՁՆԿՃՆՇՏՆ ՋՍԺՋՌՊԹՌԽՋՇԾՌՀԽՁՇԼՁՆԽՌՊԹՆՋՈՇՊՌԽՊՇԾՌՀԽ ՌՀՑՊՇՁԼԿՄԹՆԼ
ԽԿ 

# ԾՄՍՇՊՁԼԽԻՀՄՇՊԹՌԽ ՈԽՊԻՀՄՇՊԹՌԽԬԢԠԡԹԻԻԽՈՌՋԹՆՑՏԹՌԽՊՁՆ ՏՀՁԻՀՌՀԽՌԽՋՌՋՈԽԻՁԽՋՋՀՇՏԻՇՆՌՊՇՄ ՋՍՊՎՁՎԹՄԹՌՄԽԹՋՌԹՋԿՇՇԼԹՋՁՆԼՁԻԹՌԽԼՁՆ ՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԴԹՋԼՁՄՍՌՁՇՆՏԹՌԽՊԹՆԹՄՑՒԽԼԾՇՊՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊԻՇՆՌԹՅՁՆԹՆՌՋ!ԶԽՋ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

       !  "        #  $           ԦՇԼՁԼԽՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՆՏԹՌԽՊ!ԫՇ 

# ԦԾՊԽԻՇՆՋՌՁՌՍՌԽԼՏԹՌԽՊՁՋՍՋԽԼՇՊՁԾ ԺԹԻՃԿՊՇՍՆԼՄԽՎԽՄՋՇԾՁՇԼՁԼԽՁՆՆԹՌՍՊԹՄ ՏԹՌԽՊԹՊԽՄԽ ՋՋՌՀԹՆսԿԩՁՇԼՁԼԽ ՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՋՊԽԻՇՅՅԽՆԼԽԼԱՀՁՋ ՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՋՁՆԹԼԼՁՌՁՇՆՌՇՌՀԽ ՊԽԻՇՅՅԽՆԼԽԼԼՁԽՌԹՊՑՋՇՍՊԻԽՇԾՁՇԼՁԼԽ ԽԿՁՆ԰ԽՊԹԪՁԻՊՇՆԱԽՋՌՎԽՋՋԽՄՌՑՈԽՅԹՌԽՊՁԹՄՋԩԿՄԹՋՋԹՉՍԹՊՁԹ 

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

# ԹՄՄ ԿՄԹՋՋ ՎԽՋՋԽՄՋՇՊԿՄԹՋՋՎԽՋՋԽՄՋՏՁՌՀՋՌԹՁՆՄԽՋՋՋՌԽԽՄ ԾՊԹՅԽՋԹՊԽԹԻԻԽՈՌԹԺՄԽԽՐԹՅՈՄԽՋԱԽՋՌՎԽՋՋԽՄՋՁՒԽՐՐԻՅԱՀԽԼԽՈՌՀՇԾՌՀԽՌԽՋՌՏԹՌԽՊՁՆԹՊԽՈՊԽՋԽՆՌԹՌՁՎԽԻՀԹՅԺԽՊՏԹՋԻՅԣՁՄՄՎՇՄՍՅԽԩԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋՇՆԽՐՈՇՋՍՊԽՋՑՋՌԽՅ

   % & '       (   )      *                '     +                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

                            !           ԥԹՊԼՆԽՋՋ
ՅԿԩԹՋԠԹԠԬ  ԴԽԽՃՄՑԻՇՆՌՊՇՄԹՆԼՌՀԽՀՁԿՀԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՌՊԽԹՌՅԽՆՌԿՊՇՍՈՇՆՄՑ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՀԹՊԼՆԽՋՋՌՇ ՅԿԩԹՋԠԹԠԬ Ոԥ  ԴԽԽՃՄՑ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՈԥչՁՆՌԽՊ ՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊ  ՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋ ՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼԡՁՋՋՇՄՎԽԼՇՐՑԿԽՆ
ՅԿԩ   ԴԽԽՃՄՑ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆ ԡԬ ՅԿԩ
 ԹՁՊՋԹՌՍՊԹՌՁՇՆ ԬԢԠԡՊԽԻՇՅՅԽՆԼՋԡԬԻՇՆԻԽՆՌՊԹՌՁՇՆ
ՅԿԩ ԹՁՊՋԹՌՍՊԹՌՁՇՆ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

                            !           ԱԽՅՈԽՊԹՌՍՊԽ
ոԠ ԴԽԽՃՄՑԞԼԼՁՌՁՇՆԹՄՌԽՅՈԽՊԹՌՍՊԽՅԽԹՋՍՊԽՅԽՆՌՋՏԽՊԽՌԹՃԽՆԺԽՌՏԽԽՆԡԹՑՋԹՆԼՌՇԽՆՋՍՊԽՌՀԹՌՌԽՅՈԽՊԹՌՍՊԽՋՏԽՊԽՏՁՌՀՁՆՌՀԽՌԹՊԿԽՌՊԹՆԿԽԼՍԽՌՇԹԻՇՆՌՊՇՄՄԽՊՅԹՄԾՍՆԻՌՁՇՆՇՆԡԹՑԱԽՅՈԽՊԹՌՍՊԽԹՄՋՇՏԹՋՅՇՆՁՌՇՊԽԼԻՇՆՌՁՆՍՇՍՋՄՑՁՆՇՆԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՊԽՈՄՁԻԹՌԽԼՍՊՁՆԿՌՀԽԹՋՋԹՑ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌԽՅՈԽՊԹՌՍՊԽչ Շ 

# Ԡ

ՁՆՌԽՊ ՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊՌՊԽԹՌՅԽՆՌ ԼՁԾԾԽՊԽՆՌՁԹՄՋՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼ Շ 

# ԠԦՇԼՁԼԽսԿԩ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՉՍԹՌՁԻՁՇԼՁԼԽՊԹՆԿԽ

 - սԿԩ
ՋՍՈՈՄԽՅԽՆՌԹՄՁՇԼՁԼԽ ՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼսԿԩ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

                            !           ԞՅՅՇՆՁԹԫՇՌՊԽՈՇՊՌԽԼ 

# ԤԽՆԽՊԹՄՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋԾՇՊԾՊԽՉՍԽՆԻՑ ՇԾՅԽԹՋՍՊԽՅԽՆՌՋԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌ ՏԹՌԽՊՉՍԹՄՁՌՑՈԹՊԹՅԽՌԽՊՋԺԽՅԽԹՋՍՊԽԼՁՆ ԹԻՇՆՌՊՇՄԹՆԼԹՌՇՆԽՌԽՋՌՁՌԽՅ ԻՇՆԻԽՆՌՊԹՌՁՇՆԹՌՄԽԹՋՌՏԽԽՃՄՑԦՆՋՌԹՌՁԻ ՊԽՆԽՏԹՄՋՑՋՌԽՅՋՏԹՌԽՊՉՍԹՄՁՌՑ ՈԹՊԹՅԽՌԽՊՋՁՆԻՄՍԼՁՆԿԹՅՅՇՆՁԹՋՀՇՍՄԼ ԺԽՅԽԹՋՍՊԽԼՂՍՋՌՈՊՁՇՊՌՇՊԽՆԽՏԹՄԦՆ ԹԼԼՁՌՁՇՆԢԭԞՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌԡԬԺԽ ՅԽԹՋՍՊԽԼԹՌԽԹԻՀԻՇՆԻԽՆՌՊԹՌՁՇՆԹՌՄԽԹՋՌ ՏԽԽՃՄՑԹՆԼՌՀԹՌՌԽՅՈԽՊԹՌՍՊԽԺԽ ՅԽԹՋՍՊԽԼԻՇՆՌՁՆՍՇՍՋՄՑ ԬԢԠԡ ՊԽԻՇՅՅԽՆԼՋՌՀԹՌ ԡԬԹՆԼՌԽՅՈԽՊԹՌՍՊԽ ԺԽՅԽԹՋՍՊԽԼԹՌՄԽԹՋՌՏԽԽՃՄՑԹՆԼՌՀԹՌՈԥ ԹՆԼՀԹՊԼՆԽՋՋԺԽՅԽԹՋՍՊԽԼԹՌՄԽԹՋՌԹՌ ՌՀԽԺԽԿՁՆՆՁՆԿԹՆԼԽՆԼՇԾՌՀԽՌԽՋՌԣՄՍՇՊՁԼԽՅԿԩ
ՏԽՄՄՏԹՌԽՊԭԽՊԻՀՄՇՊԹՌԽԫՇՌԯԽՈՇՊՌԽԼԠՀՄՇՊԹՌԽԫՇՌԯԽՈՇՊՌԽԼԞՄՃԹՄՁՆՁՌՑ
ՅԿԩԹՋԠԹԠԬ  ԴԽԽՃՄՑԻՇՆՌՊՇՄԹՆԼՌՀԽՀՁԿՀԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՌՊԽԹՌՅԽՆՌԿՊՇՍՈՇՆՄՑԠՇՆԼՍԻՌՁՎՁՌՑ
ս԰ԻՅ ԬՆԡԹՑԹԻՇՆՌՊՇՄՄԽՊՅԹՄԾՍՆԻՌՁՇՆԽԼԹՆԼՌՀԽՌԽՅՈԽՊԹՌՍՊԽՋՁՆՋԽՎԽՊԹՄՌԽՋՌԻՀԹՅԺԽՊՋՏԽՊԽՋՄՁԿՀՌՄՑԺԽՄՇՏՌՀԽԼԽՋՁՊԽԼՊԹՆԿԽՊԹՆԿՁՆԿԾՊՇՅՌՇոԠԱՀԽԻՇՆՌՊՇՄՄԽՊՏԹՋԻՇՊՊԽԻՌԽԼՇՆՌՀԽՋԹՅԽԼԹՑԹՆԼՌԽՅՈԽՊԹՌՍՊԽՋՏԽՊԽԻՇՆԾՁՊՅԽԼԱՀՁՋՄԽՋՋՌՀԹՆոԠՎԹՊՁԹՆԻԽՀԹԼ ՆՇԹՈՈԹՊԽՆՌԹԼՎԽՊՋԽՁՅՈԹԻՌՇՆՌՀԽՌԹԼՈՇՄԽՋ ԯԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼԹՋԹՎԽՊԹԿԽՇԾՅՁՆՁՅՍՅԹՆԼՅԹՐՁՅՍՅՎԹՄՍԽՋ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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'     (   %  &        )  *           ԴԹՋԹՊԹՆԿԽԾՁՆԼԽՊԻՇՆԼՍԻՌԽԼ!ԶԽՋԦԾՑԽՋՏՀԹՌՏԹՋՌՀԽՅԽՌՀՇԼԾՇՊԼԽՌԽՊՅՁՆՁՆԿՌՀԽՀՁԿՀԽՋՌՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽՊԹՆԿԽԾՁՆԼԽՊ!ԫՇՅՁՆԹՄՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽՋԽՄԽԻՌԽԼԺՑՌՀԽ԰ՈՇՆՋՇՊԺԹՋԽԼՇՆՌՀԽԹԻՍՌԽՀՊԩԠԾՇՊԵԽՆՇՈՍՋՇԾՅԿԹՁԩԫՇՅՁՆԹՄՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՁՆՌՀԽՊԹՆԿԽԾՁՆԼԽՊԹՆԼՅԿԹՁԩ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌ ՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽ ՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾՌՀԽՌԽՋՌ ԻՇՅՈՇՍՆԼՅԿԩՇՊ ԼԽՅՇՆՋՌՊԹՌԽՋԹԼԽՉՍԹՌԽԽՎՁԼԽՆԻԽ ՇԾՌՇՐՁԻՁՌՑ
ԽԿ  ՅՇՊՌԹՄՁՌՑ ՏՀՁԻՀԽՎԽՊԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌ԰ՈԽԻՁԽՋ 

# ԵԽՆՇՈՍՋՄԹԽՎՁՋԩՁԾԽՋՌԹԿԽԫԣՋՌԹԿԽԱԽՋՌԼՍՊԹՌՁՇՆԼԹՑՋ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋԴԹՌԽՊՌԽՅՈԽՊԹՌՍՊԽՋՏԽՊԽՏՁՌՀՁՆՌՀԽչոԠՊԹՆԿԽԽՋՌԹԺՄՁՋՀԽԼԾՇՊՌՀԽՌԽՋՌԡՁՋՋՇՄՎԽԼՇՐՑԿԽՆԻՇՆԻԽՆՌՊԹՌՁՇՆՋՊԽՅԹՁՆԽԼ ՇԾՋԹՌՍՊԹՌՁՇՆ
ՅԿԩԪԽԹՋՍՊԽՅԽՆՌՋՇԾՈԥՊԹՆԿԽԼԾՊՇՅՌՇԼՍՊՁՆԿՌՀԽՌԽՋՌԪԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽԹՆԼՅԿԹՁԩՏՀՁԻՀՊԽՈՊԽՋԽՆՌԹՆԼՇԾՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋՊԽՋՈԽԻՌՁՎԽՄՑ԰ԹՅՈՄԽՋՇԾՌՀԽՅԿԹՁԩՌԽՋՌՋՇՄՍՌՁՇՆԻՇՄՄԽԻՌԽԼԼՍՊՁՆԿՌՀԽՌԽՋՌՀԹԼՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՌՀԹՌՊԹՆԿԽԼԾՊՇՅՌՇՇԾՌՀԽՆՇՅՁՆԹՄ԰ԹՅՈՄԽՋԻՇՄՄԽԻՌԽԼԼՍՊՁՆԿՌՀԽՌԽՋՌԾՊՇՅՌՀԽԾՁՎԽՀՁԿՀԽՊԻՇՆԻԽՆՌՊԹՌՁՇՆՌԽՋՌՋՇՄՍՌՁՇՆՋՀԹԼՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՌՀԹՌՊԹՆԿԽԼՇԾՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋԦՌՁՋԺԽՄՁԽՎԽԼՌՀԹՌՌՀՁՋՎԹՊՁԹԺՁՄՁՌՑՏԹՋԼՍԽՌՇԹԼՋՇՊՈՌՁՇՆՇԾՌՀԽՅԹՌԽՊՁԹՄՌՇԺՁՇՄՇԿՁԻԹՄՅԹՌԽՊՁԹՄՁՆՌՀԽՌԽՋՌԻՀԹՅԺԽՊԹՌՌՀԽՅԿԹՁԩԻՇՆԻԽՆՌՊԹՌՁՇՆԯԽՈՇՊՌԽԼՎԹՄՍԽՋԾՊՇՅՌՀՁՋՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՏԽՊԽԽՐՌՊԹՈՇՄԹՌԽԼԺԽԻԹՍՋԽՌԽՋՌՅԽԹՋՍՊԽՅԽՆՌՋՏԽՊԽՄԽՋՋՌՀԹՆՌՀԽՄՁՅՁՌՇԾՉՍԹՆՌՁՌԹՌՁՇՆՌՀՊՇՍԿՀՇՍՌՌՀԽՋՌՍԼՑԱԹԼՈՇՄԽՋՍՊՎՁՎԹՄՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԿՊՇՍՈՏԹՋԱԹԼՈՇՄԽՋՍՊՎՁՎԹՄՁՆՌՀԽԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԹՋԹՆԼՊԽՋՈԽԻՌՁՎԽՄՑԥՇՏԽՎԽՊՌՀԽՊԽՏԽՊԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼՋՍԺՄԽՌՀԹՄՋՁԿՆՋՇԾՌՇՐՁԻՁՌՑ 

# ՁԽՋՍՊԾԹԻՁՆԿԹՆԼՋՏՁՅՅՁՆԿՁՆԻՁՊԻՄԽՋԹՋՏԽՄՄԹՋՊԽԼՍԻՌՁՇՆՋՁՆԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽՏԽՌՏԽՁԿՀՌՌՇՌԹՄՄԽՆԿՌՀ԰ԳԩԹՆԼԥԩԩԹՅՇՆԿՌԹԼՈՇՄԽՋՁՆՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԴԽՌՏԽՁԿՀՌՏԹՋՌՀԽՅՇՋՌՋԽՆՋՁՌՁՎԽԽՆԼՈՇՁՆՌՅԽԹՋՍՊԽԼՏՁՌՀՊԽԼՍԻՌՁՇՆՋՁՆՏԽՌՏԽՁԿՀՌԹՅՇՆԿՌԹԼՈՇՄԽՋՁՆՌՀԽԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԬՎԽՊԹՄՄՌՀԽՊԽՏԹՋԹՆԹՈՈԹՊԽՆՌՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՇՆԿՊՇՏՌՀԹՆԼԼԽՎԽՄՇՈՅԽՆՌՁՆՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈԦՆՌՀԽԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՅԽԹՆՏԽՌՏԽՁԿՀՌՏԹՋՊԽԼՍԻԽԼԺՑՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽԻՇՆՌՊՇՄԴՀՁՄԽՌՀԽՊԽՏԽՊԽՆՇԻՇՊՊԽՋՈՇՆԼՁՆԿԼԽԻՊԽԹՋԽՋՁՆՌՀԽՄԽՆԿՌՀՈԹՊԹՅԽՌԽՊՋՌՀԽԽԾԾԽԻՌՇՆՏԽՁԿՀՌՁՆՌՀԽԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԹՋԻՇՆՋՁԼԽՊԽԼՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՌՀԽ ԼՍՊԹՌՁՇՆ ՇԾ ՌՀԽ ԼԽԾՁՆՁՌՁՎԽՌԽՋՌԺԽԼԹՑՋԪԽՌՀՇԼԾՇՊՋԽՄԽԻՌՁՆԿՌՀԽՀՁԿՀԽՋՌՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌԯԽՋՍՄՌՋՇԾՌՀԽՊԹՆԿԽԾՁՆԼԽՊԹՆԼԹԻՍՌԽՀՊԩԠՎԹՄՍԽԾՇՊԵԽՆՇՈՍՋՇԾՅԿԹՁԩ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾ ՌՀԽՌԽՋՌԻՇՅՈՇՍՆԼՅԿԩՇՊ ԼԽՅՇՆՋՌՊԹՌԽՋԹԼԽՉՍԹՌԽԽՎՁԼԽՆԻԽՇԾՌՇՐՁԻՁՌՑ

ԽԿ ՅՇՊՌԹՄՁՌՑՏՀՁԻՀԽՎԽՊ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌԯԽԾԽՊԽՆԻԽՋՌՍԼՑԻՁՌԹՌՁՇՆ
ՁԾԹՈՈՄՁԻԹԺՄԽԯԹՆԿԽԾՁՆԼԽՊՋՌՍԼՑԪԯԦԡ԰ԽՈԹՊԹՌՁՇՆՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋԹՆԼ 

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

# ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՇԾ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԹՆԼԹԻՇՆՌՊՇՄՈՄՍՋՋՇՄՎԽՆՌ ԻՇՆՌՊՇՄՁԾԹՈՈՊՇՈՊՁԹՌԽ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ԩԬԮՅԿԹՁԩ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՇՊԻՀԽՅՁԻԹՄՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽՄՇՏՌՀԽԩԬԮԹՆԹՄՑՋԽՋԺԽ ԻՇՆԼՍԻՌԽԼՇՆՌՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋԩԽՎԽՄՇԾԼԽՌԽԻՌՁՇՆ
ԩԬԡԫՇՌՊԽՈՇՊՌԽԼ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ԡԹՑՋԹՆԼԹՆԼՇՆԡԹՑՋԹՆԼԡԹՑՋԹՅՈՄԽՋԾՊՇՅՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՏԽՊԽՋՍՋՈԽԻՌ 

# ՁԽՄՇՏԱՀԽՄՇՏԹՆԹՄՑՌՁԻԹՄՊԽՋՍՄՌՋՏԽՊԽԺԽՄՁԽՎԽԼՌՇԺԽԼՍԽՌՇՋԹՅՈՄՁՆԿՇՊԹՆԹՄՑՌՁԻԹՄԽՊՊՇՊԞԼԼՁՌՁՇՆԹՄՋԹՅՈՄԽՋՏԽՊԽԻՇՄՄԽԻՌԽԼԾՊՇՅՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՌՏՇԼԹՑՋՄԹՌԽՊ
ԡԹՑԹՆԼՏԽՊԽԻՇՆՋՁԼԽՊԽԼՌՇԺԽՊԽՈՊԽՋԽՆՌԹՌՁՎԽՇԾՌՀԽԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽՌԽՋՌԻՀԹՅԺԽՊՋՇՆԡԹՑԹՆԼՍՋԽԼՁՆՌՀԽԻԹՄԻՍՄԹՌՁՇՆՇԾՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺՑՌՀԽՋՌՍԼՑԹՍՌՀՇՊ 

# ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՌԽՋՌՁՌԽՅ ԻՇՆԻԽՆՌՊԹՌՁՇՆԺԽՅԽԹՋՍՊԽԼՁՆՇՆԽՌԹՆՃԹՌ ԽԹԻՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼ ԽՎԽՊՑՏԽԽՃՌՀԽՊԽԹԾՌԽՊԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆԻՇՆՌՊՇՄ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ՁԾԹՈՈՄՁԻԹԺՄԽԫԞ 

# ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԽՍՋԽՇԾԹ ԻՇՆԻՍՊՊԽՆՌՋՇՄՎԽՆՌԻՇՆՌՊՇՄՏՀԽՆԹ ՋՇՄՍԺՁՄՁՒՁՆԿԹԿԽՆՌՁՋՍՋԽԼ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՈԽՊՌԽՋՌՁՌԽՅՌՊԽԹՌՅԽՆՌՄԽՎԽՄ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋԫՍՅԺԽՊՇԾՄԹՊՎԹԽՈԽՊՌՊԽԹՌՅԽՆՌԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԽՉՍԹՄՄՑԼՁՎՁԼԽԼԺԽՌՏԽԽՆՊԽՈՄՁԻԹՌԽՋԴԹՋԹՋՇՄՎԽՆՌՍՋԽԼ!ԫՇ԰ՇՄՎԽՆՌՌՑՈԽ
ՁԾԹՈՈՄՁԻԹԺՄԽԫԞԪԹՐՁՅՍՅՋՇՄՎԽՆՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁԾԹՈՈՄՁԻԹԺՄԽԫԞ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՋՇՄՎԽՆՌՆՇՌ ԽՐԻԽԽԼՅՄԩ 

# ԬԢ ԠԡՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌՋՇՄՎԽՆՌՀԹՎԽՆՇԽԾԾԽԻՌՇՆՋՍՊՎՁՎԹՄՆՇՊ ՈՊՇԼՍԻԽԹՆՑՇՌՀԽՊԹԼՎԽՊՋԽԽԾԾԽԻՌՋԹՆԼՌՀԹՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՆՇՌԺԽԿՊԽԹՌԽՊՌՀԹՆՅՄԩ 

#  ԥՍՌԻՀՁՆՋՇՆԱԥ԰ՀՁՄՄԹԺԽԽՊԫԴՁՆՌԽՊԪԧԭՁԻՃԾՇՊԼԡԟ
ԞԻՍՌԽԹՆԼԻՀՊՇՆՁԻԽԾԾԽԻՌՋՇԾԻԹՊՊՁԽՊՋՇՄՎԽՆՌՋՁՆԹՉՍԹՌՁԻՇՊԿԹՆՁՋՅՋԞԻՊՁՌՁԻԹՄՊԽՎՁԽՏԯԽՎՁԽՏԞՉՍԹՌՁԻԱՇՐՁԻՇՄՇԿՑՈՈ- ԬԢԠԡ
ԤՍՁԼԹՆԻԽԡՇԻՍՅԽՆՌՇՆԞՉՍԹՌՁԻԱՇՐՁԻՁՌՑԱԽՋՌՁՆԿՇԾԡՁԾԾՁԻՍՄՌ԰ՍԺՋՌԹՆԻԽՋԹՆԼԪՁՐՌՍՊԽՋԢՆՎՁՊՇՆՅԽՆՌԹՄԥԽԹՄՌՀԹՆԼ԰ԹԾԽՌՑԭՍԺՄՁԻԹՌՁՇՆՋ԰ԽՊՁԽՋՇՆԱԽՋՌՁՆԿԹՆԼԞՋՋԽՋՋՅԽՆՌԫՇԭԹՊՁՋԣՊԹՆԻԽ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

       !  "        #  $           ԴԹՋԹՈՇՋՁՌՁՎԽԻՇՆՌՊՇՄՍՋԽԼ!ԫՇԭՇՋՁՌՁՎԽԻՇՆՌՊՇՄ
ՁԾԹՈՈՄՁԻԹԺՄԽԫԞԭՇՋՁՌՁՎԽԻՇՆՌՊՇՄԻՇՆԻԽՆՌՊԹՌՁՇՆ
ՋՁԾԹՈՈՄՁԻԹԺՄԽԫԞԭՀՇՌՇՈԽՊՁՇԼՀՊՋՄՁԿՀՌՀՊՋԼԹՊՃ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՈՀՇՌՇՈԽՊՁՇԼ ՄՁԿՀՌԼԹՊՃԩՁԿՀՌՁՆՌԽՆՋՁՌՑԹՌՏԹՌԽՊ0ՋՋՍՊԾԹԻԽ-ԨՄՍՐ 

# ԢԭԞՊԽԻՇՅՅԽՆԼՋՄՁԿՀՌՁՆՌԽՆՋՁՌՑ -  ԨՄՍՐ
ԹՌՏԹՌԽՊ0ՋՋՍՊԾԹԻԽ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ՎՁԹ ՊԽՎԽՊՋԽՇՋՅՇՋՁՋԽՎԽՊՑՌՇԼԹՑՋԼՍՊՁՆԿՌՀԽՌԽՋՌԱՀԽ ՋՇՄՍՌՁՇՆ ՏԹՋ ԹԼՂՍՋՌԽԼ ՌՇ  ԹԻՌՁՎԽՁՆԿՊԽԼՁԽՆՌ ԼՍՊՁՆԿ ՈՊԽՈԹՊԹՌՁՇՆ ԺԹՋԽԼ ՇՆ ՌՀԽՌԽՋՌ ՋՍԺՋՌԹՆԻԽ ՈՍՊՁՌՑ 
 ԱՀԽ ԩՋՌՇԻՃՋՇՄՍՌՁՇՆՏԹՋՈՊԽՈԹՊԽԼՁՆԹԩԿՄԹՋՋՌԹՆՃ԰ՁՆԻԽՅԹՄԹՌՀՁՇՆՁՋՋՍԺՂԽԻՌՌՇՋՁԿՆՁԾՁԻԹՆՌՀՑԼՊՇՄՑՋՁՋ ԹՌ Ոԥ % ՌՀԽ ՋՌՇԻՃՋ ՏԽՊԽԹԻՁԼՁԾՁԽԼՌՇԹՌԹՊԿԽՌՈԥՇԾԹՈՈՊՇՐՁՅԹՌԽՄՑՍՋՁՆԿ  ՈՀՇՋՈՀՇՊՁԻ ԹԻՁԼ 
ԥԭԬ ՌՇՅԹՁՆՌԹՁՆ ՋՌԹԺՁՄՁՌՑ ՇՎԽՊ ՌՀԽ ՅԹՐՁՅՍՅ ՋՌՇԻՃՊԽՆԽՏԹՄ ՁՆՌԽՊՎԹՄ ՇԾ ԹՈՈՊՇՐՁՅԹՌԽՄՑ ՀՇՍՊՋԱՀԽՈԥՇԾՌՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋՏԹՋԻՇՆԾՁՊՅԽԼԹՌ ՌՀԽ ԺԽԿՁՆՆՁՆԿ ԹՆԼ ԽՆԼ ՇԾ ԽԹԻՀ ՋՌՇԻՃՊԽՆԽՏԹՄՈԽՊՁՇԼԹՆԼՊԹՆԿԽԼԾՊՇՅՌՇ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԫՇՌԼԽՌԽՊՅՁՆԽԼ ԰ԡ 

# ԰ՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ

 

# ԞՄՋՇՊԽԾԽՊՊԽԼՌՇԹՋ՟ՏԽՌՏԽՁԿՀՌՠՁՆՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽ

԰ԹՅՈՄՁՆԿԾՊՇՅՌՀԽԹՎԹՁՄԹԺՄԽՌԹԼՈՇՄԽՋԾՇՊՀՁՋՌՇՈԹՌՀՇՄՇԿՑՏԹՋԼՇՆԽՌՇՋԽՄԽԻՌՋՌԹԿԽՅԹՌԻՀԽԼՁՆԼՁՎՁԼՍԹՄՋ ՏՀԽՆ ՈՇՋՋՁԺՄԽ  ԱՀԽՊԽ ՏԽՊԽ ՆՇ ՊԽՈՄՁԻԹՌԽՋ ՏՁՌՀ ՅՇՊԽ ՌՀԹՆ ԾՁՎԽ ՌԹԼՈՇՄԽՋ ԹՌ ՌՀԽ ԻՇՆՌՊՇՄ ՅԽԼՁԹՆ ԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԱՀԽՊԽԾՇՊԽԹՄՄՌԹԼՈՇՄԽՋԹՌՌՀԹՌՋՌԹԿԽՏԽՊԽ ՋԽՄԽԻՌԽԼԾՊՇՅԽԹԻՀՊԽՈՄՁԻԹՌԽԴՀԽՆ ՌՀԽՊԽՏԽՊԽԾԽՏԽՊՌՀԹՆԾՁՎԽՌԹԼՈՇՄԽՋԹՌՌՀԹՌՋՌԹԿԽՁՆԹ ՊԽՈՄՁԻԹՌԽԹԼԼՁՌՁՇՆԹՄՌԹԼՈՇՄԽՋՏԽՊԽ ՊԹՆԼՇՅՄՑ ՋԽՄԽԻՌԽԼ
ՍՋՁՆԿԹՊԹՆԼՇՅՆՍՅԺԽՊԿԽՆԽՊԹՌՇՊԾՊՇՅՌՀԽԼԽՎԽՄՇՈՅԽՆՌ ԹՄՋՌԹԿԽՋՂՍՋՌԹԺՇՎԽԹՆԼՂՍՋՌԺԽՄՇՏ ՌՀԽՅԽԼՁԹՆԻՇՆՌՊՇՄԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽ 

# ՁԽ ԫԣԹՆԼԦՆԹԾԽՏԻԹՋԽՋՋԽԻՌՁՇՆՁՆԿՇԾՌՀԽՀԽԹԼՋ
ՇԾՌՀԽՌԹԼՈՇՄԽՋԼՁԼՆՇՌՈՊՇՎՁԼԽՋՄՁԼԽՋՌՀԹՌԹՄՄՇՏԽԼԾՇՊԹԻԻՍՊԹՌԽԹՋՋԽՋՋՅԽՆՌՇԾՌՀԽՋՁՒԽՇԾՌՀԽՌՀՑՊՇՁԼ ԿՄԹՆԼՋԦՆՋՍԻՀԻԹՋԽՋՌՀԽՋԽՋՄՁԼԽՋՏԽՊԽՋԽՌԹՋՁԼԽԹՆԼԹԼԼՁՌՁՇՆ ԹՄՌԹԼՈՇՄԽՋՏԽՊԽՋԽՄԽԻՌԽԼՁՆԹԻԻՇՊԼԹՆԻԽ ՏՁՌՀ ՌՀԽ ՈՊԽՎՁՇՍՋՄՑ ԼԽՋԻՊՁԺԽԼ ՋԻՀԽՅԽ ԹՆԼ ՌՀԽՋԽ ԹԼԼՁՌՁՇՆԹՄ ՌԹԼՈ ՇՄԽՋ ՏԽՊԽ ՈՊՇԻԽՋՋԽԼ ՌՇ ՈՊՇՎՁԼԽ ՊԽՈՄԹԻԽՅԽՆՌՋՄՁԼԽՋ

ԱՀԽՊԽՏԽՊԽՆՇԼՁԹԿՆՇՋՌՁԻԹԺՆՇՊՅԹՄՁՌՁԽՋՇԺՋԽՊՎԽԼԺՑՀՁՋՌՇՈԹՌՀՇՄՇ ԿՁԻԹՄԽՐԹՅՁՆԹՌՁՇՆՇԾՌՀԽՌՀՑՊՇՁԼՋՇԾ ՌԹԼՈՇՄԽՋՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈՏՀԽՆԻՇՅՈԹՊԽԼՌՇՌՀԽՆԽԿԹՌՁՎԽԻ ՇՆՌՊՇՄԹՌՌՀԽՌԽՊՅՁՆԹՌՁՇՆՇԾՌՀԽ ԼԹՑՌԽՋՌ    

       



   



 

  







 



    





 

  


# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

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#  ԱՀՑՊՇՁԼԿՄԹՆԼԿՊՇՋՋՈԹՌՀՇՄՇԿՑԹՆԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑԹՊԽԿՊԹԼԽԼ

- ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑԫՇՌ
ՊԽՅԹՊՃԹԺՄԽԪՁՄԼԪՇԼԽՊԹՌԽ԰ԽՎԽՊԽ԰ԽԽԬԢԠԡԫՇԾՇՊ ՊԽԾԽՊԽՆԻԽ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ  

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# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ԱԹԼՈՇՄԽՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԿԽՆԽՊԹՄՄՑ ԹՈՈԽԹՊԽԼՆՇՊՅԹՄՌՀՊՇՍԿՀՇՍՌՌՀԽՌԽՋՌ ԬՆԽՌԹԼՈՇՄԽՁՆԽԹԻՀՇԾՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁ ԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԹՋՇԺՋԽՊՎԽԼ ՄՑՁՆԿՇՆՌՀԽԺՇՌՌՇՅՇԾՌՀԽՌԹՆՃՇՆԡԹՑԺՍՌԹՈՈԽԹՊԽԼՆՇՊՅԹՄԺՑ ԡԹՑԭԽՊՁՇԼՁԻՇԺՋԽՊՎԹՌՁՇՆՋՇԾԹ ԾԽՏՌԹԼՈՇՄԽՋՌՀԹՌՎՁՋՍԹՄՄՑԹՈՈԽԹՊԽԼՋՅԹՄՄԽՊՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀ ԽԻՇՆՌՊՇՄՌԹԼՈՇՄԽՋՏԽՊԽՆՇՌԽԼՁՆՌՀԽ ՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈԺՍՌՌՀԽՋՁՒԽՏԹՋ ՆՇՌԻՇՆԾՁՊՅԽԼԺՑՅԽԹՋՍՊԽՅԽՆՌՋՇԾ ՌՀԽՌԹԼՈՇՄԽՋ ԹՌՌՀԽՌՁՅԽ ՇԾ ՇԺՋԽՊՎԹՌՁՇՆԟԽԿՁՆՆՁՆԿՇՆ ԡԹՑ ՇԾՌՀԽՌԽՋՌ ԹՆ ՁՆԻՊԽԹՋՁՆԿ ՆՍՅԺԽՊ ՇԾ ՌԹԼՈՇՄԽՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՀԹԼԻ ՍՊՎԽԼՌԹՁՄՋԥՇՏԽՎԽՊՌՀԽՆՍՅԺԽՊՋՇԾ ՌԹԼՈՇՄԽՋ ՁՆՌՀԽՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈՋՏՁՌՀ ԻՍՊՎԽԼ ՌԹՁՄՋՏԽՊԽ ԻՇՅՈԹՊԹ ԺՄԽ ՌՇՌՀԽ ՆՍՅԺԽՊՋ ՁՆՌՀԽ ԻՇՆՌՊՇՄ ԿՊՇՍՈ  ԱՀԽ ՋՌՍԼՑ ԹՍՌՀՇՊ ՆՇՌԽԼՌՀԹՌ ՁՆԾԽԽԼՁՆԿՌՊՁԹՄՋ ՁՌ ՀԹՋ ԺԽԽՆ ՋՀՇՏՆ ՌՀԹՌԾԽԽԼՁՆԿ ՊԹՌԽՋ ԹՄՇՆԽ

ԻՇՆՌՊՁԺՍՌԽՌՇՌՀԽԹՅՇՍՆՌՇԾԻՍՊՎԹՌՍՊԽՇԺՋԽՊՎԽԼ

ԦՆԻՁԼԽՆՌԼԹՌԹՈՊՇՎՁԼԽԼՁՆԱԹԺՄԽԹՊԽԾՇՊԡԹՑՇԺՋԽՊՎԹՌՁՇՆՋ ՇՆՄՑԹՋՌՀԽՊԽՁՋՆՇՏԹՑՌՇԼԽՌԽՊՅՁՆԽ ՌՀԽՌՇՌԹՄՆՍՅԺԽՊՇԾՁՆԻՁԼԽՆԻԽԾՊՇՅՌՀԽԼԹՌԹՈՊՇՎՁԼԽԼ

 

 

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# ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ ԱԹՁՄԻՍՊՎԹՌՍՊԽ  


 ԱԹՁՄԻՍՊՎԹՌՍՊԽ  


 ԱԹՁՄԻՍՊՎԹՌՍՊԽ  


 ԱԹՁՄԻՍՊՎԹՌՍՊԽ  


 

# ԫՇՌԽՌՀԹՌԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌ
ՍՆԹԺՄԽՌՇՋՌԹԿԽՁՋՊԽՈՇՊՌԽԼՈՊԽՎՁՇՍՋՄՑՁՆ   



 

# ԹՆԼՆՇՌ

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# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ   

# ՇԾ 



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ 











      

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# ԰ՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋԽՋՏԽՊԽՈԽՊԾՇՊՅԽԼՌՇԽՎԹՄՍԹՌԽԼՁԾԾԽՊԽՆԻԽՋԺԽՌ ՏԽԽՆՌՊԽԹՌՅԽՆՌԹՆԼԻՇՆՌՊՇՄԿՊՇՍՈՋԾՇՊ ԽԹԻՀՇԾՌՀԽԾՇՄՄՇՏՁՆԿԺՁՇՄՇԿՁԻԹՄԽՆԼՈՇՁՆՌՋՏՀԽՆՈՇՋՋՁԺՄԽՋՍՊՎ ՁՎԹՄԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԺՇԼՑՏԽՁԿՀՌ ՏՀՇՄԽԺՇԼՑՄԽՆԿՌՀՋՆՇՍՌՌՇՎԽՆՌՄԽՆԿՌՀՋԹՆԼՀՁՆԼՄՁՅԺՄԽՆԿՌՀ
ՆՇՊՅԹՄՁՒԽԼԺՑԼՁՎՁԼՁՆԿԺՑՌՀԽ԰Գԩ
԰ՁՆԻԽՌՀԽՊԽՏԽՊԽՆՇԹԺՆՇՊՅԹՄՁՌՁԽՋՁՆՌՀԽՌՀՑՊՇՁԼՀՁՋՌՇՄՇԿՑՆՇ ՋՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋԽՋՏԽՊԽՈԽՊԾՇՊՅԽԼՇՆ ՀՁՋՌՇՄՇԿՑՊԽՋՍՄՌՋ

ԪՇՋՌԽՆԼՈՇՁՆՌՋՏԽՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԻՇՅՈՄԽՅԽՆՌԹՊՑՋՌԹՌՁՋՌՁԻԹՄԹՈՈՊՇԹԻՀԽՋԦԾՊԽՋՈՇՆՋԽՋԹՈՈԽԹՊԽԼՌՇ ԺԽՅՇՆՇՌՇՆՁԻ
ՌՊԽՆԼՁՆԿՁՆՇՆԽԼՁՊԽԻՌՁՇՆԹՋՌԽՈԼՇՏՆԧՇՆԻՃՀԽԽՊ ԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌՏԹՋՍՋԽԼՌՇ ԽՎԹՄՍԹՌԽՌՊԽՆԼՋՁՆՌՀԽՊԹՆՃՋՇԾՊԽՈՄՁԻԹՌԽՅԽԹՆՋՌՇԼԽՌԽՊՅՁՆԽՈՇ ՋՋՁԺՄԽԻՇՆԻԽՆՌՊԹՌՁՇՆՊԽՋՈՇՆՋՁՎԽՌՊԽՆԼՋ ԹՅՇՆԿՌՀԽՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԞՄՄԽՆԼՈՇՁՆՌՋԽՐԻԽՈՌԾՇՊՋՍՊՎՁՎԹՄԹ ՆԼԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԼԹՌԹՏԽՊԽԹՄՋՇ ԹՆԹՄՑՒԽԼԺՑՈԽՊԾՇՊՅՁՆԿՈԹՁՊ ՏՁՋԽԻՇՅՈԹՊՁՋՇՆՋՍՋՁՆԿԡՍՆՆԽՌՌ0ՋՅՍՄՌՁՈՄԽԻՇՅՈԹՊՁՋՇՆՌԽՋՌՌՇԼԽ ՌԽՊՅՁՆԽ ՏՀՁԻՀՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԼՁԾԾԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑԾՊՇՅՌՀԽԻՇՆՌՊՇՄ ԿՊՇՍՈԡԹՌԹԾՇՊ ԽՆԼՈՇՁՆՌՋԹՆԹՄՑՒԽԼԺՑ ԡՍՆՆԽՌՌ0ՋՌԽՋՌՏԽՊԽԽՎԹՄՍԹՌԽԼԾՇՊՆՇՊՅԹՄՁՌՑՍՋՁՆԿ԰ՀԹՈՁՊՇ

 ԴՁՄՃ0ՋՌԽՋՌԹՆԼԾՇՊՀՇՅՇԿԽՆԽՁՌՑՇԾՎԹՊՁԹՆԻԽ
ՍՋՁՆԿԩԽՎԽՆԽ 0ՋՌԽՋՌ  

# ԱՀԽԼԹՌԹԾՇՊՌՀՁՋՋՌՍԼՑՈԹՋՋԽԼՌՀԽԹՋՋՍՅՈՌՁՇՆՋԾՇՊՆՇ ՊՅԹՄՁՌՑԹՆԼ ՀՇՅՇԿԽՆԽՁՌՑՇԾՎԹՊՁԹՆԻԽ

԰ՍՊՎՁՎԹՄԹՆԼԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԼԹՌԹՏԽՊԽՆՇՌԹՅԽՆԹԺՄԽՌՇՌՀԽ ՋՌԹՌՁՋՌՁԻԹՄՅԽՌՀՇԼՋՍՋԽԼԾՇՊԹՆԹՄՑՋՁՋ ՇԾՌՀԽՇՌՀԽՊԽՆԼՈՇՁՆՌՋ԰ՍՊՎՁՎԹՄՏԹՋԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽՋՌԽՈԼՇ ՏՆԧՇՆԻՃՀԽԽՊԽԱԽՊՋՌՊԹՌՊԽՆԼՌԽՋՌԹՆԼ

ԣՁՋՀԽՊ 0 ՋԢՐԹԻՌՌԽՋՌԦՆԹԻԻՇՊԼԹՆԻԽՏՁՌՀՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋՇԾԬԠ԰ԭԭԹՆԹՄՑՋՁՋՇԾՅԽՌԹՅՇՊՈՀՁԻ ՋՌԹԿԽՏԹՋՈԽՊԾՇՊՅԽԼՍՋՁՆԿՌՀԽՅՍՄՌՁՉՍԹՆՌՁՄԽԹՆԹՄՑՋՁՋԼԽՎԽՄՇՈԽԼ ԺՑԱ԰ՈՊՁՆԿԽՊԹՆԼԧԤՊԽԽՆԹՆԼ ՍՋՁՆԿՌՀԽՋՌԽՈԼՇՏՆԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌՇՆՋՌԹԿԽՉՍԹՆՌՁՄԽՋՁՆՌՀԽՊԽՈՄՁԻԹՌԽԻՀԹՅԺԽՊՋ

԰ՌԹՌՁՋՌՁԻԹՄՌԽՋՌՋՍՋԽԼՌՇԽՎԹՄՍԹՌԽՌՊԽԹՌՅԽՆՌԽԾԾԽԻՌՋՏԽՊԽՈԽՊԾՇ ՊՅԽԼԹՌԹԻՇՆԾՁԼԽՆԻԽՄԽՎԽՄՇԾ 



#  ՍՆՄԽՋՋՇՌՀԽՊՏՁՋԽՆՇՌԽԼՏՁՌՀ԰Ԟ԰ՋՇԾՌՏԹՊԽ  ԱՀԽՊԽՏԽՊԽՆՇՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԽԾԾԽԻՌՋՇՆՋՍՊՎՁՎԹՄՁՆ ԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽ ԻՇՆՌՊՇՄԼՍՊՁՆԿՌՀԽՌԽՋՌ 

# Ո 

ԞԻԻՇՊԼՁՆԿՌՇՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌՌՀԽՊԽՏԹՋՆՇՋ ՁԿՆՁԾՁԻԹՆՌՌՊԽՆԼՁՆՌՀԽԡԹՑԺՇԼՑ ՄԽՆԿՌՀԼԹՌԹ
Ո ԡՍՆՆԽՌՌ0ՋՌԽՋՌՋՀՇՏԽԼՌՀԹՌՌՀԽՁՆԻՊԽԹՋԽՁՆ ԡԹՑԺՇԼՑՄԽՆԿՌՀՁՆՌՀԽ    

       



   



 

  







 



    





 

  


# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ   

# ՇԾ 



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ՅԿԹՁԩՆՇՅՁՆԹՄ
ՅԿԹՁԩՅԽԹՆՅԽԹՋՍՊԽԼՌՊԽԹՌՅԽ ՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽ ՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՏԹՋՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌ 

# Ո  

# ԱՀԽԼՁԾԾԽՊԽՆԻԽԾՊՇՅՌՀԽԻՇՆՌՊՇՄՏԹՋՆՇՌ
ԻՇՆՋՁԼԽՊԽԼՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼՋՁՆԻԽՌՀԽՊԽՏԽՊԽՆՇԼՁԾԾԽՊԽ ՆԻԽՋՁՆՆՇՊՅԹՄՁՒԽԼՀՁՆԼՄՁՅԺՄԽՆԿՌՀՇՊ ՋՌԹԿԽ ՇԾ ԼԽՎԽՄՇՈՅԽՆՌ ՇՆ ԡԹՑ  ԦՌ ՁՋ ԹՄՋՇ ԺԹՋԽԼ ՇՆ Թ ՊԽՄԹՌՁՎԽՄՑ  ՋՅԹՄՄ ՋԹՅՈՄԽ ՋՁՒԽ ՇԾ  ԾՊՇԿՋ ԞԼԼՁՌՁՇՆԹՄՄՑՌՀԽԼՁԾԾԽՊԽՆԻԽՏԹՋՆՇՄՇՆԿԽՊԹՈՈԹՊԽՆՌՇՆԡԹՑ ԱՀԽԡԹՑՁՆԻՊԽԹՋԽՁՆԺՇԼՑՄԽՆԿՌՀ ՁՆՌՀԽՅԿԹՁԩՆՇՅՁՆԹՄ
ՅԿԹՁԩՅԽԹՆՅԽԹՋՍՊԽԼՌՊ ԽԹՌՅԽՆՌԿՊՇՍՈՊԽՄԹՌՁՎԽՌՇՌՀԽԻՇՆՌՊՇՄ ՏԹՋՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌ 

# Ո  

# ԾՇՊԺՇՌՀՌՀԽԧՇՆԻՃՀԽԽՊԽ ԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌԹՆԼԡՍՆՆԽՌՌ0ՋՌԽՋՌ ԥՇՏԽՎԽՊՌՀԽՁՆԻՊԽԹՋԽՏԹՋՋՄՁԿՀՌ
ՏԹՋՆՇՌՋՍՈՈՇՊՌԽԼԺՑԹՆՁՆԻՊԽԹՋԽՁՆՇՌՀԽՊԿՊՇՏՌՀՈԹՊԹՅԽՌԽՊՋ
ԹՆԼՌՀԽՊԽԾՇՊԽՏԹՋՆՇՌԻՇՆՋՁԼԽՊԽԼՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼ

ԞԻԻՇՊԼՁՆԿՌՇՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌՌՀԽՊԽՏԹՋՆՇՋ ՁԿՆՁԾՁԻԹՆՌՌՊԽՆԼՁՆՌՀԽԡԹՑՏԽՌՏԽՁԿՀՌ ԼԹՌԹ
Ո ԥՇՏԽՎԽՊԡՍՆՆԽՌՌ0ՋՌԽՋՌՁՆԼՁԻԹՌԽԼՌՀԽՊԽՏԹՋԹ ՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌՁՆԻՊԽԹՋԽՁՆ ԡԹՑՏԽՌՏԽՁԿՀՌՁՆՌՀԽՅԿԹՁԩՆՇՅՁՆԹՄ
ՅԿԹՁ ԩՅԽԹՆՅԽԹՋՍՊԽԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈ ՁՆ ԻՇՅՈԹՊՁՋՇՆ ՌՇՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ 

# Ո  

#   ԩՁՃԽՌՇՌԹՄ ՄԽՆԿՌՀՅԽԹՋՍՊԽՅԽՆՌՋ ՇՆԡԹՑ  ՌՀՁՋ
ԼՁԾԾԽՊԽՆԻԽՁՆՏԽՌՏԽՁԿՀՌԾՊՇՅՌՀԽԻՇՆՌՊՇՄՏԹՋՆՇՌԻՇՆՋՁԼԽՊԽԼՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼՋՁՆԻԽՌՀԽՊԽՏԽՊԽ ՆՇԼՁԾԾԽՊԽՆԻԽՋՁՆՆՇՊՅԹՄՁՒԽԼՀՁՆԼՄՁՅԺՄԽՆԿՌՀՇՊՋՌԹԿԽՇԾԼԽՎԽՄ ՇՈՅԽՆՌՇՆԡԹՑԦՌՁՋԹՄՋՇԺԹՋԽԼՇՆ ԹՊԽՄԹՌՁՎԽՄՑՋՅԹՄՄՋԹՅՈՄԽՋՁՒԽՇԾԾՊՇԿՋԹՆԼՌՀԽԼՁԾԾԽՊԽՆԻԽՏ ԹՋՆՇՄՇՆԿԽՊԹՈՈԹՊԽՆՌՇՆԡԹՑԱՀԽՊԽ ՏԽՊԽՆՇՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԽԾԾԽԻՌՋՇՆԡԹՑՏԽՌՏԽՁԿՀՌՋ ՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇ ՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ 

# Ո 

ԞԻԻՇՊԼՁՆԿՌՇՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌՌՀԽՊԽՏԹՋՆՇՋ ՁԿՆՁԾՁԻԹՆՌՌՊԽՆԼՁՆՌՀԽԡԹՑՋՆՇՍՌՌՇ ՎԽՆՌՄԽՆԿՌՀԼԹՌԹ
Ո ԥՇՏԽՎԽՊԡՍՆՆԽՌՌ0ՋՌԽՋՌՁՆԼՁԻԹՌԽԼ ՌՀԽՊԽՏԹՋԹՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌ ՁՆԻՊԽԹՋԽ ՁՆ ԡԹՑ  ՋՆՇՍՌՌՇՎԽՆՌ ՄԽՆԿՌՀ ՁՆ ՌՀԽ  ՅԿ ԹՁԩ ՆՇՅՁՆԹՄ 
 ՅԿ ԹՁԩ ՅԽԹՆ ՅԽԹՋՍՊԽԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ  

# Ո  

# ԱՀՁՋԼՁԾԾԽՊԽՆԻԽՁՆՋՆՇՍՌ
ՌՇՎԽՆՌՄԽՆԿՌՀՇՆԡԹՑՁՋԻՇՆՋՁՋՌԽՆՌՏՁՌՀՌՀԽԼՁԾԾԽՊԽՆԻԽՋՋԽԽՆ ՁՆՄԽՆԿՌՀԹՆԼՏԽՌՏԽՁԿՀՌԹՆԼՄՁՃԽ ՌՀՇՋԽՇՌՀԽՊՈԹՊԹՅԽՌԽՊՋՏԹՋՆՇՌԻՇՆՋՁԼԽՊԽԼՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԱ ՀԽՊԽՏԽՊԽՆՇՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌ ԽԾԾԽԻՌՋՇՆԡԹՑՋՆՇՍՌՌՇՎԽՆՌՄԽՆԿՌՀՋՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈ ՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ



# Ո 

ԱՀԽՊԽՏԽՊԽՆՇՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԽԾԾԽԻՌՋՇՆՆՇՊՅԹՄՁՒԽԼՀ ՁՆԼՄՁՅԺՄԽՆԿՌՀՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆ ԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ 

# Ո ՇՆԡԹՑՇՊ    

       



   



 

  







 



    





 

  


# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ 

 

# ՇԾ 



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ԱՀԽՊԽ ՏԽՊԽ ՆՇ ՋՌԹՌՁՋՌՁԻԹՄՄՑ ՋՁԿՆՁԾՁԻԹՆՌ ԽԾԾԽԻՌՋ ՇՆ ԼԽՎԽՄՇՈՅԽՆՌԹ Մ ՋՌԹԿԽ ՁՆ ԹՆՑ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ՁՆ ԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ 

# Ո ՇՆԡԹՑՋՇՊ

ԱՀԽՊԽՏԽՊԽՆՇՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՇԺՋԽՊՎԽԼԺՑՀՁՋՌՇՈԹՌՀՇՄ ՇԿՁԻԹՄԽՐԹՅՁՆԹՌՁՇՆՇԾՌՀԽՌՀՑՊՇՁԼՋՇԾ ՌԹԼՈՇՄԽՋՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈԹՌՌԽՊՅՁՆԹՌՁՇՆՇԾՌՀԽԼԹՑՌԽ ՋՌ

ԬՎԽՊԹՄՄՌՀԽՊԽՏԽՊԽՆՇԹՈՈԹՊԽՆՌՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՇՆՌԹ ԼՈՇՄԽԿՊՇՏՌՀԹՆԼԼԽՎԽՄՇՈՅԽՆՌՇԺՋԽՊՎԽԼ

ՁՆՌՀՁՋՌԽՋՌ

      

     





  

    





  





! 

# ԱՀԽՊԽՎՁԽՏԽՊՎՁՋՍԹՄՄՑԹՆԹՄՑՒԽԼԼԹՌԹԾՇՊՅՇՊՌԹՄՁՌՑԺԽԻԹՍՋԽՌՀԽ ԼԹՌԹԼՁԼՆՇՌՅԽԽՌ ՌՀԽԹՋՋՍՅՈՌՁՇՆՋԾՇՊՌՀԽԣՁՋՀԽՊ0ՋԢՐԹԻՌՌԽՋՌ ԱՀԽ ՊԽՅԹՁՆՁՆԿԽՆԼՈՇՁՆՌՋՏԽՊԽԹՆԹՄՑՒԽԼՁՆԠԢԱԦ԰ԬՆ ԡԹՑ԰ԳԩԥԩԩՆՇՊՅԹՄՁՒԽԼԥԩԩ
ԥԩԩ԰ԳԩԹՆԼՏԽՌՏԽՁԿՀՌՏԽՊԽ ՆՇՌԻՇՆՋՁՋՌԽՆՌՏՁՌՀԹՅՇՆՇՌՇՆՁԻ ԻՇՆԻԽՆՌՊԹՌՁՇՆՊԽՋՈՇՆՋԽԱՀԽՋԽԼԹՌԹՏԽՊԽՆՇՊՅԹՄՄՑԼՁՋՌՊՁԺՍՌԽԼ
԰ ՀԹՈՁՊՇ ԴՁՄՃ0Ջ

 



# ՏՁՌՀԽՉՍԹՄ ՎԹՊՁԹՆԻԽ
ԩԽ ՎԽՆԽ0ՋՌԽՋՌ  

# ԹՆԼՌՀԽՊԽԾՇՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԡՍՆՆԽՌՌ0ՋՌԽՋՌԬՆԡԹՑ ԥԩԩԹՆԼ

ՆՇՊՅԹՄՁՒԽԼ ԥԩԩ ԽՐՀՁԺՁՌԽԼ Թ ՅՇՆՇՌՇՆՁԻ ՌՊԽՆԼ ԹՆԼ ՏԽՊԽ ՌՀԽՊԽԾՇՊԽ Թ ՆԹՄՑՒԽԼ ՍՋՁՆԿ ՌՀԽ ԧՇՆԻՃՀԽԽՊԽ ԱԽՊՈՊՋՌՊԹ ՌԽՋՌ ԡԹՌԹ ԾՇՊ ԼԽՎԽՄՇՈՅԽՆՌԹՄ ՋՌԹԿԽ ՏԽՊԽ ԹՆԹՄՑՒԽԼ ՍՋՁՆ Կ ՆՇՆՈԹՊԹՅԽՌՊՁԻ ՌԽՋՌՁՆԿ 
ՅԹՆՆ ՏՀՁՌՆԽՑ ԞՄՄ ՊԽՅԹՁՆՁՆԿ ԡԹՑ  ԽՆԼՈՇՁՆՌՋ ՏԽՊԽ ԹՆԹՄՑՒԽԼ ՍՋՁՆԿ ԡՍՆՆԽՌՌ0Ջ ՌԽՋՌ ԹԾՌԽՊ ՅԽԽՌՁՆԿ ՌՀԽ
ԹՋՋՍՅՈՌՁՇՆՋ ԾՇՊ ՆՇՊՅԹՄՁՌՑ ԹՆԼ ՀՇՅՇԿԽՆԽՁՌՑ ԲՆՄԽՋՋ ՇՌՀԽՊՏՁՋԽ ՁՆԼ ՁԻԹՌԽԼ ԽԾԾԽԻՌՋ ՏԽՊԽ ԻՇՆՋՁԼԽՊԽԼ ՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌ 

# Ո ԱՀԽՊԽՏԽՊԽՇՆՄՑ


ԹՆԼ
  ՌԹԼՈՇՄԽՋԿՊԽԹՌԽՊՌՀԹՆՋՌԹԿԽ 

# ՁԽ ՋՌԹԿԽՋՌՇՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼ
  ԹՆԼ  ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈՋ ՊԽՋՈԽԻՌՁՎԽՄՑ  ԱՀԽՊԽԾՇՊԽ ՊԽՈՄՁԻԹՌԽ ՅԽԹՆՋ ԹՆԼ ՅԽԼՁԹՆՋ ԾՇՊ ԺՇԼՑՏԽՁԿՀՌ ԰Գԩ ԹՆԼ ԥԩԩ 
ՁՆԻՄՍԼՁՆԿ ՆՇՊՅԹՄՁՒԽԼ ԥԩԩ ՏԽՊԽ ԻԹՄԻՍՄԹՌԽԼ ԽՐԻՄՍԼՁՆԿ ԹՄՄ
ՌԹԼՈՇՄԽՋԹՌԫԣՋՌԹԿԽ  

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ՈՈՊԽՋՈԽԻՌ ՁՎԽՄՑ԰ՁԿՆՁԾՁԻԹՆՌՁՆԻՊԽԹՋԽՋՁՆՌՀԽՋԽ
ԽՆԼՈՇՁՆՌՋՏԽՊԽՆՇՌՇԺՋԽՊՎԽԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌ    

       



   



 

  







 



    





 

  


# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ   

# ՇԾ 



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ԿՊՇՍՈՋ ԹՆԼ ՌՀԽՊԽԾՇՊԽ ԹՊԽ ՆՇՌ ԻՇՆՋՁԼԽՊԽԼ ՌՊԽԹՌՅԽՆՌ ՊԽՄԹՌԽԼ  ԱՀԽ ՊԽ ՏԽՊԽ ՆՇ ՋՁԿՆՁԾՁԻԹՆՌ ԼՁԾԾԽՊԽՆԻԽՋ ԺԽՌՏԽԽՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԾՇՊԹՆՑՇՌՀԽՊԽՆԼՈՇՁՆՌՋ                                            ԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽ  ՇԾ   

# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

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ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄ

 

#  ԢԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌ 1 2

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x ԱՀԽԾՄՇՏՌՀՊՇՍԿՀՊԹՌԽՇԾՅԩՅՁՆԽՐԻԽԽԼՋՌՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇ ՅՅԽՆԼԹՌՁՇՆՇԾՅԩՅՁՆ

ԱՀԽԾՇՄՄՇՏՁՆԿՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹՏԽՊԽՆՇՌՅԽՌ

x ԱՀԽԠԳԾՇՊՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆ ՇԾՌՀԽՄՇՏՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ 

# ՁԽ ՅԿ ԹՁԩՏԹՋԽՐԻԽԽԼՁՆԿՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆՇԾՅԹՁՆՌ ԹՁՆՁՆԿԹԠԳՇԾ 5 

# ՇՎԽՊ ՌՀԽԼԹՑՌԽՋՌԥՇՏԽՎԽՊՁՌՋՀՇՍՄԼԺԽՆՇՌԽԼՌՀԹՌՌՀՁՋԠԳՏԹՋ ԻԹՄԻՍՄԹՌԽԼԺՑՌՀԽ ՊԽՎՁԽՏԽՊ ՍՋՁՆԿԡԹՑՋԹՅՈՄԽՋԦԾԡԹՑՋԹՅՈՄԽՋԹՊԽՍՋԽԼՁՆՄՁԽՍՇԾԡԹ ՑՋԹՅՈՄԽՋՌՀԽԠԳՇԾ ԼՇԽՋՆՇՌԽՐԻԽԽԼՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆ

x ԴԹՌԽՊՌԽՅՈԽՊԹՌՍՊԽԼՁԼՆՇՌԼՁԾԾԽՊԺՑՅՇՊԽՌՀԹՆւԠԺԽՌՏԽԽՆՌԽՋՌ ՎԽՋՋԽՄՋԹՌԹՆՑՇՆԽՌՁՅԽ ԼՍՊՁՆԿՌՀԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼՏՁՌՀՌՀԽԽՐԻԽՈՌՁՇՆՇԾՅԽԹՋՍՊԽՅԽՆՌՋ ՌԹՃԽՆՇՆԡԹՑԹՆԼԡԹՑ ՏՀԽՆՌՀԽԼՁԾԾԽՊԽՆԻԽՏԹՋԹՆԼւԠՊԽՋՈԽԻՌՁՎԽՄՑԴԹՌԽՊ ՌԽՅՈԽՊԹՌՍՊԽՏԹՋՅԹՁՆՌԹՁՆԽԼ ՏՁՌՀՁՆչւԠՏՁՌՀՌՀԽԽՐԻԽՈՌՁՇՆՇԾԡԹՑՏՀԽՆՌԽՅՈԽՊԹՌՍ ՊԽՋՁՆՋՇՅԽՌԽՋՌՎԽՋՋԽՄՋ ՏԽՊԽԹՋՄՇՏԹՋւԠԱՀԽՋԽՄՁՅՁՌՋԽՐԻԽԽԼՌՀԽՌԽՅՈԽՊԹՌՍՊԽՈԽՊԾ ՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆՇԾչ ւԠՏՁՌՀՁՆՌԽՊՊԽՈՄՁԻԹՌԽՁՆՌԽՊՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋՆՇՌԽՐԻ ԽԽԼՁՆԿւԠԞԻԻՇՊԼՁՆԿՌՇՌՀԽ    

       



   



 

  







 



    





 

  


# ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ   

# ՇԾ 



# ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ ՋՌՍԼՑԹՍՌՀՇՊՌՀԽՋԽՋՄՁԿՀՌԼՁԾԾԽՊԽՆԻԽՋՁՆՌԽՅՈԽՊԹՌՍՊԽՏԽՊԽՇԾՋ ՀՇՊՌԼՍՊԹՌՁՇՆԹՆԼԼՁԼՆՇՌ ԹՈՈԽԹՊՌՇԹԼՎԽՊՋԽՄՑՁՅՈԹԻՌՌՀԽՋՌՍԼՑ

ԴՀՁՄԽՌՀԽՋԽԼԽՈԹՊՌՍՊԽՋԾՊՇՅԿՍՁԼԽՄՁՆԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹԹՆԼԼԽՎՁԹՌՁՇՆՋԾՊՇՅԿՍՁԼԽՄՁՆԽ ՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋՏԽՊԽՇԺՋԽՊՎԽԼՌՀԽՑ ԼՁԼՆՇՌՋՍԾԾՁԻՁԽՆՌՄՑՁՅՈԹԻՌ ՌՀԽԹԺՁՄՁՌՑՌՇՁՆՌԽՊՈՊԽՌՌՀԽՊԽՋՍՄՌՋՇԾ ՌՀԽԹՋՋԹՑԱՀԽԹՋՋԹՑ     



 

# ՌՀԽԢԡ԰ԭԱՁԽՊԱԽՋՌԬՊԼԽՊՊԽՉՍՁՊԽՅԽՆՌՋԾՇՊԹՆԞՅՈՀՁԺՁԹՆ ԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ



    



# ԣՇՊ ՌՀԽ ՆՇՅՁՆԹՄ  ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ՌՀԽ ՋՌՍԼՑ ԹՍՌՀՇՊ ԻԹՄ ԻՍՄԹՌԽԼ Թ ՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՇԾՅԿԹՁԩՍՋՁՆԿԡԹՑՋԹՅՈՄԽՋՁՆՄՁԽՍՇԾԡԹՑՋԹՅՈՄԽՋԞԻԻՇՊԼՁՆԿՌՇՌՀԽ ՋՌՍԼՑ ԹՍՌՀՇՊ ԡԹՑ  ՋԹՅՈՄԽՋ ԾՊՇՅ ՌՀԽ  ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ  ՏԽՊԽ ՋՍՋՈԽԻՌ 

# ՁԽ  ՄՇՏ ԹՆԼ

ՌՀԽՊԽԾՇՊԽՆՇՌՁՆԻՄՍԼԽԼՁՆՌՀԽԻԹՄԻՍՄԹՌՁՇՆՇԾՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆԱՀԽՄՇՏԹՆԹՄՑՌՁԻԹՄՊԽՋՍՄՌՋ ՏԽՊԽԺԽՄՁԽՎԽԼՌՇԺԽԼՍԽՌՇՋԹՅՈՄՁՆԿՇՊԹՆԹՄՑՌՁԻԹՄԽՊՊՇՊԞԼԼՁՌ ՁՇՆԹՄՋԹՅՈՄԽՋՏԽՊԽԻՇՄՄԽԻՌԽԼԾՊՇՅՌՀԽ ՅԿԹՁԩՌՊԽԹՌՅԽՆՌՌՏՇԼԹՑՋՄԹՌԽՊ
ԡԹՑԹՆԼՏԽՊԽԻՇՆՋ ՁԼԽՊԽԼՌՇԺԽՊԽՈՊԽՋԽՆՌԹՌՁՎԽՇԾՌՀԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՁՆ ՌՀԽ ՌԽՋՌ ԻՀԹՅԺԽՊՋ ՇՆ ԡԹՑ  ԹՆԼ ՍՋԽԼ ՁՆ ՌՀԽ ԻԹ ՄԻՍՄԹՌՁՇՆ ՇԾ ՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺՑՌՀԽՋՌՍԼՑԹՍՌՀՇՊ԰ՁՆԻԽԹՆՇՆՋՈԽԻՁԾՁԻՊԹՌՁՇ ՆԹՄԽՏԹՋԿՁՎԽՆԾՇՊՌՀԽՄՇՏՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾՌՀԽՆՇՅՁՆԹՄՅԿԹՁԩՌՊԽԹՌՅԽՆՌՇՆԡԹՑ ՌՀԽՊԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼԹՅԽԹՆ ՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՇԾՅԿԹՁԩՍՋՁՆԿԡԹՑՋԹՅՈՄԽՋ

ԱՀԽՊԽՎՁԽՏԽՊ0ՋՊԽՋՍՄՌՋՏԽՊԽԺԹՋ ԽԼՇՆՊԽՈՄՁԻԹՌԽ ՅԽԹՆՋԹՆԼՅԽԼՁԹՆ ՋԻԹՄԻՍՄԹՌԽԼԺՑԽՐԻՄՍԼՁՆԿՌԹԼՈՇՄԽՋ ԹՌԫԣՋՌԹԿԽ  

# ԾՇՊԺՇԼՑՏԽՁԿՀՌՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀԹՆԼՀՁՆԼՄԽԿՄԽՆԿՌՀ
ՁՆԻՄՍԼՁՆԿՆՇՊՅԹՄՁՒԽԼՀՁՆԼ ՄԽԿՄԽՆԿՌՀ ԱՀԽՊԽԾՇՊԽՌՀԽՊԽՎՁԽՏԽՊ0ՋՊԽՋՍՄՌՋԹՊԽՊԽՈՇՊՌԽԼՁՆՌՀԽԢՐԽԻՍՌՁ ՎԽ԰ՍՅՅԹՊՑԹՆԼԠՇՆԻՄՍՋՁՇՆՋ

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ԤՍՁԼԽՄՁՆԽՋԬԭԭԱ԰ԫՍՅԺԽՊ ԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋ
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# ԬԢԠԡԤՍՁԼԽՄՁՆԽՋԾՇՊԱԽՋՌՁՆԿՇԾ
ԠՀԽՅՁԻԹՄՋԤՍՁԼԽՄՁՆԽ ԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ ԞԼՇՈՌԽԼ԰ԽՈՌԽՅԺԽՊ

ԨԽՆԼԹՄՄԱԷԹՆԼԴԟԫՁՐՇՆԞՆԹՄՑՌՁԻԹՄԪԽՌՀՇԼԳԽՊՁԾՁԻԹՌ ՁՇՆՋԾՇՊՌՀԽԡԽՌԽՊՅՁՆԹՌՁՇՆՇԾԪԹՄԹՌՀՁՇՆՁՆ ԣՊԽՋՀՏԹՌԽՊԴՁՄԼՄՁԾԽԦՆՌԽՊՆԹՌՁՇՆԹՄԩՌԼԭՊՇՂԽԻՌԫՍՅԺԽՊԠ ԲՆՈՍԺՄՁՋՀԽԼՊԽՈՇՊՌՈՊԽՈԹՊԽԼԾՇՊ ԠՀԽՅՁՆՇՎԹԞ԰ԧԹՆՍԹՊՑ

ԞԭԥԞԞԴԴԞԴԢԣ԰ՌԹՆԼԹՊԼԪԽՌՀՇԼՋԾՇՊՌՀԽԢՐԹՅՁՆԹՌՁՇՆՇԾ ԴԹՌԽՊԹՆԼԴԹՋՌԽՏԹՌԽՊՌՀ
ԢԼՁՌՁՇՆԞՅԽՊՁԻԹՆԭՍԺՄՁԻՀԽԹՄՌՀԞՋՋՇԻՁԹՌՁՇՆԞՅԽՊՁԻԹՆԴԹՌԽՊԴՇՊ ՃՋԞՋՋՇԻՁԹՌՁՇՆԴԹՌԽՊԢՆՎՁՊՇՆՅԽՆՌ ԣԽԼԽՊԹՌՁՇՆԴԹՋՀՁՆԿՌՇՆԡԠ

ԭԽՊՋՇՆԹՄԠՇՅՅՍՆՁԻԹՌՁՇՆԡՊԱՁՅՇՌՀՑ԰ՈՊՁՆԿԽՊԹՆԼԡՊԧՇՀՆԤՊԽԽՆ ԫՇՎԽՅԺԽՊԡՊԱՁՅՇՌՀՑ ԰ՈՊՁՆԿԽՊԴՁՄԼՄՁԾԽԦՆՌԽՊՆԹՌՁՇՆԹՄ ԩՌԼԢԹՋՌՇՆԪԡԲ԰Ԟ ԡՊԧՇՀՆԤՊԽԽՆԢԦԡՍՈԬՆՌԼԽԫԽՅՇՍՊՋ ԹՆԼԠՇՅՈԹՆՑԴՁՄՅՁՆԿՌՇՆԡԢԲ԰Ԟ

ԱՀԽ԰Ԟ԰԰ՑՋՌԽՅԾՇՊԴՁՆԼՇՏՋԳԽՊՋՁՇՆ԰Ԟ԰ԦՆՋՌՁՌՍՌԽԦՆԻԠԹՊՑԫՇՊՌՀԠԹՊՇՄՁՆԹ



 





 





 





 



   

  



























 

  



  

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# DATA EVALUATION RECORD

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

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012
(Revisions to MRID 48617503 to in clude Saturation binding data; Main study was originally reviewed under TA 2-05-2012)
Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer Signature: Sandra Hastings Date: 02/22/2012

Secondary Reviewer Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 2/23/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/26/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/26/2012

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).

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I 

# Page 1of17 MALATHION I 

# 057701


# Primary Reviewer: Kit Farwell, D.V.M. Health Effects Division, Office of Pesticide Programs Secondary Reviewer: Greg Akerman, Ph.D. Health Effects Division, Office of Pesticide Programs OCSPP 890.1150/ OECD None



# Signature:**_..;-::~,_.~::::::::=­** 

# Date:

_ __....,G3........_l

..... 

# 1r,o.-,μJ,-.r-:

__ Template version 08/2011



# DATA EVALUATION RECORD

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

PC CODE: 057701 DP BARCODE: D396178

TXR#: 0052348 CAS No.: 121-75-5

TEST MATERIAL (PURITY): Malathion (96.0% a.i.)

SYNONYMS: Fyfanon Technical; Butanedioic acid, ((dimethoxyphosphinoyhioyl) thio)-, diethyl ester

CITATION: Willoughby, J.A. (2011). Malathion: Androgen Receptor Binding (Rat Prostate Cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study Number: 9148V100372ARB, November 4, 2011. MRID 48617503. Unpublished.

Willoughby, J.A. (2012). Supplemental Information

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

SPONSOR: Cheminova A/S, P.O. Box 9, DK-7620 Lemvig, Denmark

TEST ORDER 

# #: EDSP-057701-44

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48617503), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct saturation and competitive binding experiments for malathion (96.0%, Batch# 9010501). The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radiolabeled reference androgen (Rl 881 ). The competitive binding assay was conducted to

measure the binding of a single concentration of [ 3 

# H]-Rl 881 (1 nM) 

# in the presence of increasing concentrations of malathion (logarithmic increase from 

# 10- 10 

# to 

# 10- 3 

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

Saturation binding data were not originally provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. The mean dissociation constant (Ki) 

# for [ 3 

# H]-R1881 was

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None


# 1.080±0.690 nM, and the estimated B max 

# was 1.071±0.167 fmol/100 μg protein for the single batch of prostate cytosol that was used in the assay. Only one of the three K d 

# values was within the range reported in the EPA validati on program (0.685 to 1.57 nM), and all B max 

# values were below the recommended range (7 to 16 fmol/100 μg protein). Although the goodness of fit for individual runs was acceptable (R 2 

# = 0.880-0.986), there was a large variation in K d 

# estimates among runs.
In the competitive binding experiment, malathion was classified as equivocal in the first two runs and a non-binder in the third run. Malathion re duced the mean specific binding of the ligand, [ 3 

# H]-R1881 to 51.6% and 50.4%, at 10 -3 

# M in the first two runs, re spectively. The results from these two runs were considered equivocal as the average specific binding at the lowest portion of the curve was between 50-75%. Precipitation was visually observed at the highest concentration tested (10

− 3 

# M) in Run 3; therefore, the highest con centration used for data evaluation in Run 3 was 10 − 4 

# M. The specific binding in the presence of malathion was consistently >98% at all concentrations ≤ 10 -4 

# M in all three runs.
The estimated log IC 50 

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

− 9.3 and

− 4.4 M, respectively. The mean RBA for the w eak positive control was 0.0022%. Confidence in these numbers is high due to the small variation. However, it is noted that the variations between Run 1 compared to Runs 2 and 3 are likel y due to the concentration differences used for R1881 and dexamethasone. The curve for dexameth asone was not fully defined in the first run and therefore the ca lculation for the IC 50 

# may be affected. The range of concentrations for R1881 in the first run fully defined the curve, bu t the concentration range was off by more than an order of magnitude.
The performance criteria were generally met; however, the bottom percent binding for dexamethasone was not achieved in the first r un. The concentrations prepared for R1881 and dexamethasone were lower than the targeted (guide line) levels because of an error in the stock preparation. Therefore, non- specific binding was not evaluated with 100X of cold (nonradiolabeled) R1881 but with 30X cold R1881, a nd the resulting curve for dexamethasone fit only two of the three criteria (H ill slope and Top plateau). All performance criteria were met for the other two runs.

Based on the results from the three runs, malathi on is classified as Equivocal 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 and Quality Assurance statements were provided.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc.
Location: 4717 Campus Drive, Kalamazoo, MI 49008 Study Director: Willoughby, J.A. Other Personnel: Rutherford, K. (Director Laboratory Operat ions); Blakeman, D. (Senior Scientist); Haines, C. (Scientist); McColley, S. (Scientist); Toole, C. (Director of Project Management) Study Period: September 10, 2011 to September 18, 2011



# 2. Test substance: Malathion
Description: Technical; pale yellowish liquid Source: Cheminova A/S Batch #: 9010501 (expiry date: December 11, 2011 Purity: 96.0% a.i. Solubility: Not reported Volatility: Not reported Stability: Not reported Storage conditions: 5 ± 5°C (refrigerator) CAS #: 121-75-5 Molecular weight: 330.36 g/mol Structure:



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


# 4. Radioactive ligand: [ 3 

# H]-R1881 Supplier: Perkin-Elmer (Boston, MA) Catalog and Lot #: NET590/ 653698 Date of production: February 24, 2011 Date of use: September 10, 2011 to September 17, 2011 Radiochemical purity: >97% Specific activity: 85.1 Ci/mmol Concentration of stock: 10 nM



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



# 6. Solvent/vehicle control: Dimethyl sulfoxide (DMSO)
Justification for choice of Pipetting and volatilization issues with ethanol so DMSO was chosen as the suitable

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None

solvent: vehicle Final Concentration: ~3.2% (v/v)



# B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: The rat ventral prostate tissue was purchased from Charles River La boratories (location not specif ied). Male Sprague Dawley rats (number not reported) were castrated at approximately 90 days of age and were euthanized <24 hours later and the ventral prostate tissues collected.

The ventral prostates were re ceived at the laboratory frozen and inspected for healthy appearance, weighed, and placed in ice-cold low-salt TEDG pH 7.4 buffer (inclusion of protease inhibitor not repor ted), homogenized, and centrifuged for 30 min at 30,000 × g at 4oC. Supernatant was pooled, discarding the resulting pellets. Protein concentration of the cytosol was determined to be 6.2 mg/mL us ing a commercially available protein kit compatible with DTT in the TEDG buffer (Bradford Method; Bio-Rad Dye Reagent Concentrate). Cytosol was divided into aliquot s (amounts not specified) and stored frozen (

− 80°C) until use.

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

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

TABLE 1. Summary of Conditions for Saturation Binding Experiment a 

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



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

# H]-R1881 (originally 85.1 Ci/mmol as manufactured on February 24, 2011) was adjusted for decay over time (adjusted specific activities were not reported), and seri al dilutions in low-salt TEDG+PMSF buffer were prepared to achieve th e final concentrations in cytosol of 0.25,0.50, 0.70, 1.0, 1.5, 2.5, 5.0, and 10 nM to determine total binding. To determine nonspecific binding, solutions of non-labeled R1881 were prepared in a similar manner to

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None



# achieve concentrations that were 100-fold greater than each re spective radiolabeled concentration, resulting in final concentr ations in cytosol of 25, 50, 70, 100, 150, 250, 500, and 1000 nM. In the absence of cytosol, th e radiation found in 7.5, 15, 21, 30, or 45 μL of 10 nM [ 3 

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

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

TABLE 2. Saturation Binding Experiment Run a ,b 

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

# Tubes 25-48 d 

# Tubes 49-72 e 

# [ 3 

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

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

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

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

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

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

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

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

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



# Following addition of triamcinolone acetonide, [ 3 

# H]-R1881, and/or R1881, the tubes were dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor for 20 hours at approximately 4°C. Samples were maintained at temperatures of approximately 4°C except during whole rack vortexing. To separa te bound from free R1881, hydroxyapatite (HAP) slurry was added to each tube and vortexed 5 times with 4-minute intervals over 20 minutes. The samples were then centrifuged, and the s upernatant was aspirate d and discarded. The samples were washed 4 times in 2 mL of ice cold TEDG+PMSF buffer, followed by vortexing and centrifugation for 3 minutes at 700 x g. Following the last wash and decanting of the Tris buffer pellets were then extracted by additional of 2 mL of ethanol. The samples were vortexed and centrifuged for 10 minutes at 1000 x g. Samples were

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None


# maintained on ice at all times between vortexing . After the final centrifugation, the ethanol supernatants were decanted into scintillation vials that each contained 14-mL portions of scintillation cocktail, and the radiation was quantified by liquid scin tillation counting. A total of three runs were performed.

3. Competitive Binding Experiment: A summary of the assay conditions for the competitive binding experiment is 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 8.7-9.7% of 1.0 nM radioligand b 

# Concentration of test substance (as serial dilutions) 10

− 10 

# to 10

− 3 

# M c 

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

# 0.5% (v/v) DTT 1 mM Sodium Molybdate 1 mM a Data were obtained from page 16 of the study report. b Values were calculated by the reviewer; data obtained from pages 54, 56 and 58 of the study report. Protein concentrations (μg/tube) were not reported. c The first two runs tested the test substance at 10

− 10 

# to 10

− 3 

# M, but in the third run the highest concentration precipitated, therefore only data for 10

− 10 

# to 10

− 4 

# M were evaluated. d The recommended PI in th e Guideline is 1 mM PMSF. The PI was identified in the study protocol as Calbiochem Protease Inhibitor Cocktail, Set III, EDTA fre e. It is a mixture of six PIs that includes 4-(2- aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBS F has similar specificity to PMSF with greater stability at lower pH.



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

# H]-R1881 (adjusted specific activity of 82.6 Ci/mmol for Run 1; 82.5 Ci/mmol for Runs 2 and 3) to the AR in the presence of increasing concentrations of a test substance.

DMSO was used as the vehicle, and “mild ” precipitation was visually observed at 10

− 3 

# M malathion only in the third run. Therefore, the highest concentration of malathion used in evaluating the data was 10

− 3 

# M for the first two runs and 10

− 4 

# M for the third run.

Little information for the saturation bi nding experiment was pr ovided; however, the reviewer was able to calculate from the comp etitive binding experiment that the amount of cytosolic protein used in the assay contained enough receptor to bind 9-10% of the [ 3 

# H]-R1881.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None



# Dilutions of the test subs tance, reference standard (R1881), weak positive control (dexamethasone), and solvent control (DMSO) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of th ree independent runs on three different days, and each run contained three replicates at each concentration plus si x replicates of the [ 3 

# H]-R1881, NSB, and solvent controls, resulting in a total of 84 samples per run. It is noted that in Runs 2 and 3 only three replicates of the [ 3 

# H]-R1881 were analyzed.

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

# Solvent Control Re ference standard c 

# Weak positive control c 

# Test Chemical None (Hot Mix) DMSO R1881 Dexamethasone Malathion Tubes 7-12 Tubes 13-18 and 19-33 d 

# Tubes 37-60 Tubes 61-84 e 

# Tubes 1-6 1×10

− 6 

# (3.3 ×10

− 8 

# ) 1×10

− 3 

# (1 ×10

− 4 

# ) 1×10

− 3 

# 1×10

− 7 

# (3.3 ×10

− 9 

# ) 1×10

− 4 

# (1 ×10

− 5 

# ) 1×10

− 4 

# 1×10

− 8 

# (3.3 ×10

− 10 

# ) 1×10

− 5 

# (1 ×10

− 6 

# ) 1×10

− 5 

# 1×10

− 9 

# (3.3 ×10

− 11 

# ) 1×10

− 6 

# (1 ×10

− 7 

# ) 1×10

− 6 

# 1×10

− 10 

# (3.3 ×10

− 12 

# ) 1×10

− 7 

# (1 ×10

− 8 

# ) 1×10

− 7 

# 1×10

− 11 

# (3.3 ×10

− 13 

# ) 1×10

− 8 

# (1 ×10

− 9 

# ) 1×10

− 8 

# -- 1×10

− 9 

# (1 ×10

− 10 

# ) 1×10

− 9 

# -- 1×10

− 10 

# (1 ×10

− 11 

# ) 1×10

− 10 

# a Data were obtained from pages 54-59 of the study report. b The [ 3 

# H]-R1881, NSB, and solvent controls were run in 6 replicates and each concentration of each chemical was run in triplicate for a total of 84 tubes per run. Tubes 1-84 contained 50 μL of triamcinolone acetonide and 30 μL [ 3 

# H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 7-84 also contained 10 μL of the solvent control, reference standard (n on-radiolabeled R-1881), weak positive control, or test substance, with the exception of Tubes 13-18 that contained 30 μL of non-radiolabeled R1881 (used to evaluate non-specific binding). Tubes 1-6 contained only 30 μl of [ 3 

# H]-R1881; Runs 2 and 3 only analyzed triplicate samples of [ 3 

# H]-R1881 (Tubes 1-3), but tube numbering was maintained as for Run 1. c Because of an error in the stock pr eparation, concentrations of R1881 and dexamethasone deviated from the targeted concentration in the first run. Actual concentrations for R1881 and dexamethasone for the first run are reported in parentheses. c Tubes 13-18 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881, except for Run #1 in which 30X of cold (non-radiolabeled) R1881 was added due to an error in the stock preparation. d Malathion was tested at 10

− 10 

# to 10

− 3 

# M in all of the runs; however, data for 10

− 3 

# M from the third run were excluded from evaluation because precipitation was visually observed.



# Sample tubes were stored 16-20 hours at 4°C to allow the reaction to reach equilibrium, and bound R1881 was separated from free R1881 by wa shing with HAP buffer and extraction with ethanol, followed by scintillation counting of bound [ 3 

# H]-R1881. A total of 3 runs were performed as described.

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

# implemented by using the ROUT procedure in Prism v. 5 with a Q value of 1.0. Receptor binding data plots were corrected for ligand depletion with the method of Swillens (1995) 2 

# . For the competitive binding

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

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None


# assay, similar methods of nonlinear regression were used to fit a curve (for R1881, the positive control, and the test substance) to th e Hill equation formula which incorporated log IC 50 

# as a parameter to be estimated (XLfitTM Model 204; IDBS, Guildford, UK). The top and bottom of the curve, Hill slope, inhibition concentration (IC 50 

# ), and standard deviations were also assessed using XLfitTM. For para meters reported from the saturation binding experiment (K d 

# and B max 

# ) and competitive binding experiment (log IC 50 

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

5. Definitions

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

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

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

− 0.6 to

− 1.4).

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

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

− 6 

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

b. Descriptors for receptor binding

B max 

# : maximal binding capacity K d 

# : dissociation constant IC 50 

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

# Relative Binding Affinity (RBA): IC 50 

# of R1881 × 100 ÷ IC 50 

# of test substance Log RBA: Log 10 

# ( IC 50 

# of R1881 × 100 ÷ IC 50 

# of test substance)

II. RESULTS

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

# for [ 3 

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

# was 1.071±0.167 fmol/100 μg protein for th e single batch of prostate cytosol that was used in the assay. Only one of the three individual K d 

# values was within the range

computer analysis. Molec. Pharmacol. 47(6):1197-1203.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None


# reported in the EPA validation program (0.685 to 1.57 nM); the mean value was above the acceptable range. Although the goodness of fit (R 2 

# = 0.880-0.986) was acceptable, there was a large variation in Kd estimates among runs.

TABLE 5. Saturation Binding Experiment of [ 3 

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

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

# R 2 

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

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

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

# (nM) 0.855 0.531 1.855 1.080±0.690 a Data were obtained from page 4 of the study report (no MRID). b The range of R 2 

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

# Goodness of fit for curve calculated for specific binding.



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

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

# H]-R1881 to the androgen receptor.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None


# FIGURE 1. Binding of [ 3 

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

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None



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

# H]-R1881 to the Androgen Receptor.

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None



# B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive binding experiments are summarized in Table 6 and shown graphically in Figures 3-5. Precipitation of malathion was visually observe d at the highest con centration tested (10

− 3 

# M) in the third run; therefore, the highest concentration used for data evaluation in the third run was 10 − 4 

# M malathion. Malathio n reduced (51.6% and 50.4%) the mean specific binding of the ligand, [ 3 

# H]-R1881, at 10 − 3 

# M in the first two runs. The specific binding in the presence of malathio n was consistently >98% at all concentrations ≤ 10 − 4 

# M in all three runs. Malathion is classified as “equivocal” as the average specific binding for [ 3 

# H]-R1881 at the lowest portion of the curve was between 50-75% for the three runs.
The estimated average log IC 50 

# s for R1881 and the weak positiv e control (dexamethasone)

were
− 9.3 and

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

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

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

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

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

# (M) R1881

− 9.9

− 9.0

− 9.0

− 9.3±0.30 b 

# Dexamethasone -4.3 -4.4 -4.6 -4.4±0.09 b 

# Malathion NA NA NA NA IC 50 

# (M) b 

# R1881 1.26 × 10

− 10 

# 1.00 × 10

− 9 

# 1.00 × 10

− 9 

# 7.09 × 10

− 10 

# (±2.91) Dexamethasone 5.01 × 10

− 5 

# 3.98 × 10

− 5 

# 2.51 × 10

− 5 

# 3.83 × 10

− 5 

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

# Dexamethasone

− 5.6

− 4.6

− 4.4

− 4.87±0.37 Malathion NA NA NA NA RBA (%) b 

# Dexamethasone 0.0003 0.0025 0.0040 0.0022±0.0011 Malathion NA NA NA NA a Data were obtained from the results discussion, page 22 of the study report. b Values are reviewer-calculated from the log IC50 values reported in the study report. For means expressed in scientific notation, the SE values in parentheses are presented in the same order of magnitude as the mean value. NR Not reported NA Not applicable R 2 

# Goodness of fit RBA Relative binding affinity

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None


# FIGURE 3. Percentage [ 3 

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

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None


# FIGURE 4. Percentage [ 3 

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

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 15 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None


# FIGURE 5. Percentage [ 3 

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

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 16 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None



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

TABLE 7. Criterion a 

# Tolerance Limit(s) b 

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

# H]-R1881 added per assay tube. ≤ 15% 8.7-9.7% X

Malathion Top (% binding) 80 to 115 105.9 to 112.9 X R1881 fitted curve parameters Top (% binding) 82 to 114 96 to 103 X Bottom (% binding)

− 2.0 to 2.0 0 X Hill Slope

− 1.2 to

− 0.8

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

− 12 to 12 NA; -1 to 7 c 

# X Hill Slope

− 1.4 to

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

# (nM) 0.685 to 1.57 nM 0.531 to 1.855 d 

# X e 

# Non-specific binding (%) ≤ 10.0 0.28-0.50 X a Data were obtained from page 25, 27, and 29 of the study report. b These values represent ranges from the validation study. c The bottom plateau level for dexamethasone in Run #1 was reported as NA (not applicable) because lower concentrations were used due to an error in the stock preparation. d MRID 48843501 e One value was below the limit, one value was within the limit, and one value was above the limit. NR Not reported


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

# H]-R1881 in a manner consiste nt with one-site binding, as indicated by a 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 for R1881.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Malathion was classified as “equivocal” in the first two valid independent runs and classi fied as a “non-binder ” in the third valid independent run. Thus, malathion has a final cl assification of “equivocal” for binding to the AR.

B. AGENCY COMMENTS: The mean K d 

# for [ 3 

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

# was 1.071 fmol/100 μg protein for the si ngle batch of prostate cytosol that was used in the assay. Only one of the three individual K d 

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

# Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 17 of 17 MALATHION / 057701 OCSPP 890.1150/ OECD None


# acceptable range. Confidence in these numbers is low based on the la rge variation in K d 

# estimates among runs.
In the competitive binding experiment, malathion was classified as equivocal in the first two runs and a non-binder in the th ird run. Malathion reduced th e mean specific binding of the ligand, [ 3 

# H]-R1881 to 51.6% and 50.4%, at 10 -3 

# M in the first two runs, respectively. The results from these two runs were considered e quivocal as the average sp ecific binding at the lowest portion of the curve was between 50-75%. Precipitation was visu ally observed at the highest concentration tested (10

− 3 

# M) in Run 3; therefore, the highest concentration used for data evaluation in Run 3 was 10

− 4 

# M. The specific binding in the presence of malathion was consistently >98% at all concentrations ≤ 10 -4 

# M in all three runs. Based on the results of the three runs, malathion is classified as equivocal for AR binding in this assay.
In the competitive binding experiment, the estimated average log IC 50 

# was
− 9.3 M for R1881 and

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

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

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

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

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

# DATA EVALUATION RECORD

MALATHION
Study Type: OCSPP 890.1200; Aromatase Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-5-2012 (MRID 48617504)

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: David A McEwen, B.S. Date: 2/02/2012
Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 2/22/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 2/24/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 2/24/2012

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).

# MALATHION I 

# 057701 Aromatase (Human Recombinant) Assay (2011) I 

# Page 1 of 13 OCSPP 890.1200 I 

# OECD None


# Primary Reviewer: Vincent Chen, M.S. Signature:**~.::::::::~~-=::::::::...** 

# __ Health Effects Division (7509P) Date: 'Zt.t 

# 'J.J"'1 

# Z..c.J.l-S Secondary Reviewer: Greg Akerman, Ph.D. Signature:

# -~4~.....:~~:====-- Risk Assessment Branch III, Health Effects Division (7509P) Date:

_ __,CA__,.i_;=-t:T

...... h..._.u..__:

__ Template version 08/2011



# DATA EVALUATION RECORD

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200

PC CODE: 057701 DP BARCODE: D396178

TXR: 0052348 CAS: 121-75-5

TEST MATERIAL (PURITY): Malathion (96% a.i.)

SYNONYMS:

IUPAC: diethyl ( dimethoxyphosphinothioylthio )succinate CAS: diethyl 2-[ ( dimethoxyphosphinothioyl)thio ]butanedioate

CITATION: Wilga, P.C. (2011) Malathion: Human Recombinant Aromatase Assay. CeeTox, Inc [4717 Campus Drive, Kalamazoo, MI 49009, US]. Laboratory Study No.: 9148V-100372AROM, 03 November 2011. MRID 48617504. Unpublished.

SPONSOR: Cheminova 

# NS, P.O. Box 9, DK-7620 Lemvig, Denmark

TEST ORDER: EDSP-057701-44

EXECUTIVE SUMMARY: In an 

# in vitro aromatase (CYP 19) assay (MRID 48617504), malathion (96% a.i., Batch#: 9010501) in dimethyl sulfoxide (DMSO; :Sl %) was incubated with human recombinant aromatase and tritiated androstenedione**([1~- H(N)]-androst-4-ene-3,17-**  

# dione; [ 3 

# H]-ASDN) for 15 minutes at 37°C to assess the potential of malathion to inhibit
aromatase activity. Malathion was tested at logarithmic concentrations from 10- 10 

# M to 10- 3 

# M (Run 1) and from 10- 9 

# M to 10- 3

- 5 

# M (Runs 2 and 3), due to precipitation observed at 10- 3 

# Min Run 1.

Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation period for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC). Three independent runs were conducted, and each run included a full activity control, a background activity control, a positive control series (10- 10 M to 10- 5 

# M) using a known inhibitor (4-hydroxyandrostenedione; 4-0H ASDN), and the test chemical series (10- 10 M to 10- 3 

# M) with 3 repetitions per concentration.

Aromatase activity in the full activity controls ranged from 0.170 to 0.23 7 nmol ·mgprotein-1 ·min-1 for the three test runs, with a mean and standard deviation of0.198 ± 

# 0.024 nmol·mg-protein- 1 ·min- 1

. Activity in the background controls ranged from 0.55% to 1.8% and

# Aromatase (Human Recombinant) A ssay (2011) / Page 2 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None


# averaged 0.75% of the full activ ity controls. The response of the full activity controls and background controls was acceptable for each run.
Results for the positive control were generally within the recommended ra nges for the top of the curve, bottom curve, hill slope, log IC 50 

# , and coefficient of variati on for replicates of each concentration within runs. Also, the coefficien ts of variations (%CVs) for replicates of each concentration of malath ion within a run were within the 15% guideline. For 4-OH ASDN, the estimated log IC 50 

# averaged –7.16 M, and the slope was –1.01.
For malathion, aromatase activity av eraged 0.170 ± 0.066 nmol·mg-protein

− 1 

# ·min

− 1 

# at the lowest tested concentration of 10 –9 

# M (the lowest concentra tions tested from Run 1, 10 –9 

# and 10 –10 

# , wereexcluded from analysis because of low % control values) and 0.133 ± 0.010 nmol·mgprotein -1 

# ·min

− 1 

# at the highest successfully tested concentration of 10

− 3.5 

# M (highest nonprecipitating dose). Precipitati on was observed in Run 1 at 10

− 3 

# M. The data for malathion were not suitable for modeling. The mean aromatase activity of 10 –3.5 

# M malathion was 64.6% of control activity, and the be st fit mean inhibition response curve was a straight line (essentially no inhibition was observed at other doses). The re sponse for malathion was considered equivocal since the average lowest porti on of the response curve across runs was between 50 and 75% activity.
Based on the data from the average response curv e, malathion is classified as Equivocal for aromatase inhibition in this assay.
The assay satisfies the EDSP Tier 1 Test Order requireme nts for an Aromatase assay (OCSPP 890.1200).

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

# Aromatase (Human Recombinant) A ssay (2011) / Page 3 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Malathion Description: Clear, colorless liquid Source: Cheminova A/S Batch # (Expiration Date): 9010501 (December 11, 2011) Purity: 96% a.i. Storage Conditions: –80°C Stability: Not reported Solvent: DMSO Solubility: Visually soluble at 10

− 3.5 

# M in 2 of 3 test runs. Highest Concentration Tested: 10

− 3 

# M; however, precipitation was obs erved at this dose in Run 1 Stock Solution Preparation: Not reported Molecular Weight: 330.4 g/mol CAS #: 121-75-5 Structure:

O O O

O S CH 2 

# CH 3 

# CH 2 

# CH 3 

# P O

O CH 3



# CH 3 

# S



# 2. Non-Labeled Substrate: Androstenedione (ASDN) CAS #: 63-05-8 Source: Steraloids, Inc. (Cat. # A6030-100) Batch # (Expiration Date): L1712 (April 2016) Purity: 99.8%



# 3. Radiolabeled Substrate: [1 β

- 3 

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

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



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


# 5. Solvent (Vehicle Control): DMSO Source: Sigma-Aldrich (Cat. # D2650) Batch # (Expiration Date): RNBB7617 (February 2013) Justification for Choice of Solvent: Acceptable vehicle by OCSPP guideline 890.1200 Concentration (% of Total Volume in Assays): ≤ 1% v/v



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

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

# Aromatase (Human Recombinant) A ssay (2011) / Page 4 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None



# 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 approximately 0.1 μ Ci.
Test chemical(s) were formulated in DMSO su ch that the total volum e of DMSO used per assay was no more than 1% v/v of the total assay volume to minimize the potential for the solvent to inhibit the enzyme. The report spec ified that DMSO was chosen over ethanol as the solvent of choice for the following reasons: 1) DMSO was listed as one of the vehicles acceptable for use in OCSPP 890.1200 guideline; 2) DMSO was not as volatile as ethanol and so evaporation was less of a concern in the assay, and 3) DMSO was more accurate to pipette because of density and viscosity.
A stock solution of the positive control substance, 4-OH ASDN, was formulated in DMSO. Fresh dilutions of the stock solution were prepar ed in the same solvent as the stock solution on the day of use. Dilutions were prepared such that the target concentrations of the positive control substance (10 –10 

# M to 10 –5 

# M; Table 4) were achieved by the addition of 20 μL of the dilution for a final assay volume of 2 mL.
Human recombinant microsomes were purchased from BD Biosciences, and stored at -80 ± 10oC for no longer than 7 months for this study. Before use of the microsomes, the batch was thawed and sub-aliquoted at 0.008 mg/mL into individual vi als before being refrozen to minimize freeze-thaw cycles to no more than one.
Other assay components sodium phosphate buffer, propylene glycol, and NADPH are reported in Table 1.

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

# H]ASDN 100 nM Propylene glycol 5% Temperature 37±2°C Incubation time 15 min



# 2. Suitability Assessments: The protein concentration of the microsomes (Lot # 03897) was supplied by the vendor as 7.4 mg/mL. Aromat ase activity of the microsomes was also provided by the vendor as 6.0 pmol/min/pmol P450. The minimum aromatase activity in the full activity control samples was determ ined to be 0.170 nmol/mg protein/min, which was greater than the minimum recomme nded aromatase activity of 0.1 nmol/mgprotein/min.

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

# Aromatase (Human Recombinant) A ssay (2011) / Page 5 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None


# 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 nmol·mg-protein –1 

# ·min –1



# . 4. Demonstration of Proficiency: No information was provided c oncerning demonstration of proficiency. However, the report stated that prior to conducting this assay using malathion; a full-scale assay consisting of three indepe ndent runs was conducte d using the positive control (4-OH ASDN), econazole, fenarimol, nitrofen, and atrazine. Proficiency was demonstrated when the positive control met the performance criteria from the guideline and by the correct classification of the four profic iency chemicals in Table 6 of the guideline. Data was generated by CeeTox a nd is maintained at CeeTox.

a. Positive Control: Data were not provided.

(1) Initial Demonstration of Laboratory Proficiency: The positive control data (new/historical data for laborator y) were not reported in rega rds to the following criteria:

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

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

 Coefficient of variation (% CV) for repl icates within each sample type and concentration of 4-OH ASDN was <15%.
Performance criteria were reported for the test runs only (Table 2), and served as guidance in identifying runs that provided parameters in the preferred ranges.

(2) Demonstration of Proficiency of Ne w Technician for Conducting Assay (when applicable): Demonstration of proficiency by a ne w technician, if applicable, was not reported.

TABLE 2. Performance Criteria for the Positive Control Parameter Lower Limit Criteria Upper Limit Crit eria Actual Lower Limit Actual Upper Limit Slope –1.2 –0.8 –1.03 –0.98 Top (%) 90 110 100.56 106.40 Bottom (%) –5 +6 0.86 1.25 Log IC 50 

# (M) –7.3 –7.0 –7.20 –7.10 a Data were obtained from page 30 of the study report.

# Aromatase (Human Recombinant) A ssay (2011) / Page 6 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None



# b. Proficiency Chemicals: Data were not provided.

TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations Econazole 24169-02-6 Inhibitor 

# Not reported Fenarimol 60168-88-9 Inhibitor 

# Not reported Nitrofen 1836-75-5 Inhibitor 

# Not reported Atrazine 1912-24-9 Non-inhibitor 

# Not reported



# 5. Determination of Aromatase Ac tivity with Test Chemical: The response of aromatase activity to the presence of eight concentrations of malathion per run, in triplicate, was tested during three independent runs. Solubility was visually assess ed (presence of cloudiness or a precipitate). Precipita tion was observed in Run 1 (small amount) at 10 –3 

# M, but the test compound was soluble at 10 –4 

# M in all runs. The full enzymatic activity ( ≥ 95% for the means of each run) was obtained at the two lowe st concentrations of malathion, defining the top of the concentration-response curve.

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

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

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

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

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

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

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

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

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

# Malathion Conc 1 b 

# 3 All test components plus malathion 1×10 –3 

# Malathion Conc 2 b 

# 3 All test components plus malathion 1×10 –3.5 

# Malathion Conc 3 3 All test components plus malathion 1×10 –4 

# Malathion Conc 4 b 

# 3 All test components plus malathion 1×10 –4.5 

# Malathion Conc 5 3 All test components plus malathion 1×10 –5 

# Malathion Conc 6 3 All test components plus malathion 1×10 –6 

# Malathion Conc 7 3 All test components plus malathion 1×10 –7 

# Malathion Conc 8 3 All test components plus malathion 1×10 –8 

# Malathion Conc 9 3 All test components plus malathion 1×10 –9 

# Malathion Conc 10 c 

# 3 All test components plus malathion 1×10 –10 

# a The complete assay contained bu ffer, propylene glycol , microsomal protein, [3H]ASDN, and NADPH. b The 10

− 3 

# M concentration of malathion was used only in Run 1 due to observed precipitation, and Runs 2 and 3 included concentrations of 10

− 3.5 

# and 10

− 4.5 

# M. c Malathion concentration 10

− 10 

# M was used only Run 1.



# C. DATA ANALYSIS

1. Raw Data: Raw data were converted to aromatas e activity (nmol/mg protein/min) 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 (2011) / Page 7 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None

TABLE 5. Raw and Calculated Data Raw/Calculated Data Included (X) DPM/mL for each portion of extract ed aqueous incubation mixture X Average DPM/mL for each aqueous portion (after extraction) X Total DPM for each aqueous portion (after extraction) X The total DPM present in the assay tube at initiation X The percentage of substrate converted to product X Total DPM after extraction corrected for background X Aromatase activity expressed in nmol/mg protein/min X Average aromatase activity in the full activity control tubes X Percentage of control activity remaining in the presence of various inhibitor concentrations X DPM Disintegrations per minute



# 2. Statistical Methods: Statistical analyses and graphica l displays were conducted using IDBS XLfit v5.2.0.0 (Guildford, UK). Basic stat istical analyses were performed on the data, which included means of replicates, standa rd deviation of the mean, standard error of the mean, and coefficient of variation. An alysis of variance (ANOVA) was not performed.
The response curve was fitted by weighted leas t squares nonlinear regression analysis with weights equal to 1/Y. Model fits were carried out using a 4-parameter regression model (XLfit v5.2.0.0, Fit Model 208) and Tukey’s Bi-Wei ght statistical analysis for outlier analysis. For each run, percent of control activity was plotted ve rsus the log (base 10) of the malathion concentration. Each run was plotted with the data’s best fit curve. Additionally, the average inhibition response curve across all runs was also plotted.

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

TABLE 6. Interpretation of Results Criteria Interpretation Data fit 4-parameter nonlinear regression model Average curve across runs crossed 50% 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 solubil ity 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 0.170 to 0.237 nmol·mg-protein

− 1 

# ·min

− 1 

# for the three test runs, with a mean and standard deviation of 0.198 ± 0.024 nmol·mg-protein

− 1 

# ·min

− 1 

# . Activity in the background controls ranged from 0.55 to 1.8% and averaged 0.75% of th e full activity controls. The response of each full activity control within a run was betw een 90 to 110% of the average full activity.

B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.203 ± 0.027 nmol·mg-protein

− 1 

# ·min

− 1 

# at the lowest tested concentration (10

− 10 

# M) and 0.002 ± 0.001 nmol·mg-protein

− 1 

# ·min

− 1 

# at the highest tested concentration (10

− 5 

# M). The mean aromatase activity of the positive control (expressed as % full control

# Aromatase (Human Recombinant) A ssay (2011) / Page 8 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None


# activity) for each concentration tested across a ll three runs is presented in Table 7, along with the overall standard deviation, standard error of the mean, and % CV. Inhibition response curves for the positive control from each run are shown in Figure 1. These results were within the recommended ranges for the top of the curve, bottom curve, hill slope, log IC 50 

# , and coefficient of variation for replicates of each concentration within runs. The coefficient of variations (CVs) for replicates within each concentration of 4-OH ASDN and malathion were within the 15% guideline, with the exception of one concentration of 4-OH ASDN (10

− 7.5 

# M; 17% CV) in Run 2.

TABLE 7. Effect of Malathion on Aromatase Activi ty (as Percent of Control) from Independent Runs a 

# Chemical Conc. Log M # Runs Overall Mean Overall SD Overall SEM Overall %CV 4-OH ASDN (positive control) –5 3 0.91 0.18 0.07 19.4 –6 3 8.07 0.54 0.22 6.7 –6.5 3 20.27 1.43 0.58 7.1 –7 3 41.55 1.58 0.64 3.8 –7.5 3 74.77 7.62 3.11 10.2 –8 3 90.86 2.63 1.07 2.9 –9 3 101.44 4.51 1.84 4.4 –10 3 102.31 1.68 0.69 1.6 Malathion –3 1 31.37 2.50 1.02 8.0 –3.5 2 64.58 3.88 1.58 6.0 –4 3 74.82 8.65 3.53 11.6 –4.5 2 95.95 2.53 1.03 2.6 –5 3 97.39 4.79 1.96 4.9 –6 3 99.60 3.93 1.60 3.9 –7 3 102.74 3.84 1.57 3.7 –8 3 101.59 4.76 1.94 4.7 –9 b 

# 2 103.15 2.83 1.16 2.7 –10 b 

# 1 ND ND ND ND a Values were calculated by the reviewers based on da ta provided on pages 42, 44 and 46 of the study report. b The values for malathion at 10

− 9 

# and 10

− 10 

# M (46.6 and 50.8% VC) in Run 1 were excluded as outliers. SD Standard Deviation SEM Standard Error of the Mean CV Coefficient of Variance ND No Data

# Aromatase (Human Recombinant) Assay (2011) / Page 9 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None


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

# Aromatase (Human Recombinant) A ssay (2011) / Page 10 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None



# C. TEST SUBSTANCE: For malathion, aromatase activity averaged 0.170 ± 0.066 nmol·mg-protein

− 1 

# ·min

− 1 

# at the lowest tested concentration of 10 –9 

# M (the lowest concentrations tested from Run 1, 10 –9 

# and 10 –10 

# , were excluded from analysis because of low % control values) and 0.133 ± 0.010 nmol·mg-protein –1 

# ·min

− 1 

# at the highest successfully tested concentration of 10

− 3.5 

# M (highest non-precipitati ng dose). Precipitation was observed in Run 1 at 10

− 3 

# M. The mean aromatase activity of malathion (expressed as % full control activity) for each concentration tested across all three runs is provided in Table 7 (presented above), along with the overall standard deviation, standard error of the mean, and %CV. Inhibition response curves for malathion from each run are shown in Figure 2, and the average inhib ition response curve across all runs is shown in Figure 3.

# Aromatase (Human Recombinant) A ssay (2011) / Page 11 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None


# FIGURE 2. Inhibition Response Curves for Malathion.

# Aromatase (Human Recombinant) A ssay (2011) / Page 12 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None


# FIGURE 3. Mean Inhibition Response Curve for Malathion.

The data for malathion were not suitable fo r modeling. The mean aromatase activity of 10 -3.5 

# M malathion (highest soluble concentratio n tested) was 64.58% of control activity, and the best fit mean inhibition response curve (Figure 3) was a strai ght line (essentially no inhibition was observed at other doses). The effect of the positive control on inhibition of aromatase activity is presented in Table 8. For 4-OH ASDN, the estimated log IC 50 

# averaged –7.16 M, and the slope was –1.01. C onfidence in these values is very high due to the small variation (<3% CV).

TABLE 8. Effect of Malathion and 4-OH ASDN on Aromatase Activity (as Percent of Control) from Independent Runs a 

# Chemical Run 1 Run 2 Run 3 Mean SD %CV Log IC 50 

# (M) Malathion NA NA NA NA NA NA 4-OH ASDN –7.10 –7.18 –7.20 –7.16 0.05 –0.74 Hill Slope Malathion NA NA NA NA NA NA 4-OH ASDN –1.01 –1.03 –0.98 –1.01 0.03 –2.50 a Data were provided on page 30 of the study report. Mean, SD, and %CV were calculated by the reviewers based on the data from the individual runs. SD Standard Deviation CV Coefficient of Variance NA Not applicable. Values for Mala thion were not suitable for modeling.



# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: Malathion was not determined to be an inhibitor of aromatase activity, but was classified as “equivocal” in its response as defined by the Guideline OCSPP 890.1200. Although a decrease in aromatase activity was identified at the very highest concentration of malathion tested (e.g., 10 -3 

# M) in the first run, solubility issues were observed so this concentration was not included in the data interpretation. In subsequent runs of the assay, 10 -3.5 

# M malathion reduced aromatase activity, but not to a level that would classify this compound as an inhibitor of aromatase activity.

# Aromatase (Human Recombinant) A ssay (2011) / Page 13 of 13 MALATHION / 057701 OCSPP 890.1200 / OECD None



# B. AGENCY COMMENTS: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.203 ± 0.027 nmol·mg-protein

− 1 

# ·min

− 1 

# at the lowest tested concentration (10

− 10 

# M) and 0.002 ± 0.001 nmol·mg-protein

− 1 

# ·min

− 1 

# at the highest tested concentration (10

− 5 

# M). These results were within the reco mmended ranges for the top of the curve, bottom curve, hill slope, log IC 50 

# , and coefficient of variation for replicates of each concentration within runs. The coefficient of variations (CVs) for replicates within each concentration of 4-OH ASDN and malathion we re generally within the 15% guideline.
For malathion, aromatase activity av eraged 0.170 ± 0.066 nmol·mg-protein

− 1 

# ·min

− 1 

# at the lowest tested concentration of 10 –9 

# M (the lowest concentrat ions tested from Run 1, 10 –9 

# and 10 –10 

# M, were excluded from analysis because of low % control values) and 0.133 ± 0.010 nmol·mg-protein –1 

# ·min

− 1 

# at the highest successfully tested concentration of 10

− 3.5 

# M (highest non-precipitating dose). Precipitation was observed in Run 1 at 10

− 3 

# M. For 4-OH ASDN, the estimated log IC 50 

# averaged –7.16 M, and the slope was –1.01. Confidence in these values is very high due to the small variation (<3% CV).

The data for malathion were not suitable for modeling. The mean aromatase activity of the highest soluble c oncentration (10 –3.5 

# M) malathion was 64.58% of control activity, and the best fit mean inhibition response curve was a straight line (essentia lly no inhibition was observed at other doses). The response for malathion was considered equivocal since the average lowest portion of the response curve across runs was between 50 and 75% activity.

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

 Proficiency chemical da ta were not provided.

 Information on the stability of th e stock solution was not reported.

# DATA EVALUATION RECORD

MALATHION
Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012
(Revisions to MRID 48617505 to include laboratory proficiency data; Main study was originally reviewed under TA 2-5-2012)

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: 7/10/2012
Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 7/16/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7/24/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 7/24/2012

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).

# MALATHION I 

# 057701 Estrogen Receptor Transcriptional Activation (2011) I 

# Page 1 of 10 OCSPP 890.1300/ OECD 455


# Primary Reviewer:**~Ki=·~t=F-=arw~e=ll=·-=D~.V~.M~.** 

# _______ _ 

# Health Effects Division Date:**--= .l.4--(9c....;...1.r.-~'-­**  

# Secondary Reviewer: Minerva Mercado-Feliciano PhD DABT**Signature' ~~~** **~ifC.==-­** 

# Health Effects Division Date:**-L .1.-** **1-L~~- --** 



# DATA EVALUATION RECORD

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



# PC CODE: 057701 

# DP BARCODE: Not Provided

TXR#: Not provided 

# CAS No.: 121-75-5



# TEST MATERIAL (PURITY): Malathion 96.0% a.i.



# SYNONYMS: Butanedioic acid, ((dimethoxyphosphinothioyl) thio)-, diethyl ester



# CITATION: Willoughby, J.A. (2011) Malathion: estrogen receptor transcriptional activation (human cell line (HeLa-9903)). CeeTox, Inc., Kalamazoo, ML Laboratory Study No.: 9148V-100372ERTA, November 4, 2011. MRID 48617505. Unpublished.



# SPONSOR: Willoughby, J.A. (2012). Supplemental Information

- Laboratory Proficiency Data for ERT A assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. CeeTox, Inc., Kalamazoo, Ml. July?? 2011. MRID 48843501. Unpublished.

Cheminova A/S, P.O. Box 9, Lemvig, Denmark



# TEST ORDER#: EDSP-057701-44



# EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID 48617505), hERa-HeLa-9903 cells cultured 

# in vitro were exposed to malathion (96.0% a.i., Batch# 9010501) at logarithmically increasing concentrations from 10- 11 

# to10-4 M in 0.1% DMSO for 24±2 hours. The experiments were performed using 96-well plates and each malathion concentration was tested in 6 wells/plate. Cells were exposed to the test agent for 24±2 hours to induce reporter (luciferase) gene products. Luciferase expression in response to activation of the estrogen receptor was measured upon addition of a luciferase substrate and detection with a luminometer.

Malathion was tested up to the limit of solubility, which was 10- 5 

# M. The mean RPCMax for malathion was 3.4% for the first run and 3.7% for the second run, and the associated PCMax was 

# 10- 1 

# M for both runs.

# Estrogen Receptor Transc riptional Activation (2011) / Page 2 of 10 MALATHION / 057701 OCSPP 890.1300/ OECD 455


# In the main assays, the responsiveness of th e cells to the very weak positive control 17 α

methyltestosterone was lower than the expected values, indicating a decrea sed sensitivity of the assay to very weak agonists. Although the conditi ons of this assay were not optimal to detect very weak activity, malathion responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 6.5-37.4% PC. Malathion wa s only able to reach a maximum of 3.4-3.7% PC when tested up to the highest concentrati on possible based on cytotoxicity. Because the RPC Max 

# < PC 10 

# in both assay runs, malathion was cons idered negative for estrogen receptor transcriptional activation in this test system.
This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor Transcriptional Activation assay (OCSPP 890.1300).

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

# Estrogen Receptor Transc riptional Activation (2011) / Page 3 of 10 MALATHION / 057701 OCSPP 890.1300/ OECD 455



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc. Location: Kalamazoo, MI Study Director: J.A. Willoughby Other Personnel: K. Rutherford (Director Laboratory Operations); D. Blakeman (Senior Scientist); C. Haines (Scientist); S. McColley (Scientist); B. Wallace (Lead Cell Culture Scientist); and C. Toole (Director of Project Management) Study Period: May 23, 2011 to November 4, 2011



# 2. Test Substance: Malathion Description: Pale yellowish liquid, MW 330.36 Source: Cheminova A/S (no catalog #) Lot/Batch #: 9010501 Exp December 11, 2011 Purity: 96.0% Solubility: NR Volatility: NR Stability: NR Storage conditions: 5±5 ° C CAS #: 121-75-5 Structure:



# 3. Reference substances 17 β -estradiol (strong estrogen; positive control) Supplier: Not reported (NR) Catalogue and Batch #: NR Purity: NR CAS # : 50-28-2



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

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



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


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

# Estrogen Receptor Transc riptional Activation (2011) / Page 4 of 10 MALATHION / 057701 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 Biores ources Cell Bank and verified to be free of mycoplasma infection using a DNA fluorochrome assay. Ce lls were maintained in Eagles Minimum Essential Medium without phenol red, supplem ented with 60 mg/L kanamycin and 10% dextran-coated charcoal-treat ed fetal bovine serum (source not reported), in an incubator under 5% CO 2 

# at 37 oC. Upon reaching 75-90% conflu ence, cells were subcultured 5 times prior to exposure to test material. Cells used in the range-finding assa y were at passage 35. New cells at passage 14 were brought out of cr yopreservation and us ed at passages 19 and 20 for the main assay.
2. Transcriptional Activation Assays: For each test, cells were plated at a density of 1×10 4 

# cells/100 μL medium/well in a 96 well plate and a llowed to attach for 3 hours (estrogenicity of 96-well plate not reported). Growth media was replaced with media containing serial log dilutions of test agent in DMSO (0.1% final concentration). Cells were incubated for 24±2 hours at 37±2 °C. Cytotoxicity was determin ed by a two-read propidium iodide (PI) uptake assay. Runs 1 and 2 for the main assay were conducted on August 23 and 25, 2011, respectively. Transcriptional activation of the estrogen receptor was determined using a proprietary CeeTox Standard Op erating Protocol (SOP). A list of reagents was provided, but the assay reagent was liste d as proprietary information, and the SOP was not provided.
a. Preliminary Test: A preliminary test at c oncentrations ranging from 10

− 3 

# to 10 − 10 

# M was conducted to determine concentrations resulti ng in insolubility and/ or cytotoxicity.
b. Proficiency Chemicals: Responsiveness of the test syst em was tested on March 5, April 12 and April 28, 2011 (MRID 48843501), using cells at passage 15, 25 and 28, respectively. Based on dates and passage number, it is unlikely the cells used for pr oficiency testing are from the same frozen stock as cells used in the main assay. Cells were tested using the following proficiency chemicals, each chemi cal tested in duplicate on separate days:

Compound CAS No. Concentration Range (M) Expected Response a 

# Notes Diethylstilbestrol (DES) 56-53-1 10

− 14 

# to 10

− 8 

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

− 14 

# to 10

− 8 

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

− 13 

# to 10

− 7 

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

− 12 

# to 10

− 5 

# Positive Cytotoxic at 0.01 b 

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

− 12 

# to 10

− 6 

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

− 11 

# to 10

− 4 

# Positive Cytotoxic at 0.1 b 

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

# 15797-52-1 10

− 12 

# to 10

− 5 

# Positive Cytotoxic at 100 μM. PC max 

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

− 11 

# to 10

− 4 

# Negative d 

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

− 11 

# to 10

− 4 

# Negative Cytotoxic at 1 mM b 

# Corticosterone 50-22-6 10

− 10 

# to 10

− 4 

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

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

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

# Estrogen Receptor Transc riptional Activation (2011) / Page 5 of 10 MALATHION / 057701 OCSPP 890.1300/ OECD 455



# c. Reference Chemicals: To ensure the stability of th e response from the cell line, six concentrations of each of the following reference chemicals were included in each plate in the current assay, along w ith the test chemical:

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

− 15 

# to 10

− 8 

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

− 13 

# to 10

− 6 

# Weak estrogen Corticosterone 50-22-6 10

− 11 

# to 10

− 4 

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

− 12 

# to 10

− 5 

# Very weak agonist


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

# for malathion was calculated, but the software used was not reported. The test material was define d as negative for inducing estrogen receptor transcriptional activation if the RPC Max 

# < PC 10 

# in at least 2 of 2 (o r 2 of 3) runs. Log EC 50 

# and Hill slope values are calculated only if a positive response is observed. Coefficients of variation (CV) were calculated for the lumi nescence data triplicates. Concentrations showing >20% cytotoxicity or evidence of in solubility 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. Precipitation was determined by visual inspection of the test materials in culture media. A sample of the 1x dosing solution was placed into the wells of a clear 96-well plate and an endoscope was used to assess precipitation in each sample. The results of the cytotoxicity test are presented in Table 1. As a result of this preliminary test, logarithmically increasing concentrations from 10 -11 

# to 10 -4 

# M were selected for the assay.

# Estrogen Receptor Transc riptional Activation (2011) / Page 6 of 10 MALATHION / 057701 OCSPP 890.1300/ OECD 455



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

# Concentration (M) % Viability b 

# Comments c 

# 10

− 3 

# 90 Precipitation observed 10

− 4 

# 94 Precipitation observed 10

− 5 

# 94 10

− 6 

# 91 10

− 7 

# 93 10

− 8 

# 96 10

− 9 

# 98 10

− 10 

# 98 E2 1nM NA VC b 

# NA a Data were obtained from page 20 of the study report. b VC = Vehicle control NA = not applicable


# B. Positive and Negative Reference Chemicals

1. Proficiency Chemicals: The laboratory proficiency a ssays were not included in the original study report, but were provided to the Agency at a later date (MRID 48843501). The responsiveness of cells to the required proficiency chemicals was performed in duplicate on different days for each chemical. The reported responses are in Table 2a. In the proficiency tests, the reference chemicals 17 β -estradiol, 17 α -estradiol and 17 α

methyltestosterone were tested concurrently w ith each run of the assay (Table 2b). In the first run, the responsiveness of 17 β -estradiol indicated decrea sed sensitivity to strong agonists, and the response to 17 α -methyltestosterone showed an increased responsiveness to very weak agonists; despite the minor deviations this run is consider ed acceptable. Run 2 was inadequate as the PC 50 

# could not be calculated for 17 α -methyltestosterone indicating a decreased sensitivity to very weak agonists. Run 3 was acceptable as 17 β -estradiol and 17 α -methyltestosterone performed within the expected range, but the Hill Slope for 17 α -estradiol was higher than expected. Th e PC-induced fold induction for the three reference chemicals was within the Guideline-re commended historical ra nge of 4- to 30-fold in Runs 1 and 3, but fold induction was 75.1- to 84.7-fold in Run 2 with no explanation given for this 3- to 4-four increase. Results of the cytotoxicity assay were not provided. Raw data were not reported, but the scales of the graphs prov ided indicate genistein and butyl paraben had maximum RTAs well above 400%.

TABLE 2a. Proficiency Chemicals Compound Expected Response Lab Response Run 1 Run 2 Run 3 Diethylstilbestrol Positive Positive Positive NA 17 α -Ethynyl estradiol Pos itive Positive Positive NA Hexestrol Positive Positive Positive NA Genistein Positive Positive Positive NA Estrone Positive Positive Positive NA Butyl paraben Positive Positive Positive NA 1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive NA Positive Positive Dibutyl phthalate Negativ e Negative Negative NA Atrazine Negative Ne gative NA Negative Corticosterone Negative Negative NA Negative NA = not applicable. The chemical was not tested at this time.

# Estrogen Receptor Transc riptional Activation (2011) / Page 7 of 10 MALATHION / 057701 OCSPP 890.1300/ OECD 455



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

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

# − 11.4 to

− 10.1

− 9.6

− 11.3

− 10.6 Run 1 Log PC 10 

# <

− 11

− 11.5

− 12.5

− 12.1 X Log EC 50 

# − 11.3 to

− 10.1

− 9.0

− 11.3

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

− 14 

# to 10

− 8 

# 10

− 14 

# to 10

− 8 

# 10

− 14 

# to 10

− 8 

# 10

− 14 

# to 10

− 8 

# X 17 α -estradiol Log PC 50 

# − 9.6 to

− 8.1

− 8.3

− 9.4

− 8.7 X Log PC 10 

# − 10.7 to

− 9.3

− 9.3

− 10.5

− 9.9 X Log EC 50 

# − 9.6 to

− 8.4

− 8.2

− 9.3

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

− 12 

# to 10

− 6 

# 10

− 12 

# to 10

− 6 

# 10

− 12 

# to 10

− 6 

# 10

− 12 

# to 10

− 6 

# X 17 α -methyltestosterone Log PC 50 

# − 6.0 to

− 5.1

− 6.2 NC

− 5.2 Run 1, 2 Log PC 10 

# − 8.0 to

− 6.2

− 8.1

− 6.3

− 7.7 Run 1 Test range (M) 10

− 11 

# to 10

− 5 

# 10

− 11 

# to 10

− 5 

# 10

− 11 

# to 10

− 5 

# 10

− 11 

# to 10

− 5 

# X



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

# , log PC 10 

# , log EC 50, 

# Hill slope) for the concurrently run reference compounds are in Table 3. Performance criteria were met for 17 β -estradiol and 17 α -estradiol; the log EC 50 

# for 17 β -estradiol was -10.0 M for Run 1, which is only just outside the ac ceptable range. For 17 α -methyltestosterone, PC 50 

# and PC 10 

# values could not be calculated for Run 1, PC 50 

# could not be calculated for Run 2, and the PC 10 

# was higher than the expected range, indicating a decrease in sensitivity of the assa y to very weak agonists. For 17 α -methyltestosterone, the mean RPC Max 

# was 6.5% for the first run and 37.4% for the second run.

TABLE 3. Performance Criteria for Reference Chemicals a


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

# − 11.4 to

− 10.1

− 10.0

− 10.3 X Log PC 10 

# <

− 11

− 11.1

− 11.4 X Log EC 50 

# − 11.3 to

− 10.1

− 10.0

− 10.2 Run 1 Hill Slope 0.7 to 1.5 1.1 1.2 X Test range 10

− 14 

# to 10

− 8 

# M 10

− 15 

# to 10

− 8 

# M 10

− 15 

# to 10

− 8 

# M X 17 α -estradiol Log PC 50 

# − 9.6 to

− 8.1

− 8.6

− 8.7 X Log PC 10 

# − 10.7 to

− 9.3

− 9.6

− 9.7 X Log EC 50 

# − 9.6 to

− 8.4

− 8.5

− 8.5 X Hill Slope 0.9 to 2.0 1.1 1.2 X Test range 10

− 12 

# to 10

− 6 

# M 10

− 13 

# to 10

− 6 

# M 10

− 13 

# to 10

− 6 

# M X Corticosterone Test range 10

− 10 

# to 10

− 4 

# M 10

− 11 

# to 10

− 4 

# M 10

− 11 

# to 10

− 4 

# M X 17 α -methyltestosterone Log PC 50 

# − 6.0 to

− 5.1 NC NC Run 1,2 Log PC 10 

# − 8.0 to

− 6.2 NC

− 5.9 Run 1,2 Test range 10

− 11 

# to 10

− 5 

# M 10

− 12 

# to 10

− 5 

# M 10

− 12 

# to 10

− 5 

# M X a Data were obtained from pages 21 and 23 of the study report. NC = not calculable

# Estrogen Receptor Transc riptional Activation (2011) / Page 8 of 10 MALATHION / 057701 OCSPP 890.1300/ OECD 455



# 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 for the ve hicle control, expresse d as arbitrary light units, was 11396 for the first run and 10408 for the second run. The mean normalized value for the positive control was 209762 for the first run and 154017 for the second run. The PC 50 

# (50% of the maximum response) for E2 in this assay was 104881 for the first run and 77009 for the second run, and the PC 10 

# (10% of the maximum response) was 20976 for the first run and 17055 for the second run.

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

# Sample Vehicle Control Positive Control b 

# Runs Mean SD Mean SD Fold Induction c 

# 1 11396 1648 209762 33693 18.4 2 10408 1653 154017 43272 14.8 a Data were obtained from pages 48 and 49 of the study report. b Positive control was 17 β -estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC)



# 2. Test Material: Relative (to the PC) transcriptio nal activation at each malathion concentration during the two assa y runs is presented in Table 5. The concentration-response curves depicting fold induction of RTA is presented in Figures 1 and 2. The mean RPC Max 

# was 3.4% for the first run and 3.7% for the second run, and the associated PC Max 

# was 10 − 7 

# M for both runs. Because the RPC Max 

# < PC 10 

# in both assay runs, malathion was considered negative for estrogen receptor transcrip tional activation in this test system.

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


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

− 4 

# --* -- 0.6 2.1 10

− 5 

# 2.2 1.6 2.2 1.4 10

− 6 

# 2.8 1.1 3.0 0.7 10

− 7 

# 3.4 1.6 3.7 1.4 10

− 8 

# 0.7 1.4

− 0.1 1.5 10

− 9 

# 1.5 1.7 0.8 1.3 10

− 10 

# 0.9 1.3 0.6 1.8 10

− 11 

# 1.4 1.4 0.2 0.8 RPC Max 

# 3.4 3.7 PC Max 

# (M) 10

− 7 

# 10

− 7 

# PC 50 

# (M) NA NA PC 10 

# (M) NA NA a Data were obtained from pages 21 and 22 of the study report. * Cytotoxicity observed NA = Not Applicable

# Estrogen Receptor Transc riptional Activation (2011) / Page 9 of 10 MALATHION / 057701 OCSPP 890.1300/ OECD 455



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

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



# FIGURE 2. Fold Induction of Relative Transcription Activation (RTA) of Malathion Compared to the Positive Control. Run 2

3. Performance Criteria: Performance criteria were generally met for 17 β -estradiol and 17 α

estradiol, although the Log EC 50 

# for 17 β -estradiol in Run 1 (-10.0 Log M) was just outside the expected range. For 17 α -methyltestosterone, PC 50 

# and PC 10 

# values could not be calculated for Run 1, the PC 50 

# could not be calculated for Run 2, and the PC 10 

# was higher than the expected range, indicating decrease d sensitivity to very weak agonists. Six replicates of each concentration, including sa mples of the positive control (PC, 1 nM E2) 0 5 10 15 20 25

VC PC10 PC

‐ 11

‐ 10

‐ 9

‐ 8

‐ 7

‐ 6

‐ 5Foldinduction



# Log [M]

0 2 4 6 8 10 12 14 16 18 20

VC PC10 PC

‐ 11

‐ 10

‐ 9

‐ 8

‐ 7

‐ 6

‐ 5Foldinduction



# Log [M]

# Estrogen Receptor Transc riptional Activation (2011) / Page 10 of 10 MALATHION / 057701 OCSPP 890.1300/ OECD 455


# and the vehicle control (VC, 0.1% DMSO) we re included in each plate, and the mean luciferase activity of the positive control was greater than 4-fold that of the mean vehicle control on each plate. Additionally, two replicates/plate incorporating the hER α antagonist, ICI 182,780 were included; cells re sponded appropriately to the an tagonist (data not shown).
Cytotoxicity was observed in the first assay at the highest concen trations tested of malathion, 17 α -estradiol, corticosterone and 17 α -methyltestosterone. Cytotoxicity should not occur at any of the require d concentrations of the reference chemicals and indicate a flaw in the system. Additionally, in the s econd assay, the cells' average response to 10

− 9 

# M of 17 β -estradiol and to 10

− 7 

# M or 10 − 6 

# M of 17 α -estradiol was greater than the positive control, 10

− 9 

# M 17 β -estradiol. The fold-induc tion corresponding to the PC 10 

# of the concurrent PC was greater than 1+2 standard deviations of the fold -induction value of the concurrent VC, and the result s were reproducible among runs. The CV was not reported.

III. DISCUSSION AND CONCLUSIONS

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

− 5 

# M, based on precipitation observed at concentrations >10

− 5 

# M. Cytotoxicity was observed with malathion at the highest concentration tested in the first run. Ther e was no cytotoxicity observed at any of the exposure concentrations tested in the range-finding study or the second run. Four runs were conducted; however, the first two runs were invalid because the data did not fit either the assay acceptance criteria or CeeTox historical da ta. The subsequent two runs of the assay using new HeLa cells were judged to be acceptable. Using these independent runs, malathion did not result in an increase in luciferase activity at any of the viable concentrations tested (RPC Max 

# <10%).

B. AGENCY COMMENTS: Malathion was tested up to the limit of cytotoxicity and solubility, 10

− 5 

# M. The mean RPC Max 

# was 3.4% for the first r un and 3.7% for the second run, and the associated PC Max 

# was 10 − 7 

# M for both runs. Performance criteria were generally met for 17 β -estradiol and 17 α -estradiol, although the EC 50 

# for 17 β -estradiol (- 10.0) was just outside the expected range in run 1. For 17 α -methyltestosterone, the PC 50 

# and PC 10 

# values could not be calculated for run 1, the PC 50 

# could not be calculated for run 2, and the PC 10 

# was higher than the acceptable range, i ndicating a decrease in sensitivity to very weak agonists. Although conditions were not optimal to detect very weak activity, malathion responses were similar to those of the negative control corticosterone and not comparable to the responses of 17 α -methyltestosterone, which was able to reach a maximum of 6.5-37.4% PC. Malathion was only able to reach a maximum of 3.4-3.7% PC when tested up to the highest concentration possible based on cytoto xicity. Because the RPC Max 

# < PC 10 

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

C. STUDY DEFICIENCIES: The following deficiencies were noted:

 The response of the cells to the reference chemicals was inappropriate.

 The source and purity of the reference compounds, DMSO and DCC-FBS were not provided.

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

EPA MRID Number 48617506

Page 1 R f 93 Version: 22 September 2011 

# Data Requirement: EPA DP Barcode D396355

OECD Data Point [............]

EPA MRID 48617506 EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test Material: Malathion Purity (%): 96.0%

Common Name Chemical Name IUPAC: Diethyl (dimethoxyphosphinothioylthio)succinate CAS Name: Diethyl 2-[(dimethoxyphosphinothioyl)thio]butanedioate

CAS No.: 121-75-5 Synonyms: Fyfanon Technical

EPA PC Code: 057701

Primary Reviewer: Dana Worcester Signature: Senior Scientist, Cambridge Environmental, Inc. Date: 5/8/12

Secondary Reviewer: Joan Gaidos Signature: Senior Scientist, Cambridge Environmental, Inc Date: 5/8/12

Primary Reviewer: Elizabeth Donovan Date:

EPA/EFED/ERB 6

Additional Reviewer(s): Justin Housenger, Biologist Date: 12/19/13

EPA/EFED/ERB 5

Additional Reviewer(s): Amy Blankinship Date:

EPA/EFED/ERB 6

Date Evaluation Completed: 6/6/15 Digitally signed by Elizabeth Donovan DN: cn=Elizabeth Donovan, o=EPA, ou=EFED, email=donovan.elizabeth@epa. gov, c=US Date: 2015.06.10 08:48:44 -04'00'

Digitally signed by JUSTIN HOUSENGER DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=JUSTIN HOUSENGER, dnQualifier=0000044455 Date: 2015.06.12 08:20:15 -04'00'



# AMY BLANKINSHIP Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=AMY BLANKINSHIP, dnQualifier=0000040917 Date: 2015.06.15 17:38:10 -04'00'

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

EPA MRID Number 48617506

Page 2 R f 93 Version: 22 September 2011 

# CITATION: Palmer, S.J., S.Z. Schneider, T.Z., Kendall and H.O. Krueger, 2011, Malathion: Fish Short-Term Reproduction Assay with the Fathead Minnow ( Pimephales promelas ), Performed by Wildlife International,
Ltd., Easton, Maryland. Lab Project No.: 232A-121. Submitted by Cheminova A/S, Arlington, Virginia. Study completed October 27, 2011.

Note: The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estro gen, 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).

Guideline recommendations are provided in italics; these recommendations should remain visible in the completed

DER.

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 Malathion

EPA MRID Number 48617506

Page 3 R f 93 Version: 22 September 2011 

# EXECUTIVE SUMMARY

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

group; ca . 5 months old) were exposed to malathion (96.0% purity) at nominal concentrations of 0 (control),
0.10, 0.32 and 1.0 mg a.i./L. The mean-measured concentrations, calculated by the study authors and excluding Day 21 recoveries, were <LOQ (0.0400), 0.08, 0.25 and 0.82 mg a.i/L, with %CVs of 4.4, 8.7, and 6.1%, respectively. The study authors excluded Day 21 samples due to suspected handling or analysis error, however, no supporting data were provided. Therefore, the reviewer calculated mean-measured concentrations, including Day 21 samples, of <LOQ (0 .0400), 0.08, 0.22, and 0.69 mg a.i/L, with %CV’s
of 7.6, 26, and 29%, respectively. EPA requires a CV for measured test concentrations of <20% over 21 days. The test system was maintained at 24.7 to 25.3 o 

# C and a pH of 7.9 to 8.2. Unless otherwise indicated, all effects are reported based on comparison to the negative (clean water) control.

Male and female survival was 100% in the negative control and in the lowest treatment concentration (0.08 mg a.i/L). There was a single male and female mortality each at the mid treatment concentration and a single
female mortality at the high treatment concentration. Observed clinical signs of toxicity included one fish at the mid concentration exhibiting a crooked spine, which was not attributed to treatment. However, in the high concentration group, 8 of 24 fish (33%) exhibited clinical signs of toxicity that included bruising on the body,
loss of color or no color in the tail, erratic swimming, lethargy, and crooked spine. These effects were observed as early as the third day of exposure and increased in incidence as the test continued. Excess feed was noted in the test chamber on Days 7 through 9 for the mid concentration and on Days 5 through 16 for the high concentration. Male body weight was decreased (not significant) by 19.8 and 17.3% in the mid and high
concentration groups, respectively; there was no effects on female body weight and length or male length.

Spawning and fecundity in controls, respectively, we re every 3 days and 26.3 eggs/female/day; fertilization success in controls was 99.3%. There was a statistically significant (p<0.05) reduction in fecundity ( Ļ 48%)
at the high concentration compared to the negative control. There were no significant effects of malathion treatment on, female and male vitellogenin (VTG), fertilization success, or male gonado-somatic index (GSI).
Female GSI exhibited a concentration-dependent monotonic increasing trend, with an increase of 21% at the

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

EPA MRID Number 48617506

Page 4 R f 93 Version: 22 September 2011 

# high concentration compared to the negative control (p<0.05; Jonckheere-Terpstra). At the high concentration,
male tubercle scores were significantly reduced compared to the negative c ontrol (10.5 vs. 25.5 in control) (p<0.01; Jonckheere-Terpstra). The study author’s also reported a statistically significant decrease in the fatpad score was reduced (2.3 versus 4.0 for control; p<0.05) compared to the control.

Malathion significantly impacted the multiple endpoints involved with the development of the testes at the highest treatment level but did not appear to affect ovarian development to the same degree, with the exception of increased oocyte atresia. In the high-concentration group males, there were treatment-related effects (p<0.05; Jonckheere-Terpstra trend test) including increased spermatogonia, altered proportion of
spermatocytes and spermatids, as well as decreases in gonad size, Sertoli cell spermatocyst number/tubule and spermatocyst size, germinal epithelium thickness, tubule diameter, efferent duct diameter, spermatozoa, and milt. Although these effects were not statistically analyzed by the reviewer, the reviewer’s observations were validated by the study authors’ statistical analysis.

The only validity criterion for the OCSPP 890.1350 guideline which was not met was maintenance of %CV at <20% for measured concentrations from all replicate chambers during the 21 day study. Performance criteria for the OCSPP 890.1350 guideline were met for reported parameters, Average daily recoveries of measured test concentrations ranged from 37.8% to 87.9% of nominal concentrations, with a pattern of decline toward test termination. The study authors used mean-measured test concentration excluding Day 21 data, while the

reviewer relied on mean-measured concentration including Day 21 data.

This assay satisfies the EDSP Tier 1 Test Order requirements for a Fish Short-Term Reproduction Assay (OCSPP Guideline 890.1350).

Results Synopsis:

Test organism age at test initiation: ca. 5 months Mean body weight at test initiation (if measured): Males, 2.7 g; Females, 1.2 g Mean length at test initiation (if measured): Not measured
Test type: Flow-throughData Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page5Rf93Version: 22 September 2011Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Malathion.Treatment(mg a.i./L)3[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMFControl NoNoNoNoNoNoNoNoNoNoNANANANA0.08 No No No No No No No No No No NANANANA0.22 No No No No No No No No No No NANANANA0.69YesNoYesNo NoYes Yes YesNo No NANANANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may beheavily weighted by the expert opinion of a board-certified pathologist.3The mean-measured concentrations were calculated by the reviewer, including the 21-day measured concentrations.

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

EPA MRID Number 48617506

Page 6 R f 93 Version: 22 September 2011 

# I. MATERIALS AND METHODS

Guideline Followed: This study was conducted according to the U.S. EPA OCSPP 890.1350: “Fish Short-Term Reproductive Assay” an d OECD 229 (2009). The following

deviations were noted:

1. Individual weight of male and female fish (n = 10 each) selected prior to adding
fish to the pre-exposure system, had a deviation of ± 21% of the estimated mean of 2.7 g for males and 1.2 g. for females. EPA recommends that individual weight of each fish selected for the test be within ± 20% of the estimated mean

of each sex.

2. Measured concentrations in test solutions at 0, 7, 14 and 21 days ranged from 37.8 to 87.9% of nominal concentrations with a pattern of decline toward test termination. The coefficient of variation ranged from 4% to 9%, excluding Day 21 samples; the Day 21 samples were much lower than nominal (37.8-72.25),
especially at the higher concentrations. The study authors suspected sample handling or analysis error and did not include them in the calculations of the mean measured; however, there is no data supporting this conclusion. Therefore, the reviewer calculated the %CV including Day 21 data and obtained %CV values of 6, 26, and 29% for nominal application rates of 0.10, 0.32 and 1.0 mg a.i/L, respectively. EPA requires a CV for measured test concentrations of <20% over 21 days.

3. The unionized ammonia and residual chlorine were not in the test water were not
reported.

4. Although the study authors reported ca. 6 total volume exchanges per day in the test system, the actual flow rate was not reported. OCSPP 890.1350 guidelines recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total
volume exchanges per day.

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

EPA MRID Number 48617506

Page 7 R f 93 Version: 22 September 2011 

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

Compliance: Signed and dated GLP, Quality Assurance, and Data Confidentiality claims
statements were provided. This st udy 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], with the following exceptions:
periodic analyses of water for potential contaminants were not performed according to GLP standards, but we re performed using a certified laboratory and standard US EPA analytical methods.

A. Test Material Malathion, CAS No. 121-75-5

Description: The test material was a pale yellowish liquid, and the water solubility was reported to be 48.2 mg/L at 25 q C. Expiration date December 11, 2011.

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

Lot No./Batch No. : 9010501 (Batch No.)

Purity: 96.0%

Impurities: None reported

Stability of Compound: The test material was reported to be stable in acidified deionized water for at least three days. Analytical verification of di luter stock fortified with 5.0, 16 and 50 mg a.i/L yielded recoveries ranging from 83.4 to 108% of nominal concentrations. Analytical verification on
the test solutions prior to test initiation at -6 and -2 days yielded recoveries ranging from 82.0 to 94.1% of nominal concentrations. Measured concentrations in test solutions at 0, 7, 14 and 21 days ranged from 37.8 to 87.9% of nominal concentrations with a pattern of decline toward test

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

EPA MRID Number 48617506

Page 8 R f 93 Version: 22 September 2011 

# termination. The coefficient of variation ranged from 4% to 9%, excluding Day 21 samples; the Day 21 samples were much lower than nominal (37.8-72.25), especially at the higher concentrations.
The study authors suspected sample handling or analysis error and did not include them in the calculations of the mean measured concentrations; however, there is no data supporting this conclusion. Therefore, the reviewer calculation included Day 21 data and obtained %CV values of 6, 26, and 29% for nominal concentrations of 0.10, 0.32 and 1.0 mg a.i/L, respectively. These

data indicate the test material was not stable under the test conditions.

Storage Conditions of

Test Chemicals: The test material was stored refrigerated ( ca . 4 q C); analytical standard stored frozen ( ca . 25 q C).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page9Rf93Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name: Fathead minnow 

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

# Pimephales promelasSpecies strain (if stated): Not reportedWere fish obtained from asingle laboratory stock?Yes Osage Catfisheries, Inc., Osage Beach,Missouri. 

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

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

test.Acclimation period:2 weeks 

# EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page10Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Pre-exposure mortalities not reported.Fish did not receive any treatment fordisease during acclimation period.No behavioral abnormalities or clinicalsigns were 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:Brine shrimp andcommercial flake food(Sera Vipan) 

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

condition.Source of food:Brine Shrimp Direct,Ogden, Utah and Sera,North AmericaFrequency of feeding:Two times/dayData Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page11Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on feeding:None.Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation: 

# ca. 5 months 

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

old.Mean weight of males at testinitiation (if determined):2.7 Based on 10 males used to stockaquaria for pre-exposure phase. 

# EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for each sex.Range of individual weights(males) at test initiation (ifdetermined):r21%Mean weight of females at testinitiation (if determined):1.2 Based on 10 females used to stockaquaria for pre-exposure phase.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page12Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsRange of individual weights(females) at test initiation (ifdetermined):r21%Mean length of males at testinitiation (if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase: 15 days 

# EPA recommends a minimum of 14 days.Were 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 tanksExtra replicates were established 

# 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page13Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber 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 3days. 

# EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Details on pre-exposure:None.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page14Rf93Version: 22 September 2011C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through

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

# , the use of a static renewal system is not recommended for this assay.Type of flow-through dilutionsystem:Continuous flow diluter 

# Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate: 

# ca. 6 volumereplacements/dayActual flow rate not reported. 

# Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine DisruptorScreening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington,D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page15Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems:Flow-splitting chamberswere used between 4replicate test vesselsSolution from mixing chamber split equallyamong replicate test vessels.Treatment recoveries prior to test initiationfrom pretest diluter were 82.0 to 94.1%.Flow rates to each replicate varied by nomore than ± 10% of the mean flow.

# Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy is within 10%.Aeration? No 

# EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (< 60%
saturation).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page16Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSource of dilution water: Well waterPrior to use, the water was sand-filtered,aerated, carbon-filtered and UV-sterilized. 

# EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?Yes All potential contaminants were confirmedto be below thedetection limit.Test vessel type/materials: Glass aquaria

# EPA and OECD recommend that watercontact portions of the system not compromise the study (e.g., 

# all glass vessels or glass vessels with stainless steel frames are acceptable examples).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page17Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel size:19 L, 41 x 21 x 25 cmhigh 

# 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 into semicircular sections. 

# EPA recommends that each tank contain three semi-circular spawning substrates, e.g., aged PVC pipe, 10 - 20 cm in length, split lengthwise.Spawning substrate size: Not reported.Additional details on exposuresystem:Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page18Rf93Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)24.7 25.7 25 ± 1°C Continuous 

# EPA recommends temperature 25±1 o 

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

# C.pH 7.98.2NotreportedWeekly 

# EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)5.78.0 Not reported Weekly 

# EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L asCaCO3)176 184 Not reported Weekly 

# EPA recommends total alkalinity > 20 mg/L as CaCO 3.Hardness [mg/L as CaCO3] 132 144 Not reported WeeklyTotal organic carbon (mg/L) Not reported Not reported <1 Once 

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

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

P g/L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page19Rf93Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsResidual chlorine (Pg/L) Not reported Not reported Not reportedNot reported 

# EPA recommends that residual chlorine in dilution water be < 10 P g/L.Conductance (սmhos/cm) 301 376 Not reported Not reported 

# General recommendations for frequency of measurements: EPA recommends that temperature, pH, and dissolved oxygen be measured in all test tanks at least weekly and that hardness and alkalinity be measured in controls and in one tank at the highest test concentration at least weekly. In addition, continuous temperature monitoring of at least one tank is encouraged.Abbreviations:NANot applicable.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page20Rf93Version: 22 September 2011D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes 

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

LC 50 

# data for the fathead minnow are unavailable.If yes, what was the method fordetermining the highest testconcentration in the rangefinder?0.20, 0.60, 2.0 mga.i/L 

# EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower)

is appropriate.Species: 

# Pimephales promelasLife stage:Not reported 

# EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in the test.Test duration:Not reported 

# EPA recommends a 96-hour exposure.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page21Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details: 

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

hr LC 50, 

# may be selected as the highest exposure concentration in the 21-day test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days 

# EPA recommends that the duration of the definitive test be 21 days.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page22Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in thedefinitive test:Based on Pilot studyresults 

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

# ), whichever concentration is lowest.Reference study citation (ifapplicable):Not reported.Separation of testconcentrations:Three fold 

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

# .Number of test concentrations:3 

# EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?NoData Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page23Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Mean measuredLimit of quantification (LOQ): 0.0400 mg a.i./L 

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

# It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two replicates per treatment level.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page24Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWas the randomized completeblock design used?Yes 

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

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

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

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

# EPA recommends 2 males per replicate.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page25Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of female fish perreplicate at test initiation:4 

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

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

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

# .Was a positive control used? NoPositive control (if applicable): NA3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testingand Assessment. No. 23. Paris, France.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page26Rf93Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsPositive control concentration(s)(if applicable):NAPhotoperiod:16 hrs light8 hrs dark 

# EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’ssurface:432 to 657 lux 

# EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page27Rf93Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Malathion.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration1(mg a.i./L)Mean CV(%)1MeasuredConcentration2(mg a.i./L)Mean CV(%)2Details orRemarksGuidelineRecommendationsControl (dilutionwater only)<0.0400 <LOQ NA <LOQ NA 

# EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Solvent control(if applicable)NA NA NA NA NATreatment1 0.1 0.08 4% 0.08 8%Treatment 20.320.259% 0.22 26%% CV maxexceeded2Treatment3 1.0 0.82 6% 0.69 29%% CV maxexceeded2DiluterStock NA NA NA NA NAAbbreviations:CVCoefficient of variation.1The study authors did not include Day 21 samples in calculationsdue to suspected handling oranalysis error (see Reviewer’s Comment).2The reviewer calculated mean measured and %CV including Day 21 samples.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page28Rf93Version: 22 September 2011E. ObservationsBiological Endpoints:Survival, fecundity, fertilization success, and clinical signs were observed daily. At test termination (Day 21),secondary sex characterization (body color, pattern, bodyshape), body weight, length, tubercle score, fatpadscore, gonadal staging and histopathology, and plasma vitellogenin were evaluated.Were raw (individual) data provided?Yes 

# EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinic al signs occur at least daily. At test termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma vitellogenin, and

plasma sex steroids (testosterone and 17ȕ 

# -estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal beha vior.II.RESULTS AND DISCUSSIONA.ResultsOne male died in the 0.25 mg a.i/L treatment, and one female died in each of the 0.22 and 0.69 mg a.i/L treatment groups. Remaining groupsexhibited 100% survival. Overall mean survival values were 100,100, 91.7 and 95.8% in the mean-measured 0 (control), 0.08, 0.22, and 0.69 mgData Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page29Rf93Version: 22 September 2011a.i/L treatment levels, respectively. Observed clinical signs of toxicity included one fish in the middle treatment group exhibiting a crooked spine, butthis effect was not attributed to treatment by the study authors as it was isolated to one fish. In the high treatment group (0.82 mg a.i/L), 8 of 24fish (33%) exhibited clinical signs of toxicity that included bruising on the body, loss of color or no color in the tail, erratic swimming, lethargy, andcrooked spine. These effects were observed as early as the third day of exposure and increased in incidence as the test continued. Excess feed wasnoted in the test chamber for the middle treatment group starting at Day 7 and persisting to Day 9. Excess feed was observed in the test chamber inthe high treatment group in all replicate tanks from Day 5 to Day 16.Table 9: Adult Fish Survival in 

# Pimephales promelasTreatment (mg a.i./L) [mean-measured]MalesFemalesn# Surviving% Survivaln# Surviving% SurvivalControl (<LOQ)8810016161000.08 8810016161000.22 87881615940.69 88100161594Abbreviations:NANot applicable.LOQ=0.0400 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page30Rf93Version: 22 September 2011Mean male body weight values were 3.15, 2.88, 2.53, and 2.60 g and female body weight values were 1.22, 1.25, 1.24 and 1.28 g in themean-measured 0 (control), 0.08, 0.22, and 0.69 mg a.i/L treatment levels, respectively. Mean male body length values were 60.6, 59.0,56.9 and 59.6 mm and female body length values were 47.9, 47.3, 47.6 and 47.4 mm in the mean-measured 0 (control), 0.08, 0.22,and0.69 mg a.i/L treatment levels, respectively.Table 10: Size at Test Termination in 

# Pimephales promelas.Treatment(mg a.i./L)[mean-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDControl (<LOQ) 4 3.15 0.467 4 1.22 0.092 4 60.6 2.14 4 47.9 1.110.084 2.88 0.338 4 1.25 0.112 4 59.0 1.68 4 47.3 1.250.224 2.53 0.427 4 1.24 0.086 4 56.9 2.46 4 47.6 0.550.694 2.60 0.219 4 1.28 0.071 4 59.6 2.14 4 47.4 1.02Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.0400 mg a.i./L.

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# Mean fecundity values were 29.3, 29.6, 26.4 and 15.2 and fertilization success was 99.3, 99.6, 99.4 and 99.2% in the mean-measured 0 (control), 0.08, 0.22, and 0.69 mg a.i/L treatment levels,
respectively.

Table 11: Fecundity and Fertilization Success in Pimephales promelas

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

# ± SD Fertilization Success (%) 2 

# ± SD

Control (<LOQ) 29.3 2.11 99.3 0.35

0.08 

# 29.6 6.47 99.6 0.18

0.22 

# 26.4 6.32 99.4 0.43

0.69 

# 15.2 4.96 99.2 0.99

Abbreviations: NA 

# Not applicable. ND 

# Not determined.

1 

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

2 

# Fertilization success (%) is calculated as the number of embryos divided by the number of eggs,
multiplied by 100. LOQ=0.0400 mg a.i./L.

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# Median male tubercle scores were 25.5, 28.3, 19.0 and 10.5 in the mean-measured 0 (control), 0.08,
0.22, and 0.69 mg a.i/L treatment levels, respectively.

Table 12a: Nuptial Tubercle Score in Pimephales promelas

.

Treatment (mg a.i./L)

[mean-measured] Males Females

n Median

Tubercle Score (Mean) 1 

# n Median

Tubercle Score

Control (<LOQ) 4 25.5 4 0

0.08 

# 4 28.3 4 0

0.22 

# 4 19.0 4 0

0.69 

# 4 10.5 4 0

Abbreviations: NA 

# Not applicable. ND 

# Not determined. SD 

# Standard deviation.

1 

# Study authors reported and analyzed only mean tubercle scores (23.8, 28.1, 19.8 and 11.4 in control, low, mid and high treatment); median male tubercle scores were calculated by the reviewer.
LOQ=0.0400 mg a.i./L.

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# Mean male fatpad scores were 4.0, 3.5, 3.8 and 2.3 in the mean-measured 0 (control), 0.08, 0.22,
and 0.69 mg a.i/L treatment levels, respectively.

Table 12b: Fatpad Score in Pimephales promelas

.

Treatment (mg a.i./L)
[ mean- 

# measured] Males Females

n Mean Fatpad

Score n Mean Fatpad

Score

Control (<LOQ) 4 4.0 4 1

0.08 

# 4 3.5 4 1

0.22 

# 4 3.8 4 1

0.69 

# 4 2.3 4 1

Abbreviations: NA 

# Not applicable. ND 

# Not determined. SD 

# Standard deviation. LOQ=0.0400 mg a.i./L.

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# Mean male GSI was 1.47, 1.56, 1.16 and 1.44 and mean female GSI was 11.9, 11.7, 12.9 and 14.4 in the mean-measured 0 (control), 0.08, 0.22, and 0.69 mg a.i/L treatment levels, respectively.

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

.

Treatment (mg a.i./L)
[ mean- 

# measured] Males Females

n Mean GSI 1


# (%) ±SD n Mean GSI 1


# (%) ±SD

Control (<LOQ) 4 1.47 0.238 4 11.9 1.71

0.08 

# 4 1.56 0.432 4 11.7 1.89

0.22 

# 4 1.16 0.160 4 12.9 1.68

0.69 

# 8 1.44 0.195 16 14.4 0.810

Abbreviations: NA 

# Not applicable.

1 

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

100. LOQ=0.0400 mg a.i./L.

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# Median male gonadal stage was 2, 2, 2, and 2 and median female gonadal stage was 3, 2, 3, and 3 in the mean-measured 0 (control), 0.08, 0.22, and 0.69 mg a.i/L treatment levels, respectively.

Table 14: Gonadal Staging in Pimephales promelas

.
Treatment (mg a.i./L)
[ mean- 

# measured] Males Females

n Median Stage 1 

# n Median Stage 2



# Control (<LOQ) 4 2 4 3

0.08 

# 4 2 4 2.3

0.22 

# 4 1.5 4 3

0.69 

# 4 2.3 4 3

Abbreviations: J 

# Juvenile. NA 

# Not applicable. ND 

# Not determined. UTS 

# Unable to stage.

1 

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

2 

# The guideline recommends the following gonadal staging scale for female fathead minnow: 0=undeveloped, 1=early development, 2=mid-development, 3=late development, 4=late development/hydrated, 5=post-ovulatory. LOQ=0.0400 mg a.i./L.

In the males, no animals in the control group or 0.08 and 0.22 mg a.i/L showed an increased proportion of spermatogonia. An increased proportion of spermatogonia were observed in the 0.69 mg a.i/L treatment group, in addition there was mild to severe secondary spermatocyte decreases in three males and mild to severe spermatid decreases in 5 males. In the females, mild atresia was observed in the control group and

all treatment levels. Severe atresia was observed in two females from the 0.69 mg a.i/L treatment group. There was no significant difference in the gonadal development stages.

The study authors also statistically analyzed histopathology effects and found statistically significant differences in males for increased spermatogonia, alte red proportion of spermato cytes and spermatids, gonad size decrease, Sertoli cell spermatocyst number/tubule and spermatocyst size decrease, decreased germinal

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# epithelium thickness, decreased tubule diameter, decreased efferent duct diameter, decreased spermatozoa, and decreased milt at the 0.69 mg a.i/L treatment (p<0.05; Jonckheere-Terpstra trend test). Additionally,
the testes of 4 of 8 fish in the 0.69 mg a.i/L treatment could not be staged using normal criteria due to moderate to severe alteration of the spermatocyte ratios including abnormal spermiation, proliferation and meiosis and maturation, and therefore could not be statistically compared.

Table 15a: Gonadal Histopathology in Male Pimephales promelas

.

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



# Severity 

# Increased Proportion of Spermatogonia Tubule Diameter

Decrease Increased

Testicular Degeneration Interstitial Cell Hypertrophy/ Hyperplasia

n Incidence n Incidence n Incidence n Incidence



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

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 1

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

0.08 0 8 8 8 8 8 8 8 8

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 8 0 8 0 8 0

0.22 0 8 8 8 8 8 8 8 6

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 2

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

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[mean-measured] Diagnostic Observations 1



# Severity 

# Increased Proportion of Spermatogonia Tubule Diameter

Decrease Increased

Testicular Degeneration Interstitial Cell Hypertrophy/ Hyperplasia

n Incidence n Incidence n Incidence n Incidence



# 0.69 0 8 3 8 5 8 5 8 8

1 8 0 8 0 8 0 8 0

2 8 4 8 2 8 2 8 0

3 8 1 8 1 8 1 8 0

4 8 0 8 0 8 0 8 0

1 

# Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. LOQ=0.0400 mg a.i./L.

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.

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



# Severity 

# Interstial Cellularity

Increase Spermatozoa

decrease Increased

Interstitial

Thickness Germinal
Epithelium

Thickness

Decrease

n Incidence n Incidence n Incidence n Incidence



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

1 8 0 8 0 8 0 8 0

2 8 1 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

0.08 0 8 8 8 7 8 8 8 8

1 8 0 8 0 8 0 8 0

2 8 0 8 1 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 8 0 8 0 8 0

0.22 0 8 6 8 6 8 7 8 8

1 8 0 8 0 8 0 8 0

2 8 2 8 2 8 1 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

0.69 0 8 3 8 4 8 8 8 4

1 8 0 8 0 8 0 8 0

2 8 4 8 3 8 0 8 1

3 8 1 8 1 8 0 8 3

4 8 0 8 0 8 0 8 0

1 

# Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. LOQ=0.0400 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page39Rf93Version: 22 September 2011Table 16a: Additional Gonadal Histopathology Observations in Male 

# Pimephales promelas.Treatment (mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityIncreaseSpermatogoniaEfferent DuctDiameter DecreaseMilt DecreaseAltered Proportions ofSpermatids(Decrease)GranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceControl(<LOQ)0 8 8888888881 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 03 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 00.080 8 8 8 8 8 8 8 8 8 71 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 13 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 0Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page40Rf93Version: 22 September 2011Treatment (mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityIncreaseSpermatogoniaEfferent DuctDiameter DecreaseMilt DecreaseAltered Proportions ofSpermatids(Decrease)GranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.220 8 8 8 8 8 8 8 8 8 71 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 13 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 00.690 8 3 8 5 8 4 8 3 8 81 8 0 8 0 8 0 8 0 8 02 8 4 8 2 8 3 8 1 8 03 8 1 8 1 8 1 8 2 8 04 8 0 8 0 8 0 8 2 8 01Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. SeeAppendix E of the test guideline for reference.LOQ=0.0400 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page41Rf93Version: 22 September 2011Table 16b: Additional Gonadal Histopathology Observations in Male 

# Pimephales promelas.Treatment (mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityPrimarySpermatocytesDecreaseSecondarySpermatocytesDecreaseSertoli CellSpermatocyst#/tubule DecreaseSertoli CellSpermatocyst SizeDecreaseGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceControl(<LOQ)0 8 8888888881 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 03 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 00.080 8 8 8 8 8 8 8 8 8 71 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 13 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 0Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page42Rf93Version: 22 September 2011Treatment (mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityPrimarySpermatocytesDecreaseSecondarySpermatocytesDecreaseSertoli CellSpermatocyst#/tubule DecreaseSertoli CellSpermatocyst SizeDecreaseGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.220 8 8 8 8 8 8 8 8 8 71 8 0 8 0 8 0 8 0 8 02 8 0 8 0 8 0 8 0 8 13 8 0 8 0 8 0 8 0 8 04 8 0 8 0 8 0 8 0 8 00.690 8 3 8 5 8 5 8 5 8 81 8 0 8 0 8 0 8 0 8 02 8 2 8 0 8 0 8 0 8 03 8 2 8 3 8 3 8 3 8 04 8 1 8 0 8 0 8 0 8 01Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. SeeAppendix E of the test guideline for reference.LOQ=0.0400 mg a.i./L.

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

EPA MRID Number 48617506

Page 43 R f 93 Version: 22 September 2011 Table 17: Gonadal Histopathology in Female Pimephales promelas

.

Treatment (mg a.i./L)

[mean-measured] Diagnostic Observations 1



# Severity 

# Increased Oocyte

Atresia Gonad Size Decrease Mature Oocyte

Increase

n Incidence n Incidence n Incidence



# Control (<LOQ) 0 16 13 16 14 16 15

1 16 0 16 0 16 0

2 16 3 16 2 16 0

3 16 0 16 0 16 1

4 16 0 16 0 16 0

0.08 0 16 14 16 11 16 16

1 16 0 16 0 16 0

2 16 0 16 5 16 0

3 16 2 16 0 16 0

4 16 0 16 0 16 0

0.22 0 15 10 15 14 15 15

1 15 0 15 0 15 0

2 15 4 15 1 15 0

3 15 1 15 0 15 0

4 15 0 15 0 15 0

0.69 0 15 9 15 14 15 14

1 15 0 15 0 15 0

2 15 4 15 1 15 1

3 15 0 15 0 15 0

4 15 2 15 0 15 0

1 

# Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. LOQ=0.0400 mg a.i./L.

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

EPA MRID Number 48617506

Page 44 R f 93 Version: 22 September 2011 Table 18: Additional Gonadal Histopathology Observations in Female Pimephales promelas

.

Treatment (mg a.i./L)
[meanmeasured] Additional Diagnostic Observations 1



# Severity 

# Early Vitellogenic oocyte decrease Late Vitellogenic oocyte decrease Granulomatous

Inflammation Decreased PostOvulatory Follicles

n Incidence n Incidence n Incidence n Incidence



# Control (<LOQ) 0 16 15 16 15 16 16 16 16

1 16 0 16 0 16 0 16 0

2 16 1 16 1 16 0 16 0

3 16 0 16 0 16 0 16 0

4 16 0 16 0 16 0 16 0

0.08 0 16 16 16 16 16 16 16 16

1 16 0 16 0 16 0 16 0

2 16 0 16 0 16 0 16 0

3 16 0 16 0 16 0 16 0

4 16 0 16 0 16 0 16 0

0.22 0 15 15 15 14 15 15 15 15

1 15 0 15 0 15 0 15 0

2 15 0 15 1 15 0 15 0

3 15 0 15 0 15 0 15 0

4 15 0 15 0 15 0 15 0

0.69 0 15 15 15 15 15 15 15 15

1 15 0 15 0 15 0 15 0

2 15 0 15 0 15 0 15 0

3 15 0 15 0 15 0 15 0

4 15 0 15 0 15 0 15 0

1 

# Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference. LOQ=0.0400 mg a.i./L.

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

EPA MRID Number 48617506

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# Mean male VTG was 970, 270, 390 and 270 and female VTG was 1.1x10 8 

# , 1.2x10 8 

# , 6.3x10 7 

# and

1.1x10 8 

# in the mean-measured 0 (control), 0.08, 0.22, and 0.69 mg a.i/L treatment levels, respectively.

Table 19: Plasma Vitellogenin in Pimephales promelas

.

Treatment (mg a.i./L)
[ mean- 

# measured] Plasma Vitellogenin (VTG)

Males Females

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

Control (<LOQ) 4 370 196 4 1.1x10 8 

# 9.8x10 7



# 0.08 

# 4 270 291 4 1.2x10 8 

# 1.5x10 8



# 0.22 

# 4 390 497 4 6.3x10 7 

# 3.1x10 7



# 0.69 

# 4 270 321 4 1.1x10 8 

# 1.1x10 8



# Abbreviations: NA 

# Not applicable. ND 

# Not determined. SD 

# Standard deviation. LOQ=0.0400 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page46Rf93Version: 22 September 2011Table 20: Plasma Sex Steroids in 

# Pimephales promelas(if measured).Treatment(mg a.i./L)[mean-measured]Plasma Testosterone (T)Plasma 17ȕ-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDControl (<LOQ) ND -- -- ND -- -- ND -- -- ND -- --0.08ND -- -- ND -- -- ND -- -- ND -- --0.22ND -- -- ND -- -- ND -- -- ND -- --0.69ND -- -- ND -- -- ND -- -- ND -- --Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.LOQ=0.0400 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page47Rf93Version: 22 September 2011One female in the 0.22 mg a.i/L treatment group showed signs of a crooked spine, and 6 males in the 0.69 mg a.i/L treatment groupsshowed signs ofbruising, erratic swimming, loss of color or no color in tail, lethargy, and crooked spine. All other male and female fish appearednormal.Table 21: Secondary Sex Characteristics and Clinical Signs in 

# Pimephales promelas.Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceTypenIncidenceControl(<LOQ)None reported 4 - None reported 4 -0.08None reported4 -None reported4 -0.22None reported4 --Crooked spine4 10.69Bruising, erratic swimming, loss of color orno color in tail, lethargy, crooked spine4 6None reported 4 -LOQ=0.0400 mg a.i./L.

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

EPA MRID Number 48617506

Page 48 R f 93 Version: 22 September 2011 

# B. Study Authors’ Analysis and Conclusions

The study authors analyzed survival, weight, length, tubercle score, fatpad score, GSI, fertility, fecundity, and VTG. Data for growth, tubercle score, GSI, and VTG were analyzed separately by sex. For all parameters, the treatments were compared to the negative control group.

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 and fertility were determined using step-down Jonckheere-Terpstra trend test and Fisher’s exact test, and histopathology severity scores and stages of individuals were analyzed using step-down Jonckheere-Terpstra trend test only. All other endpoints (length, weight, GSI, fecundity, VTG, tubercle score, fatpad score, and gonad stage) were analyzed by ANOVA and pair-wise comparisons using Dunnett’s multicomparison test or the Wilcoxon rank-sum test. 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 using the transformed data. 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 method s recommended in the guideline.

Adult survival (range 91.7-100%) was not significant ly different compared to the controls (100%).
There was a statistically significant decrease in fecundity in the 0.69 mg a.i/L treatment group compared to the control (p<0.05; Dunnett’s test and Jonckheere-Terpstra trend test). There was a
statistically significant decrease in the fatpad sc ore at the 0.69 mg a.i/L compared to the control (p<0.05; Jonckheere-Terpstra trend test. There was also a significant decrease in the tubercle score at the 0.69 mg a.i/L compared to the control (p<0.05; Dunnett’s test). There was a statistically significant increase in the female GSI at the 0.69 mg a.i/L compared to the control (p<0.05; Jonckheere-Terpstra trend test). There were no significant effects determined for female body weight
and length, female and male VTG, fertilizat ion success, male GSI, and male length.

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

EPA MRID Number 48617506

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# The study authors also statistically analyzed histopathology effects and found statistically significant differences in males for increased spermatogonia, altered proportion of spermatocytes and spermatids, gonad size decrease, Sertoli cell spermatocyst number/tubule and spermatocyst size decrease, decrease germinal epithelium thickness, decrease tubule diameter, decrease efferent duct diameter,decrease spermatozoa, and decrease milt at the 0.69 mg a.i/L treatment (p<0.05; JonckheereTerpstra trend test). Additionally, the testes of 4 of 8 fish in the 0.69 mg a.i/L treatment could not
be staged using normal criteria due to moderate to severe alteration of the spermatocyte ratios, including abnormal spermiation, proliferation and meiosis and maturation, and therefore could not be statistically compared. In females, there was a significant increase in oocyte atresia (p<0.05; Jonckheere-Terpstra trend test). Although not analyzed statistically, there were generalized signs of
toxicity in males at 0.69 mg a.i/L treatment level that included 6 incidence of bruising, erratic swimming, loss of color or no color in tail, lethargy, and crooked spine. There were no significant differences in survival, fertility, female weight, male and female length, male GSI, and male and female

VTG.

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), male gonadal somatic index (GSI), male testosterone, and fertility data were not consistent
with a monotonic concentration-response. All data were tested for normality using Shapiro-Wilks test and for homogeneity of variance using Levene’s test. Data which met the assumptions of normality and homogeneity of variance were then analyzed using the parametric Dunnett’s test. Data
that failed parametric assumptions were analyzed using the non-parametric Mann-Whitney U test. Female GSI, fecundity, and male tubercle score data exhibited monotonic trends and were analyzed using the non-parametric Jonckheere-Terpstra test, as recommended by the OCSPP 890.1350 guideline. The reviewer compared all malathion-treated groups to the negative control group.

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

EPA MRID Number 48617506

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# Female GSI exhibited a concentration-dependent monotonic increasing trend of 21%, compared to the negative control, at the highest treatment level (p<0.05; Jonckheere-Terpstra). Male tubercle
scores were significantly reduced and fecundity was significantly reduced 48% at the highest treatment level compared to the negative control (p<0.01; Jonckheere-Terpstra). The study authors
did not dismiss the possibility of a treatment-induced reduction, but did not quantify that food
consumption was also reduced at affected levels.

Histopathology, gonadal staging, and secondary sex characteristics were adversely indicated at the highest treatment level, but these effects were no t statistically analyzed by the reviewer. However, reviewer’s observations were validated by the study authors’ statistical analysis.

Conclusions:

Female GSI followed a concentration-dependent increase (p<0.05) of 21% at the highest treatment
level, compared to the negative control. Additionally, male tubercle scores were significantly reduced (p<0.01) (10.5 vs. 25.5) and fecundity was reduced 48% (p<0.01) in the highest treatment group.
The study authors also reported a statistically significant decrease in the fatpad score at the highest treatment compared to the control (p<0.05; Jonckheere-Terpstra trend test). There were also
reductions in male body weight at the two highest treatment levels of 20% and 17%, respectively although these reductions were no t determined to be statistically significant. Malathion also significantly impacted the multiple endpoints involved with the development of the testes at the highest treatment level (p<0.01) but did not appear to affect ovarian development to the same degree, with the exception of increased oocyte atresia (p = 0.042). It is noted however that the effects generally only occurred at the highest treatment concentration (0.69 mg a.i/L), where there was also a high incidence (8 of 24 fish) exhibiting a variety of clinical signs of toxicity that included
bruising, loss of coloration in the tail, erratic swimming, lethargy, and crooked spine. Additionally, reduced feeding was indicated by an excess of feed in the test chambers of the highest treatment

concentration.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page51Rf93Version: 22 September 2011Table 22: Reproductive and HPG Endpoints1,2for Male 

# Pimephales promelasin the FSTRA with Malathion.Treatment(mg a.i./L)3[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.p% Diff.p% Diff.pControl (<LOQ) 25.5 NA 0 NA NA 0 NA NA NA NA NA0.08 28.30.8456.10.939 No -27.21 0.955 NA NA NA NA0.22 19.00.151-21.360.297 No 5.441.00 NA NA NANA0.6910.50.006 -2.71 0.994Yes-25.85 0.961 NA NA NA NAStatistical Test Jonckheere-Terpstra Dunnett’sNADunnett’sNANAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusionsregarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3The mean-measured concentrations were calculated by the reviewer, including the Day 21 measured concentrationsLOQ=0.0400 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page52Rf93Version: 22 September 2011Table 23: Reproductive and HPG Endpoints1,2for Female 

# Pimephales promelasin the FSTRA with Malathion.Treatment(mg a.i./L)3[mean-measured]FecundityFert. SuccessTubercle ScoreGSIGonadal Stagingand Histo.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.pMedianp% Diff.pEffect? (Yes/No)% Diff.p% Diff.p% Diff.pControl (<LOQ) 0 NA 0 NA 0 NA 0 NA NA NA NA NA NA NA NA0.080.94 0.557 0.35 0.728 0.0 NA -1.12 0.614 No 5.19 0.494 NA NA NA NA0.22 -10.06 0.416 0.13 0.980 0.0 NA 8.21 0.249 No -43.6 0.889 NA NA NA NA0.69-48.250.013 - 0.08 0.995 0.0 NA20.840.016 Yes -2.63 1.00 NA NA NA NAStatistical TestJonckheereTerpstraDunnett’sJonckheere-TerpstraJonckheere-TerpstraNA Mann Whitney NA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3The mean-measured concentrations were calculated by the reviewer, including the Day 21 measured concentrationsLOQ=0.0400 mg a.i./L.

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

EPA MRID Number 48617506

Page 53 R f 93 Version: 22 September 2011 Table 24: Growth Endpoints 1,2 

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

Treatment (mg a.i./L) 3


# [mean-measured] Body Weight Length

Males Females Males Females

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


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



# 0.08 -8.42 0.639 1.84 0.972 -2.68 0.584 -1.15 0.792



# 0.22 -19.78 0.090 1.43 0.987 -6.19 0.069 -0.57 0.964



# 0.69 -17.32 0.147 4.49 0.743 -1.65 0.844 -0.89 0.886



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

Abbreviations: Diff. 

# Difference. NA 

# Not applicable. ND 

# Not determined.

1 

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

control.

2 

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

3 

# The mean-measured concentrations were calculated by the reviewer, including the Day 21 measured concentrations. LOQ=0.0400 mg a.i./L.

E. Study Deficiencies



# Individual weight of male and female fish (n = 10 each) selected prior to adding fish to the pre-exposure
system, had a deviation of ± 21% of the estimated mean of 2.7 g for males and 1.2 g. for females. EPA recommends that individual weight of each fish selected for the test be within ± 20% of the estimated mean of each sex. This deficiency did not impact the ability to interpret the results of the assay.

Measured concentrations in test solutions at 0, 7, 14 and 21 days ranged from 37.8 to 87.9% of nominal concentrations with a pattern of decline toward test termination. The coefficient of variation ranged from 4% to 9%, excluding Day 21 samples; the Day 21 samp les were much lower than nominal (37.8-72.25),
especially at the higher concentrations. The study authors suspected sample handling or analysis error and did not include them in the calculations of the mean measured; however, there is no data supporting this conclusion. Therefore, the reviewer calculated the %CV including Day 21 data and obtained %CV

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

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# values of 6, 26, and 29% for nominal application rates of 0.10, 0.32 and 1.0 mg a.i/L, respectively. These data indicate the test material was not stable under the test conditions. EPA requires a CV for measured test concentrations of <20% over 21 days. Excluding the Day 21 test concentrations also substantially affected the mean-measured concentrations obtained by the study authors. Excluding the Day 21 data, the mean-measured concentrations were 0.08, 0.26 and 0.82 mg a.i/L; including the Day 21 data, the mean-measured concentrations were 0.08, 0.22, and 0.69 mg a.i/L. The reviewer-calculated mean-measured concentrations, including the Day 21 data, were used in the Executive Summary and

Conclusions.

All other performance and validity criteria were met.

F. Reviewer’s Comments



# The reviewer’s and the study authors’ results were in agreement regarding effects on female GSI, male
tubercle score, and fecundity . Effects on male body weight were not determined to be significant based on the data being more appropriate for Dunnett’s test, which determined no significant differences. 

# The reviewer and study authors agreed on the subjectively-determined effects on secondary sex characteristics, gonad development, and histopathology. The histopathology was further verified by the study authors through statistical analysis.

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 te st 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 unionized ammonia and residual chlorine was not reported. Guidel ine 890.1350 recommends that residual chlorine in

dilution water be <10 P g/L and requires that unionized ammonia in the dilution water be <1 P g/L.

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

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# Although the study authors reported ca. 6 total volume exchanges per day in the test system, the actual flow rate was not reported. OCSPP 890.1350 guide lines recommended flow-thr ough rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.

III. REFERENCES

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

2. Organization for Economic Cooperation and Development. 2009. OECD Guidelines for Testing of Chemicals,

Guideline 229. Fish Short Term Reproduction Assay. Adopted 7 September 2009.

3. Boyd, C.E. 1979. Water Quality in warmwater fish ponds . Agricultural Experiment Station, Auburn University,
Auburn, Alabama.

4. Kendall, T.Z. and W.B. Nixon. 2011. Analytical Method Verification for the Determination of Malathion in Freshwater. Wildlife International, Ltd. Project Number: 232C-120. Unpublished report prepared for Cheminova A/S. January 18, 2011.

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

D.C.

6. Organization for Economic Cooperation and Development. 2006. Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application. OECD ENV/JM/MONO(2008)18.

7. The SAS System for Windows. 1999 - 2001. Version 8.2. SAS Institute, Inc. Cary, North CarolinaData Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page56Rf93Version: 22 September 2011APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:Endpoint Monotonic? Parametric? EDSP EFED CommentsFemale body weight NoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale body weight No; neither themean normedianYes Dunnett’s:n.s. p>0.05Jonckheere:Dose 2 p=0.014Dose 3 p=0.011Dunnett’s:n.s. p>0.05William’s:Dose 2 p=0.029Dose 3 p=0.030EDSP and EFED same conclusions, no effectReductions from control were 8, 20, and 17% at themeasured 0.08, 0.25, and 0.82 mg a.i/L levels; this doesnot follow a linear, monotonic pattern. It should be noted,however, that Jonckheere’s and William’s test detectedsignificant reductions at the two highest treatment levels,where reductions in wet weight were 17-20% of control.The study authors did not dismiss the possibility of atreatment-induced reduction at the 0.25 and 0.82 mg a.i/Llevels and observed, but did not quantify, that foodconsumption was also reduced at affected levels.Female body length NoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectMale body length NoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale VTG NoNoMannWhitney:n.s. p>0.05MannWhitney:n.s. p>0.05EDSP and EFED same conclusions, no effectData Evaluation Record onthe Fish Short-Term Reproduction Assay with MalathionEPA MRID Number 48617506Page57Rf93Version: 22 September 2011Endpoint Monotonic? Parametric? EDSP EFED CommentsMale VTGNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale GSI Yes, medianswere forincreasing trendYes Jonckheere:Dose 3 p=0.016William’s:Dose 3 p=0.030EDSP and EFED same conclusions, significant promotion(p<0.05) of 21% at the top dose.Male GSINoNoMannWhitney:n.s. p>0.05MannWhitney:n.s. p>0.05EDSP and EFED same conclusions, no effectFemale tuberclescoreNA NA NA NA No scoreMale tubercle score YesYesJonckheere:Dose 3 p=0.006William’s:Dose 3 p=0.002EDSP and EFED same conclusions; significant reduction atDose 3 (p<0.01; 52% lower than control)Fecundity Yes Yes Jonckheere:Dose 3 p=0.013William’s:Dose 3 p=0.001EDSP and EFED same conclusions; significant reduction atDose 3 (p<0.05; 48% lower than control)Fertility No Yes Dunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05EDSP and EFED same conclusions, no effectFtestosterone NA NA NA NA NAMtestosterone NA NA NA NA NAFestradiol NA NA NA NA NAMestradiol NA NA NA NA NA

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# 057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.913 0.130 0.202 0.893 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.23 0.09 0.05 7.73 1.07, 1.38 Dose1 4 1.25 0.11 0.06 9.05 1.07, 1.43 Dose2 4 1.24 0.09 0.04 7.12 1.10, 1.38 Dose3 4 1.28 0.07 0.04 5.52 1.17, 1.39

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.22 1.12 1.35 . . Dose1 1.23 1.14 1.40 101.84 -1.84 Dose2 1.22 1.17 1.37 101.43 -1.43 Dose3 1.27 1.21 1.37 104.49 -4.49

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

*** 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 0.24 0.864

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 1.23 . 1.25 . . . . .

. Dose1 1.25 0.972 1.25 0.726 . . . .

.

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

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# Dose2 1.24 0.987 1.25 0.760 1.000 . . .

. Dose3 1.28 0.743 1.25 0.778 0.959 0.939 . .

.

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

*** 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 1.31 0.727

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 1.22 . . Dose1 1.23 0.889 0.614 Dose2 1.22 0.889 0.615 Dose3 1.27 0.413 0.848

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 0.24 0.864

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 -1.23 . -1.23 . . . . .

. Dose1 -1.25 0.972 -1.25 0.454 . . . .

. Dose2 -1.24 0.987 -1.25 0.483 1.000 . . .

.

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# Dose3 -1.28 0.743 -1.28 0.277 0.959 0.939 . .

.

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

*** 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 1.31 0.727

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 -1.22 . . Dose1 -1.23 0.889 0.386 Dose2 -1.22 0.889 0.385 Dose3 -1.27 0.413 0.152

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.931 0.253 1.333 0.310 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3.15 0.47 0.23 14.83 2.40, 3.89 Dose1 4 2.88 0.34 0.17 11.75 2.34, 3.42 Dose2 4 2.53 0.43 0.21 16.87 1.85, 3.20 Dose3 4 2.60 0.22 0.11 8.51 2.25, 2.96

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# Level Median Min Max %of Control(means) %Reduction(means) Ctrl 3.14 2.69 3.63 . . Dose1 2.81 2.58 3.34 91.58 8.42 Dose2 2.46 2.09 3.10 80.22 19.78 Dose3 2.54 2.42 2.92 82.68 17.32

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 2.29 0.130

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

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

Ctrl 3.15 . 3.15 . . . . .

. Dose1 2.88 0.639 2.88 0.201 . . . .

. Dose2 2.53 0.090 2.56 0.029 0.553 . . .

. Dose3 2.60 0.147 2.56 0.030 0.722 0.991 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 6.16 0.104

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

Level Median MannWhit p-value Jonckheere p-value Ctrl 3.14 . . Dose1 2.81 0.346 0.124 Dose2 2.46 0.156 0.014 Dose3 2.54 0.156 0.011

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL

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# Williams Dose2 Jonckheere Dose2

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*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 2.29 0.130

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

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

Ctrl -3.15 . -2.79 . . . . .

. Dose1 -2.88 0.639 -2.79 0.946 . . . .

. Dose2 -2.53 0.090 -2.79 0.960 0.553 . . .

. Dose3 -2.60 0.147 -2.79 0.967 0.722 0.991 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 6.16 0.104

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

Level Median MannWhit p-value Jonckheere p-value Ctrl -3.14 . . Dose1 -2.81 0.346 0.876 Dose2 -2.46 0.156 0.986 Dose3 -2.54 0.156 0.989

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

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# Jonckheere >highest dose (no sign. differences)

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.931 0.249 1.569 0.248 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 47.88 1.11 0.55 2.32 46.11, 49.64 Dose1 4 47.33 1.26 0.63 2.66 45.32, 49.33 Dose2 4 47.60 0.57 0.29 1.20 46.69, 48.51 Dose3 4 47.45 1.03 0.52 2.18 45.80, 49.10

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 47.50 47.00 49.50 . . Dose1 47.40 46.00 48.50 98.85 1.15 Dose2 47.55 47.00 48.30 99.43 0.57 Dose3 47.25 46.50 48.80 99.11 0.89

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.21 0.886

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 47.88 . 47.88 . . . . .

.

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# Dose1 47.33 0.792 47.46 0.346 . . . .

. Dose2 47.60 0.964 47.46 0.370 0.981 . . .

. Dose3 47.45 0.886 47.45 0.376 0.998 0.997 . .

.

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

*** 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 0.52 0.915

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 47.50 . . Dose1 47.40 0.676 0.281 Dose2 47.55 1.000 0.384 Dose3 47.25 0.676 0.337

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

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.21 0.886

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 -47.88 . -47.56 . . . . .

. Dose1 -47.33 0.792 -47.56 0.750 . . . .

.

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# Dose2 -47.60 0.964 -47.56 0.783 0.981 . . .

. Dose3 -47.45 0.886 -47.56 0.801 0.998 0.997 . .

.

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

*** 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 0.52 0.915

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 -47.50 . . Dose1 -47.40 0.676 0.719 Dose2 -47.55 1.000 0.616 Dose3 -47.25 0.676 0.663

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.936 0.306 0.197 0.896 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 60.63 2.14 1.07 3.52 57.23, 64.02 Dose1 4 59.00 1.68 0.84 2.85 56.32, 61.68 Dose2 4 56.88 2.46 1.23 4.33 52.96, 60.79

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# Dose3 4 59.63 2.14 1.07 3.58 56.23, 63.02

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 60.75 58.00 63.00 . . Dose1 59.00 57.00 61.00 97.32 2.68 Dose2 56.00 55.00 60.50 93.81 6.19 Dose3 59.00 58.00 62.50 98.35 1.65

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

*** 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 2.23 0.137

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 60.63 . 60.63 . . . . .

. Dose1 59.00 0.584 59.00 0.179 . . . .

. Dose2 56.88 0.069 58.25 0.087 0.514 . . .

. Dose3 59.63 0.844 58.25 0.090 0.975 0.306 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 4.72 0.193

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 60.75 . . Dose1 59.00 0.346 0.124 Dose2 56.00 0.154 0.014 Dose3 59.00 0.570 0.140

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# 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 2.23 0.137

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 -60.63 . -58.83 . . . . .

. Dose1 -59.00 0.584 -58.83 0.928 . . . .

. Dose2 -56.88 0.069 -58.83 0.945 0.514 . . .

. Dose3 -59.63 0.844 -59.63 0.867 0.975 0.306 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 4.72 0.193

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 -60.75 . . Dose1 -59.00 0.346 0.876 Dose2 -56.00 0.154 0.986 Dose3 -59.00 0.570 0.860

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.806 0.003 1.797 0.201 USE NON-PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.1231E8 98502706 49251353 87.71 -4.443E7,2.6905E8 Dose1 4 1.1814E8 1.5281E8 76405278 129.35 -1.25E8,3.6129E8 Dose2 4 63312500 30817995 15408998 48.68 14274193,1.1235E8 Dose3 4 1.0936E8 1.0734E8 53671009 98.16 -6.145E7,2.8016E8

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 70036500 50165000 2.5901E8 . . Dose1 46727500 32267000 3.4684E8 105.19 -5.19 Dose2 66932500 24851000 94534000 56.37 43.63 Dose3 74262500 25090000 2.6382E8 97.37 2.63

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

*** 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 0.22 0.878

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

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# p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5

Ctrl 1.1231E8 . 1.1523E8 . . . . .

. Dose11.1814E8 1.000 1.1523E8 0.600 . . . .

. Dose263312500 0.854 86335250 0.466 0.885 . . .

. Dose31.0936E8 1.000 86335250 0.482 0.999 0.927 . .

.

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

*** 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 0.42 0.936

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 70036500 . . Dose1 46727500 0.494 0.193 Dose2 66932500 0.889 0.279 Dose3 74262500 1.000 0.426

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 0.22 0.878

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 Malathion

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# Ctrl -1.123E8 . -9.792E7 . . . . .

. Dose1-1.181E8 1.000 -9.792E7 0.661 . . . .

. Dose2-6.331E7 0.854 -9.792E7 0.696 0.885 . . .

. Dose3-1.094E8 1.000 -1.094E8 0.653 0.999 0.927 . .

.

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

*** 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 0.42 0.936

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

Level Median MannWhit p-value Jonckheere p-value Ctrl -7.004E7 . . Dose1 -4.673E7 0.494 0.807 Dose2 -6.693E7 0.889 0.721 Dose3 -7.426E7 1.000 0.574

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.898 0.074 0.893 0.473 USE PARAMETRIC TESTS

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

***

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

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# BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 367.50 199.56 99.78 54.30 49.95, 685.05 Dose1 4 267.50 295.68 147.84 110.53 -202.99, 737.99 Dose2 4 387.50 499.62 249.81 128.94 -407.51, 1182.51 Dose3 4 272.50 320.25 160.12 117.52 -237.08, 782.08

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 355.00 160.00 600.00 . . Dose1 205.00 20.00 640.00 72.79 27.21 Dose2 210.00 20.00 1110.00 105.44 -5.44 Dose3 160.00 30.00 740.00 74.15 25.85

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

*** 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 0.13 0.940

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 367.50 . 367.50 . . . . .

. Dose1 267.50 0.955 327.50 0.514 . . . .

. Dose2 387.50 1.000 327.50 0.546 0.960 . . .

. Dose3 272.50 0.961 272.50 0.462 1.000 0.964 . .

.

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

*** 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 0.75 0.862

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

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

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# Ctrl 355.00 . . Dose1 205.00 0.678 0.282 Dose2 210.00 0.678 0.304 Dose3 160.00 0.494 0.304

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 0.13 0.940

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 -367.50 . -317.50 . . . . .

. Dose1 -267.50 0.955 -317.50 0.667 . . . .

. Dose2 -387.50 1.000 -330.00 0.681 0.960 . . .

. Dose3 -272.50 0.961 -330.00 0.700 1.000 0.964 . .

.

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

*** 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 0.75 0.862

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 -355.00 . .

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

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# Dose1 -205.00 0.678 0.718 Dose2 -210.00 0.678 0.696 Dose3 -160.00 0.494 0.696

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.947 0.438 0.519 0.677 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 11.88 1.73 0.87 14.61 9.11, 14.64 Dose1 4 11.74 1.90 0.95 16.19 8.72, 14.77 Dose2 4 12.85 1.67 0.84 13.00 10.19, 15.51 Dose3 4 14.35 0.81 0.41 5.65 13.06, 15.64

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 11.70 10.00 14.10 . . Dose1 11.90 9.27 13.90 98.88 1.12 Dose2 12.65 11.10 15.00 108.21 -8.21 Dose3 14.45 13.30 15.20 120.84 -20.84

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 2.30 0.129

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

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

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# 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 11.88 . 12.70 . . . . .

. Dose1 11.74 0.999 12.70 0.841 . . . .

. Dose2 12.85 0.723 12.70 0.869 0.759 . . .

. Dose3 14.35 0.114 12.70 0.883 0.147 0.559 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 6.02 0.111

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 11.70 . . Dose1 11.90 0.889 0.386 Dose2 12.65 0.678 0.721 Dose3 14.45 0.103 0.984

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 2.30 0.129

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

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

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

Ctrl -11.88 . -11.81 . . . . .

. Dose1 -11.74 0.999 -11.81 0.608 . . . .

. Dose2 -12.85 0.723 -12.85 0.257 0.759 . . .

. Dose3 -14.35 0.114 -14.35 0.030 0.147 0.559 . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 6.02 0.111

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 -11.70 . . Dose1 -11.90 0.889 0.614 Dose2 -12.65 0.678 0.279 Dose3 -14.45 0.103 0.016

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.967 0.791 5.006 0.018 USE NON-PARAMETRIC TESTS

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

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# *****************************************************************************

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.48 0.24 0.12 16.14 1.10, 1.85 Dose1 4 1.57 0.43 0.22 27.71 0.88, 2.25 Dose2 4 1.16 0.16 0.08 13.81 0.91, 1.41 Dose3 4 1.44 0.19 0.10 13.54 1.13, 1.74

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.48 1.22 1.72 . . Dose1 1.55 1.12 2.05 106.10 -6.10 Dose2 1.09 1.07 1.40 78.64 21.36 Dose3 1.45 1.19 1.66 97.29 2.71

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

*** 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 1.58 0.245

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 1.48 . 1.52 . . . . .

. Dose1 1.57 0.939 1.52 0.676 . . . .

. Dose2 1.16 0.297 1.30 0.245 0.220 . . .

. Dose3 1.44 0.994 1.30 0.254 0.909 0.522 . .

.

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

*** 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 5.36 0.147

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

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

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# Level Median MannWhit p-value Jonckheere p-value Ctrl 1.48 . . Dose1 1.55 0.889 0.614 Dose2 1.09 0.156 0.054 Dose3 1.45 0.889 0.288

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 1.58 0.245

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 -1.48 . -1.40 . . . . .

. Dose1 -1.57 0.939 -1.40 0.733 . . . .

. Dose2 -1.16 0.297 -1.40 0.767 0.220 . . .

. Dose3 -1.44 0.994 -1.44 0.720 0.909 0.522 . .

.

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

*** 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 5.36 0.147

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 Malathion

EPA MRID Number 48617506

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# Level Median MannWhit p-value Jonckheere p-value Ctrl -1.48 . . Dose1 -1.55 0.889 0.386 Dose2 -1.09 0.156 0.946 Dose3 -1.45 0.889 0.712

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.00 0.00 0.00 . . , . Dose1 4 0.00 0.00 0.00 . . , . Dose2 4 0.00 0.00 0.00 . . , . Dose3 4 0.00 0.00 0.00 . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.00 0.00 0.00 . . Dose1 0.00 0.00 0.00 . . Dose2 0.00 0.00 0.00 . . Dose3 0.00 0.00 0.00 . .

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

*** 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 . . . . . .

.

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

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

Ctrl 0.00 . . . . . . .

. Dose1 0.00 . . . . . . .

. Dose2 0.00 . . . . . . .

. Dose3 0.00 . . . . . . .

.

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

*** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 0.00 1.000

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

Level Median MannWhit p-value Jonckheere p-value Ctrl 0.00 . . Dose1 0.00 1.000 . Dose2 0.00 1.000 . Dose3 0.00 1.000 .

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose1

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

*** 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 . . . . . .

.

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

EPA MRID Number 48617506

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# 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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) )

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

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

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# Test Stat P-value Test Stat P-value 0.980 0.967 0.301 0.824 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 23.75 5.36 2.68 22.58 15.22, 32.28 Dose1 4 28.13 4.33 2.16 15.39 21.24, 35.01 Dose2 4 19.75 3.38 1.69 17.11 14.37, 25.13 Dose3 4 11.38 6.02 3.01 52.91 1.80, 20.95

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 25.50 16.00 28.00 . . Dose1 28.25 23.00 33.00 118.42 -18.42 Dose2 19.00 16.50 24.50 83.16 16.84 Dose3 10.50 5.00 19.50 47.89 52.11

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 8.54 0.003

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

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

Ctrl 23.75 . 25.94 . . . . .

. Dose1 28.13 0.470 25.94 0.813 . . . .

. Dose2 19.75 0.535 19.75 0.171 0.124 . . .

. Dose3 11.38 0.010 11.38 0.002 0.002 0.124 . .

.

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

*** 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 9.10 0.028

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

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# 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 25.50 . . Dose1 28.25 0.413 0.845 Dose2 19.00 0.410 0.151 Dose3 10.50 0.103 0.006

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 8.54 0.003

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

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

Ctrl -23.75 . -20.75 . . . . .

. Dose1 -28.13 0.470 -20.75 0.872 . . . .

. Dose2 -19.75 0.535 -20.75 0.897 0.124 . . .

. Dose3 -11.38 0.010 -20.75 0.909 0.002 0.124 . .

.

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

*** 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 9.10 0.028

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

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# Level Median MannWhit p-value Jonckheere p-value Ctrl -25.50 . . Dose1 -28.25 0.413 0.155 Dose2 -19.00 0.410 0.849 Dose3 -10.50 0.103 0.994

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.919 0.165 2.222 0.138 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 29.33 2.11 1.05 7.19 25.97, 32.68 Dose1 4 29.60 6.47 3.24 21.87 19.30, 39.90 Dose2 4 26.38 6.32 3.16 23.96 16.32, 36.43 Dose3 4 15.18 4.96 2.48 32.69 7.28, 23.07

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 28.50 27.90 32.40 . . Dose1 29.20 23.30 36.70 100.94 -0.94 Dose2 29.05 17.00 30.40 89.94 10.06 Dose3 17.20 7.80 18.50 51.75 48.25

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 6.65 0.007

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

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

Ctrl 29.33 . 29.46 . . . . .

. Dose1 29.60 1.000 29.46 0.599 . . . .

. Dose2 26.38 0.771 26.38 0.283 0.822 . . .

. Dose3 15.18 0.007 15.18 0.001 0.010 0.047 . .

.

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

*** 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 7.32 0.062

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

Level Median MannWhit p-value Jonckheere p-value Ctrl 28.50 . . Dose1 29.20 1.000 0.500 Dose2 29.05 1.000 0.385 Dose3 17.20 0.067 0.013

DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose3 Jonckheere Dose3

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 6.65 0.007

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

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

Ctrl -29.33 . -25.12 . . . . .

. Dose1 -29.60 1.000 -25.12 0.919 . . . .

. Dose2 -26.38 0.771 -25.12 0.937 0.822 . . .

. Dose3 -15.18 0.007 -25.12 0.946 0.010 0.047 . .

.

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

*** 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 7.32 0.062

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

Level Median MannWhit p-value Jonckheere p-value Ctrl -28.50 . . Dose1 -29.20 1.000 0.500 Dose2 -29.05 1.000 0.615 Dose3 -17.20 0.067 0.987

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility )

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

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

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# 0.865 0.023 3.318 0.057 USE PARAMETRIC TESTS

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 99.25 0.35 0.18 0.35 98.69, 99.81 Dose1 4 99.60 0.18 0.09 0.18 99.31, 99.89 Dose2 4 99.38 0.43 0.22 0.44 98.68, 100.07 Dose3 4 99.18 0.99 0.49 1.00 97.60, 100.75

Level Median Min Max %of Control(means) %Reduction(means) Ctrl 99.25 98.90 99.60 . . Dose1 99.60 99.40 99.80 100.35 -0.35 Dose2 99.45 98.80 99.80 100.13 -0.13 Dose3 99.60 97.70 99.80 99.92 0.08

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

*** 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 0.42 0.743

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 99.25 . 99.43 . . . . .

. Dose1 99.60 0.728 99.43 0.750 . . . .

. Dose2 99.38 0.980 99.38 0.741 0.944 . . .

. Dose3 99.18 0.995 99.18 0.554 0.727 0.959 . .

.

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

*** 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 1.67 0.643

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

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# 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 99.60 0.283 0.904 Dose2 99.45 0.780 0.697 Dose3 99.60 0.484 0.731

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 0.42 0.743

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 -99.25 . -99.25 . . . . .

. Dose1 -99.60 0.728 -99.38 0.444 . . . .

. Dose2 -99.38 0.980 -99.38 0.473 0.944 . . .

. Dose3 -99.18 0.995 -99.38 0.489 0.727 0.959 . .

.

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

*** 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 1.67 0.643

MannWhit - testing each trt median signif. different from control

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

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# Jonckheere - test assumes dose-response relationship, testing INCREASING trend

Level Median MannWhit p-value Jonckheere p-value Ctrl -99.25 . . Dose1 -99.60 0.283 0.096 Dose2 -99.45 0.780 0.303 Dose3 -99.60 0.484 0.269

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR13 ( F testosterone (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST

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

*** BASIC SUMMARY STATISTICS 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

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# Numerator df Denominator df F-stat P-value 3 12 0.42 0.743

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 0.42 0.743

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR14 ( M testosterone (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , .

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# 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 0.42 0.743

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 0.42 0.743

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

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR15 ( F 17b-estradiol (ng/mL) )

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

EPA MRID Number 48617506

Page 91 R f 93 Version: 22 September 2011 

# TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl . . . . . Dose1 . . . . . Dose2 . . . . . Dose3 . . . . .

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.42 0.743

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 0.42 0.743

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

EPA MRID Number 48617506

Page 92 R f 93 Version: 22 September 2011 

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

Level Median MannWhit p-value Jonckheere p-value Ctrl . . . Dose1 . . . Dose2 . . . Dose3 . . . Jonckheere Dose1

057701 Malathion 890 1350 48617506 ANALYSIS RESULTS FOR VARIABLE VAR16 ( M 17b-estradiol (ng/mL) )

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01Levenes test for homogeneity of variance(absolute residuals) -- alphalevel=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST

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

*** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 0 . . . . . , . Dose1 0 . . . . . , . Dose2 0 . . . . . , . Dose3 0 . . . . . , .

Level Median Min Max %of Control(means) %Reduction(means) Ctrl . . . . . Dose1 . . . . . Dose2 . . . . . Dose3 . . . . .

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

*** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 0.42 0.743

MannWhit - testing each trt median signif. different from control

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

EPA MRID Number 48617506

Page 93 R f 93 Version: 22 September 2011 

# 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 0.42 0.743

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

# DATA EVALUATION RECORD

MALATHION
Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-05-2012 (MRID 48617507)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer Signature: Kelly Luck, M.S. Date: 2/27/2012
Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 3/01/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 3/05/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 3/05/2012

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).

# MALA TRION/ 057701


# Primary Reviewer: Kit Farewell, D.V.M. Health Effects Division Secondary Reviewer: Greg Akerman, Ph.D. Health Effects Division In vivo Hershberger Assay (2011) I 

# Page 1 of 18 OCSPP 890.1400/ OECD 441


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# Template version 10/2011 

# DATA EVALUATION RECORD

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

PC CODE: 057701 DP BARCODE: D396178

TXR#:0052348 CAS#: 121-75-5

TEST MATERIAL (PURITY): Malathion (96.0% a.i.)

SYNONYMS: Fyfanon® Technical; 2-[( dimethoxyphosphinothioyl)thio ]butanedioic acid, 1,4- diethyl ester

CITATION: Barnett, J. F. (2011) Oral (gavage) hershberger bioassay of malathion in male
rats. Charles River Laboratories, Preclinical Services, Horsham, PA. Laboratory Project ID 20012264, November 3, 2011. MRID 48617507. Unpublished.

SPONSOR: Cheminova A/S, P.O. Box 9, Lemvig, Denmark

TEST ORDER#: EDSP-057701-44

EXECUTIVE SUMMARY: In a Hershberger Assay (MRID 48617507) screening for androgenic activity, malathion (96.0% a.i., batch/lot 9010501) in com oil was administered daily via oral gavage to groups of eight 60-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 1000 mg/kg/day (limit dose). An 

# androgenic positive control group consisted of eight castrated rats exposed to 0.4 mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c.) injection.

To screen for potential anti-androgenic activity, malathion (96.0% a.i., batch/lot 9010501) in

com oil was administered daily via oral gavage to groups of eight 60-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 100, 300, or 1000 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive control group consisted of eight castrated rats exposed to 0.4 mg/kg/day TP and 3 mg/kg/day flutamide (Fn. TP alone was used as the anti-androgenic negative control.

For both components of the assay, body weights and food consumption were determined daily. The animals were dosed for 10 consecutive days and terminated approximately 24 hours after the final dose. At necropsy, the five androgen-dependent tissues, liver, and kidneys were collected and weighed. Brain and red blood cell (RBC) samples were also collected from rats in the androgen agonist assay for determination of cholinesterase (ChE) activity.

# In vivo Hershberger Assay (201 1) / Page 2 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441



# There were no treatment-related clin ical signs of toxicity or gross pathological findings in either assay.
In the androgen agonist assay, there were no tr eatment-related effects of malathion on accessory sex organ weights. At 1000 mg/kg/day, absolu te and relative (to body weight) liver weights were increased (p ≤ 0.01) by 29% and 32%, respectively, comp ared to controls; absolute and relative kidney weights were also increased (p ≤ 0.05) in this group ( ↑ 18% and ↑ 16%). Relative liver and kidney weights were increased (p ≤ 0.05) at 300 mg/kg/day ( ↑ 9% and ↑ 7%, respectively). At 1000 mg/kg/day, br ain ChE activity was inhibited (p ≤ 0.01) by 39%, while RBC ChE activity was inhibited (p ≤ 0.01) by 22-88% at 100 mg/kg/day and above.
Percent CVs for the vehicle control and 1000 mg /kg/day group of the androgen agonist assay were compared to the performan ce criteria in the Guidelines. The %CV values were less than the maximum permissible values for all accesso ry sex organs, with the exception of ventral prostate (CV = 50.5% for control; maximum %CV = 45) and seminal vesicles (CV = 41.6% for control and 42.8% for 1000 mg/kg/day group; co mpared to maximum %CV = 40). The study author noted that the same prosec tor dissected these organs for ev ery rat in the androgen agonist assay.
In the anti-androgen assay, there were no treatment -related effects for ma lathion on the weights of accessory sex organs. At 1000 mg/kg/day, ab solute and relative (to body weight) liver weights were increased (p ≤ 0.01) by 21% and 27%, respectively ; relative kidney weights were also increased by 13% (p ≤ 0.01) at this dose. Absolute and re lative liver weight s were increased (p ≤ 0.05) by 14% and 10%, respective ly, at 100 mg/kg/day. The pe rformance criteria indicated that this assay was performing as expected.
Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. Malathion was negative for androgenicity and anti-androgenicity in the Hershberger assay.
The assay satisfies the EDSP Tier 1 Test Order requireme nts for a Hershberger assay (OCSPP 890.1400).

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

# In vivo Hershberger Assay (201 1) / Page 3 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories, Preclinical Services
Location: Horsham, PA Study Director: J. F. Barnett Other Personnel: A. Hoberman (Testing Facility Mana gement); M. Vane man (Director of Operations); J. Lech (Senior Manager Technical Operations); J. Yeager (Senior Manager Technical Resources ); J. Gulbinski (Senior Manager Laboratory Sciences); J. Sarsoza (Princ ipal Investigator Dose Formulation Analysis) Study Period: July 26 - November 3, 2011



# 2. Test Substance: Malathion Description: Clear to yellowish liquid Source: Cheminova A/S (Lemvig, Denmark) Lot/Batch #: 9010501 (expiration date 11/12/2014) Purity: 96.0% Stability: Stable in vehicle for up to 40 hours at room temperature and for up to 13 days at 2-8 °C CAS #: 121-75-5 Structure:



# 3. Reference Androgen: Testosterone propionate (TP) Supplier: PCCA (Houston, TX) Lot/Batch #: C141154 (expiration date 5/19/2013) Purity: 99.7% CAS #: 57-85-2


# 4. Reference Anti-androgen: Flutamide (FT) Supplier: TCI America (Portland, OR) Lot/Batch #: 7EDOD (expiration date 1/11/2012) Purity: 99.8% CAS #: 13311-84-7



# 5. Solvent/Vehicle Control: Corn oil Supplier: Charkit Chemical Corporation (Norwalk, CT) Lot/Batch #: M-631 (expiration date 3/18/2012) Rationale (if other than water): Appropriate vehicle for malathion formulations Final concentration: Not applicable S

O O CH 3 

# O O CH 3



# P S

O CH

3 

# O CH

3

# In vivo Hershberger Assay (201 1) / Page 4 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441



# 6. Test Animals: Species: Rat (males only) Strain: Sprague Dawley [Crl:CD(SD)] Age/weight at dose initiation: Post-natal day (PND) 60/ 210.5 – 326.7 g Source: Charles River Laboratorie s, Inc. (Portage, MI) Housing: Rats were housed two/cage in clear pl astic nesting boxes w ith heat-treated laboratory-grade pine shavings Diet: Harlan Teklad 2018c meal food, ad libitum Phytoestrogen content ≤ 300 ppm genistein equivalents Water: Reverse-osmosis deionized tap water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 19-25 oC 30-70% minimum 10/hr 12 hrs light/12 hrs dark Acclimation period: Rats were castrated at supplier on P ND 43 and received at the study facility at PND 49; the postcastration acclimatio n period was 17 days (11-12 days at the study facility).



# B. STUDY DESIGN

1. In life dates: Androgen Agonist Assay: Start: 8/06/2011 End: 8/16/2011 Anti-androgen Assay: Start: 8/20/2011 End: 8/30/2011

2. Study Design: In a Hershberger Assay conducted to screen for pot ential androgenic activity, the test substance was administered daily via oral gavage to castrated male rats. Positive androgenic activity is defined as a signif icant increase in two or more target organ weights compared to the vehicle control. To screen for the po tential anti-androgenic activity, the test substance was also administered daily via oral gavage to castrated male rats in conjunction with a daily dose of TP (0.4 mg /kg/day) by s.c. injection. Anti-androgenic activity is indicated by a statistically signifi cant decrease in two or more target organ weights of the treated groups (test substance + TP) compared to the TP-only control group. For both assays, the animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose admi nistration for organ weight measurements.

3. Study Schedule: Sexually mature male rats were castr ated on PND 43 at the supplier and allowed at least 7 days for recovery and regression of accessory sex organ weights prior to initiation of dosing. The dose administrati on period was from PND 60 through PND 70. Rats were euthanized on PND 70 approximate ly 24 hours after the last dose and necropsied for organ weight measurements.

4. Animal Assignment: Animals were assigned to the te st groups noted in Table 1 using computer-generated, weight-ordered randomi zation procedures. At randomization, the weight variation of the rats did not exceed  20% of the mean weight in each group.

# In vivo Hershberger Assay (201 1) / Page 5 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441



# TABLE 1. Study Design a 

# Test group Dose (mg/kg/day) # of Males Androgen Agonist Assay Vehicle control (negative control) 0 8 Low 100 8 Mid 300 8 High 1000 8 Testosterone propionate (TP), positive control b 

# 0.4 8 Anti-Androgen Assay Vehicle control (+TP b 

# ) 0 8 Low (+TP b 

# ) 100 8 Mid (+TP b 

# ) 300 8 High (+TP b 

# ) 1000 8 Flutamide + TP b 

# , positive control 3 8 a Data were obtained from Text Tables 9 and 10 on pages 27 and 28 of the study report. b TP administered once daily by s.c. on all test days at 0.4 mg/kg/day.



# 5. Dose Selection Rationale: The dose levels were selected based on the results from a rangefinding study (MRID No. 48617508) in Sprague Dawl ey rats. A summary of this study is included as Appendix I to this DER.

6. (a) Dose Preparation: Dose formulations were prepared at least weekly (three times for the androgen agonist assay and two time s for the anti-androgen assay) by mixing appropriate amounts of test substance with co rn oil. Dose formulations were stored refrigerated (2-8 °C) until use. Doses were adjusted daily based on individual body weight measurements. Homogeneity was tested in all three dose levels at the first preparation for each assay, by collecting samples from the top, middle, and bottom of each formulation. During the study, samples of dose formulations at each level were analyzed, twice for the androgen agonist assay and once for the anti -androgen assay, for achieved concentration. The stability of the test substance in corn oil was tested in a separate study (MRID No. 48617512); a summary of this study is incl uded as Appendix II to this DER.
( b) Dose Analysis:

Results
Homogeneity (%CV): 0.3-1.2%
Stability (% of Day 0): 98.8-100.5% following storag e for 40 hours at 20-25 °C 97.2-99.0% following storage fo r 13 days at 2-8 °C.

Concentration (% of nominal): 93.1-103.9%
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: The malathion test formulations were administered to the animals daily via oral gavage (5 mL/ kg bw) for 10 days. TP was give n via s.c. injection at 0.5

# In vivo Hershberger Assay (201 1) / Page 6 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441


# mL/kg bw, and FT was administer ed via oral gavage at 5 mL /kg bw. Dose volumes were adjusted daily based on the concurrent body weight measurement.

8. Statistics: Clinical observations and other prop ortional data were analyzed using the variance test for homogeneity of the binomial distributi on. Continuous data (e.g., body weights, body weight gains, food consump tion values, and organ weight data) were analyzed using Bartlett’s test of homogeneity of variances an d the analysis of variance (ANOVA), when appropriate [i.e ., Bartlett’s test was not si gnificant (p>0.001)]. If the ANOVA was significant (p ≤ 0.05), Dunnett’s test was used to identify the statistical significance of the individual groups. If the ANOVA was not appropriate [i.e., Bartlett’s Test was significant (p ≤ 0.001)], the Kruskal-Wallis test was used ( ≤ 75% ties). In cases where the Kruskal-Wallis test was statistically significant (p ≤ 0.05), Dunn’s method of multiple comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fish er’s exact test was used to analyze the data. Data from the evaluation for pr eputial separation were analyzed using Fisher’s exact test. Absolute organ weights were also analyzed by analysis of covariance (ANCOVA), using the body weight from the first day of treatment as the covariate. Relative organ weight comparisons were not made.

ChE values for RBC and brains were evaluated as separate dependent variables in one-way ANOVA. In the event that the ANOVA was significant (p ≤ 0.05), Dunnett’s test was used to identify the statistical signi ficance of the individual groups.

C. METHODS

1. Clinical Examinations: The rats were checked for viabil ity at least twice daily during the study and were observed for general appearan ce daily during the dosing period. Post-dose observations were made daily 1-2 ho urs following dose administration.

2. Body Weight: Animals were weighed at rando mization, daily thr oughout the dosing period, and at termination.

3. Food Consumption: Food consumption was recorded daily per nesting box during the dosing period and on the day of terminati on. Values were reported in terms of g/animal/day.

4. Serum Hormone Measurements (Optional): Serum hormone measurements were not conducted.

5. Dissection and Measurement of Tissue and Organ Weights: On PND 70 (approximately 24 hours after the final administration of the test substance), surviving animals were euthanized by isoflurane/oxygen anesthet ization following by sodium pentobarbital injection (rats in the 0, 1 00, 300, and 1000 mg/kg/day dose groups of the androgen agonist assay) or by carbon dioxide asphyxiat ion (all other rats). The rats were subjected to a gross necropsy. The five mandatory androgen-depe ndent organs (ventral prostate, seminal vesicles, levator ani-bulbocavernosus (LABC) , Cowper’s gland, and glans penis) were excised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the

# In vivo Hershberger Assay (201 1) / Page 7 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441


# standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400). Additionally, the liver and kidneys were weighed.

6. Cholinesterase Evaluations: Evaluations of ChE activity we re conducted for rats in the 0, 100, 300, and 1000 mg/kg/day dose groups of the a ndrogen assay. At study termination, the selected rats were anesthetized with isof lurane/oxygen, and blood was collected from the inferior vena cava for ChE activity determ inations. After collection of blood and euthanasia, the brain was collected, weighed, a nd analyzed for ChE activity. Samples were analyzed within 3.25 hours of collection.

II. RESULTS

A. OBSERVATIONS

1. Mortality: One animal in the 1000 mg/kg/day dose group (androgen agonist assay; Animal No. 3239) was found dead on Day 6 of dosing. This rat lost weight on Days 4 to 5 and the food consumption for the rat’s nesting box was reduced on Days 5 to 6. Gross examination of this animal at necropsy revealed missing skin; all other tissues examined appeared normal. All other rats survived until scheduled sacrifice. Because no clinical signs were observed, and all other rats survived until te rmination, the death of this rat was not considered to be related to malathion dosing.

2. Clinical signs of toxicity: No treatment-related adverse c linical signs of toxicity were observed in animals for any dose groups.

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data for the androgen agonist assay are pres ented in Table 2. B ody weights and overall (Days 1-11) body weight gains in all malath ion treatment groups were comparable to controls throughout the duration of the assa y. Daily body weight gains were decreased (p ≤ 0.05) on Days 3-4 ( ↓ 89%) and 4-5 ( ↓ 59%) in the 1000 mg/kg/day group, but were similar to controls during the remainder of the treatment period. Overall body weight gains were increased (p ≤ 0.05) by 19% in the positive control group.

# In vivo Hershberger Assay (201 1) / Page 8 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441



# TABLE 2. Selected Group Mean Body Weights and Cumu lative Body Weight Gains (g) in the Androgen Agonist Assay a



# Study Day # Dose (mg/kg/day)

Vehicle Control Positive Control Vehicle + TP (0.4) Malathion (100) Malathion (300) Malathion (1000)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1 8 265.9 29.5 8 261.7 21.2 8 266. 1 18.1 8 268.6 29.7 8 265.7 22.4 4 8 286.0 32.2 8 288.3 24.1 8 287. 8 19.3 8 287.4 31.0 8 279.1 23.3 7 8 304.8 36.5 8 307.9 25.6 8 306. 1 20.8 8 304.5 36.2 7 297.3 28.7 11 8 328.5 37.8 8 336.2 29.3 8 331. 2 24.6 8 327.7 38.9 7 319.5 32.8 B ody Weight Gain (Days 1-11) 8 62.5 10.1 8 74.6* ( ↑ 19) 11.4 8 65.1 11.9 8 59.1 11.8 7 52.8 11.5

a Data were obtained from Tables 2 and 3 on pages 56 and 58 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



# Selected body weight and body weight gain data for the anti-androgen assay are presented in Table 3. Body weights in all malathion treatment groups were comparable to controls throughout treatment. Overall body we ight gains were increased (p ≤ 0.05) by 14% in rats in the 100 mg/kg/day dose group, and were decreased (p ≤ 0.01) by 14% in the 1000 mg/kg/day dose group. Daily body weight gains were increased (p ≤ 0.05 or p ≤ 0.01) on Days 2-3 in the 100 mg/kg/day group and on Days 1-2 in th e 1000 mg/kg/day group. Body weights and overall body weight gains were in the positive control group were comparable to the negative control.

TABLE 3. Selected Group Mean Body Weights an d Cumulative Body Weight Gains (g) in the Anti-Androgen Assay a



# Study Day # Dose (mg/kg/day)

Vehicle Control + TP (0.4) Positive Control TP + FT (0.4 + 3.0) Malathion + TP (100 + 0.4) Malathion + TP (300 + 0.4) Malathion + TP (1000 + 0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1 8 282.1 16.9 8 282.6 20.5 8 282. 6 19.3 8 278.9 22.0 8 277.6 15.2 4 8 306.4 18.3 8 303.8 21.8 8 310. 4 19.3 8 303.0 20.7 8 300.5 16.7 7 8 332.4 21.8 8 327.7 24.6 8 338. 6 21.3 8 328.7 24.5 8 324.1 17.2 11 8 360.6 25.5 8 354.1 27.8 8 371. 9 21.3 8 352.2 24.6 8 345.2 21.2 B ody Weight Gain (Days 1-11) 8 78.5 9.6 8 71.4 10.4 8 89.4* ( ↑ 14) 7.6 8 73.3 8.6 8 67.6** ( ↓ 14) 9.0

a Data were obtained from Tables 10 and 11 on pages 72 and 74 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 ** Significantly different from controls at p ≤ 0.01



# C. FOOD CONSUMPTION: Food consumption data for the androgen agonist assay are presented in Table 4. Daily mean food consumption was increased (p ≤ 0.05) in the 100 mg/kg/day group on Days 1-2 ( ↑ 12%) and Days 8-9 ( ↑ 11%); average food consumption over the duration of the study in this group was co mparable to the control. Daily mean food

# In vivo Hershberger Assay (201 1) / Page 9 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441


# consumption was decreased (p ≤ 0.05) in the 1000 mg/kg/day group on Days 3-4 ( ↓ 9%) and Days 4-5 ( ↓ 10). Food consumption was measured for each nesting box, and then divided by two to calculate the consumption in terms of g/rat/day. Based on the individual data, the study authors did not account for the deat h of one rat when calculating the food consumption for the 1000 mg/kg/day group for Da ys 6 through 11. The values reported by the study authors are included in Table 6; in addition, the reviewers have re-calculated the means for the dosing day interval s affected by the incorrect calcu lations. Statistical analyses were not conducted by the reviewers.
Food consumption in the 300 mg/kg/day group wa s comparable to the controls over the duration of the study. In the positive cont rol group, mean daily food consumption was increased (p ≤ 0.05) at several intervals; the average daily food consumption over Days 1-11 was increased (p ≤ 0.01) by 14% compared to the negative control.

TABLE 4. Food Consumption (g/rat/d ay) in the Androgen Agonist Assay a



# Study Day

Interval Dose (mg/kg/day)

Vehicle Control Positive Control Vehicle + TP (0.4) Malathion (100) Malathion (300) Malathion (1000)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1-2 8 18.9 2.4 8 22.4* ( ↑ 19) 0.2 8 21.2* ( ↑ 12) 1.5 8 20.8 2.7 8 17.9 1.5

2-3 8 20.4 0.8 8 22.2** ( ↑ 9) 0.9 8 22.0 1.8 8 21.1 1.0 8 19.8 2.5

3-4 8 20.0 1.9 8 20.8 1.5 8 21.5 1.6 8 20.1 1.0 8 18.2* ( ↓ 9) 1.4

4-5 8 21.4 1.2 8 21.4 2.1 8 22.8 2.4 8 22.9 1.4 8 19.2* ( ↓ 10) 1.4

5-6 8 19.9 1.4 8 21.6 2.0 8 19. 8 2.3 8 18.8 2.1 8 16.9 5.0 6-7 8 20.6 1.9 8 24.6** ( ↑ 19) 2.1 8 21.8 1.1 8 22.0 1.7 8 b 

# 19.8 [21.1] b 

# 3.9

7-8 8 21.0 1.2 8 24.6** ( ↑ 17) 1.0 8 20.9 2.1 8 21.0 2.4 8 b 

# 17.0** c 

# [19.4] b 

# 3.2

8-9 8 19.5 0.5 8 24.4** ( ↑ 25) 1.7 8 21.6* ( ↑ 11) 1.8 8 19.0 1.5 8 b 

# 17.5 [20.0] b 

# 3.7

9-10 8 21.1 0.9 8 26.1** ( ↑ 24) 0.8 8 23.0 4.0 8 23.0 2.0 8 b 

# 20.2 [23.1] b 

# 3.6

10-11 8 19.8 1.4 8 22.6** ( ↑ 14) 1.5 8 20.8 1.4 8 18.2 0.9 8 b 

# 16.8* c 

# [19.1] b 

# 3.7

(Days 1-11) 8 20.3 1.1 8 23.1** ( ↑ 14) 0.7 8 21.6 1.6 8 20.7 1.2 8 b 

# 18.4* c 

# [19.5] b 

# 2.2

a Data were obtained from Table 4 on page 60 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. b It is clear from the food consumption data reported in Appendix 8 on page 267 of the study report that the study author did n ot account for the death of one rat in this group on Day 6. The mean food consumption values ha ve been recalculated by the reviewers and are included in brackets. c The study author concluded that this value was significantl y different from the control; however, because it was calculated incorrectly, the percent difference from the control is not included. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01

# In vivo Hershberger Assay (201 1) / Page 10 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441


# Selected food consumption data for the anti-a ndrogen agonist assay are presented in Table 5. The average daily food consumption over Da ys 1-11 was comparable to the negative control for all malathion treatm ent groups as well as the posi tive control. Daily mean food consumption was increased (p ≤ 0.05) in the 100 mg/kg/day group on Days 8-9 ( ↑ 5%). Daily mean food consumption was decreased (p ≤ 0.05 or p ≤ 0.01) on Days 10-11 in the 300 mg/kg/day group ( ↓ 14%) and in the 1000 mg/kg/day group ( ↓ 19%); however, these single instances were considered incidental. Daily food consumption was decreased (p ≤ 0.05) on Days 5-6 in the posi tive control group ( ↓ 6%).

TABLE 5. Selected Food Consumption (g /rat/day) in the Anti-Androgen Assay a



# Study Day

Interval Dose (mg/kg/day)

Vehicle Control + TP (0.4) Positive Control TP + FT (0.4 + 3.0) Malathion + TP (100 + 0.4) Malathion + TP (300 + 0.4) Malathion + TP (1000 + 0.4)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD
1-2 8 21.6 1.3 8 20.1 1.7 8 21. 1 1.8 8 19.9 0.6 8 20.5 1.4 5-6 8 22.8 1.1 8 21.4** ( ↓ 6) 0.8 8 23.2 1.2 8 21.9 1.7 8 22.5 2.6

8-9 8 22.9 1.2 8 22.8 0.8 8 24.1* ( ↑ 5) 1.4 8 23.1 0.6 8 22.0 1.2

10-11 8 21.5 1.1 8 21.1 0.9 8 22.1 2.3 8 18.4** ( ↓ 14) 0.8 8 17.4** ( ↓ 19) 2.9

(Days 1-11) 8 22.5 1.1 8 21.8 0.7 8 23.0 1.2 8 22.0 1.2 8 21.9 1.2 a Data were obtained from Table 12 on page 76 of the study re port. 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 ** Significantly different from controls at p ≤ 0.01



# D. SERUM HORMONE CONCE NTRATIONS (OPTIONAL): Serum hormone concentration measurements were not conducted.

E. ORGAN WEIGHTS: Accessory sex organ, liver, and kidney weights for the androgen agonist assay are presented in Table 6. There were no malathion treatme nt-related effects in accessory sex organs. In the 1000 mg/kg/day group, absolute and relative (to body weight) liver weights were increased (p ≤ 0.01) by 29% and 32%, respectiv ely, compared to controls; absolute and relative kidney weights were also increased (p ≤ 0.05) in this group ( ↑ 18% and ↑ 21%). Relative liver and kidney weights were increased (p ≤ 0.05) in the 300 mg/kg/day group ( ↑ 9% and ↑ 7%). Rats in the positive control group had increased (p ≤ 0.01) organ weights as follows: 1007% in seminal vesicl es; 862% in ventral prostate; 193% in LABC; 532% in Cowper’s glands; 43% in glans penis; and 22% in kidneys; re lative kidney weight was also increased ( ↑ 19%).

Percent CVs for the vehicle control and 1000 mg/kg/day group were compared to the performance criteria in the Guidelines. Th e %CV values were less than the maximum permissible values for all accessory sex organs, with the exception of ventral prostate (CV = 50.5% for control; maximum %C V = 45) and seminal vesicles (CV = 41.6% for control and 42.8% for 1000 mg/kg/day group; compared to maximum %CV = 40). The study author

# In vivo Hershberger Assay (201 1) / Page 11 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441


# noted that the same prosector dissected these organs for every rat in the androgen agonist assay.In vivoHershberger Assay (2011) / Page 12 of 18MALATHION/ 057701OCSPP 890.1400/ OECD 441TABLE 6. Accessory Sex Organ, Liver,and Kidney Weights from AndrogenAgonist Assay in Sprague Dawley RatsaOrganDose (mg/kg/day)Vehicle ControlPositive Control Vehicle + TP(0.4)Malathion (100) Malathion (300) Malathion (1000)N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)Seminalvesicles (g)8 0.0615 0.0256 41.6 80.6808**(↑1007)0.1483 21.8 8 0.0656 0.0211 32.2 8 0.0621 0.0306 49.3 7 0.0538 0.0230 42.8Ventral prostate(g)8 0.0190 0.0096 50.5 80.1828**(↑862)0.0228 12.5 8 0.0166 0.0032 19.3 08 0.0182 0.0036 19.8 7 0.0198 0.0074 37.4LABC (g) 8 0.2232 0.0338 15.1 80.6540**(↑193)0.1254 19.2 8 0.1957 0.0215 11.0 8 0.2235 0.0726 32.5 7 0.1956 0.0362 18.5Cowper’sglands (g)7 0.0063 0.0026 41.3 80.0398**(↑532)0.0050 12.6 8 0.0072 0.0044 61.1 8 0.0054 0.0020 37.0 7 0.0052 0.0022 42.3Glans penis (g) 8 0.07 0.01 14.3 80.10**(↑43)0.01 10.0 8 0.08 0.01 12.5 8 0.07 0.01 14.3 7 0.07 0.01 14.3Liver (g) 8 13.6 1.9 14.0 8 14.4 1.913.2 8 14.2 1.3 9.2 8 14.8 2.0 13.5 717.5**(↑29)3.7 21.1Liver, relativeto terminalbody weight(%)8 4.135 0.136 3.3b8 4.274 0.258 6.0b8 4.300 0.178 4.1b84.502**(↑9)0.190 4.2b75.443**(↑32)0.635 11.7bKidneys (g) 8 2.0288 0.2618 12.9 82.4824**(↑22)0.3415 13.8 8 2.1092 0.1877 8.9 8 2.1672 0.2746 12.7 72.3955*(↑18)0.2573 10.7Kidney, relativeto terminalbody weight(%)8 0.617 0.042 6.8b80.736**(↑19)0.060 8.2b8 0.636 0.031 4.9b80.661*(↑7)0.027 4.1b70.748**(↑21)0.022 2.9ba Data were obtained from Table 6 on page 64 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses.b Calculated by the reviewers.N Number of animals in the groupSD Standard DeviationCV Coefficient of Variation* Significantly different from controls at p≤0.05** Significantly different from controls at p≤0.01

# In vivo Hershberger Assay (201 1) / Page 13 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441


# Accessory sex organ, liver, and kidney weight s for the anti-androgen assay are presented in Table 7. In the 100 mg/kg/day group, vent ral prostate weights were increased (p ≤ 0.01) 38% compared to the control; however, ventral pr ostate weights in th e 300 or 1000 mg/kg/day dose groups were similar to controls. There we re no malathion treatmen t-related effects in seminal vesicles, LABC, Cowper’s glands, or glans penis. In the 1000 mg/kg/day group, absolute and relative (to body weight ) liver weights were increased (p ≤ 0.01) 21% and 27%, respectively, and the relative kidne y weights were also increased by ↑ 13% (p ≤ 0.01). Absolute and relative liver weights were increased (p ≤ 0.05) by 14% and 10% in the 100 mg/kg/day group, respectively; absolute and re lative liver weights in the 300 mg/kg/day group were similar to the controls. Rats in the positive control group had decreased (p ≤ 0.01) accessory sex organ weights as follows: 75% in seminal vesicles; 68% in ventral prostate; 46% in LABC; and 64% in Cowper’s glands.

Percent CVs for the vehicle control and 1000 mg/kg/day group were compared to the performance criteria in the Guidelines. Th e %CV values were less than the maximum permissible values for all accessory sex organs.
There were no treatment-related gross pa thological findings in either assay.In vivoHershberger Assay (2011) / Page 14 of 18MALATHION/ 057701OCSPP 890.1400/ OECD 441TABLE 7. Accessory Sex Organ, Liver,and Kidney Weights from Anti-AndrogenAgonist Assay in Sprague Dawley RatsaOrganDose (mg/kg/day)Vehicle Control + TP(0.4)Positive ControlTP + FT(0.4 + 3.0)Malathion + TP (100 + 0.4)Malathion + TP (300 + 0.4)Malathion + TP (1000 + 0.4)N Mean SD CV(%)N Mean SD CV(%)N Mean SD CV(%)N Mean SD CV(%)N Mean SD CV(%)Seminalvesicles (g)8 0.7122 0.1333 18.7 80.1815**(↓75)0.0552 30.4 8 0.7233 0.1482 20.5 8 0.7874 0.1108 14.1 8 0.6852 0.1382 20.2Ventralprostate (g)8 0.1456 0.0251 17.2 80.0465**(↓68)0.0132 28.4 80.2013**(↑38)0.0560 27.8 8 0.1730 0.033719.5 8 0.1856 0.0255 13.7LABC (g) 8 0.6304 0.0531 8.4 80.3414**(↓46)0.0588 17.2 8 0.6202 0.0765 12.3 8 0.6543 0.0668 10.2 8 0.5966 0.0923 15.5Cowper’sglands (g)8 0.0441 0.0106 24.0 70.0159**(↓64)0.0056 35.2 8 0.0498 0.0056 11.2 8 0.0502 0.0089 17.7 8 0.0468 0.0031 6.6Glans penis (g) 8 0.1162 0.0186 16.0 80.1008 0.0157 15.6 8 0.1138 0.0132 11.68 0.1146 0.0115 10.0 8 0.1083 0.0144 13.3Liver (g) 8 16.0 1.4 8.8 8 15.7 1.4 8.9 818.2*(↑14)1.7 9.3 8 16.8 1.5 8.9 819.4**(↑21)2.0 10.3Liver, relativeto terminalbody weight(%)8 4.435 0.205 4.6b8 4.421 0.175 4.0b84.884*(↑10)0.332 6.8b8 4.759 0.302 6.3b85.625**(↑27)0.426 7.6bKidneys (g) 8 2.7033 0.2286 8.5 8 2.6104 0.2359 9.0 82.8570 0.2892 10.1 8 2.7188 0.2198 8.1 8 2.9338 0.2968 10.1Kidney,relative toterminal bodyweight (%)8 0.749 0.042 5.6b8 0.739 0.065 8.8b8 0.768 0.043 5.6b8 0.772 0.041 5.3b80.849**(↑13)0.068 8.0ba Data were obtained from Table 14 on page 80 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses.b Calculated by the reviewers.N Number of animals in the groupSD Standard DeviationCV Coefficient of Variation* Significantly different from controls at p<0.05** Significantly different from controls at p<0.01

# In vivo Hershberger Assay (201 1) / Page 15 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441



# F. CHOLINESTERASE ACTIVITY: ChE activity data for th e androgen agonist assay are presented in Table 8. At 1000 mg/kg/day, brain ChE activity was inhibited (p ≤ 0.01) by 39%, while RBC ChE activity was inhibited (p ≤ 0.01) by 22-88% at 100 mg/kg/day and above. ChE activity was not determined in rats in the anti-androgen assay.

TABLE 8. Cholinesterase Activity in the Androgen Agonist Assay a



# Matrix Dose (mg/kg/day) Vehicle Control Malathion (100) Malathion (300) Malathion (1000) N Mean SD N Mean SD N Mean SD N Mean SD Brain (U/g) 7 12.695 0.750 8 12. 520 0.596 7 11.689 0.899 7 7.728** ( ↓ 39) 1.325

RBC (U/mL) 8 1.242 0.132 8 0.974** ( ↓ 22) 0.180 8 0.482** ( ↓ 61) 0.042 7 0.148** ( ↓ 88) 0.031

a Data were obtained from Text Tables 15 and 16 on page 42 of the study report. Percent differences from controls are included in parentheses. N Number of animals in the group SD Standard Deviation ** Significantly different from controls at p ≤ 0.01



# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Malathion did not have a positive response for either androgenic or antiandrogenic activity in the Hershberger bioass ay at dose levels as high as 1000 mg/kg bw/day (the highest dose le vel tested and the limit dose as specified in the test guidelines) even in the presence of overt toxicity (body weight and feed consumption effects and increased liver a nd kidney weights) a nd statistically and biologically significant RBC and brain ChE inhibition.

B. AGENCY COMMENTS: One animal in the 1000 mg/ kg/day dose group in the androgen agonist assay was found dead on Day 6 of dos ing. Because all other rats survived until termination, the death of this rat was not consid ered to be related to malathion dosing. No other treatment-related clinical signs of t oxicity were observed in animals for any dose group. There were no treatment-rela ted gross pathological findings.
In the androgen agonist assa y, body weights and overall (Days 1-11) body weight gains in all malathion treatment groups were comparable to controls throughout the duration of the assay. Overall body weight gains were increased (p ≤ 0.05) by 19% in the positive control group. Daily mean food consumption was decreased (p ≤ 0.05) in the 1000 mg/kg/day group on Days 3-4 ( ↓ 9%) and Days 4-5 ( ↓ 10). Average daily food consumption in the 100 and 300 mg/kg/day groups was comparable to contro ls over the duration of the study. In the positive control group, average daily food c onsumption over Days 1-11 was increased (p ≤ 0.01) by 14% compared to the negative contro l. There were no malathion treatmentrelated effects in accessory sex organs. In the 1000 mg/kg/day group, absolute and relative (to body weight) liver weights were increased (p ≤ 0.01) by 29% and 32%, respectively, compared to the control; absolute and rela tive kidney weights were also increased (p ≤ 0.05) in this group ( ↑ 18% and ↑ 16%). Relative liver and kidney weights were increased (p ≤ 0.05) in the 300 mg/kg/day group ( ↑ 9% and ↑ 7%). Rats in the positive control group had

# In vivo Hershberger Assay (201 1) / Page 16 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441


# increased (p ≤ 0.01) organ weights as follows: 1007% in seminal vesicles; 862% in ventral prostate; 193% in LABC; 532% in Cowper’s glands; 43% in glans penis; and 22% in kidneys; relative kidney weight was also increased (19%). At 1000 mg/kg/day, brain ChE activity was significantly inhibited (p ≤ 0.01) by 39%, while RBC ChE activity was inhibited (p ≤ 0.01) by 22-88% at 100 mg /kg/day and above.

Percent CVs for the vehicle control and 1000 mg/kg/day group of the androgen agonist assay were compared to the performance criteri a in the Guidelines. The %CV values were less than the maximum permissible values for all accessory sex organs , with the exception of ventral prostate (CV = 50.5% for contro l; maximum %CV = 45) and seminal vesicles (CV = 41.6% for control and 42.8% for 1000 mg/kg/day group; compared to maximum %CV = 40).
In the anti-androgen assay, body we ights in all malathion treatme nt groups were comparable to controls throughout treatment. Over all body weight gains were increased (p ≤ 0.05) by 14% in rats in the 100 mg/kg/day dose group, and were decreased (p ≤ 0.01) by 14% in the 1000 mg/kg/day dose group. Body weights and overall body weight gains were in the positive control group were comparable to the negative control. The average daily food consumption over Days 1-11 was comparable to the negative control for all malathion treatment groups as well as the positive control. There were no treatment-related effects in the accessory sex organs. In the 1000 mg /kg/day group, absolute and relative (to body weight) liver weights were increased (p ≤ 0.01) by 21% and 27%, re spectively; relative kidney weights were also increased (p ≤ 0.01) in this group ( ↑ 13%). Absolute and relative liver weights were increased (p ≤ 0.05) 14% and 10% in the 100 mg/kg/day group, respectively; absolute and rela tive liver weights in the 300 mg/ kg/day group were similar to the controls. Rats in the positive control group had decreased (p ≤ 0.01) accessory sex organ weights as follows: 75% in seminal vesicles ; 68% in ventral prostate; 46% in LABC; and 64% in Cowper’s glands. The performance crite ria indicated that this assay was performing as expected.
Since statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights, malathion is consider ed to be negative for androgenicity and antiandrogenicity in the Hershberger assay.

C. STUDY DEFICIENCIES: None

# In vivo Hershberger Assay (201 1) / Page 17 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441



# APPENDIX I
Because this is a range-finding study, only a summary is provided to confirm the adequacy of the dose selection rationale used in the Hers hberger assay (MRID 48617507). The range-finding study was conducted to determine the maximum tolerated dose.
In a dose range-finding toxicity study (MRID 48617508), malathion [96.0% a.i.; Batch (Lot) No. 9010501] in corn oil was administered via daily oral gavage in a dose volume of 5 mL/kg body weight to groups of 8 male and 8 female Sprague Dawley [Crl:CD®(SD)] rats at doses of 0, 800, or 1000 mg/kg/day. Male rats were approximately 54 days old and female rats were approximately 52 days old at rece ipt at the study facility; rats we re acclimated for approximately 3 days prior to study initiation. Males rats were dosed for 10 consecutive days and female rats were dosed for 3 consecutive days; animals were terminated approximately 24 hours after the final dose administration and ki dneys and livers were collected and weighed. Statistical analyses, other than calculations of means and standard deviati ons, were not conducted.
All animals survived to the scheduled study term ination. Excess salivation (slight) was observed in one male rat in each of the 800 and 1000 mg/kg bw/day dose groups. These observations were not considered to be test substance-rela ted because they only occurred on one occasion and did not persist. There were no adverse clinical signs observed in the female rats. No gross lesions were observed in the male or female rats at the time of necropsy.
Body weight gains were decreased in the males at ≥ 800 mg/kg/day ( ↓ 10-20%), and in females at 1000 mg/kg/day ( ↓ 44%). Daily and terminal body weights were generally similar across all groups throughout the dosing period. Absolute and relative food consum ption values were generally comparable across all groups during the st udy. Slight decreases ( ↓ 5-8%) in absolute overall food consumption were observed for ma les (Days 1-11) and females (Days 1-4) at ≥ 800 mg/kg/day.

Absolute and relative liver weights were increased in males in the 800 ( ↑ 24% and 27%, respectively) and 1000 mg/day dose groups ( ↑ 25% and 31%, respectively) and in females in the 800 ( ↑ 8% both weights) and 1000 ( ↑ 11% and 14%, respectively) mg/kg/day dose groups. Absolute and relative kidney weights in males a nd females in both dose groups were comparable to controls.
Based on these findings, dose levels of 100, 300, and 1000 mg/kg/day were selected for the Hershberger and the uterotrophic assays.

COMPLIANCE: A signed and dated GLP Compliance statement was provided, which stated that the study did not meet the re quirements of 40 CFR Part 160.

# In vivo Hershberger Assay (201 1) / Page 18 of 18 MALATHION/ 057701 OCSPP 890.1400/ OECD 441



# APPENDIX II
Because this is a method validation study, only a summary is provided. The analytical method for determination of malathion in corn oil dos e formulations was desc ribed, and validation and stability data were provided in the submission (MRID 48617512).
The method for determination of malathion in corn oil involves dilution of samples in acetone followed by analysis by HPLC/UV. Precision of the method was evaluated by analyzing 6 replicates of low (1 mg/mL) and high (201 mg/mL) concentration fortified samples; the relative standard deviations of these analyses were 0.8% and 1.3%, respectively. These analyses were also used to evaluate accuracy ; the mean percent errors were

− 1.8% and

− 1.1%, respectively, for the low and high concentrated fortified samples.
The stability of calibration sta ndards and final dilutions of 1 mg/mL and 200 mg/mL fortified samples at 15 °C was evaluated by re-analyzi ng the standards and samples 38 and 37 hours, respectively, following the initial injections; the percent difference betwee n the initial and final results ranged from

− 2.3% to 5.3%

. 

# Stability of these samples was also evaluated following storage at 2-8 °C for 14 days protected from light. For the calibration standards and the 1 mg/mL fortified sample, the percent difference between the initial and final concentrations ranged from

− 2.7% to 2.5%. For the 201 mg/mL for tified sample, the percent difference between the initial and final re sult was 24%; a second fortified sa mple was prepared and stored at 2-8 °C for 15 days and the percent difference between the initial and final analyses was 14.2%. The study author noted that a positive differen ce is not an indication of instability.
Stability was evaluated for 1 mg/mL and 200 mg/m L dose formulations prepared in com oil. The dose formulations were analyzed prior to storage and following storage at 20-25 °C, protected from light, for 40 hours an d at 2-8 °C, protected from li ght, for 13 days. The percent difference between the final stability samp le results and initial results ranged

− 2.8% to 0.5%.
Based on these findings, it was concluded that the method was sufficiently reproducible and accurate to support dose formulation analyses. Dose formulations in corn oil, protected from light, were found to be stable for 40 hours at 20-25 °C and for 13 days at 2-8 °C.

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

# DATA EVALUATION RECORD

MALATHION
Study Type: OCSPP 890.1450, Female Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-16-2012 (MRID 48685701)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer: Signature: Rebecca L. Bryan, B.S. Date: 8/30/2012
Secondary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 9/12/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 9/14/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 9/14/2012

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).

# MALATHION/ 057701 Female Pubertal Assay (2012) I 

# Page 1 of22 OCSPP 890.1450/ OECD None 

# Primary Reviewer: Myron S. Ottley, Ph.D. 

# Risk Assessment Branch 3, Health Effects Division (7509P) Secondary Reviewer: Elizabeth Mendez, Ph.D. 

# Health Effects Division

DATA EVALUATION RECORD Signature:**-~+--:--t~,...,...--=-­** 

# Date:

# Signatur~.=--:::;~~~~~~ Date:

_ __ -fP.,μ:21.flUZ--



# STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None.



# PC CODE: 057701 

# DP BARCODE: D404274

TXR#: 0052348 

# CAS No: 121-75-5



# TEST MATERIAL (PURITY): Malathion Technical (95.8-96.0%)



# SYNONYMS: Fyfanon®; Diethyl 2-[( dimethoxyphosphinothioyl)thio ]butanedioate



# CITATION: Barnett, J.F. (2012) A Pubertal Development and Thyroid Function Study of Malathion by Oral Gavage in Peripubertal Male and Female Rats. Charles River Laboratories, Horsham, PA. Laboratory Project ID: 20012266, June 18, 2012. MRID 48685701. Unpublished.



# SPONSOR: Barnett, J.F. (2012) Oral (Gavage) Repeated Dose 14-Day Dose Range-Finding Study of Malathion in Rats. Charles River Laboratories, Horsham, PA. Laboratory Project ID: 20014321, May 1, 2012. MRID 48685702. Unpublished.

Cheminova A/S, P.O. Box 9, DK-7620 Lemvig, Denmark



# TEST ORDER#: EDSP-057701-44



# EXECUTIVE SUMMARY: In a Female Pubertal Assay (MRID 48685701), 20 Crl:CD(SD) Sprague Dawley rats/dose group were treated daily via oral gavage with malathion (95.8-96.0% purity, Lot #s 9010501 or D2014-0SJ-MLT-01-S) in com oil at doses ofO, 250, 500 or 1000/750 mg/kg/day from post-natal day (PND) 22 to 42. The 1000 mg/kg/day dose level was decreased
to 750 mg/kg/day on PND 24 due to mortality and clinical observations at the high-dose. Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight on the day of attainment of VO was recorded. After sacrifice on PND 42, blood was collected for clinical chemistry analyses, including total thyroxine (T 4) 

# and thyroid stimulating hormone (TSH), which were determined using radioimmunoassays. Kidney, liver, adrenal gland, thyroid, pituitary, uterus, and ovary weights were recorded, and microscopic examinations

were performed on the thyroid, ovaries, uterus, and kidneys. In addition to the main study groups, additional groups of eight female rats/dose were used in a cohort that examined blood and brain cholinesterase (ChE) activity at 2 hours after the last dose on PND 42.

Two animals in the high-dose group from the main study were found dead on PND 25 and 41, and four animals in the high-dose group from the cholinesterase cohort were found dead between

# Female Pubertal Assay (2012) / Page 2 of 22 MALATHION/ 057701 OCSPP 890.1450/ OECD None



# PNDs 23-25. All other rats survived until schedule d sacrifice. Treatment-related clinical signs observed in the high-dose group included e ffects on gait (decreased motor activity and prostration), irregular respiration (dypsnea), ne rvous system (whole body tremors, tremors in the head, slight excess salivation) , and general toxicity (urine stained abdominal fur, chromodacryorrhea, laceration on the neck and s cab on the neck). In the low- and mid-dose groups, sporadic clinical signs included slight excess salivation, urine stained abdominal fur, and/or chromodacryorrhea.
There were no statistically signifi cant effects on age or weight at VO, mean age at first vaginal estrous, mean cycle length, number of females cy cling, number of females regularly cycling, or cycle status at necropsy. However, there were apparent dose-related trends toward decreased percentages in the number of anim als cycling and the number of an imals regularly cycling in the malathion-treated rats.

Serum T 4 

# levels in the low-, mid- and high-dose groups were increased (p ≤ 0.01) by 19, 16 (NS), and 25%, respectively, compared to the control. However, serum TSH levels were not affected by treatment, and there were no ch anges in thyroid weight or hist ology. Therefore, the biological significance of the changes in T 4 

# levels is unclear.
Compared to control, mean liver weight in the high-dose group was increased (p ≤ 0.01) by 16%, and the relative liver weight (to body) was increased (p ≤ 0.01) by 6% and 14% in the mid- and high-dose groups, respectively. In the mid- and high-dose groups, mean kidney weights were increased (p ≤ 0.01) by 7% and 12%, respectively, and re lative kidney weights were increased (p ≤ 0.01) by 7% and 11%, respectively. Given that the weight change s in liver and kidneys were minor in magnitude and there were no adverse hi stopathological findings in the liver or kidneys, these weight increases are considered to be adaptive in nature.

In addition, both absolute and relative pituitary weights were decreased (p ≤ 0.01, 29 to 31%) in the mid- and high-dose groups, but these changes were not associated with any histological changes in the pituitary glands. There were no other significant eff ects on organ weights, including the uterus and ovaries, and there were no microscopic ch anges in any of the examined organs.

There were a number of treatment -related changes in clinical chemistry parameters that were concomitant with the changes in liver weight; these included increases in GGT (NS, 94 to 128%), albumin (p ≤ 0.01 or 0.05; 4 to 8%), and globulin (p ≤ 0.01 or 0.05; 5 and 11%), and dosedependent decreases in the A/G ratio (p ≤ 0.01; 7 to 18%). In addition, there were decreases (p ≤ 0.01) in total bilirubin (17 to 31%) and triglyceride (40 and 53 %). Finally, other treatmentrelated changes were also noted in cholesterol, creatinine, phosphorus, calcium, potassium, and urea nitrogen; however, the releva nce of these changes is unclear as similar changes were not observed in the male pubertal rat assay.
In the ChE cohort (250, 500 and 750 mg/kg/day), ChE activity was dose-dependently inhibited (p ≤ 0.01 or 0.05) in the brain by 9, 24, and 42%, respectively, and in the RBC by 74, 89, and 97%, respectively. The high dose (750 mg/kg/da y) was considered adequate based on the mortality, clinical signs a nd cholinesterase inhibition observed at 1000 mg/kg/day.

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# The assay satisfies the EDSP Tier 1 Test Order require ments for a Female Pubertal Assay (OCSPP 890.1450).

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

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# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories
Location: Horsham, PA Study Director: J.F Barnett Other Personnel: J. Steinke, R.M. Lueke (Clinical Chemistry) S. Polivy (Hormone Analysis) K.P. Keenan (Pathology) Study Period: September 1, 2011 through June 18, 2012



# 2. Test Substance: Malathion Description: Clear, light, pale yellowish liquid Source: Cheminova A/S (Lemvig, Denmark) Lot #: 9010501 (expires November 12, 2014) and D2014-OSJ-MLT-01-S (expires September 28, 2013) Purity: 96.0% (Lot # 9010501) and 95.8% (Lot # D2014-OSJ-MLT-01-S) Stability:

Storage: Stable in corn oil for up to 40 hours at room temperature and for up to 13 days refrigerated (protected from light). 2-8°C, protected from light CAS #: 121-75-5 Structure:



# S

O O CH 3 

# O O CH 3



# P S

O CH

3 

# O CH

3



# 3. Vehicle:

Description: Source: Lot #: Storage: 

# Corn oil Clear, yellowish liquid Charkit Chemical Corporation (Norwalk, CT) M-631 (expires March 18, 2012) Room temperature



# 4. Test Animals: Species: Rat (time-mated females) Strain: Sprague-Dawley (Crl:CD[SD]) Age/Weight at Study Initiation: Main study phase: 22 days old/ 48.1-74.6 g; Cholinesterase phase: 22 days old/ 42.6-73.7 g. Source: Charles River Laboratorie s, Inc., Portage, MI Housing: Rats were housed two/cage in clear plastic nesting boxes with heat-treated laboratory-grade pine shavings (Northeastern Products, lot 5222411). Diet: Harlan Teklad 2018c meal feed (lot 2018CM072211MA), ad libitum Phytoestrogen content was ≤ 300 ppm of genistein equivalents (aglycone) or daidzein Water: Reverse-osmosis filtered (on site), ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 19-25°C 30-70% ≥ 10 fresh air changes/hour 14 hrs light/ 10 hrs dark

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# B. STUDY DESIGN

1. In-Life Dates: Start: 10/4/2011 End: 10/26/2011

2. Mating: Time-mated pregnant dams were receiv ed from the supplier on gestation day 8 and were allowed to deliver naturally and rear their pups to PND 21. Litter size was standardized to 8 pups (4 males/4 females, if possible) on PND 4.

3. Animal Assignment: Following weaning on PND 21, the rats from the extreme ends of the weight range were eliminated. Animals were assigned to the test groups noted in Table 1 using a randomization procedure designed to en sure equal stratifica tion of body weights. Littermates were not assigned to the same treatment group.

TABLE 1. Study Design a 

# Test group Dose (mg/kg/day) # of Females b 

# Control 0 20 + 8 Low 250 20 + 8 Mid 500 20 + 8 High 1000/750 c 

# 20 + 8 a Data were obtained from page 34 of the study report. b For the cholinesterase phase of the study, an additional 8 female s for each dose group were tested. c The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/day due to mortality and clinical observations. This dose was decreased at PND 22 (2 rats), 23 (2 rats) or PND 24 (16 rats) for the main study females, and at PND 24 for the cholinesterase phase females.



# 4. Dose Selection Rationale: The dose levels used in this study were chosen based on the results of a preliminary 14-day range-finding study (MRID 48685702, submitted concurrently) in juvenile Sprague Dawley ra ts. A summary of this study is included as Appendix I to this DER.

5. Dose Preparation and Analysis: Dose formulations were prepared weekly by suspending the appropriate amounts of test substance in corn oil. The test substance formulations were divided into aliquots for daily dispensation and stored refrigera ted (2-8 °C), protected from light. The prepared doses were allowed to warm to room temperature and stirred for at least 30 minutes prior to dose administration. The doses were also stirred continuously during dose administration.
The first bulk test substance, Lot # 9010501, was used for dose preparation from test initiation to October 20, 2012. Samples from th e top, middle and bottom of all dose levels on the first day of dosing were collected fo r concentration and homogeneity analyses. Additional samples from the middle of the reduced concentration high dose material (150 mg/mL) were collected on th e third day of preparation.
A second lot of the bulk test substance (Lot # D2014-OSJ-MLT-01-S) was used beginning on October 21, 2011 until test termination. Samp les from the top, middle and bottom of all dose levels from the first and last day of dose preparation using the second lot were collected for concentration and homogeneity an alyses. Additional samples from the middle

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# of the reduced concentration high dose material (150 mg/mL) were collected from the first and last day of dose prepar ation from the second lot.
The stability of the test substance in corn oil was tested in a previously submitted study (MRID No. 48617512, previously reviewed for 48617507.DER); a summary of this study is included as Appendix II to this DER.

Results of Dose Analysis
Homogeneity (% RSD): 0.2-1.2%
Stability (% of initial): 98.8-100.5% following stor age for 40 hours at 20-25 °C 97.2-99.0% following storage for 13 days at 2-8 °C (MRID No. 48617512).

Concentration (% of nominal): 97.3-102.6%.
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 oral gavage, from PND 22 through PND 42 or 43, in a volume of 5 mL/kg body weight. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: The following parameters were anal yzed statistically: body weights, body weight gains, age and body weight at VO, te rminal body weights, orga n weights, clinical chemistry and hormonal analyses. The data were analyzed using Bartlett’s test of Homogeneity of Variances and Analysis of Variance (ANOVA). If the data were not significant, an ANOVA test was pe rformed; a Dunnett’s test wa s conducted if the results of the ANOVA were significant. If the results of Bartlett’s test were significant, a KruskalWallis test was performed, followed by a D unn’s Method of Multiple Comparisons.
The following parameters were analyzed by analysis of covariance (ANCOVA), using bodyweight on PND 21 as the covariate: termin al body weights, organ weights (pituitary, liver, kidneys, adrenals), initial body weight , final body weight, body we ight gain, day of first vaginal estrous, cycle length, and age a nd body weight at VO. If the results of ANCOVA were significant, ANOVA was used. A modified Bonf erroni correction was used for each pairwise comparison.
Cycling status (cycling vs. not cycling) and percent of animals cyc ling regularly in the control and treatment groups were analyzed us ing the Variance Test for Homogeneity of the Binomial Distribution.

The RBC and brain ChE data were evaluated as separate vari ables in one-way ANOVA. A Dunnett’s test was conducte d if the results of the ANOVA were significant. Significance was denoted at p ≤ 0.05 and p ≤ 0.01.

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# C. METHODS

1. Mortality and Clin ical Examinations: All animals were examined twice daily for viability and once daily for general appearan ce prior to dose administration. Post-dose clinical examinations were c onducted within 2 hours after dose administration and at the end of the normal working day.

2. Body Weight: Animals were weighed on the day of randomization, daily prior to dosing and on the day of euthanasia. Mean body wei ght gains were calculated for PND 22 to PND 42 or 43 for the main study animals.

3. Food Consumption: Food consumption was recorded daily per nesting box during the dosing period and on the day of terminati on. Values were reported in terms of g/animal/day.

4. Vaginal Opening: Beginning on PND 22, all animals were examined daily for onset of VO. The appearance of a small “pin hole”, a vaginal thread, and complete VO were recorded. The age and body weight on the day at which the vaginal lumen was first observed to open was recorded for each animal . Examination of the females was continued daily until VO was observed or until euthanasia.

5. Estrous Cyclicity: Beginning on the day VO was obs erved, vaginal lavages were performed daily, and the slides were evaluate d microscopically to de termine the stage of estrus of each female until necropsy. The age of first vaginal estrus after VO was recorded. The average cycle length was calculated and repo rted for complete estrous cycles. At the end of the study, the overall pattern of each fe male was characterized as regularly cycling, irregularly cycling, not cyc ling, or insufficient data.

6. Sacrifice and Pathology: On PND 42 or 43, all females were euthanized by decapitation. Animals were euthanized beginning at least 2 hours following dose administration that day, and all sacrifices were completed by 1300 hour s. Immediately following decapitation, trunk blood samples were taken for T 4 

# , TSH, and serum chemistry analyses. The samples for T 4 

# , TSH, and serum chemistry were immediately froz en and shipped frozen for analysis. Serum samples were stored frozen until analysis. Necropsies were performed on all females found dead or that survived to the scheduled sacrifi ce. Any tissues with gross lesions were saved in 10% neutral-buffered formalin for possi ble future histopathological examination.

a. Hormone Analysis: T 4 

# and TSH levels were determined with radioimmunoassay kits and analyzed with a Wallac 1470 Gamma Counter.

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

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ELECTROLYTES OTHER
X Calcium X Albumin X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Cholesterol X Potassium X Globulins X Sodium X Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein X Cholinesterase (ChE)-ChE animals only X Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Albumin globulin ratio (A/G Rati o, by calculation) X Alanine aminotransferase (A LT/also SGPT) Bile acids 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*+ X Liver* XX Kidneys (paired)*+ X Adrenals (paired)* X Cervix X Pituitary* * Weights required base d on guideline 890.1450 + Histopathological examination re quired based on Guideline 890.1450


# All organs except the thyroid/ trachea were weighed prior to fixation. Paired organs (kidneys, adrenals, and ovaries) were weighed together. The uterus was weighed “wet” and then again following removal of the fl uid in the lumen (blotted weight).
The ovaries (left or right not specified), uterus, and kidneys were fixed in neutral-buffered 10% formalin, rinsed, and stored in 70% etha nol prior to embedding. The thyroid was fixed in neutral-buffered 10% formalin for at leas t 24 hours. The thyroid gland was dissected from the trachea, blotted, weighed, and pl aced in 70% ethanol until histopathological processing. The liver, adrenals, and pituitary were placed in neutral- buffered 10% formalin for potential future histopathologic examination.
After fixation, protocol-specified tissues were shipped to Charles River, Pathology Associates, Maryland for proces sing. Tissue samples from the thyroid, kidney, ovary, and uterus were routinely processe d into paraffin blocks, secti oned, stained with hematoxylin and eosin, and examined microscopically.

# Female Pubertal Assay (2012) / Page 9 of 22 MALATHION/ 057701 OCSPP 890.1450/ OECD None


# Thyroid sections were subjectively evaluated fo r follicular epithelial cell height and colloid area, using a five point gradi ng scale (1 = shortest/smallest; 5 = tallest/largest), and any abnormalities/lesions were noted. At least two se ctions of the thyroid were examined. Five random sections of an ovary were evaluated for follicular development and any abnormalities/lesions (such as atrophy). The ova ry sections were evaluated on a five point grading scale (1=markedly reduced number or absent; 3=normal; 5=markedly increased number) for primordial, primary, secondary, ter tiary, and atretic follic les and growth phase, mature, and regressive corpora lutea. Uter ine histology documented cases of uterine hyperor hypotrophy as characterized by changes in uter ine horn diameter and myometrial stromal, or endometrial gland development. The stage of the estrous cycle of the female at the time of necropsy was taken into account in the fi nal histological assessment, as ovarian and uterine cellular cha nges are dependent upon endocrine status.

7. Cholinesterase Assay: Animals from the cholinesterase cohort were terminated by sodium pentobarbital injection on PND 42, approximate ly 2 hours after dose administration. The ChE activity was determined from blood and brain samples collected from up to eight rats/group. Blood samples were collected from th e inferior vena cava and stored under cold packs until being process and analyzed. The brain samples were collected, weighed, and stored in saline on wet ice until being process and analyzed. RBC and brain samples were analyzed for cholinesterase activity on the same day as collection (within 90 minutes of euthanasia) at the testing facility. Mean Ch E activity and percentage of ChE inhibition was calculated.

II. RESULTS

A. MORTALITY: Two animals in the high-dose group in the main study (Animal Nos. 4780 and 4773) were found dead on PND 25 (4 doses) and 41 (20 doses). Prior to death, both animals displayed slight exce ss salivation, and head and body tremors, but body weight and food consumption parameters were normal. Findings were unremarkable at necropsy for these animals. All other rats survived until scheduled sacrifice.

In the cholinesterase cohort, one female in the control group was f ound dead after the first dose, presumably due related to a dosing error; this female was replaced. Four females in the high-dose group were found dead between P NDs 23 and 25 (after 2 to 4 doses). These animals displayed variou s clinical signs, including slight excess salivation and tremors.

B. CLINICAL SIGNS OF TOXICITY: Treatment-related clinical signs were observed for the high-dose rats that were f ound dead or subsequently recovere d (Table 2). These clinical signs included effects on gait (d ecreased motor activity and prostate), irregular respiration (gasping), nervous system (whole body tremor s, tremors in the head, slight excess salivation), and general toxicity (urine stained abdominal fur, chromodacryorrhea, laceration on the neck and scab on the neck). In the lo w- and mid-dose groups, sp oradic clinical signs included slight excess salivati on, urine stained abdominal fur, and/or chromodacryorrhea.

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# TABLE 2. Incidence of Clinical Observations a



# Observation Dose Level

Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b 

# # Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined Urine stained abdominal fur 0 20 2 20 5 20 8 20

Excess salivation 0 20 1 20 1 20 10 20 Chromodacryorrhea 0 20 0 20 1 20 1 20 Decreased motor activity 0 20 0 20 0 20 2 20 Tremors 0 20 0 20 0 20 3 20 Gasping 0 20 0 20 0 20 1 20 Neck laceration 0 20 0 20 0 20 1 20 Neck scab 0 20 0 20 0 20 1 20 Prostate 0 20 0 20 0 20 1 20 a Data were extracted from pages 437-440 of the study report. b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/ day by PND 24 due to mortalit y and clinical observations.



# C. FOOD CONSUMPTION: There were no significant differe nces in food consumption in the treated groups relative to controls.

D. GENERAL GROWTH AND VAGINAL OPENING: Body weights, body weight gains, and age and weight at attainme nt of VO are presented in Tabl e 3. There were no significant differences in mean age or body weight at VO, or in mean initial and final body weights in the treated groups relative to controls.
Guideline criteria in the controls for age and body weight at VO were met; however, the %CV for the final body weight mean (9.0%) wa s slightly greater th an the performance criteria maximum of 8.93%.

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# TABLE 3. General Growth and Vaginal Opening (VO) a



# Parameter Evaluated Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b



# N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 22; g) U 20 61.7 6.2 10.0 20 61.4 6.2 10.1 20 60.7 6.4 10.5 20 62.0 6.5 10.5
A 20 61.4 -- -- 20 61.5 -- -- 20 61.4 -- -- 20 61.6 -- -- Body weight at vaginal opening (g) U 20 129.2 14.9 11.5 20 127.6 16.2 12.7 19 126.2 17.5 13.9 19 132.7 17.8 13.4 A 20 128.7 -- -- 20 127.3 -- -- 19 127.2 -- -- 19 132.5 -- --
Final body weight (g) U 20 172.6 15.5 9.0 20 175.3 15.9 9.1 20 172.5 16.4 9.5 18 176.2 14.0 7.9
A 20 172.1 -- -- 20 175.3 -- -- 20 173.8 -- -- 18 175.4 -- -- Final body weight (% of control) U 20 NA 20 101.6 20 99.9 18 102.1
Body weight gain (final – initial; g) U 20 111.0 12.8 11.5 20 113.9 11.6 10.2 20 111.9 12.5 11.2 18 114.3 10.0 8.7 A 20 110.7 -- -- 20 113.9 -- -- 20 112.4 -- -- 18 113.9 -- -- Age at vaginal opening, first day (PND) U 20 32.6 2.4 7.4 20 32.6 3.5 10.7 19 32.8 3.3 10.1 19 33.4 2.9 8.7 A 20 32.6 -- -- 20 32.6 -- -- 19 32.7 -- -- 19 33.4 -- -- Age at vaginal opening, complete (PND) U 20 33.8 1.9 5.6 20 33.6 2.8 8.3 20 34.5 3.7 10.7 19 34.9 3.2 9.2 A 20 33.8 -- -- 20 33.7 -- -- 20 34.5 -- -- 19 34.9 -- -- Proportion unopened (#/N) 0/20 0/20 1/20 0/18 a Data were obtained from page 65 and Ta ble 5 on pages 101-102 of the study report b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/ day by PND 24 due to mortalit y and clinical observations. U Unadjusted for body weight on PND 22 A Adjusted for body weight on PND 21 N Number of animals examined SD Standard Deviation CV Coefficient of Variation



# E. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 4. Compared to the control, mean liver weight in the high-dose group was increased (p ≤ 0.01) by 16%, and the relative liver weight (to body) was increased (p ≤ 0.01) by 6% and 14% in the mid- and high-dose group. In the mid- and high-dose gr oups, mean kidney weights were increased (p ≤ 0.01) by 7% and 12%, respectively, and rela tive kidney weights were increased (p ≤ 0.01) by 7% and 11%, respectively. In addition, mean pituitary weights for both the mid and high-dose groups were decreased (p ≤ 0.01) by 29%, and the relative pituitary weights were decreased (p ≤ 0.01) by 31%. There were no other sign ificant effects on organ weights.
Guideline criteria were met for control organ weights, except the %CV for mean pituitary weight (30.5%) was greater than the pe rformance criteria maximum of 26.97%.

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# TABLE 4. Organ Weights at Necropsy a



# Organ Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b



# N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%)

Liver (g) U 20 8.82 1.10 12.5 20 9.31 0. 95 10.2 20 9.37 1.12 12.0 18 10.24** ( ↑ 16) 1.00 9.8

A 20 8.80 -- -- 20 9.31 -- -- 20 9.42 -- -- 18 10.21 -- --

R 20 5.04 0.31 6.1 20 5.22 0.29 5.6 20 5.34** ( ↑ 6) 0.39 7.3 18 5.76** ( ↑ 14) 0.29 5.1

Kidneys (g) U 20 1.64 0.15 9.1 20 1.70 0.15 8.8 20 1.76* ( ↑ 7) 0.20 11.4 18 1.84** ( ↑ 12) 0.19 10.3

A 20 1.63 -- -- 20 1.70 -- -- 20 1.77 -- -- 18 1.84 -- --

R 20 0.94 0.07 7.1 20 0.95 0.05 5.1 20 1.01** ( ↑ 7) 0.07 6.7 18 1.04** ( ↑ 11) 0.07 6.6

Pituitary (mg) U 20 9.5 2.9 30.5 20 8.2 2.1 25.6 20 6.7** ( ↓ 29) 2.2 32.8 18 6.7** ( ↓ 29) 2.6 38.8

A 20 9.5 -- -- 20 8.3 -- -- 20 6.7 -- -- 18 6.7 -- --

R 20 5.50 1.69 30.8 20 4.66 1.25 26.7 20 3.79** ( ↓ 31) 1.11 29.4 18 3.79** ( ↓ 31) 1.40 37.1

Adrenals (mg) U 20 48.7 8.2 16.8 20 47.2 7.8 16.5 20 43.8 5.5 12.6 18 45.9 8.0 17.4 A 20 48.6 -- -- 20 47.2 -- -- 20 44.0 -- -- 18 45.8 -- -- R 20 0.026 0.006 23.1 20 0.025 0.006 24.0 20 0.024 0.005 20.8 18 0.026 0.006 23.1 Ovaries (mg) U 20 69.0 15.1 21.9 20 70.9 13.2 18.6 20 68.7 14.9 21.7 18 66.6 12.9 19.4 A 20 68.8 -- -- 20 70.9 -- -- 20 69.0 -- -- 18 66.3 -- -- Uterus, wet (mg) U 20 334.4 58.8 17.6 20 350.8 135.6 38.7 20 375.5 171.2 45.6 17 317.6 116.8 36.8 A 20 334.1 -- -- 20 350.8 -- -- 20 376.4 -- -- 17 316.9 -- -- Uterus, blotted (mg) U 20 318.4 52.2 16.4 20 317.1 87.5 27.6 20 322.0 102.6 31.9 18 299.4 77.1 25.8

A 20 318.2 -- -- 20 317.1 -- -- 20 322.6 -- -- 18 299.1 -- -- Thyroid, fixed (mg) U 20 20.76 3.82 18.4 20 19.53 3.66 18.7 20 19.04 3.46 18.2 18 17.94 2.89 16.1 A 20 20.72 -- -- 20 19.53 -- -- 20 19.13 -- -- 18 17.87 -- -- a Data were obtained from Table 6 on page 109- 111 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/ day by PND 24 due to mortalit y and clinical observations. U Unadjusted for body weight on PND 21 A Adjusted for 100 g body weight on PND 21 N Number of animals examined SD Standard Deviation CV Coefficient of Variation R Organ-to-body weight ratio (relative to body weight) * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



# F. ESTROUS CYCLICITY: Estrous cycle data are presen ted in Table 5. There were no statistically significant effects on mean age at first vaginal estrous, mean cycle length. However, there were apparent dose-related tr ends toward decreased percentages in the number of animals cycling and regularly cycling.

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# TABLE 5. Estrous Cyclicity a



# Treatment

Groups N Mean Age at First Vaginal Estrus (PND) Mean Cycle Length (days) Cycling (%) Regularly Cycling

(%) Not Cycling Cycle Status at Necropsy (# Females)

N Diestrus Proestrus Metestrus Estrus Vehicle 20 36.3 4.0 100.0 80.0 0 20 7 6 5 2 Malathion 250 mg/kg/day 19 35.9 4.6 90.0 60.0 2 20 9 3 4 4

Malathion 500 mg/kg/day 18 35.8 4.9 89.5 47.4 2 20 9 7 3 1

Malathion 1000/750 mg/kg/day b 

# 17 37.6 4.5 73.7 47.4 5 18 4 6 6 2

a Data were obtained from Table 5 on page 103 and page 661 of the study report. b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/ day by PND 24 due to mortalit y and clinical observations. N Number of animals



# G. HORMONE LEVELS AND CLINICAL CHEMISTRY: Mean hormone levels and clinical chemistry values are presented in Table 6. Compared to the control, serum T 4 

# concentrations were increased in the low-, mid-, and high-dose (p ≤ 0.05 or ≤ 0.01) by 19% (p ≤ 0.05), 16% (NS), and 25%, respectively. Serum TSH concentrations were not affected by treatment.
Compared to the control group, GGT levels in the low-, mid- and high-dose groups were increased (NS) by 128, 94, and 96%, respectively. Albumin levels in the low-, mid- and high-dose groups were decreased (p ≤ 0.01) by 4, 8, and 8%, respectively, and globulin levels in the mid- and high-dose groups were increased (p ≤ 0.05) by 5 and 11%, respectively. In addition, the A/G ratio in the low-, mid-, high-dose groups was decreased (p ≤ 0.01) by 7, 12, and 18%, respectively. These changes in clini cal chemistry parameters were concurrent with the increased liver we ights for all dose groups.

Total bilirubin levels in the low-, mi d-, and high-dose groups were decreased (p ≤ 0.01) by 17, 31, and 27%, respectively, and triglyceride leve ls in the mid- and high-dose groups were decreased (p ≤ 0.01) by 40 and 53%, respectively. Glucos e levels in the low- and mid-dose groups were also decreased (p ≤ 0.05) by 8 and 6%, but this effect was not dose dependent.
Although significant changes were observed in th e following clinical chemistry parameters, the values were within the historical control ra nges and are not considered to be biologically relevant because corresponding changes we re not observed in treated males (MRID 48685701). In the low-, mid- and high-dose groups, cholesterol was increased (p ≤ 0.05) by 11, 13, and 18%, respectively, creatinine was decreased (p ≤ 0.05) by 9, 17, and 22%, and phosphorus was increased (p ≤ 0.01) by 13, 15, and 14%. In the mid- and high-dose groups, calcium was decreased (p ≤ 0.05) by 3-4%, potassium was decreased (p ≤ 0.01) by 10 and 15%, respectively, and urea nitrogen was decreased (p ≤ 0.01) by 17 and 23%, respectively.
The alanine aminotransferase, aspartate ami notransferase, chloride, sodium, and total protein treated group levels were sim ilar to those in the control group.

The Guideline criterion was met for serum T 4 

# level in the control group.

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# TABLE 6. Hormone Levels and Clinical Chemistry a



# Parameter Evaluated Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b



# N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Hormones Serum T 4 

# , Total (μg/dL) 20 4.11 0.83 20.2 20 4.88* ( ↑ 19) 1.01 20.8 20 4.75 ( ↑ 16) 0.62 13.0 18 5.12** ( ↑ 25) 1.31 25.5

Serum TSH (ng/mL) 20 1.89 0.65 34.2 20 2.26 0.84 37.0 2 0 2.49 1.37 55.0 18 2.14 0.70 32.8

Clinical Chemistry A/G ratio 19 1.57 0.08 4.8 19 1.46** ( ↓ 7) 0.06 3.9 19 1.38** ( ↓ 12) 0.06 4.1 17 1.29** ( ↓ 18) 0.06 4.6

Albumin (g/dL) 19 3.38 0.14 4.0 19 3.26* ( ↓ 4) 0.13 4.1 19 3.12** ( ↓ 8) 0.13 4.1 17 3.10** ( ↓ 8) 0.21 6.8

Alkaline phosphatase (U/L) 19 403 123 30.4 19 398 84.8 21.3 19 389 102.6 26.4 17 298** ( ↓ 26) 98.1 33.0

ALT (U/L) 19 73.5 10.5 14.3 19 72.2 11.4 15.8 19 73.9 12.2 16.5 17 75.1 11.4 15.2 AST (U/L) 19 216 43.9 20.3 19 234 55.9 23.9 19 213 40.5 19.0 17 210 27.9 13.3 Calcium (mg/dL) 19 10.8 0.32 3.0 19 10.8 0.32 3.0 19 10.5* ( ↓ 3) 0.42 4.0 17 10.4** ( ↓ 4) 0.32 3.1

Chloride (mEq/L) 19 97.7 2.8 2.9 19 96.5 2.4 2.5 19 96.6 2.4 2.5 17 95.6 2.3 2.4

Cholesterol (mg/dL) 19 92.4 11.8 12.8 19 103* ( ↑ 11) 15.6 15.1 19 104* ( ↑ 13) 13.0 12.5 17 109** ( ↑ 18) 13.0 11.9

Creatinine (mg/dL) 19 0.248 0.03 13.7 19 0.230* ( ↓ 7) 0.02 10.0 19 0.212** ( ↓ 15) 0.03 11.8 17 0.198** ( ↓ 20) 0.02 11.1

GGT (U/L) 17 0.684 1.19 173 17 1.56 ( ↑ 128) 1.38 88.3 19 1.33 ( ↑ 94) 1.31 98.0 17 1.34 ( ↑ 96) 0.78 58.3

Globulin (g/dL) 19 2.16 0.15 6.7 19 2.24 0.11 4.9 19 2.27* ( ↑ 5) 0.14 6.0 17 2.40** ( ↑ 11) 0.17 7.2

Glucose (mg/dL) 19 145 11.0 7.6 19 133** ( ↓ 8) 10.4 7.8 19 136* ( ↓ 6) 7.9 5.8 17 143 20.7 14.5

Phosphorus (mg/dL) 19 9.23 0.74 8.0 19 10.4** ( ↑ 13) 0.63 6.1 19 10.6** ( ↑ 15) 0.95 8.9 17 10.5** ( ↑ 14) 0.68 6.5

Potassium (mEq/L) 19 7.27 0.67 9.3 19 7.10 0.78 11.0 19 6.53** ( ↓ 10) 0.59 9.1 17 6.16** ( ↓ 15) 0.39 6.4

Sodium (mEq/L) 19 136 3.5 2.6 19 135 2.7 2.0 19 136 3.8 2.8 17 136 2.7 2.0

# Female Pubertal Assay (2012) / Page 15 of 22 MALATHION/ 057701 OCSPP 890.1450/ OECD None



# TABLE 6. Hormone Levels and Clinical Chemistry a



# Parameter Evaluated Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b



# N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Total bilirubin (mg/dL) 19 0.154 0.04 25.3 19 0.128** ( ↓ 17) 0.03 19.5 19 0.107** ( ↓ 31) 0.02 17.8 17 0.113** ( ↓ 27) 0.02 18.6

Total protein (g/dL) 19 5.54 0.26 4.7 19 5.50 0.22 4.0 19 5.39 0.25 4.6 17 5.50 0.36 6.5

Triglyceride (mg/dL) 19 157 62.2 39.5 19 142 54.3 38.3 19 93.8** ( ↓ 40) 29.9 31.9 17 74.5** ( ↓ 53) 24.3 32.6

Serum urea nitrogen (mg/dL) 19 17.5 2.0 11.4 19 15.5** 1.9 12.3 19 14.5** ( ↓ 17) 2.4 16.6 17 13.4** ( ↓ 23) 1.7 12.7

a Data were obtained from Tables 7 & 8 on pages 113 & 115-118 of the study report. b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/day by PND 24 due to mortality and clinical observations. c Percent difference from controls, calculated by the reviewers, is included in parentheses. N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



# G. GROSS PATHOLOGY: No treatment-related gross findings were observed.

H. HISTOPATHOLOGY: The assessment of colloid area and follicular cell height in the thyroid is presented in Table 7. No tr eatment-related findings were observed.

# Female Pubertal Assay (2012) / Page 16 of 22 MALATHION/ 057701 OCSPP 890.1450/ OECD None



# TABLE 7. Evaluation of Colloid Area and Follicular Cell Height in 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 20 20 1 20 20 2 0 20 2 0 20 3 0 20 3 0 20 4 0 20 4 0 20 5 0 20 5 0 20

Malathion 250 mg/kg/day 1 20 20 1 19 20 2 0 20 2 1 20 3 0 20 3 0 20 4 0 20 4 0 20 5 0 20 5 0 20

Malathion 500 mg/kg/day 1 20 20 1 19 20 2 0 20 2 1 20 3 0 20 3 0 20 4 0 20 4 0 20 5 0 20 5 0 20

Malathion 1000/750 mg/kg/day c 

# 1 18 18 1 17 18 2 0 18 2 1 18 3 0 18 3 0 18 4 0 18 4 0 18 5 0 18 5 0 18 a Data were obtained from page 661 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. c The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/day by PND 24 due to mortality and clinical observations. O Number Observed E Number Examined



# The incidence of histopathological findings of the ovaries, uterus, pituitary, liver and kidneys are presented in Table 8, and no trea tment-related findings were observed.
The number of primary follicles in the combined left and right ovaries (mean of 28 primary follicles) were increased (p ≤ 0.05) in the high-dose group compar ed to control (mean of 20 primary follicles), and the numbe r of atretic follicles in the left ovary were significantly decreased (p ≤ 0.05) for the low- and mid-dose groups (m eans of 3 atretic follicles) compared to control (mean of 4.7 atretic follicles). However, thes e effects are not considered treatment-related.

# Female Pubertal Assay (2012) / Page 17 of 22 MALATHION/ 057701 OCSPP 890.1450/ OECD None



# TABLE 8. Incidence of Histpathological Le sions of the Ovaries, Uterus and Kidney a



# Findings Dose Level (mg/kg/day)

Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b


# # Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined 

# Ovaries (Left and Right) Unremarkable 20 20 20 20 20 20 18 18 Uterus Unremarkable 20 20 20 20 20 20 18 18 Pituitary c 

# Unremarkable 20 20 20 20 18 19 17 18 Cyst, Pars Distalis 0 20 0 20 1 20 1 18 Liver Unremarkable 20 20 20 20 19 20 17 18 Infiltration, mononuclear cell, minimal 0 20 0 20 1 20 1 18 Kidney (Right) Unremarkable 16 20 17 20 14 20 13 18 Fibrosis, focal, minimal 0 20 0 20 1 20 0 18 Hydronephrosis, minimal 0 20 0 20 0 20 1 18 Mineralization, tubular, minimal 2 20 1 20 1 20 3 18 Infiltration, mononuclear cell, interstitial, focal, minimal 1 20 0 20 1 20 1 18 Cyst, epithelial, minimal 0 20 0 20 2 20 0 18 Dilation, tubular, minimal 1 20 0 20 0 20 0 18 Basophilia, tubular, minimal 1 20 2 20 3 20 1 18 a Data were obtained from pages 660-661 of the study report. b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/day by PND 24 due to mortality and clinical observations. c One pituitary in the 500 mg/kg/day group was not examined because it wa s not found at trimming.



# I. CHOLINESTERASE ACTIVITY : There were dose-dependent reductions in RBC and brain ChE levels in all dose groups (Table 9) . In the low-, mid-, and high-dose groups, RBC ChE levels were decreased (p ≤ 0.05) by 74, 89, and 97%, respectively, and brain ChE levels were decreased (p ≤ 0.05) by 9, 24, and 42%, respectively.

# Female Pubertal Assay (2012) / Page 18 of 22 MALATHION/ 057701 OCSPP 890.1450/ OECD None



# Table 9. Cholinesterase Activity a



# Parameter Study Day Dose Group
Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b 

# N Mean SD N Mean SD N Mean SD N Mean SD Brain weight (g) 42 8 1.82 0.06 8 1.78 0.03 8 1.79 0.06 4 c 

# 1.77 0.07
RBC ChE

levels (U/L) 42 8 1.27 0.13 8 0.330** ( ↓ 74.1) 0.085 3 c 

# 0.139** ( ↓ 89.1) 0.057 1 c 

# 0.035 ( ↓ 97.3) –

Brain ChE

levels (U/L) 42 8 12.4 0.464 8 11.3* ( ↓ 8.9) 0.655 8 9.48** ( ↓ 23.6) 1.21 4 c 

# 7.18** ( ↓ 42.1) 1.57

a Data were obtained from page 69, Table 13 on page 137 and Table 15 on page 141 of the study report. Percent inhibition is shown in parentheses. b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/day by PND 24 due to mortality and clinical observations. c Excludes rats that were found dead, had unconfirmed results, or had negative results. N No. of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: Mortality occurred in female rats in the high-dose group, and test substance-related adverse clinical signs were obs erved primarily in the midand high-dose groups. These adverse clinical signs were observed in rats that subsequently recovered, were found dead, or we re euthanized in extremis pr ior to scheduled termination. The adverse clinical signs incl uded the following: effects on ga it, excess salivation, irregular respiration, decreased moto r activity, tremors, as well as additional signs of general toxicity. Due to mortality and adverse clinical signs of toxicity observed in several female rats dosed with 1000 mg/kg/day within the first two days of dosing, the high-dose level was reduced to 750 mg/kg/day beginning on PND 24 in the chol inesterase cohort and on PNDs 22 to 24 for the females in the main study.
Statistically significan tly reductions were observed in RBC and brain ChE activity in all malathion-treated female dose groups.
In the low-, mid- and high-dose groups, statis tically significant incr eases (119%, 115%, and 124%) were observed in T 4 

# levels; however, the significance of this finding is not clear because the increases were not clearly dose dependent, and the T 4 

# values in each dose group were within the Testing Facil ity's historical control range.
Changes in serum chemistry that were presumed to be related to test substance administration included: an increase in GGT at >250 mg/ kg/day; a statistically significant increase in globulin at >500 mg/kg/day; statistically significant reductions in albumin and the albumin/globulin ratio at >250 mg/kg/day; st atistically significan t reductions in total bilirubin levels at >250 mg/kg/da y; and statistically significant decreases in the triglyceride levels at >500 mg/kg/day.
Slight increases or statistically significant incr eases in absolute and relative kidney and liver weights were seen in the mid- and high-dose gr oups of both sexes, but no histopathological

# Female Pubertal Assay (2012) / Page 19 of 22 MALATHION/ 057701 OCSPP 890.1450/ OECD None


# findings were was observed in these tissues. These slight changes reflect the adaptive, metabolic and execratory responses expected in the livers and kidneys of rats given high doses of malathion. A minimal, statistically significant decreas e in the absolute and relative pituitary weight was seen in the mid- and high-dose females; howev er, no histopathological findings were observed in the pituitary. No ot her test article-relate d organ weight changes were noted that were different from their re spective controls. Th ere were no correlating morphological data, thereby suggesting the kidne y and liver weight changes were likely adaptive changes to the mid- a nd high-doses and not considered adverse or toxicologically relevant.

B. AGENCY COMMENTS: Two animals from the high-dose group in the main study were found dead on PND 25 and 41, and four animals in th e high-dose from the cholinesterase cohort were found dead between PNDs 23-25. All other rats survived until scheduled sacrifice. The treatment-re lated clinical signs observed in the high-dose group included effects on gait (decreased motor activity and pr ostate), irregular re spiration (dypsnea), nervous system (whole body tremors, tremors in the head, slight excess salivation), and general toxicity (urine staine d abdominal fur, chromodacryorrh ea, laceration on the neck and scab on the neck). In the low- and mid-dose gr oups, sporadic clinical signs included slight excess salivation, urine stained abdominal fur, and/or chromodacryorrhea. The dose on the high-dose group was decreased from 1000 to 750 mg/kg/day on PNDs 22-24 due to mortality and clinical observations.
There were no statistically si gnificant effects on mean age and weight at VO, age at first vaginal estrous, mean cycle lengt h, number of females cycling, number of females regularly cycling, and cycle status at necropsy. Although not statistic ally significant, there were apparent dose-related trends to ward decreased percentages in the number of animals cycling and the number of animals regularly cyc ling for the malathion-treated rats.

Serum T 4 

# concentrations in the low-, mid- and high-dose groups were increased (p ≤ 0.01) by 19, 16 (NS), and 25%, respectively, compared to the control. However, serum TSH concentrations were not affect ed by treatment, and there were no changes in thyroid weight or histology. Therefore, the biological significan ce of the changes in T 4 

# concentrations is unclear.
Compared to control, absolu te and relative liver and ki dney weights were significantly increased in a dose-dependent manner. Given that the chan ges were minor in magnitude ( ≤ 16% increase) and there were no adverse histopathological findi ngs in the liver or kidneys, these weight increases are considered to be adap tive in nature. In a ddition, both absolute and relative pituitary weights were decreased (p ≤ 0.01) in the mid and high-dose groups, but these changes were not associated with any histolog ical changes in the pituitary glands. There were no other significant eff ects on organ weights; includin g the uterus and ovaries; and there were not microscopic changes in any of the examined organs.

There were a number of treatment -related changes in clinical chemistry parameters that were concomitant with the changes in liver weight; these included increases in GGT (NS, 94 to 128%), albumin (p ≤ 0.01 or 0.05; 4 to 8%), and globulin (p ≤ 0.01 or 0.05; 5 and 11%), and dose-dependent decreases in the A/G ratio (p ≤ 0.01; 7 to 18%). In addition, there were decreases (p ≤ 0.01) in total bilirubin (17 to 31%) and triglyceride ( 40 and 53%). Significant

# Female Pubertal Assay (2012) / Page 20 of 22 MALATHION/ 057701 OCSPP 890.1450/ OECD None


# treatment-related changes were also noted in cholesterol levels ( ↑ 11-18%), creatinine ( ↓ 9- 22%), phosphorus levels ( ↑ 13-15%), calcium levels ( ↓ 3-4%), potassium levels ( ↓ 10 and 15%), and urea nitrogen ( ↓ 17 and 23%). However, the releva nce of these changes is unclear as the values were within the historical control ranges for th e laboratory and because similar changes were not observed in the rela ted male pubertal assay (MRID 48685701).
In the ChE cohort (250, 500 and 750 mg/kg/day) , the ChE activity was dose-dependently inhibited (p ≤ 0.01 or 0.05) in the brain by 9, 24, and 42%, respectively, and in the RBC by 74, 89, and 97%, respectively. The high dose (750 mg/ kg/day) was considered adequate. based on the mortality, clinical signs and Ch E data observed at 1000 mg/kg/day.

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

 The control %CV for the final body weight mean (9.0%) was greater than the performance criteria maximum of 8.93%

 The control mean %CV for pituitary weight (30.5%) was greater than the performance criteria maximum of 26.97%.

# Female Pubertal Assay (2012) / Page 21 of 22 MALATHION/ 057701 OCSPP 890.1450/ OECD None



# APPENDIX I
Because this is a range-finding study, only a summary is provided to confirm the adequacy of the dose selection rationale used in the Female Pubertal Assay.
In a dose range-finding assay (MRID 48685702), ma lathion (96.0%; Batch #9010501) in corn oil was administered daily via oral gavage in a dos e volume of 5 mL/kg to eight juvenile female (PND 22 to 35) and eight male (PND 23 to 36) Sp rague Dawley [Crl:CD(SD)] rats/dose group at doses of 0, 250, 450, or 600 mg/kg/day. The animal s were dosed for 14 consecutive days, and necropsied one day after the final dose admini stration. Body weights and food consumption were determined daily. The brain and liver were collected and weighed. Blood and brain samples were collected and analyzed for cholineste rase activity. Only the results for the female rats are discussed in this appendix; the results for the male rats are summarized in the DER for MRID 48685701B (concurre ntly reviewed).

All rats survived to scheduled termination. Clinical findings in cluded at least one female with excess salivation in each of the 450 and 600 mg/ kg/day groups, one 450 mg/kg/day female with ungroomed coat, and one 600 mg/kg/day female with urine-stained abdominal fur and soft or liquid feces. Mean body weights, body weight gains, and food consumption in the 250, 450, and 600 mg/kg/day groups were comparable to the cont rols. The brain and liv er weights (absolute and relative to final body weight) we re comparable to the controls.
The brain cholinesterase activity was reduced (p ≤ 0.01) in the 450 and 600 mg/kg/day females by 16.3% and 14.6%, respectively. Red blood cell cholinesterase activity was reduced (p ≤ 0.01) in the 250, 450 and 600 mg/kg/day females by 56.5, 74.1, and 77.7%, respectively.

# Female Pubertal Assay (2012) / Page 22 of 22 MALATHION/ 057701 OCSPP 890.1450/ OECD None



# APPENDIX II
Because this is a method validation study, only a summary is provided. The analytical method for determination of malathion in corn oil dos e formulations was desc ribed, and validation and stability data were provided, in a previous submission (MRID 48617512).
The method for determination of malathion in corn oil involves dilution of samples in acetone followed by analysis by HPLC/UV. Precision of the method was evaluated by analyzing 6 replicates of low (1 mg/mL) and high (201 mg/mL) concentration fortified samples; the relative standard deviations of these analyses were 0.8% and 1.3%, respectively. These analyses were also used to evaluate accuracy ; the mean percent errors were

− 1.8% and

− 1.1%, respectively, for the low and high concentrati on fortified samples.
The stability of calibration sta ndards and final dilutions of 1 mg/mL and 200 mg/mL fortified samples at 15°C was evaluated by re-analyzing th e standards and samples at 38 and 37 hours, respectively, after the initial inj ections; the percent difference betw een the initial and final results ranged from

− 2.3% to 5.3%

. 

# Stability of these samples was also evaluated following storage at 2- 8 °C for 14 days protected from light. For th e calibration standards a nd the 1 mg/mL fortified sample, the percent difference between the in itial and final concentrations ranged from

− 2.7% to 2.5%. For the 200 mg/mL fortified sample, the pe rcent difference between the initial and final result was 24%; a second fortified sample was prep ared and stored at 2-80 °C for 15 days and the percent difference between the init ial and final analyses was 14.2% . The study author noted that a positive difference is not an indication of instability.
Stability was evaluated for the 1 mg/mL and 200 mg/m L dose formulations prepared in corn oil. The dose formulations were analyzed prior to an d after storage at 20-25 °C, protected from light, for 40 hours and at 2-8 °C, protected from light, for 13 days. The percent difference between the final stability sample results and initial results ranged from

− 2.8% to 0.5%.
Based on these findings, it was concluded that the method was sufficiently reproducible and accurate to support dose formulation analyses. Dose formulations in corn oil, protected from light, were found to be stable for 40 hours at 20-25 °C and for 13 days at 2-8 °C.

# DATA EVALUATION RECORD

MALATHION
Study Type: OCSPP 890.1500, Male Pubertal Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-16-2012 (MRID 48685701)

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: 9/7/2012
Primary Reviewer: Signature: Scott D. Studenberg, Ph.D., D.A.B.T. Date: 9/12/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 9/14/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 9/14/2012

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).

# MALATIDON/ 057701 Male Pubertal Assay (2012)/ Page 1 of21 OCSPP 

# 890.1500 I 

# OECD 

# None 

# Primary Reviewer: Myron S. Ottley, Ph.D. 

# Signature: 

# (/ltt40'f/lY

Risk Assessment Branch 3, Health Effects Division Date: 

# 6 0 't(J 

# I] 

# Secondary Reviewer: -=-J-=ohn==L=ic=c=-=io=n=e,,_,P'--"h=.D=-:-.

_______ 

# Signature:**---7"~....,....._,,.---,,--~** 

# Health Effects Division**Date:----"¥---""-~.:.__....,"--**



# DATA EVALUATION RECORD

STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500



# PC CODE: 057701 

# DP BARCODE: D404274

TXR#: 0052348 

# CAS No: 121-75-5



# TEST MATERIAL (PURITY): Malathion Technical (95.8-96.0% purity)



# SYNONYMS: Fyfanon®; Diethyl 2-[( dimethoxyphosphinothioyl)thio ]butanedioate



# CITATION: Barnett, J.F. (2012) A Pubertal Development and Thyroid Function Study of Malathion by Oral Gavage in Peripubertal Male and Female Rats. Charles River Laboratories, Horsham, PA. Laboratory Project ID: 20012266, June 18, 2012. MRID 48685701. Unpublished.



# SPONSOR: Barnett, J.F. (2012) Oral {Gavage) Repeated Dose 14-Day Dose Range-Finding Study of Malathion in Rats. Charles River Laboratories, Horsham, PA. Laboratory Project ID: 20014321, May 1, 2012. MRID 48685702. Unpublished.

CheminovaA/S, P.O. Box 9, DK-7620 Lemvig, Denmark



# TEST ORDER#: EDSP-057701-44



# EXECUTIVE SUMMARY: In 

# a Male Pubertal Assay (MRID 48685701), 20 Crl:CD(SD) Sprague-Dawley rats/dose group were treated daily via oral gavage with malathion technical (95.8-96.0% purity, Lot #s 9010501 or D2014-0SJ-MLT-01-S) in com oil at doses of 0, 250, 500 or 1000/750 mg/kg/day from post-natal day (PND) 23 to 53. Due to mortalities and clinical observations of toxicity, the initial high-dose level (1000 mg/kg/day) was decreased to 750 mg/kg/day by PND 24. Animals were examined for preputial separation (PPS) daily beginning

on PND 30, and age and weight on the day of attainment was recorded. Following sacrifice on PND 53, blood was collected for clinical chemistry analyses, including total thyroxine 

# (T 4), testosterone, and thyroid stimulating hormone (TSH). Testosterone, T4, and TSH were analyzed using radioimmunoassay kits. Kidney, liver, adrenals, pituitary, thyroid, and urogenital organ weights were recorded, and microscopic examinations were performed on the thyroid, kidney, testis and epididymis. In 

# addition to the main study groups, additional groups of 8 male rats/dose

were used in a cohort that examined blood and brain cholinesterase (ChE) activity at 2 hours after the last dose on PND 53.

# Male Pubertal Assay (2012)/ Page 2 of 21 MALATHION/ 057701 OCSPP 890.1500 / OECD None


# In the main study, 14 rats from the high-dose gr oup were found dead during the study, from PND 25 (3 doses) to PND 53 (31 doses), and one rat was sacrificed in extremis due to adverse clinical observations on PND 32. Two animals in the mid-dose group (500 mg/kg/day) were also found dead on PNDs 45 and 50. In addition, 6 rats from the high-dose group in the ChE cohort were found dead between PNDs 24 and 49 (2 to 27 doses) . All other rats survived until scheduled sacrifice. In the mid- and high-dose groups, th e observed clinical signs included effects on gait ( e.g. , ataxia, lost righting reflex, and decreased motor activity), ir regular respiration (dyspnea), nervous system ( e.g. , whole body tremors, tremors in the head, extreme excess salivation, and lacrimation), and general toxicity ( e.g. , urine-stained abdominal fur, ungroomed coat, chromodacryorrhea, dehydration, and chromorhi norrhea). In the low-dose group, sporadic clinical signs included slight excess salivation, ungroomed coat , and urine-stained abdominal fur.

There were no treatment-related effects on fina l body weight, overall body weight gain, and body weight at PPS. Although the first day of PPS was delayed (p ≤ 0.01) by 1.4 to 1.6 days in the lowand mid-dose groups, there was no effect on fi rst day of PPS in the high-dose group.
Compared to the control group, absolu te liver weights were increased (p ≤ 0.05) by 21% in the mid- and high-dose groups, and relative (to body) liver weights were increased (p ≤ 0.01) by 10, 22, and 26% in the low-, mid- and high-dose groups , respectively. Absolute kidney weight in the mid-dose group was increased (p ≤ 0.01) by 12%, and relative kidney weights were increased (p ≤ 0.01) by 6, 14, and 16% in the low-, mid-, and high-dose groups, respectiv ely. The liver and kidney weight effects are treatment related and ar e likely an adaptive response to malathion. The mean LABC weights for the mid- and high-dose groups were also decreased (p ≤ 0.01) by 14% and 17%, respectively, but there were no other ch anges in weights of accessory sex tissues. There were no other significan t effects on organ weights.

Serum T 4 

# concentrations in the low-, mid-, and high-dose groups were increased (p ≤ 0.01) by 41, 35, and 32%, respectively, but these cha nges were not dose-dependent, the T 4 

# levels were within the range of historical control values, and ther e were no related changes in TSH levels. In addition, there were no changes in thyroid weight or any microscopi c findings in the thyroids of malathion-treated rats. Therefore, the bi ological relevance of the changes in T 4 

# levels is unclear. However, there were clear, dose-dependent decreases (p ≤ 0.01) in testosterone levels of 60, 78, and 90% in the low-, mid-, and high-dose groups, respectively, which may correlate with the observed delay in PPS and the reductions in weight of the LABC.
In all malathion-treated animals, albumin was decreased (p ≤ 0.01) by 5-6%, globulin was increased (p ≤ 0.01) by 8-17%, and the A/G ratio was decreased (p ≤ 0.01) by 13-19%. There were also dose-dependent decreases (p ≤ 0.01) in total bilirubi n levels of 16-36% and in triglycerides of 33-68%, and cholesterol levels were increased (p ≤ 0.05) by 11 and 19% in the mid- and highdose groups. These changes were concurrent with the increases in liver weights and likely reflect adaptive changes in liver metabolism. Although significant changes were observed in other clinical chemistry parameters, the values we re generally within the historical control ranges and are not considered to be biologically relevant.

In the ChE cohort, the ChE levels on PND 53 in the low- and mid-dose groups were decreased (p ≤ 0.01) dose-dependently by 79 and 88% in red blood cells and by 17 and 35% in brain tissue. ChE levels were not eval uated in the high-dose group.

# Male Pubertal Assay (2012)/ Page 3 of 21 MALATHION/ 057701 OCSPP 890.1500 / OECD None


# Based on the ChE data, and the mortality and c linical signs, both the mi d- and high-doses (500 and 750 mg/kg/day) of malathion were clearly ex cessive for immature ma le rats; however, the low-dose level was adequate.
The assay satisfies the EDSP Tier 1 Test Or der requirements for a Male Pubertal Assay (OCSPP 890.1500).

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

# Male Pubertal Assay (2012)/ Page 4 of 21 MALATHION/ 057701 OCSPP 890.1500 / OECD None



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories
Location: Horsham, PA Study Director: J.F Barnett Other Personnel: J. Steinke, R.M. Lueke (Clinical Chemistry) S. Polivy (Hormone Analysis) K.P. Keenan (Pathology) Study Period: September 1, 2011 through June 18, 2012



# 2. Test Substance: Malathion Description: Clear, light, pale yellowish liquid Source: Cheminova A/S (Lemvig, Denmark) Lot #: 9010501 (expires November 12, 2014) and D2014-OSJ-MLT-01-S (expires September 28, 2013) Purity: 96.0%(Lot # 9010501) and 95.8%(Lot # D2014-OSJ-MLT-01-S) Stability:

Storage: Stable in corn oil for up to 40 hours at room temperature and for up to 13 days refrigerated (protected from light). 2-8°C, protected from light CAS #: 121-75-5 Structure:



# S

O O CH 3 

# O O CH 3



# P S

O CH

3 

# O CH

3



# 3. Vehicle: Description: Source: Lot #: Storage: 

# Corn oil Clear, yellowish liquid Charkit Chemical Corporation (Norwalk, CT) M-631 (expires March 18, 2012) Room temperature



# 4. Test Animals: Species: Rat (time-mated females) Strain: Sprague-Dawley (Crl:CD[SD]) Age/Weight at Study Initiation: Main study phase: 23 days old/ 58.5-84.8 g; Cholinesterase phase: 23 days old/ 57.5-73.4 g. Source: Charles River Laboratorie s, Inc., Portage, MI Housing: Rats were housed two/cage in clear plastic nesting boxes with heat-treated laboratory-grade pine shavings (Northeastern Products, lot 5222411). Diet: Harlan Teklad 2018c meal feed (lot 2018CM072211MA), ad libitum Phytoestrogen content was ≤ 300 ppm of genistein equivalents (aglycone) or daidzein Water: Reverse-osmosis filtered (on site), ad libitum Environmental Conditions: Temperature: Humidity: Air changes: Photoperiod: 19-25°C 30-70% ≥ 10 fresh air changes/hour 14 hrs light/ 10 hrs dark

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# B. STUDY DESIGN

1. In-Life Dates: Start: 10/5/2011 End: 11/6/2011

2. Mating: Time-mated pregnant dams were receiv ed from the supplier on gestation day 8 and were allowed to deliver naturally and rear their pups to PND 21. Litter size was standardized to 8 pups (4 males/4 females, if possible) on PND 4.

3. Animal Assignment: Following weaning on PND 21, the rats from the extreme ends of the weight range were eliminated. Animals were assigned to the test groups noted in Table 1 using a randomization procedure designed to en sure equal stratifica tion of body weights. Littermates were not assigned to the same treatment group.

TABLE 1. Study Design a 

# Test group Dose (mg/kg/day) # of Males b 

# Control 0 20 + 8 Low 250 20 + 8 Mid 500 20 + 8 d 

# High 1000/750 c 

# 20 + 8 e 

# a Data were obtained from page 34 of the study report. b For the cholinesterase phase of the study, an additional 8 males for each dose group were tested. c The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/day due to mortality and clinical observations. This dose was decreased at PND 23 (18 rats) or 24 (2 rats) for the main study males, and at PND 23 for the cholinesterase phase males. d At PND 51, two 500 mg/kg/day male rats from the cholinesterase phase were reassigned to the main study to replace 500 mg/kg/day animals that died. e At PND 53, two 1000/750 mg/kg/day male rats from the cholinesterase phase were reassigned to the main study to replace 1000/750 mg/kg/day animals that died.



# 4. Dose Selection Rationale: The dose levels used in this study were chosen based on the results of a preliminary 14-day range-finding study (MRID 48685702, submitted concurrently) in juvenile Sprague Dawley ra ts. A summary of this study is included as Appendix I to this DER.

5. Dose Preparation and Analysis: Dose formulations were prepared weekly by suspending the appropriate amounts of test substance in corn oil. The test substance formulations were divided into aliquots for daily dispensation a nd stored refrigerated (2-8°C), protected from light. The prepared doses were allowed to warm to room temperature and stirred for at least 30 minutes prior to dose administration. The doses were also stirred continuously during dose administration.

The first bulk test substance, lot # 9010501, was used for dose preparation from test initiation to October 20, 2012. Samples from th e top, middle and bottom of all dose levels on the first day of dosing were collected fo r concentration and homogeneity analyses. Additional samples from the middle of the reduced concentration high dose material (150 mg/mL) were collected on th e third day of preparation.

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# A second lot of the bulk test substance (Lot # D2014-OSJ-MLT-01-S) was used beginning on October 21, 2011 until test termination. Samp les from the top, middle and bottom of all dose levels from the first and last day of dose preparation using the second lot were collected for concentration and homogeneity anal yses. Additional samples from middle of the reduced concentration high dose material (1 50 mg/mL) were colle cted on the first and last day of dose preparation from the second lot.
The stability of the test substance in corn oil was tested in a previously submitted study (MRID No. 48617512, previously reviewed for 48617507.DER); a summary of this study is included as Appendix II to this DER.

Results of Dose Analysis
Homogeneity (% RSD): 0.2-1.2%
Stability (% of initial): 98.8-100.5% following stor age for 40 hours at 20-25 °C 97.2-99.0% following storage for 13 days at 2-8 °C (MRID No. 48617512).

Concentration (% of nominal): 97.3-102.6%.
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 or 54, in a volume of 5 mL/kg body weight. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. Dosing was performed between 0700 and 0900 hours daily.

7. Statistics: The following parameters were anal yzed statistically: body weights, body weight gains, age and body weight at PPS, te rminal body weights, organ weights, clinical chemistry and hormonal analyses. The data were analyzed using Bartlett’s test of Homogeneity of Variances and Analysis of Variance (ANOVA). If the data were not significant, an ANOVA test was pe rformed; a Dunnett’s test wa s conducted if the results of the ANOVA were significant. If the results of Bartlett’s test were significant, a KruskalWallis test was performed, followed by a D unn’s Method of Multiple Comparisons.
The following parameters were analyzed by analysis of covariance (ANCOVA), using bodyweight on PND 21 as the covariate: termin al body weights, organ weights (pituitary, liver, kidneys, and adrenals), initial body we ight, final body weight, body weight gain, and age and body weight at PPS. If the resu lts of ANCOVA were si gnificant, ANOVA was used. A modified Bonferroni correction was used for each pairwise comparison.

The RBC and brain ChE data were evaluated as separate vari ables in one-way ANOVA. A Dunnett’s test was conducted if the results of the ANO VA were significant.
Significance was denoted at p ≤ 0.05 and p ≤ 0.01.

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# C. METHODS

1. Mortality and Clin ical Examinations: All animals were examined twice daily for viability and once daily for general appearan ce prior to dose administration. Post-dose clinical examinations were c onducted within 2 hours after dose administration and at the end of the normal working day.

2. Body Weight: Animals were weighed on the day of randomization, daily prior to dosing and on the day of euthanasia. Mean body wei ght gains were calculated for PND 23 to PND 53 or 54 for the main study animals.

3. Food Consumption: Food consumption was recorded daily per nesting box during the dosing period and on the day of terminati on. Values were reported in terms of g/animal/day.

4. Preputial Separation (PPS): Beginning on PND 30, all animals were examined daily for onset of PPS. The appearance of a partial or a persistent thread of tissue between the glans and prepuce was recorded. Age and weight at on the day of completion of PPS were recorded.

5. Sacrifice and Pathology: On PND 53 or 54, all males were euthanized by decapitation. Animals were euthanized beginning at least 2 hours following dose administration that day, and all sacrifices were completed by 1300 hour s. Immediately following decapitation, trunk blood samples were taken for T 4 

# , TSH, testosterone, and serum chemistry analyses. The samples for T 4 

# , TSH, testosterone, and serum chemistry were immediately frozen and shipped frozen for analysis. Serum samples were stored frozen until analysis. Necropsies were performed on all males found dead or that survived to the sche duled sacrifice. Any tissues with gross lesions were saved in 10% neutral-buffered formalin for possible future histopathological examination.

a. Hormone Analysis: T 4 

# , TSH, and testosterone leve ls were determined with radioimmunoassay kits and analyzed with a Wallac 1470 Gamma Counter.

b. Clinical Chemistry: The following CHECKED (X) parameters were examined.

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ELECTROLYTES OTHER
X Calcium X Albumin X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Cholesterol X Potassium X Globulins X Sodium X Glucose
ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein X Cholinesterase (ChE)-ChE animals only X Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Albumin globulin ratio (A/G Rati o, by calculation) X Alanine aminotransferase (A LT/also SGPT) Bile acids X Aspartate aminotransfera se (AST/also SGOT) Sorbitol dehydrogenase (SDH) X Gamma glutamyl transferase (GGT) Glutamate dehydrogenase * Recommended for the pubertal assay in ma le rats based on Guideline 890.1500



# c. Organ Weights and Histopathology: The following CHECKED (X) tissues were collected and weighed. The (XX) organs, in addition, were subject ed to histological examination.

UROGENITAL OTHER XX Testes (left and right separately)* + 

# XX Thyroid* + 

# XX Epididymides (left and right separately)* + 

# X Liver* X Seminal vesicle plus coagulating glands (with fluid)* X Adrenals (paired)* X Ventral prostate* X Pituitary* X Dorsolateral prostate* X Levator ani-bulbocavernosus (LABC) muscle complex* XX Kidneys (paired)* + 

# * Weights required base d on guideline 890.1500 + Histopathological examination re quired based on Guideline 890.1500


# All organs except the thyroid/tr achea were weighed prior to fi xation. Kidneys and adrenals were weighed together. Follo wing weighing, the testis and epididymis were fixed in Bouin’s solution for no more than 24 hours. The thyroid (with parathyroid) was collected with the trachea and fixed in neutral-buffered 10% formalin fo r at least 24 hrs. Following fixation, the thyroid was dissected free of the trachea and weighed. Kidneys were fixed for at least 24 hours in neutral-buffered 10% formalin, and then placed in 70% ethanol until histological processing. All ot her tissues listed above were placed in neutral-buffered 10% formalin for possible future histopathologic examination.
After fixation, protocol-specified tissues were shipped to Charles River, Pathology Associates, Maryland, for processing. Tissue samples from the epididymis, kidney, testis, and thyroid were routinely processed into paraffin bloc ks, sectioned, stained with hematoxylin and eosin, and examined microscopically.

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# Thyroid sections were subjectively evaluated fo r follicular epithelial cell height and colloid area, using a five point gradi ng scale (1 = shortest/smallest; 5 = tallest/largest), and any abnormalities/lesions were noted. At least tw o sections of the thyroid were examined.

6. Cholinesterase Assay: Animals from the cholinesterase cohort were terminated by sodium pentobarbital injection on PND 53, approximate ly 2 hours after dose administration. The ChE activity was determined from blood and brain samples collected from up to 8 rats/group. Blood samples were collected from th e inferior vena cava and stored under cold packs until being process and analyzed. The brain samples were collected, weighed, and stored in saline on wet ice until being process and analyzed. RBC and brain samples were analyzed for cholinesterase activity on the same day as collection (within 90 minutes of euthanasia) at the testing facility. Mean Ch E activity and percentage of ChE inhibition was calculated.

II. RESULTS

A. MORTALITY: In the main study, 14 rats in the high-dose group were found dead from PND 25 (3 doses) to PND 53 (31 doses), and one rat was sacrificed in extremis due to adverse clinical observations on PND 32. Tw o animals in the mid-dose group were also found dead on PNDs 45 and 50. In the cholinestera se cohort, 6 rats in the high-dose group were found dead between PNDs 24 and 49 (2 to 27 doses). One death in this subset was attributable to a gavage error. All othe r rats survived until scheduled sacrifice.

B. CLINICAL SIGNS OF TOXICITY: Treatment-related clinical signs of toxicity were observed in rats from both the mid- and hi gh-dose groups that were found dead, euthanized, or subsequently recovered during testing (Table 2). These clinical signs included effects on gait ( e.g. , ataxia, lost righting reflex, and decrea sed motor activity), irregular respiration (dyspnea), nervous system ( e.g. , whole body tremors, tremors in the head, slight, moderate, and extreme excess salivation and lacr imation), and general toxicity ( e.g. , urine-stained abdominal fur, ungroomed coat, chromod acryorrhea, dehydration, chromorhinorrhea, missing left eye, and missing portion of skin). In the low-dose group, sporadic clinical signs included slight excess salivation, ungroomed coat, and urine-stained abdominal fur.

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# TABLE 2. Incidence of Clinical Observations a



# Observation Dose Level

Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b 

# # Observed # Examined # Observed # Examined # Observed # Examined # Observed # Examined Sparse hair coat, back 1 20 0 20 0 20 0 20 Excess salivation 0 20 3 20 13 20 16 20 Ungroomed coat 0 20 1 20 2 20 6 20 Urine stained abdominal fur 0 20 3 20 6 20 9 20

Chromorhinorrhea 0 20 0 20 1 20 0 20 Chromodacryorrhea 0 20 0 20 0 20 7 20 Decreased motor activity 0 20 0 20 1 20 3 20 Low carriage 0 20 0 20 1 20 0 20 Tremors 0 20 0 20 2 20 7 20 Prostate 0 20 0 20 1 20 0 20 Lacrimation 0 20 0 20 1 20 2 20 Portion of skin missing 0 20 0 20 0 20 1 20 Ataxia 0 20 0 20 0 20 1 20 Lost righting reflex 0 20 0 20 0 20 1 20 Dehydration 0 20 0 20 0 20 1 20 Dyspnea 0 20 0 20 0 20 2 20 Left eye missing 0 20 0 20 0 20 1 20 a Data were extracted from pages 381-387 of the study report. b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/ day by PND 24 due to mortalit y and clinical observations.



# C. GENERAL GROWTH AND PREPUTIAL SEPARATION: Body weights, body weight gains, age and weight at attainment of PPS are presented in Table 3. There were no treatment-related effects on fi nal body weights, overall body we ight gain, or age or body weight at PPS. The first day of PPS for animals in the low and mid-dose groups was delayed (p ≤ 0.01) by 1.4 to 1.6 days compared to cont rol, but no effect on first day of PPS was observed for the high-dose group.
The mean body weight at wean ing (61.5 g) was above the perf ormance criteria acceptable range (45.5-59.8 g). The mean CV value fo r body weight at PPS (8.7%) was above the performance criteria maximum of 7.6%.

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# TABLE 3. General Growth and Preputial Separation (PPS) a



# Parameter Evaluated Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b



# N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) Initial body weight (PND 23; g) U 20 70.2 5.8 8.3 20 71.5 6.8 9.5 22 c 

# 69.7 6.2 8.9 22 c 

# 70.2 6.9 9.8 A 20 70.2 -- -- 20 71.0 -- -- 22 69.8 -- -- 22 70.5 -- -- Body weight at PPS (g) U 20 223.0 19.4 8.7 19 236.1 24.0 10.2 19 g 

# 236.5 33.0 14.0 15 e 

# 225.5 26.0 11.5

A 20 223.7 -- -- 19 235.5 -- -- 19 g 

# 236.9 -- -- 15 e 

# 224.7 -- -- Final body weight (g) U 20 324.2 20.4 6.3 20 325.0 32.6 10.0 20 e 

# 321.1 34.3 10.7 8 e 

# 303.4 45.0 14.8 A 20 324.7 -- -- 20 324.0 -- -- 20 320.7 -- -- 8 305.5 -- -- Final body weight (% of control) U NA 100.2 99.0 93.6
Body weight gain (final – initial; g) U 20 254.0 17.4 6.9 20 253.5 28.9 11.4 20 251.2 30.6 12.2 8 233.2 39.6 17.0 A 20 254.3 -- -- 20 252.8 -- -- 20 251.0 -- -- 8 234.6 -- --

First Day of PPS (PND) U 20 41.0 1.1 2.7 20 42.4** ( ↑ 1.4 days) 1.6 3.8 20 d 

# 42.6** ( ↑ 1.6 days) 1.6 3.8 15 e 

# 41.5 1.3 3.1

A 20 41.0 -- -- 20 42.4 -- -- 20 42.5 -- -- 15 e 

# 41.6 -- -- Age at PPS (PND) U 20 42.2 1.3 3.1 19 f 

# 43.2 1.6 3.7 19 g 

# 43.6 2.5 5.7 15 e 

# 43.0 1.8 4.2 A 20 42.1 -- -- 19 f 

# 43.2 -- -- 19 g 

# 43.6 -- -- 15 e 

# 43.0 -- -- Proportion unseparated (#/N) 0/20 0/20 0/20 0/8 a Data were obtained from Table 1 on pages 81 and 82 of the study report. b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/ day by PND 24 due to mortalit y and clinical observations. c Included the two rats that were reassigned from the cholinesterase phase to the main study. d Excludes rats that were reassigned from the cholinesterase phase to the main study. e Excludes animals found dead or euthan ized due to clinic al observations. f Day of maturity could not be determined for one rat. g One rat was excluded because te sting was discontinued before pr eputial separation occurred. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 N Number of animals examined SD Standard Deviation CV Coefficient of Variation NA Not applicable ** Significantly different from controls at p ≤ 0.01.



# D. ORGAN WEIGHTS: Organ weights at necropsy are presented in Table 4. Compared to the control, mean liver weights for both th e mid- and high-dose groups were increased (p ≤ 0.05) by 21%, and the relative (to body) liver weights were increased (p ≤ 0.01) by 10, 22, and 26% for the low-, mid- and high-dose groups , respectively. The mean kidney weights for the mid-dose group was increased (p ≤ 0.01) by 12%, and the relative kidney weights were increased (p ≤ 0.01) by 6, 14, and 16% for the low- , mid- and high-dose groups, respectively.
The mean LABC weights for the mid- a nd high-dose groups were decreased (p ≤ 0.01) by 14% and 17%, respectively, but there were no other significant changes in weights of accessory sex tissues or other organs.
Guideline criteria were met for organ weights in the controls, except as follows: the mean control absolute liver weight (15.44 g) was slightly above the performance criteria acceptable range (9.990-15.350 g); and the mean cont rol absolute thyroid weight (0.0326 g) was slightly above the performance criteria acceptabl e range (0.014-0.026 g).

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# TABLE 4. Organ Weights at Necropsy a



# Organ Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b



# N Mean SD CV (%) N Mean SD CV (%) N c 

# Mean SD CV (%) N c 

# Mean SD CV (%)
Liver (g) U 20 15.44 1.49 9.7 20 17.00 2.52 14.8 20 18.72 ** ( ↑ 21) 3.18 17.0 7 18.64* ( ↑ 21) 4.05 21.8

A 20 15.47 -- -- 20 16.91 -- -- 20 18.68 -- -- 7 18.89 -- --

R 20 4.70 0.258 5.5 20 5.18** ( ↑ 10) 0.357 6.9 20 5.75** ( ↑ 22) 0.477 8.3 7 5.91** ( ↑ 26) 0.507 8.6
Kidneys (g) U 20 2.49 0.19 7.6 20 2.64 0.32 12.1 20 2.79** ( ↑ 12) 0.34 12.2 7 2.74 0.37 13.4

A 20 2.49 -- -- 20 2.63 -- -- 20 2.79 -- -- 7 2.77 -- --

R 20 0.758 0.038 5.0 20 0.806** ( ↑ 6) 0.043 5.3 20 0.863** ( ↑ 14) 0.054 6.2 7 0.879** ( ↑ 16) 0.046 5.3
Pituitary (mg) U 20 10.4 1.5 14.4 20 10.0 1.7 17.0 2 0 9.7 1.1 11.3 7 10.2 2.4 23.5 A 20 10.4 -- -- 20 10.0 -- -- 20 9.7 -- -- 7 10.3 -- -- R 20 3.18 0.450 14.2 20 3.07 0.348 11. 3 20 3.00 0.294 9.8 7 3.27 0.674 20.6 Adrenals (mg) U 20 49.9 7.6 15.2 20 50.7 10.4 20.5 2 0 51.2 7.6 14.8 7 55.2 7.4 13.4 A 20 49.9 -- -- 20 50.6 -- -- 20 51.2 -- -- 7 55.6 -- -- R 20 0.016 0.005 31.3 20 0.015 0.005 33. 3 20 0.017 0.005 29.1 7 0.019 0.004 20.0 Seminal vesicle + coagulating gland, with fluid (mg) U 20 654.5 124.5 19.0 20 598.2 157.6 26.3 2 0 575.5 165.0 28.7 7 525.2 90.8 17.3

A 20 654.6 -- -- 20 597.8 -- -- 20 575.3 -- -- 7 526.5 -- --
Seminal vesicle + coagulating gland, without fluid (mg) U 20 311.6 43.6 14.0 20 290.2 51.7 17.8 2 0 280.3 57.9 20.7 7 266.2 45.2 17.0

A 20 311.8 -- -- 20 289.7 -- -- 20 280.0 -- -- 7 267.9 -- -- Ventral prostate (mg) U 20 260.5 47.4 18.2 20 239.2 61.8 25.8 2 0 227.7 51.9 22.8 7 201.0 51.9 25.8 A 20 260.7 -- -- 20 238.7 -- -- 20 227.4 -- -- 7 202.7 -- -- Dorsolateral prostate (mg) U 20 210.6 44.8 21.3 20 182.3 35.8 19.6 2 0 200.3 46.2 23.1 7 178.2 34.3 19.2

A 20 210.7 -- -- 20 182.0 -- -- 20 200.2 -- -- 7 178.9 -- --
LABC (mg) U 20 736.2 90.5 12.3 20 692.4 82.7 11.9 20 636.7** ( ↓ 14) 85.2 13.4 7 612.6** ( ↓ 17) 96.4 15.7

A 20 736.8 -- -- 20 690.7 -- -- 20 636.0 -- -- 7 617.8 -- -- Epididymis, left (mg) U 20 232.9 22.3 9.6 20 237.6 22.8 9.6 20 235.2 23.2 9.9 7 214.6 40.8 19.0 A 20 233.1 -- -- 20 237.1 -- -- 20 234.9 -- -- 7 216.3 -- -- Epididymis, right (mg) U 20 237.7 24.2 10.2 20 245.0 26.2 10.7 2 0 236.9 27.1 11.4 7 225.2 31.9 14.2

A 20 237.9 -- -- 20 244.4 -- -- 20 236.7 -- -- 7 226.8 -- --

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# TABLE 4. Organ Weights at Necropsy a



# Organ Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b



# N Mean SD CV (%) N Mean SD CV (%) N c 

# Mean SD CV (%) N c 

# Mean SD CV (%) Testis, left (mg) U 20 1504.2 117.3 7.8 20 1506.7 125.8 8.3 20 1554.2 117.2 7.5 7 1528.8 123.6 8.1 A 20 1505.1 -- -- 20 1504.1 -- -- 20 1553.1 -- -- 7 1536.8 -- -- Testis, right (mg) U 20 1495.1 99.9 6.7 20 1523.5 114.0 7.5 20 1547.4 134.2 8.7 7 1541.4 98.2 6.4 A 20 1496.0 -- -- 20 1520.8 -- -- 20 1546.3 -- -- 7 1549.8 -- -- Thyroid, fixed (mg) U 20 32.6 6.84 21.0 20 32.3 5.64 17.5 2 0 30.9 7.42 24.0 7 28.3 4.58 16.2 A 20 32.7 -- -- 20 32.2 -- -- 20 30.9 -- -- 7 28.5 -- -- a Data were obtained from Table 2 on pages 89-92 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentheses. b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/ day by PND 24 due to mortalit y and clinical observations. c Excludes animals found dead or euthanized due to clin ical observations, and includes the rats that were reassigned from the cholinesterase phase to the main study. U Unadjusted for body weight on PND 21 A Adjusted for body weight on PND 21 R Organ-to-body weight ratio (relative to body weight) N Number of animals examined SD Standard Deviation CV Coefficient of Variation ** Significantly different from controls at p ≤ 0.01.



# E. CLINICAL CHEMISTRY AND HORMONE LEVELS: Mean hormone levels and clinical chemistry values are presented in Ta ble 5. In the low-, mi d-, and high-dose groups, serum T 4 

# concentrations were increased (p ≤ 0.01) by 41, 35, and 32%, respectively, and testosterone levels were decreased (p ≤ 0.01) by 60, 78, and 90%, respectively. Serum TSH levels were not affected by treatment.
In all malathion-treated animals, albumin was decreased (p ≤ 0.01) by 5-6% and globulin was increased (p ≤ 0.01) by 8-17%; therefore, the A/G ratio was decreased (p ≤ 0.01) by 13-19%. Compared to the control, there were increa ses (NS) in GGT levels of 470, 51, and 179% in the low-, mid-, and high-dose groups. In the low-, mid-, and high-dose groups, total bilirubin levels were decreased (p ≤ 0.01) by 16, 30, and 36%, respec tively, and triglycerides were decreased (p ≤ 0.01) by 33, 65, and 68%, respectively. Th ese clinical chemistry effects were concurrent with increases in live r weights observed in all dose groups.
Although significant changes were observed in th e following clinical chemistry parameters, the values were within the historical control ra nges and are not considered to be biologically relevant. Calcium levels in all dose groups were decreased (p ≤ 0.05) by 3%. In the mid- and high-dose groups, potassium levels were decreased (p ≤ 0.01) by 7 and 9%, respectively; chloride levels were increased (p ≤ 0.05) by 2 and 3%; cholestero l levels were increased (p ≤ 0.05) by 11 and 19%; glucose le vels were increased (p ≤ 0.05) by 18 and 39%; aspartate aminotransferase was decreased (p ≤ 0.01) by 27% and 33%; and alkaline phosphatase was decreased (p ≤ 0.01) by 19% and 36%.
The alanine aminotransferase, total protei n, urea nitrogen, creatinine, phosphorus, and sodium treated group concentrations we re similar to control values.

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# The mean control TSH level (2.780) was belo w the performance criteria acceptable range (4.212-24.112). Guideline criteria for serum T 4 

# and testosterone leve ls were met in the control.

TABLE 5. Hormone Levels and Clinical Chemistry a



# Parameter Evaluated Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b



# N Mean SD CV (%) N Mean SD CV (%) N c 

# Mean SD CV (%) N c 

# Mean SD CV (%) Hormones Serum T 4 

# , Total (μg/dL) 20 5.12 0.642 12.5 20 7.24** ( ↑ 41) 1.02 14.1 20 6.92** ( ↑ 35) 1.28 18.5 7 6.76** ( ↑ 32) 0.979 14.5

Serum TSH (ng/mL) 20 2.78 1.46 52.4 20 4.01 1.76 43.9 20 3.41 1.22 35.7 7 3.26 2.58 79.1

Testosterone (ng/mL) 20 2.15 1.19 55.3 20 0.862** ( ↓ 60) 0.551 63.9 20 0.478** ( ↓ 78) 0.529 111 7 0.213** ( ↓ 90) 0.172 80.8

Clinical Chemistry A/G ratio 17 d 

# 1.49 0.123 8.2 20 1.30** ( ↓ 13) 0.078 6.0 20 1.20** ( ↓ 19) 0.070 5.8 7 1.21** ( ↓ 19) 0.051 4.2

Albumin (g/dL) 17 d 

# 3.39 0.120 3.5 20 3.20** ( ↓ 6) 0.174 5.4 20 3.18** ( ↓ 6) 0.136 4.3 7 3.21* ( ↓ 5) 0.145 4.5

Alkaline phosphatase (U/L) 17 d 

# 514 87.9 17.1 20 525 146 27.7 20 415* ( ↓ 19) 113 27.2 7 330** ( ↓ 36) 95.3 28.9

ALT (U/L) 17 d 

# 64.8 8.7 13.4 20 63.9 10.7 16.7 20 60.9 8.5 14.0 7 62.9 13.7 21.8 AST (U/L) 17 d 

# 212 37.8 17.8 20 195 44.1 22.6 20 154** ( ↓ 27) 26.3 17.1 7 143** ( ↓ 33) 27.8 19.4

Calcium (mg/dL) 17 d 

# 10.50 0.317 3.0 20 10.18** ( ↓ 3) 0.345 3.4 20 10.15** ( ↓ 3) 0.325 3.2 7 10.18* ( ↓ 3) 0.203 2.0

Chloride (mEq/L) 17 d 

# 93.8 2.1 2.2 20 94.8 2.9 3.1 20 95.6* ( ↑ 2) 2.7 2.8 7 97.0* ( ↑ 3) 2.5 2.6

Cholesterol (mg/dL) 17 d 

# 90.6 8.7 9.6 20 95.7 13.8 14.4 20 101* ( ↑ 11) 15.2 15.0 7 108** ( ↑ 19) 16.1 14.9

Creatinine (mg/dL) 17 d 

# 0.255 0.038 14.9 20 0.259 0.028 10.8 20 0.266 0.028 10.5 7 0.247 0.015 6.1

GGT (U/L) 17 d 

# 0.090 0.135 150 20 0.513 ( ↑ 470) 0.895 175 20 0.136 ( ↑ 51) 0.221 163 7 0.251 ( ↑ 179) 0.276 110

Globulin (g/dL) 17 d 

# 2.28 0.156 6.8 20 2.47** ( ↑ 8) 0.171 6.9 20 2.67** ( ↑ 17) 0.227 8.5 7 2.66** ( ↑ 17) 0.166 6.2

Glucose (mg/dL) 17 d 

# 125 9.0 7.2 20 123 7.9 6.4 20 147** ( ↑ 18) 38.2 26 7 174** ( ↑ 39) 58.3 34

Phosphorus (mg/dL) 17 d 

# 9.51 0.726 7.6 20 9.49 0.902 9.5 20 9.92 0.814 8.2 7 9.81 0.364 3.7

Potassium (mEq/L) 17 d 

# 6.65 0.459 6.9 20 6.33 0.599 9.5 20 6.16** ( ↓ 7) 0.493 8.0 7 6.05* ( ↓ 9) 0.360 6.0

Sodium (mEq/L) 17 d 

# 137 3.5 2.6 20 137 3.3 2.4 20 138 3.5 2.5 7 139 2.8 2.0

Total bilirubin (mg/dL) 17 d 

# 0.159 0.037 23.3 20 0.133** ( ↓ 16) 0.024 18.0 20 0.111** ( ↓ 30) 0.020 18.0 7 0.101** ( ↓ 36) 0.011 10.9

Total protein (g/dL) 17 d 

# 5.66 0.192 3.4 20 5.67 0.306 5.4 20 5.85 0.344 5.9 7 5.87 0.289 4.9

Triglyceride (mg/dL) 17 d 

# 465 190 40.8 20 312** ( ↓ 33) 193 61.7 20 165** ( ↓ 65) 105 64.0 7 150** ( ↓ 68) 65.5 43.7

Serum urea nitrogen (mg/dL) 17 d 

# 15.1 1.7 11.3 20 14.3 2.5 17.5 20 13.4 2.3 17.2 7 13.3 1.1 8.3

a Data were obtained from Tables 3 on page 94 and Table 4 on pages 96-99 of the study report. Percent difference from controls, calculated by the reviewers, is included in parentheses.

# Male Pubertal Assay (2012)/ Page 15 of 21 MALATHION/ 057701 OCSPP 890.1500 / OECD None

b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/ day by PND 24 due to mortalit y and clinical observations. c Excludes animals found dead or euthanized due to clin ical observations, and includes the rats that were reassigned from the cholinesterase phase to the main study. d Rats with moderately or severely he molyzed serum samples were not analyzed. N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



# F. GROSS PATHOLOGY AND HISTOPATHOLOGY: At the scheduled euthanasia, no treatment-related macroscopic findings were noted. Slight to moderate dilation in the pelvis of the right kidney was observed in two animals in the vehicle control group, two animals in the low-dose group, and one animal each in the mid- and high-dose groups. These findings were not considered treatment related.
The incidences of histopathological findings of the thyroid gland are presented below in Table 6. There were no treatmen t-related findings in the thyr oid gland, epididymides, right kidney, liver, testis, or pituitary.

# Male Pubertal Assay (2012)/ Page 16 of 21 MALATHION/ 057701 OCSPP 890.1500 / OECD None



# TABLE 6. Evaluation of Colloid Area and Follicular Cell Height in 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 15 20 1 18 20 2 5 20 2 2 20 3 0 20 3 0 20 4 0 20 4 0 20 5 0 20 5 0 20

Malathion 250 mg/kg/day 1 20 20 1 20 20 2 0 20 2 0 20 3 0 20 3 0 20 4 0 20 4 0 20 5 0 20 5 0 20

Malathion 500 mg/kg/day 1 20 20 1 17 20 2 0 20 2 3 20 3 0 20 3 0 20 4 0 20 4 0 20 5 0 20 5 0 20

Malathion 1000/750 mg/kg/day c 

# 1 7 7 1 7 7 2 0 7 2 0 7 3 0 7 3 0 7 4 0 7 4 0 7 5 0 7 5 0 7 a Data were obtained from page 661 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. c The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/day by PND 24 due to mortality and clinical observations. O Number Observed E Number Examined



# G. Cholinesterase Activity: There were dose-dependent redu ctions in RBC and brain ChE activity in the low and mid-dos e groups (Table 7). No animals from the high-dose group were evaluated for RBC and brain ChE activ ity. In the low- and mid-dose groups, RBC ChE levels were decreased (p ≤ 0.05) by 79 and 88%, respectively, and brain ChE levels were decreased (p ≤ 0.05) by 17 and 35%, respectively.

# Male Pubertal Assay (2012)/ Page 17 of 21 MALATHION/ 057701 OCSPP 890.1500 / OECD None



# Table 7. Cholinesterase Activity a



# Parameter Study Day Dose Group

Vehicle Control Malathion 250 mg/kg/day Malathion 500 mg/kg/day Malathion 1000/750 mg/kg/day b 

# N Mean SD N Mean SD N Mean SD N Mean SD Brain weight (g) 53 8 2.00 0.064 8 2.00 0.060 6 c 

# 2.02 0.050 0 c 

# -- -- RBC ChE levels (U/L) 53 7 1.18 0.145 7 0.245** ( ↓ 79.2) 0.061 6 c 

# 0.145** ( ↓ 87.7) 0.035 0 c 

# -- -- Brain ChE levels (U/L) 53 8 14.2 0.737 8 11.7 ( ↓ 17.4) 0.850 6 c 

# 9.25** ( ↓ 34.6) 1.79 0 c 

# -- -- a Data were obtained from page 69, Table 12 on page 135 and Table 14 on page 139 of the study report. Percent inhibition is shown in parentheses. b The dose level was decreased from 1000 mg/kg/day to 750 mg/kg/day by PND 24 due to mortality and clinical observations. c Excludes rats that were found dead or reassigned to main study. N No. of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05. ** Significantly different from controls at p ≤ 0.01.



# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Mortality occurred in ma le rats at both the midand high-doses. Malathion-relate d adverse clinic al signs observed in the male rats occurred primarily in the mid- and high-dose groups. The adverse clinical signs included the following: effects on gait, excess salivation, ir regular respiration, decreased motor activity, tremors, as well as additional signs of gene ral toxicity. Due to mortality and adverse clinical signs of toxici ty observed in several rats dosed at 1000 mg/kg/day within the first two days of dosing, the high-dose level was reduced to 750 mg/kg/day for male rats beginning on PND 24 in the cholinesterase subset and PNDs 23 and 24 for the male rats in the main study.
Significant reductions in RBC and brain ChE activity was observed in all malathion-treated dose groups available for evaluation (low- and mid-dose groups). St atistically significant decreases in mean testosterone levels were observed in all malathion-treated groups. Although the mean values for all four dose gr oups were within the acceptable range for control male Sprague-Dawley ra ts specified in the OCSPP test guideline, the decreases in testosterone were considered to be test substance related. The toxicological relevance of this finding in the absence of any other anti-a ndrogenic findings in the peripubertal male rat is not clear. The effect of malathion on serum testosterone levels has been previously reported. It is possible that th e decreased testosterone levels are due to alterations in the hepatic metabolism of testosterone by th e mixed function oxidases (Cytochrome P450 dependent enzymes), which malathion has been found to inhibit. Decreases in testosterone production have been found in the testes of ra ts with inhibited microsomal cytochrome P450-dependent enzymes 17-hydroxylase and 17, 20-ly ase. Alternatively, malathion at these high and systemically toxic dose levels could be overwhelming metabolic processes, as it too is a substrate for theses enzymes, in the liver resulting in altered testosterone levels.
In the low-, mid- and high-dose groups, statisti cally significant increases were observed in mean T 4 

# levels (132-141%); however, the significan ce of this finding in male rats is not clear because the increases were not clearly dose dependent and the T 4 

# values were within

# Male Pubertal Assay (2012)/ Page 18 of 21 MALATHION/ 057701 OCSPP 890.1500 / OECD None


# the Testing Facility's historical control range. In addition, the mean and CV values for all four dose groups were within the acceptable range for control male Sprague-Dawley rats specified in the OCSPP test gui delines. Because malathion has been shown to be an inhibitor of mixed function oxida ses, the increases seen in th is study could be related to effects of malathion on the hepatic metabolism of T 4 

# , reducing excretion of T 4 

# , and/or malathion could be overwhelming metabolic pro cesses in the liver at these dose levels and altering thyroid hormone metabolism.
Changes in serum chemistry that were pres umed to be related to test substance administration included an increase in GGT at >250 mg/kg/day; a si gnificant increase in globulin at >250 mg/kg/day; si gnificant reductions in albumin and the A/G ratio at >250 mg/kg/day; statistically significan t reductions in total bilirubin levels in male rats at >250 mg/kg/day; and signifi cant decreases in the triglycerid e levels at >250 mg/kg/day.
Slight increases or statistically significant incr eases in absolute and relative kidney and liver weights were seen in the mid- and high-dose gr oups of both sexes, but no histopathological findings were associated with these weight changes. These slight changes reflect the adaptive, metabolic and execratory responses ex pected in the livers and kidneys of rats given high doses of malathion. No other test article-related organ weight changes were noted that were different from their resp ective controls. There were no correlating morphological data, thereby suggesting the kidne y and liver weight changes were likely adaptive changes to the mid a nd high doses and not considered adverse or toxicologically relevant.

Based on these data, administration of malathion to peripubertal male and rats at dose levels of ≤ 750 mg/kg/day did not result in any direct an drogenic or antiandroge nic effects in male rats or any effect on pubertal develo pment or thyroid function in males.

AGENCY COMMENTS: In the main study, 14 rats in the high-dose group were found dead during the study, from PND 25 (3 doses) to PND 53 (31 doses), and one rat was sacrificed due to adverse clin ical observations on PND 32. Two animals in the mid-dose group were found dead on PNDs 45 and 50. In the cholineste rase cohort, 6 rats in the highdose group were found dead between PNDs 24 and 49 (2 to 27 doses), with one death being attributed to gavage error. All other rats survived until scheduled sacrifice.
Treatment-related clinical signs were observed for the mid- and high-dose rats that were found dead, euthanized, or subsequently recove red. These clinical signs included effects on gait, irregular respiration, ne rvous system, and general toxici ty. In the low-dose group, sporadic clinical signs incl uded slight excess salivation, ungr oomed coat, and urine stained abdominal fur.
There were no treatment-related effects on final body weight, overall body weight gain, and body weight at PPS. Although the first day of PPS was delayed (p ≤ 0.01) by 1.4 to 1.6 days in the low- and mid-dose groups, there was no effect on first day of PPS in the high-dose group.
Compared to the control group, absolu te liver weights were increased (p ≤ 0.05) by 21% in the mid- and high-dose groups, and the rela tive (to body) liver weight was increased

# Male Pubertal Assay (2012)/ Page 19 of 21 MALATHION/ 057701 OCSPP 890.1500 / OECD None


# (p ≤ 0.01) by 10, 22, and 26% in the low-, mid- a nd high-dose groups, respectively. Absolute kidney weight in the mid- dose group was increased (p ≤ 0.01) by 12%, and relative kidney weight was increased (p ≤ 0.01) by 6, 14, and 16% in the low-, mid-, and high-dose groups, respectively. The liver and kidney weight eff ects were considered to be treatment related and are likely an adaptive response to malath ion treatment. The mean LABC weights for the mid- and high-dose groups were decreased (p ≤ 0.01) by 14% and 17%, respectively, but there were no other changes in weights of accesso ry sex tissues. No ot her significant effects on organ weights we re reported.

Serum T 4 

# concentrations in the low-, mid-, and high-dose groups were increased (p ≤ 0.01) by 41, 35, and 32%, respectively, but these changes were not dose-dependent, the T 4 

# levels were within the range of histor ical control values, and there were no related changes in TSH levels. In addition, there were no changes in weight or mi croscopic findings in thyroid glands of malathion-treated ra ts. Therefore, the biological relevance of the changes in T 4 

# levels is unclear. There were cl ear, dose-dependent decreases (p ≤ 0.01) in testosterone levels of 60, 78, and 90% in the low-, mid-, and high-dose groups, respectively, which may correlate with the observed delay in PPS and the reductions in weight of the LABC.
In all malathion-treated animals, albumin was decreased (p ≤ 0.01) by 5-6%, globulin was increased (p ≤ 0.01) by 8-17%, and the A/G ratio was decreased (p ≤ 0.01) by 13-19%. There were also dose-dependent decreases (p ≤ 0.01) in total bilirubin levels of 16-36% and in triglycerides of 33-68%, and choles terol levels were increased (p ≤ 0.05) by 11 and 19% in the mid- and high-dose groups. The above change s were concurrent with the increases in liver weights and likely reflect adaptive changes in liver metabolism. Although significant changes were observed in other selected clin ical chemistry parameters, the values were generally within the historical control ranges and are not c onsidered to be biologically relevant.

In the cholinesterase cohort, the ChE levels on PND 53 were decreased (p ≤ 0.01) dosedependently by 79 and 88% in red blood cells and by 17 and 35% in brain tissue. Based on the ChE data, and the mortality and clinical signs, both the mid- and high-doses (500 and 750 mg/kg/day) were clearly excessive for im mature male rats. Cholinesterase was not evaluated at the high dose.

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

 The mean body weight at weaning (61.5 g) was above the performance criteria acceptable range (45.5-59.8 g).

 The mean CV value for body weight at PPS (8.7%) was above the performance criteria maximum of 7.6%.

 The mean control absolute liver weight ( 15.44 g) was slightly above the performance criteria acceptable range (9.990-15.350 g).

 The mean control absolute thyroid wei ght (0.0326 g) was slightly above the performance criteria acce ptable range (0.014-0.026 g).

 The mean control TSH level (2.780) was below the performance criteria acceptable range (4.212-24.112).

# Male Pubertal Assay (2012)/ Page 20 of 21 MALATHION/ 057701 OCSPP 890.1500 / OECD None



# APPENDIX I
Because this is a range-finding study, only a summary is provided to confirm the adequacy of the dose selection rationale used in the Male Pubertal Assay.
In a dose range-finding assay (MRID 48685702), ma lathion (96.0%; Batch #9010501) in corn oil was administered daily via oral gavage in a dos e volume of 5 mL/kg to eight juvenile female (PND 22 to 35) and eight male (PND 23 to 36) Sp rague Dawley [Crl:CD(SD)] rats/dose group at doses of 0, 250, 450, or 600 mg/kg/day. The animal s were dosed for 14 consecutive days and necropsied one day after the final dose admini stration. Body weights and food consumption were determined daily. The brain and liver were collected and weighed. Blood and brain samples were collected and analyzed for cholines terase activity. Only the results for the male rats are discussed in this appendix; the results for the female rats are summarized in the DER for MRID 48685701A (concurre ntly reviewed).

All rats survived to scheduled termination. Clinical findings in cluded at least one male with excess salivation in each of the dose groups, and one 250 mg/kg/day male with a nodule proximal to the hock on the right hindlimb. M ean body weights, body weight gains, and food consumption in the 250, 450, and 600 mg/kg/day groups were comparable to the controls. The brain and liver weights (absolut e and relative to final body weight) were comparable to the controls.
The brain cholinesterase activity was reduced (p ≤ 0.01) in the 250, 450 and 600 mg/kg/day males by 9.3, 15.8, and 16.0%, respectively. Red blood ce ll cholinesterase ac tivity was reduced (p ≤ 0.01) in the 250, 450 and 600 mg/kg/day males by 67.4, 76.7, and 79.6%, respectively.

COMPLIANCE: It was stated that the study was not performed in comp liance with GLPs.

# Male Pubertal Assay (2012)/ Page 21 of 21 MALATHION/ 057701 OCSPP 890.1500 / OECD None



# APPENDIX II
Because this is a method validation study, only a summary is provided. The analytical method for determination of malathion in corn oil dos e formulations was desc ribed, and validation and stability data were provided in a previous submission (MRID 48617512).
The method for determination of malathion in corn oil involves dilution of samples in acetone followed by analysis by HPLC/UV. Precision of the method was evaluated by analyzing 6 replicates of low (1 mg/mL) and high (201 mg/mL) concentration fortified samples; the relative standard deviations of these analyses were 0.8% and 1.3%, respectively. These analyses were also used to evaluate accuracy ; the mean percent errors were

− 1.8% and

− 1.1%, respectively, for the low and high concentrated fortified samples.
The stability of calibration sta ndards and final dilutions of 1 mg/mL and 200 mg/mL fortified samples at 15 °C was evaluated by re-analyzi ng the standards and samples 38 and 37 hours, respectively, following the initial injections; the percent difference between the initial and final results ranged from

− 2.3% to 5.3%

. 

# Stability of these samples was also evaluated following storage at 2-8 °C for 14 days protected from light. For the calibration standards and the 1 mg/mL fortified sample, the percent difference between the initial and final concentrations ranged from

− 2.7% to 2.5%. For the 200 mg/mL for tified sample, the percent difference between the initial and final re sult was 24%; a second fortified sa mple was prepared and stored at 2-8 °C for 15 days and the percent difference between the initial and final analyses was 14.2%. The study author noted that a positive differen ce is not an indication of instability.
Stability was evaluated for 1 mg/mL and 200 mg/m L dose formulations prepared in com oil. The dose formulations were analyzed prior to storage and following storage at 20-25 °C, protected from light, for 40 hours an d at 2-8 °C, protected from li ght, for 13 days. The percent difference between the final stability samp le results and initial results ranged

− 2.8% to 0.5%.
Based on these findings, it was concluded that the method was sufficiently reproducible and accurate to support dose formulation analyses. Dose formulations in corn oil, protected from light, were found to be stable for 40 hours at 20-25 °C and for 13 days at 2-8 °C.

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

# DATA EVALUATION RECORD

MALATHION
Study Type: OCSPP 890.1550, Steroidogenesis Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-5-2012 (MRID 48617509)

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: 2/27/2012
Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 03/06/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 03/08/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 03/08/2012

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

.

# The US EPA Endocrine Disruptor 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).

# MALA THION/057701


# Primary Reviewer: Kit Farewell. D.V.M. Health Effects Division Secondary Reviewer: John Liccione, Ph.D. Health Effects Division Steroidogenesis Assay (2011) I 

# Page 1 of 14 OCSPP 890.1550/ OECD None


# Signature: 

# C2o_-<**~** 

# @2**K~**

# Date:~-

Signatu •

# ~~ 

# :=E**~on** Date: · P 

# 5,.-

DATA EVALUATION RECORD

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550

PC CODE: 057701 DP BARCODE: D396178

TXR#:0052348 CAS No.: 121-75-5

TEST MATERIAL (PURITY): Malathion 96.0% a.i.

SYNONYMS: Butanedioic acid, ((dimethoxyphosphinothioyl) thio)-, diethyl ester

CITATION: Wagner, H 

# (2011) Malathion: H295R steroidogenesis assay. CeeTox, Inc, Kalamazoo, Ml. Laboratory Report No.: 9148V-100372STER, Nov 3, 2011. MRID 48617509. Unpublished.

SPONSOR: Cheminova A/S, P.O. Box 9, Lemvig, Denmark

TEST ORDER#: EDSP-057701-44

EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48617509), H295R cells cultured in vitro in 24-well plates were incubated with malathion (96% purity, Lot# 9010501) at concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μMin triplicate for 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentration of 0.1 %.

Testosterone and estradiol levels were measured using HPLC/MS-MS. Four independent experiments were performed, although only Runs 2, 3 and 4 were reported. The media from Run 1 was not analyzed for testosterone and estradiol concentrations because one of the forskolin concentrations on the Quality Control (QC) plate was different from the concentration specified in the Test Guideline. A QC plate was run concurrently with each independent run of the test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentrations; positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production.

Guideline acceptability recommendations and requirements were met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls. It 

# was stated that all assays were performed at CeeTox by personnel that have demonstrated proficiency performing this assay, however, proficiency records were not provided.

The only significant changes noted in hormone production of malathion treated cells were a decrease in testosterone production at 100 μM of malathion (p<0.05; 0.81-fold change) in Run 4

# Steroidogenesis Assay (2011) / Page 2 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None



# and an increase in estradiol production at 10 μM of malath ion in Run 2 (p<0.05; 1.12-fold change). The validity of the obser ved decrease in testosterone is questionable as precipitation of malathion was observed at 100 μ M in Runs 2 and 3, and there was no other concentration dependent effects noted in test osterone production. Similarly for the observed increase in estradiol, the effect was observed at 10 μ M in only one of three runs, and no concentration dependent changes in estradio l production were observed.
Based on hormone response in at least three inde pendent runs, malathion treatment did not result in statistically significant and re producible alterations in testosterone or estradiol production.
The assay satisfies the EDSP Tier 1 Test Order requi rements for a Steroidogenesis assay (OCSPP 890.1550).

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

# Steroidogenesis Assay (2011) / Page 3 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc.
Location: Kalamazoo, MI Study Director: Hillary Wagner Other Personnel: Colleen Toole, Ph.D., Director of Project Management Karen Rutherford, B.S., Direct or of Laboratory Operations David Blakeman, B.S., Senior Sc ientist/Endocrine Group Leader Briana Wallace, B.S., Lead Cell Culture Scientist Steven McColley, M.S., Scientist Cameron Haines, B.S., Scientist Francisca Wong, M.S., Scientist Joey Burgam, B.S., Associate Scientist Lisa Blakeman, B.S., Associate Scientist Kenneth Lewis, Ph.D., Principa l Investigator (OpAns, LLC) Lisa St. John-Williams, M.E., Analyst (OpAns, LLC) Study Period: May 23, 2011 - November 3, 2011



# 2. Test Substance: Malathion Description: Pale yellowish liquid Lot / Batch # (Expiration Date): Lot # 9010501 (Dec 11 2011) Purity: 96% Solubility (in Solvent): NR Volatility: NR Stability: 3 years Storage conditions: 0-10 ° C CAS #: 121-75-5 Molecular weight: 330.4 Structure:



# 3. Positive Control: Forskolin Description: White powder, MW410.5 Source: Sigma Aldrich, St. Louis, MO Lot / Batch # (Expiration Date): 109K50571V (July 2016) Purity: 98% Solubility (in Solvent): NR Storage conditions: Ambient CAS #: 66575-29-9


# 4. Negative Control: Prochloraz Description: White powder, MW 376.67 Source: Sigma Aldrich, St. Louis, MO Lot / Batch # (Expiration Date): SZE6220X (Aug 8 , 2012) Purity: 99.1 Solubility (in Solvent): NR Storage conditions: Ambient CAS #: 67747-09-5

# Steroidogenesis Assay (2011) / Page 4 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None



# 5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) Description: NR Source: Sigma Aldrich, St Louis, MO (date not provided) Lot / Batch # (Expiration Date): RNBB7617 Purity: NR Storage conditions: NR CAS #: NR Justification for choice of solvent: NR Final concentration: (% volume in assay) 0.05%



# 6. Other Materials: 22R-Hydroxycholesterol Description (molecular weight): White powder (402.65) Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): 089K4132, 060M4098 (not provided) Purity: 99.0% Storage conditions: Room temperature Solvent used: Ethanol, final concentratio n in assay, 0.025% v/v CAS #: 17954-98-2



# 7. Stock Medium: Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture Description: Source: Sigma-Aldrich, St. Louis, MO Lot / Batch # (Expiration Date): 021M8304 (date not provided) Sodium bicarbonate: Sigma-Aldrich, Lot#068K0105 Nu-Serum: Lot # 81515; tested for background hor mone concentrations by supplier ITS+ premix: Lot # 05245, 09233



# 8. Test Cells: H295R human adrenocortical carcinom a cells (ATCC CLR-2128; lot#7635054) at passage 7.5 - 8.5 were incubated in the st ock medium at the time of plating, dosing, and harvest. Incubation conditions were at 5% CO 2 

# and 37 ± 2°C for approximately 24 hours prior to exposure.
The following performance criteria were met (indicated by an “x”):

X Cell passage identifier. Cell Passage #: 7.5-8.5 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: An interference test was not conducted as the hormone levels were analyzed by LC-MS/MS.

b. Hormone Extraction: Hormone extraction was not performed.

c. Laboratory Proficiency Test: No laboratory proficiency test data were reported.

# Steroidogenesis Assay (2011) / Page 5 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None



# 2. Test Solutions: Malathion was dissolved in DMSO to make a 200 mM stock and then serially diluted 1:10 in DM SO. 22R-Hydroxycholesterol (S igma Aldrich, Lot #s 089K4132, 060M4098) was dissolved in ethanol to make a 40 mM stock and then diluted in supplemented medium to a final concentration of 10 μ M. Malathion was then diluted 1:2000 in supplemented medium containing 10 μ M 22R-hydroxycholesterol to prepare mastermix solutions. Forskolin and prochloraz were dissolved in DMSO to make 100 mM solutions and then serially diluted in DMSO. Forskolin and prochloraz were then diluted 1:2000 in supplemented medium containing 10 μ M 22R-hydroxycholesterol. When added to the cell culture plates, these diluti ons yielded final concentrations of 1 μ M and 10 μ M for forskolin, 0.1 μ M and 1 μ M for prochloraz and 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μ M for malathion.

3. Cell Plating and Preincubation: H295R cells were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for seven or eight additional passages. The cells were then seeded in 24-well plates using 1 mL of cells at a concentration of 300,000 cells/mL, yielding approximately 50-60% confluency at 24 hours. The seeded plates were incubated for 24 hours at 37°C in a 5% CO 2 

# atmosphere.

4. Exposure: The cells were checked microscopi cally for good attachment and proper morphology, and the medium was removed a nd replaced with medium containing the concentrations of the test materials presented in Table 1. The final concentration of DMSO in the medium was 0.05% v/v.

TABLE 1. Dosing Schematic for the Exposure of H295R Cells to Malathion(Final Concentrations in μM) a 

# 1 2 3 4 5 6 A DMSO DMSO DMSO 0.1 0.1 0.1 B 100 100 100 0.01 0.01 0.01 C 10 10 10 0.001 0.001 0.001 D 1 1 1 0.0001 0.0001 0.0001 a Data were obtained from page 16 of the study report. Dosing was calculated based on a to tal volume of 1 mL per well.



# A concurrent QC plate was included with each of the three independent runs of the test chemical plate 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 Po sitive Controls (Final Concentrations in μM) a 

# 1 2 3 4 5 6 A Blank b 

# Blank Blank Background c 

# Background Background B DMSO DMSO DMSO DMSO + MeOH d 

# DMSO + MeOH DMSO + MeOH C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochlor az 0.1μM Prochloraz 0.1μM Prochloraz 0.1μM D Forskolin 10μM Forskolin 10μM Forskolin 10μM Proc hloraz 1μM Prochloraz 1μM Prochloraz 1μM a Data were obtained from page 16 of the study report. Dosing was calculated based on a to tal volume of 1 mL per well. b Blank wells received medium co ntaining 22R-hydroxycholesterol. c Background wells received medium only. d MeOH = 70% methanol was added to these wells for 30 mi nutes at room temperature following medium removal.

# Steroidogenesis Assay (2011) / Page 6 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None



# Following dosing, the plates were incubated for 48±2 hours under the conditions previously described. The medium from each well was re moved, split into two equal volume aliquots, and frozen at

− 80 °C until hormone measurements.

5. Cell Viability/Cytotoxicity Assay: Cell viability was determined using the MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium br omide] test immediately after removal of the culture medium. Following media remova l and/or methanol treatment, 0.5 mL of 0.5 mg/mL MTT solution in supplemented medium containing 10 μ M 22R-hydroxycholesterol was added to each well of the test chemical a nd QC plates. The plates were incubated at 37±2 °C in a 5% CO 2 

# incubator for 3 hours. Followi ng incubation the MTT solution was removed and 0.5 mL isopropanol was added to each well. Plates were incubated at room temperature for 20 minutes with shaking. Fo llowing this incubation, absorbance at 570 mm and 650 nm were measured, and the absorbance at 650 nm was subtracted from the absorbance at 570 nm to calculate the MTT value for each well.

6. Hormone Measurement System: The concentration of eac h hormone (testosterone and estradiol) was evaluated in triplicate in the culture medium derived from each well using HPLC/MS-MS at OpANS, LLC. The method dete ction limit is 100 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 within 30% of nominal concentration) NA Hormone cross-reactivity (antibody-based assays only; ≤ 30% of basal production of the respective hormone) X Solvent control within 75% range be low maximum response on standard curve NA Test compound tested for interf erence with meas urement system



# C. DATA ANALYSIS: Mean values (pg/mL) and standard deviations for testosterone and estradiol were calculated for each concentrati on of the test chemical, reference chemicals, and the solvent controls. Re lative changes in testosterone and estradiol production were calculated using the equation below:
Relative Change = [Hormone] in each well ÷ [H ormone] of mean solvent (vehicle) control

For forskolin induction of testosterone, rela tive change was calculated both with and without subtracting the background hormone pr oduction from the forskolin-treated wells and blank and solvent control wells. Th is was done because of the high background production of testosterone and the small dynamic range for forskolin induction. Background hormone production was calculated from three wells with cells on the QC plate that received no 22R-hydroxycholes terol at the time of exposure. All concentrations that exhibited greater than 20% cytotoxicity in th e MTT cell viability assay were excluded from further analysis. Concentrations where precipitation was observed were also excluded from further analysis. Normality of the data was evaluated using Shapiro-Wilk’s test. Homogeneity of the variances between the tr eatment groups was evaluated using Levene’s test. If the p-values were greater than 0.05 in both tests, statistical significance between each treatment group and the control group was evaluated using Dunnett’s test. If the pvalues were less than or equal to 0.05 in ei ther the normality or the homogeneity test, a log

# Steroidogenesis Assay (2011) / Page 7 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None


# transformation was performed on the data to at tempt to approximate a normal distribution. If, following the transformation, p-values were greater than 0.05 in both the normality and homogeneity tests, Dunnett’s test was perf ormed on the transformed data to evaluate statistical significance between the each treatment group and th e control group. If following the log transformation, p-values were less than or equal to 0.05 in either the normality or the homogeneity test, the non-tr ansformed data set was anal yzed using the nonparametric Kruskal-Wallis test followed by Dunn’s test to evaluate statistical significance between each treatment group and the control gr oup. Data analysis was con ducted in Microsoft Excel. Statistics were calculated us ing Unistat 6.0 Light for Excel. Graphs were generated using Microsoft Excel.

II. RESULTS

A. TEST COMPOUND: Precipitation of ma lathion was observed at 100 μM in Runs 2 and 3. No precipitation or cytotoxicity was observed at concentrations up to 10 μM. The % CVs of replicate wells of the solv ent control within a plate were 2.56-7.78% for absolute testosterone concentrations and 1.34-5.11% for absolute estradiol concentrations. The between-plate CVs for solvent controls base d on absolute concentration were 20% for testosterone and 17% for estradiol. Guideline recommendations were met.
The hormone concentrations following exposure to malathion are presented in Table 3, and the fold-changes in hormone production for malath ion-treated cells compared to the solvent control are shown graphically in Figures 1 and 2. In Run 4, there was a statistically significant decrease in testosterone production at 100 μM ( 0.81-fold change), and in Run 2 there was a statistically signi ficant increase in es tradiol production at 10 μM (1.12-fold change). There were no other st atistically significant changes.

# Steroidogenesis Assay (2011) / Page 8 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None



# TABLE 3. Mean (±SD) Hormone Concentrations Fo llowing Treatment with Malathion for 48 Hours. a


# Nominal Concentration (μM) Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean ± SD Statistical Significance
Testosterone (pg/mL) Fold Difference DMSO 2849 1917 2396 1.00 1.00 1.00 1.00 0 0.0001 3035 1873 2219 1.07 0.98 0.93 0.99 0.07 0.001 2803 1883 2250 0.98 0.98 0.94 0.97 0.02 0.01 2800 1912 2267 0.98 1.00 0.95 0.98 0.03 0.1 2940 1943 2306 1.03 1.01 0.96 1.00 0.04 1 3122 2007 2322 1.10 1.05 0.97 1.04 0.07 10 2661 1872 2261 0.93 0.98 0.94 0.95 0.03 100 1886 1621 1934 NA NA 0.81 * NA NA Run 4 Estradiol (pg/mL) Fold Difference DMSO 252 178 216 1.00 1.00 1.00 1.00 0 0.0001 239 173 199 0.95 0.98 0.92 0.95 0.03 0.001 240 170 205 0.95 0.96 0.95 0.95 0.01 0.01 239 174 206 0.95 0.98 0.95 0.96 0.02 0.1 241 170 212 0.96 0.96 0.98 0.97 0.01 1 262 189 225 1.04 1.07 1.04 1.05 0.02 10 281 * 198 247 1.12 1.11 1.14 1.12 0.02 Run 2 100 231 198 245 NA NA 1.13 NA NA a Data were obtained from Tables 8 and 9 on pages 29 and 30 and Appendices 2-4 on pages 55-57 of the study report. NA Not applicable; concentration excl uded because precipitation was observed 

# *p ≤ 0.05

# Steroidogenesis Assay (2011) / Page 9 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None


# FIGURE 1. Change in Testosterone Producti on Relative to Malathion Concentration.

# Steroidogenesis Assay (2011) / Page 10 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None


# FIGURE 2. Change in Estradiol Production Relative to Malathion Concentration.

B. CYTOTOXICITY: Results from the cytotoxicity testing for malathion and the reference compounds are summarized in Table 4 and pres ented graphically in Figure 3. Malathion was not cytotoxic to the H295R ce lls at any concentration tested.

# Steroidogenesis Assay (2011) / Page 11 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None



# TABLE 4. Mean (±SD) MTT Cell Viability Results af ter Treatment with Forskolin, Prochloraz, or Malathion for 48 Hours. a


# Compound Concen. (μM) Cell Viability – Run 2 Cell Viabilit y – Run 3 Cell Viability – Run 4 Mean SD Mean SD Mean SD Blank NA 101.6 1.27 100.3 0.99 99.8 3.20 Background NA 97.9 3.85 97.8 2.50 101.9 4.39 SC + Methanol NA 5.9 1. 38 6.3 1.35 7.3 1.37 Forskolin 1 b 

# 107.1 2.34 103.6 0.90 101.7 2.89 Forskolin 10 105.5 1.25 102.3 5.08 104.8 0.52 Prochloraz 0.1 101.8 0.52 99.8 1.99 99.3 2.38 Prochloraz 1 103.0 1.88 97.7 1.82 100.4 1.08 Malathion 0.0001 96.2 1.69 95.1 1.18 91.91 0.69 Malathion 0.001 96.8 1.22 95.7 0.59 94.3 1.20 Malathion 0.01 95.6 0.74 96.9 1.79 94.4 0.63 Malathion 0.1 97.2 1.55 98.8 1.29 94.3 1.32 Malathion 1 96.1 0.11 98.9 3.69 93.0 0.63 Malathion 10 97.9 0.92 96.0 1.50 96.4 1.20 Malathion 100 94.3 5.25 101.6 b 

# 6.32 97.1 1.43 a Data were obtained from pages 23 and 24 of the study report. b MTT outlier point removed. S ubtracted value for the outlier well was 0.024. Image taken of this well indicates that cells are present and adherent. SC = Solvent control

# Steroidogenesis Assay (2011) / Page 12 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None


# FIGURE 3. Cell Viability Relative to Malathion Concentration

C. QC PLATE: The hormone concentrations after expos ure to the reference chemicals, SC, and blank samples, as well as the fold differen ce change relative to SC (individual and mean ± SD) for the three assay runs are presente d in Table 5. The minimal basal hormone productions levels (500 pg/mL for testosterone and 40 pg/mL for estradiol) were met in both blank and SC wells. Basal hormone pr oduction in SC wells was >22-fold minimum detection level (MDL) for testosterone and >21- fold MDL for estradiol. Compared to SC, 10 μM forskolin on average induced test osterone by 1.80-fold (instead of ≥ 2-fold as per the

# Steroidogenesis Assay (2011) / Page 13 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None


# guideline) and estradiol by 10.75-fold. Howeve r, when the background is subtracted, 10 μM forskolin induced testosterone an aver age of 2.63-fold. Compared to SC, 1 μM prochloraz on average inhibited the synthesis of testosterone to 0.44-fold and estradiol to 0.77-fold. Thus, the guideline requirements were met indicating that the assay was sensitive to induction and inhibition of testosterone and estradiol. The variability (%CV) between the runs (calculated by the reviewer) based on the absolute hormone concen trations in the SC were 16% for testosterone and 10% for estr adiol, which meets the guideline criteria of ≤ 30% for the assay. The %CVs within each run for the QC plates were 2.33 to 6.95% for testosterone and 3.99 to 5.75% for estradiol.

TABLE 5. Hormone Concentrations Following Treat ment with Forskolin or Prochloraz for 48 Hours. a



# Compound Conc. (μM) Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Mean b 

# ± SD b 

# Testosterone (pg/mL) Fold Difference Background NA 1188 1120 1229 0.45 0.59 0.52 0.52 0.07 Blank NA 2725 1927 2311 1.04 1.02 0.98 1.01 0.03 DMSO NA 2625 1896 2351 1.00 1. 00 1.00 1.00 0.00 Forskolin 1 4298 2482 3078 1.64 c 

# 1.31 c 

# 1.31 c 

# 1.42 c 

# 0.18 Forskolin 10 5898 3054 3596 2.25 d 

# 1.61 d 

# 1.53 d 

# 1.80 d 

# 0.39 Prochloraz 0.1 2019 1534 1807 0.77 0.81 0.77 0.78 0.02 Prochloraz 1 1335 724 995 0.51 0.38 0.42 0.44 0.07 Estradiol (pg/mL) Fold Difference Background NA 61 65 73 0.27 0.35 0.33 0.32 0.04 Blank NA 231 194 219 1.03 1.05 0.99 1.02 0.03 DMSO NA 225 185 220 1.0 1.0 1.0 1.00 0.00 Forskolin 1 1673 1151 1559 7.43 6.23 7.08 6.91 0.62 Forskolin 10 2634 1750 2436 11.70 9.48 11.06 10.75 1.14 Prochloraz 0.1 154 157 167 0.69 0.85 0.76 0.77 0.08 a Data were obtained from page 25 and 26 of the study report. b Calculated by reviewers. c When the background is subtracted, the valu es are 2.16-, 1.76-, and 1.65-fold difference for Runs 2, 3, and 4, respectively, with a mean of 1.86-fold change. d When the background is subtracted, the valu es are 3.28-, 2.49-, and 2.11-fold difference for Runs 2, 3, and 4, respectively, with a mean of 2.63-fold change.



# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: No cytotoxicity greater than 20% was observed at any of the concentrations tested in any of the assay runs. No solubility issues were observed at the time of exposure. Pr ecipitate was noted upon observa tions of the wells under the microscope after the 48- hour incubation in the 100 μ M wells in runs 2 and 3. The suitable highest concentration of malathi on that could be tested was 100 μ M in Run 4 and 10 μ M in Runs 2 and 3 because of precipitate observed. A statistically significant reduction of testosterone was observed at 100 μ M in Run 4. No statistically significant alterations in testosterone were observed at any concentration in Run 2 or 3. A statistically significant increase in estradio l was observed at 10 μ M in Run 2. No statistically significant alterations in estradiol were observed at a ny concentration in Run 3 or 4.

B. AGENCY COMMENTS: Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxi city, adequate production of testosterone and

# Steroidogenesis Assay (2011) / Page 14 of 14 MALATHION/057701 OCSPP 890.1550/ OECD None


# estradiol, acceptable reproducib ility (low %CV), and appropria te induction and inhibition with positive controls.
There was a single decrease in testosterone production at 100 μM of malathion (p<0.05; 0.81-fold change) in Run 4, however, in Runs 2 and 3 this concentra tion of malathion was not be evaluated due to obser ved precipitation of the test compound. No other significant changes for concentration dependent changes were observed in test osterone production. There was a single increase in estradiol production observed at 10 μM of malathion in Run 2 (p<0.05; 1.12-fold change); but similar signif icant changes were not observed in the other two runs at this concentrati on and no other significant changes were noted at the other concentrations tested.

C. STUDY DEFICIENCIES: The following deficiency was no ted that is not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

 The results of the proficienc y test were not reported.

# DATA EVALUATION RECORD

MALATHION
Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-05-2012 (MRID 48617510)

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

Arlington, VA 22202

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

Durham, NC 27713

Primary Reviewer Signature: Kelly Luck, M.S. Date: 02/29/2012
Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 03/06/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 03/07/2012
Quality Assurance: Signature: Jack D. Early, M.S. Date: 03/07/2012

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).

# MALATHION/ 057701


# Primary Reviewer: Sheila Healy. Ph.D. 

# Health Effects Division Jn vivo 

# Uterotrophic Assay (2011) I Page 1 of 10 OCSPP 890.1600/ OECD 440



# Secondary Reviewer:**---'J""""'e=ss~R=o""""w"""lan=d'---** 

# ________ 

# Signature:**-4~==~~==~-** 

# Health Effects Division Date:

# -~G ....

# ~""'-f-f-~~l-<=---- Temptate version 09/2011 

# DATA EVALUATION RECORD

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440



# PC CODE: 057701 

# DP BARCODE: D396178

TXR#: 0052348 

# CAS#: 121-75-5



# TEST MATERIAL (PURITY): Malathion (96.0% a.i.)



# SYNONYMS: Fyfanon® Technical; 2-[(dimethoxyphosphinothioyl)thio]butanedioic acid, 1,4- diethyl ester; diethyl 2-[ ( dimethoxyphosphinothioyl )thio ]butanedioate



# CITATION: Barnett, J. F. (2011) Oral (gavage) uterotrophic bioassay of malathion in female rats. Charles River Laboratories, Horsham, PA. Laboratory Project ID: 20012263, November 3, 2011. MRID 48617510. Unpublished.



# SPONSOR: Cheminova A/S, Lemvig, Denmark



# TEST ORDER#: EDSP-057701-44



# EXECUTIVE SUMMARY: In a uterotrophic assay (MRID 48617510) conducted to screen for potential estrogenic activity, malathion (96.0% a.i., Batch/lot 9010501) in com oil was administered daily via oral gavage to groups of eight ovariectomized female Sprague Dawley
rats at dose levels of 0 (vehicle), 100, 300, or 1000 (limit dose) mg/kg/day on post-natal days (PND) 68-70. A positive control group was treated with a daily dose of 17a-ethynyl estradiol (EE) at 5 μg/kg/day by subcutaneous (s.c.) injections. Body weights and food consumption were determined daily. All animals were terminated and necropsied on PND 71 approximately 24 hours after the final dose to determine wet and blotted uterine weights and liver weights. Brain and red blood cell (RBC) samples were also collected from rats in the vehicle control and malathion treatment groups for determination of cholinesterase (ChE) activity.

All animals survived until scheduled termination. No clinical signs of toxicity were observed in animals for any dose group.
Body weights in all malathion treatment groups were comparable to controls over the duration of the study. At 1000 mg/kg/day, the rats displayed a body weight loss (p::;0.05) during Days 2-3 (-0.8 g treated vs. +2.9 g controls), resulting in a decrease in overall (Days 1-4) body weight gains (NS) of 35%. Additionally at this dose, daily mean food consumption was decreased (p::;0.01) during Days 2-3 U10%) and Days 3-4 U8%), leading to decreased (p::;0.01) average food consumption over the duration of the study U6%). Uterine weights in the malathion
treatment groups were comparable to controls. Absolute and relative liver weights were increased (p::;0.01) at 1000 mg/kg/day (j24 and 25%); relative (to body) liver weights were also

# In vivo Uterotrophic Assay (2011) / Page 2 of 10 MALATHION/ 057701 OCSPP 890.1600/ OECD 440


# increased (p ≤ 0.05) at 300 mg/kg/day ( ↑ 10%). These increases were attributed to malathion treatment. Brain ChE activity was significantly inhibited (p ≤ 0.01) at 1000 mg/kg/day (16% inhibition), and RBC ChE activity was significantly inhibited (p ≤ 0.01) in rats at 100 mg/kg/day and above (20-69% inhibition).
In the positive control group, overall body weight gain was decreased (p ≤ 0.01; ↓ 85%); however, body weights in this group were comparable to the vehicle control over the duration of the study. Mean food consumption was increased (p ≤ 0.01) on Days 1-2 ( ↑ 10%) and decreased (p ≤ 0.01) on Days 2-3 ( ↓ 10%); average food consumption over the dur ation of the study was comparable to the control. Absolute wet and blotted uterine weights were increased (p ≤ 0.01) by 509% and 196%, respectively. The increased uterine we ights were in the expected range.
No statistically significant changes were seen in uterine weight in this assay. Malathion is negative in the uterotrophic assay.
The assay satisfies the EDSP Tier 1 Test Order require ments for a uterotrophic assay (OCSPP 890.1600).

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

# In vivo Uterotrophic Assay (2011) / Page 3 of 10 MALATHION/ 057701 OCSPP 890.1600/ OECD 440



# I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Charles River Laboratories Location: Horsham, PA Study Director: J. F. Barnett Other Personnel: A. Hoberman (Testing Facility Mana gement); M. Vane man (Director of Operations); J. Lech (Senior Manager Technical Operations); J. Yeager (Senior Manager Technical Resources ); J. Gulbinski (Senior Manager Laboratory Sciences); J. Sarsoza (Princ ipal Investigator Dose Formulation Analysis) Study Period: August 1 - November 3, 2011



# 2. Test Substance: Malathion Description: Clear to yellowish liquid Source: Cheminova A/S (Lemvig, Denmark) Lot/Batch #: 9010501 (expiration date 11/12/2014) Purity: 96.0% Stability: Stable in vehicle for up to 40 hours at room temperature and for up to 13 days at 2-8 °C CAS #: 121-75-5 Structure:



# 3. Reference Estrogen: 17 α -ethynyl estradiol (EE) Supplier Sigma-Aldrich (St. Louis, MO) Lot/Batch #: 011M1252V (expiration date 08/05/2012) Purity: 99% CAS #: 57-63-6



# 4. Solvent/Vehicle Control: Corn oil Supplier Charkit Chemical Corporation (Norwalk, CT) Lot/Batch #: M-631 (expiration date 3/18/2012) Rationale (if other than water) Appropriate vehicle for malathion formulations Final concentration Not applicable



# 5. Test Animals: Species: Rats (ovariectomized females) Strain: Sprague Dawley [Crl:CD(SD)] Age/weight at dose initiation: 68 days old/ 218.1-297.9 g females Source: Charles River Laboratorie s, Inc. (Portage, MI) Housing: Rats were housed two/cage in clear pl astic nesting boxes w ith heat-treated laboratory-grade pine shavings Diet: Harlan Teklad 2018c meal food, ad libitum Phytoestrogen content ≤ 300 ppm genistein equivalents Water: Reverse-osmosis deionized tap water, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 19-25 oC 30-70% minimum 10/hr 12 hrs light/12 hrs dark Acclimation period: 12 days S

O O CH 3 

# O O CH 3



# P S

O CH

3 

# O CH

3

# In vivo Uterotrophic Assay (2011) / Page 4 of 10 MALATHION/ 057701 OCSPP 890.1600/ OECD 440



# B. STUDY DESIGN

1. In Life Dates: Start: August 14, 2011 End: August 17, 2011

2. Study Design: Following a 12-day acclimation period, ovariectomized female rats (surgery performed by supplier at PND 49) were ad ministered the test substance from PND 68 through 70. Rats were euthanized approxima tely 24 hours later on PND 71 and necropsied for uterine weight measurements.

3. Animal Assignment: Animals were assigned to the te st groups noted in Table 1 using computer-generated, weight-ordered randomizati on procedures. The weight variation of the rats did not exceed  20% of the mean weight in each group, and there were no significant 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 0 8 Low 100 8 Mid 300 8 High 1000 8 17 α -ethynyl estradiol (EE), Reference Estrogen 5 μg/kg/day 8 a Data were obtained from Text Table 4 on page 22 of the study report.



# 4. Dose Selection Rationale: The dose levels were selected based on the results of a range-finding study (MRID No. 48617508) in Spra gue Dawley rats. A summary of this study is included as Appendix I to the DER for MRID 48617507 (concurrently reviewed). The uterotrophic assay study design include s a limit-dose group of test animals.

5. Dose Preparation and Analysis: Dose formulations were pr epared once prior to initiation of dosing by initially dissolving the appropriate amounts of test substance in corn oil. The reference estrogen (EE) dose formulation was prepared daily by mixing with corn oil. Homogeneity and achieved concentration were tested in all three dose levels at dose preparation, by collecting samples from the t op, middle, and bottom of each formulation. The stability of the test substance in corn oil was tested in a separate study (MRID No. 48617512); a summary of this study is include d as Appendix II to the DER for MRID 48617507 (concurrently reviewed).

Results of Dose Analysis

Homogeneity (%RSD): 0.3-3.2%

Stability (% of Day 0): Stable following storage for 40 hours at 20-25 °C Stable following storage for 13 days at 2-8 °C.

Concentration (% of nominal): 97.5-101.4%

# In vivo Uterotrophic Assay (2011) / Page 5 of 10 MALATHION/ 057701 OCSPP 890.1600/ OECD 440


# 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 te st formulations or vehicle daily via oral gavage for three consecutive days in a dose volume of 5 mL /kg body weight. Rats in the positive control group were administered EE daily via two s.c. injections in a dose volume of 2.5 μg/kg body weight. Dose vol umes were adjusted daily based on the concurrent body wei ght measurement.

7. Statistics: Clinical observations and other prop ortional data were analyzed using the variance test for homogeneity of the binomial distributi on. Continuous data (e.g., body weights, body weight gains, and food consumpti on) were analyzed usi ng Bartlett’s test of homogeneity of variances and the analysis of variance (ANOVA), wh en appropriate [i.e., Bartlett’s test was not significant (p >0.001)]. If the ANOV A was significant (p ≤ 0.05), Dunnett’s test was used to identify the statis tical significance of th e individual groups. If the ANOVA was not appropriate [i.e., Ba rtlett’s Test was significant (p ≤ 0.001)], the Kruskal-Wallis test was used ( ≤ 75% ties). In cases where the Kruskal-Wallis test was statistically significant (p ≤ 0.05), Dunn’s method of multiple comparisons was used to identify the statistical significance of the indivi dual groups. If there were greater than 75% ties, Fisher’s exact test was used to analyze the data. Wet and blotted uterine weights and liver weights were analyzed by analysis of covariance (ANCOVA), using the terminal body weight as the covariate.

ChE values for RBC and brains were evaluated as separate dependent variables in one-way ANOVA. In the event that the ANOVA was significant (p ≤ 0.05), Dunnett’s test was used to identify the statistical signi ficance of the individual groups.

C. METHODS

1. Clinical Examinations: The rats were checked for viabil ity at least twice daily during the study and were observed for general appearan ce daily during the dosing period. Post-dose observations were made 1-2 hours following dose administration.

2. Body Weight: Animals were weighed at rando mization, daily thr oughout the dosing period, and at termination.

3. Food Consumption: Food consumption was recorded daily per nesting box during the dosing period and on the day of termination. Valu es were reported in terms of g/animal/day.

4. Necropsy and Measurement of Uterine Weight: On PND 71 (approximately 24 hours after final administration of the test substance), animals were anesthetized with isoflurane/oxygen, allowing blood co llection. Animals were then euthanized with sodium pentobarbital (rats in th e 0, 100, 300, and 1000 mg/kg/day dose groups) or by carbon dioxide asphyxiation (rats in th e positive control group). All animals were subjected to a gross necropsy. Dissection of the uterus was performed according to the U.S. EPA Guideline. Briefly, the vagina was removed ju st below the cervix in order to retain the luminal fluid in the uterus. The “wet” uter us (i.e., containing th e luminal fluid) was weighed. Subsequently, the uterine horns were cut longitudinally and gently blotted with

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# moist filter paper to remove the luminal flui d while preventing desiccation and the blotted uterus was weighed. In addition, the liver was collected and weighed.

5. Microscopic Examination (Optional): Microscopic examinations were not conducted.

6. Cholinesterase Evaluations: Cholinesterase evaluations we re conducted for rats in the 0, 100, 300, and 1000 mg/kg/day dose groups. At study termination, the selected rats were anesthetized with isoflurane/ oxygen, and blood was collected from the inferior vena cava for cholinesterase activity determinations. After collection of blood and euthanasia, the brain was collected, weighed, and analyzed for cholin esterase activity. Samples were analyzed within 4 hours of collection.

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.

3. Estrous Cycling and Vaginal Patency: Evaluation of estrous cycle was performed for five consecutive days before initiati on of dosing; the evaluations conf irmed that all rats were not cycling and had been properly ovariectomized. On the day of euthanasia, it was confirmed that all rats had achie ved vaginal patency.

B. BODY WEIGHT AND WEIGHT GAIN: Body weight and body weight gain data are presented in Table 2. Body weights were comparab le to controls in al l dose groups over the duration of the study. In the 1000 mg/kg/day group, there was a sta tistically significant (p ≤ 0.05) body weight loss from Days 2 to 3 (loss of 0.8 g compared to a gain of 2.9 g in the control); overall (Days 1-4) body weight gains were also de creased (not significant [NS]) by 35%. Body weights gains in the 100 and 300 mg/kg/day dose groups were similar to controls over the duration of the study. In the positive control group, there was a statistically significant (p ≤ 0.01) body weight loss from Days 2 to 3 (loss of 2.7 g) and decreased body weight gain from Days 3 to 4 ( ↓ 58%), leading to decreased (p ≤ 0.01) overall body weight gain ( ↓ 85%).

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# TABLE 2. Group Body Weights and Body Weight Gains (g) In the Estrogen Agonist Assay a



# Study Day # Dose (mg/kg/day) Vehicle Control Malathion (100) Malathion (300) Malathion (1000) Estrogen EE (5 μ g /k g /da y ) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD Body Weights 1 8 254.8 21.6 8 256.1 20.2 8 258.6 22.0 8 257.8 20.6 8 256.0 18.5 2 8 263.4 22.5 8 260.6 20.4 8 263.6 21.2 8 264.4 21.5 8 258.5 16.9 3 8 266.2 22.7 8 264.0 20.5 8 267.2 21.6 8 263.6 20.7 8 255.8 15.2 4 8 273.3 23.3 8 272.0 21.6 8 276.6 24.0 8 269.8 22.0 8 258.8 16.7 Body Weight Gains 1-2 8 8.6 9.4 8 4.5 2.2 8 5.0 1.3 8 6.6 2.0 8 2.5 2.5 2-3 8 2.9 3.0 8 3.4 1.2 8 3.6 3.1 8

− 0.8* 3.4 8

− 2.7** 2.8 3-4 8 7.1 3.2 8 8.0 3.0 8 9.5 3.6 8 6.2 3.3 8 3.0** ( ↓ 58) 2.5

Overall (Days 1-4) 8 18.6 9.1 8 16.0 3.4 8 18.0 2.9 8 12.0 ( ↓ 35) 3.8 8 2.8** ( ↓ 85) 2.6

a Data were obtained from Table 2 on page 42 of the study report. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01



# C. FOOD CONSUMPTION: Food consumption data are presented in Table 3. Daily mean food consumption was decreased (p ≤ 0.05) in the 1000 mg/kg/ day group on Days 2-3 ( ↓ 10%) and Days 3-4 ( ↓ 8%), leading to decreased (p ≤ 0.01) average food consumption over the duration of the study ( ↓ 6%). Daily mean and overall average food consumption in the 100 and 300 mg/kg/day dose groups was comparable to the controls. In the positive control group, mean food consumption was increased (p ≤ 0.01) on Days 1-2 ( ↑ 10%) and decreased (p ≤ 0.01) on Days 2-3 ( ↓ 10%); average food consumption ove r the duration of the study was comparable to the control.

TABLE 3. Food Consumption (g/rat/d ay) in the Estrogen Agonist Assay a



# Study Day # Dose (mg/kg/day) Vehicle Control Malathion (100) Malathion (300) Malathion (1000) Estrogen EE (5 μ g /k g /da y ) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1-2 8 17.1 1.2 8 16.0 1.8 8 16. 9 1.2 8 17.1 0.4 8 18.8** ( ↑ 10) 0.6



# 2-3 8 18.7 0.5 8 18.9 0.7 8 18.8 0.6 8 16.9** ( ↓ 10) 0.6 8 16.8** ( ↓ 10) 1.8



# 3-4 8 17.4 1.0 8 18.2 1.4 8 17.6 1.2 8 16.0* ( ↓ 8) 0.5 8 18.8 3.2


# Overall Average (Days 1-4) 8 17.7 0.4 8 17.7 0.9 8 17.8 0.6 8 16.7** ( ↓ 6) 0.3 8 18.1 1.4



# a Data were obtained from Table 3 on page 44 of the study report. N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01

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# D. PATHOLOGY

1. Uterine and Liver Weights: Uterine and liver weight data are presented in Table 4. Uterine weights in the malathi on treatment groups were comparab le to controls. Absolute wet and blotted uterine we ights were increased (p ≤ 0.01) in the positive control group by 509% and 196%, respectively. The positive cont rols elicited the expected response.
Absolute and relative liver weights were increased (p ≤ 0.01) in the 1000 mg/kg/day group ( ↑ 24% and ↑ 25%, respectively), and relative liv er weights were increased (p ≤ 0.05) in the 300 mg/kg/day group ( ↑ 10%). These increases were consis tent with the increases in liver weights observed in the range-finding study (MRID No. 48617508) and were attributed to malathion treatment.
At necropsy, a red, tan, and/or firm mass was observed on the right or left ovarian stub in one rat in the vehicle contro l group, three rats in the 300 mg /kg/day dose group, one rat in the 1000 mg/kg/day dose group, and one rat in the positive control group. These observations were not attributed to treatment with the test substance or positive control because they were presumed to be residual ov arian stubs. However ovarian stubs were not an apparent source of endogenous estrogen as no estrous was observed in any treatment animal throughout the study.

TABLE 4. Uterine and Liver Weights from Estrogen Agonist Assay in SD Rats a



# Parameter Dose (mg/kg/day) Vehicle Control Malathion (100) Malathion (300) Malathion (1000) Estrogen EE (5 μg/kg/day) N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD Terminal BW (g) 8 273.3 23.3 8 272.0 21.6 8 276.6 24.0 8 269.8 22.0 8 258.8 16.7 Uterine Weights Wet, absolute (mg) 8 104.2 11.1 8 102.0 11.4 8 100.7 10.1 8 107.9 9.5 8 634.5** ( ↑ 509) 152.8

Wet, relative (%) 8 0.036 0.005 8 0.036 0.005 8 0.038 0.005 8 0.039 0.004 8 0.246** 0.065 Blotted, absolute (mg) 8 101.3 11.0 8 98.4 10.8 8 97. 5 9.6 8 104.1 8.3 8 299.9** ( ↑ 196) 27.6

Blotted, relative (%) 8 0.036 0.005 8 0.035 0.005 8 0.038 0.005 8 0.039 0.004 8 0.116** 0.015 Liver Weights Absolute (g) 8 10.5 1.2 8 11.1 0.8 8 11.7 1.5 8 13.0** ( ↑ 24) 1.6 Not measured

Relative (%) 8 3.857 0. 447 8 4.095 0.261 8 4.227* ( ↑ 10) 0.281 8 4.802** ( ↑ 25) 0.301 Not measured

a Data were obtained from Table 5 on page 48 of the study report. BW Body weight N Number of animals in the group SD Standard Deviation * Significantly different from controls at p ≤ 0.05 ** Significantly different from controls at p ≤ 0.01

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# 2. Microscopic Examination (Optional): Microscopic examinations were not conducted.

E. CHOLINESTERASE ACTIVITY: Data on ChE activity are presented in Table 5. ChE activity was significantly inhibited (p ≤ 0.01) in the brains of ra ts in the 1000 mg/kg/day group (16% inhibition), and ChE activity in RBC was significantly inhibited (p ≤ 0.01) in rats in the 100 (20% inhibition), 300 (38% inhibition), and 1000 (69% inhibition) mg/kg/day groups.

TABLE 5. Cholinesterase Activity in the Estrogen Agonist Assay a



# Matrix Dose (mg/kg/day) Vehicle Control Malathion (100) Malathion (300) Malathion (1000) N Mean SD N Mean SD N Mean SD N Mean SD Brain (U/g) 8 13.645 0.865 8 13. 405 1.057 8 13.326 0.805 8 11.503** ( ↓ 16) 1.041

RBC (U/mL) 8 1.330 0.135 8 1.062** ( ↓ 20) 0.157 8 0.821** ( ↓ 38) 0.149 8 0.416** ( ↓ 69) 0.116

a Data were obtained from Text Tables 9 and 10 on page 33 of the study report. Percent differences from controls are included in parentheses. N Number of animals in the group SD Standard Deviation ** Significantly different from controls at p ≤ 0.01



# III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Malathion did not have a positive response for estrogenic activity in the uterot rophic bioassay at dose levels as high as 1000 mg/kg bw/day (the limit dose as specified in the test guideline s) even in the presen ce of overt toxicity (body weight and feed consumption effects and in creased liver weights) and statistically and biologically significant RBC a nd brain ChE inhibition.

B. AGENCY COMMENTS: All animals survived until scheduled termination. No clinical signs of toxicity were observed in anim als for any dose group. Body weights in all malathion treatment groups were comparable to controls over the duratio n of the study. At 1000 mg/kg/day, the rats disp layed a body weight loss (p ≤ 0.05) during Days 2-3 (

− 0.8 g treated vs. +2.9 g controls), re sulting in a decrease in overall (Days 1-4) body weight gains (NS) of 35%. In the positive control group, overall body weight gain was decreased (p ≤ 0.01; ↓ 85%); body weights in this group were comp arable to the vehicle control over the duration of the study. Daily mean food consumption was decreased (p ≤ 0.01) at 1000 mg/kg/day on Days 2-3 ( ↓ 10%) and Days 3-4 ( ↓ 8%), leading to decreased (p ≤ 0.01) average food consumption over the duration of the study ( ↓ 6%). Daily mean and overall average food consumption in the 100 and 300 mg/kg/day gr oups was comparable to the controls. In the positive control group, mean fo od consumption was increased (p ≤ 0.01) on Days 1-2 ( ↑ 10%) and decreased (p ≤ 0.01) on Days 2-3 ( ↓ 10%); average food consumption over the duration of the study was comparable to the c ontrol. Uterine weights in the malathion treatment groups were comparable to controls . Absolute wet and blotted uterine weights were increased (p ≤ 0.01) in the positive control group by 509% and 196%, respectively. The increased uterine weights were in the expected range.

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# Absolute and relative liver weights were increased (p ≤ 0.01) at 1000 mg/kg/day ( ↑ 24 and 25%), and relative liver weights were increased (p ≤ 0.05) at 300 mg/kg/day ( ↑ 10%). These increases were attributed to malathion treatm ent. ChE activity was significantly inhibited (p ≤ 0.01) in the brains of rats in the 1000 mg/kg/day group (16% inhibition), and ChE activity in RBC was significantly inhibited (p ≤ 0.01) in rats at 100 (20% inhibition), 300, (38% inhibition), and 1000 ( 69% inhibition) mg/kg/day.
No statistically significant changes were seen in uterine weight in this assay. Malathion is negative in the uterotrophic assay.

C. STUDY DEFICIENCIES: None