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 1 of 294
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 other scientifically relevant information (OSRI)) 

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 ! "

 ԭԹՄՅԽՊ ԰ԧ ԰Է ԰ԻՀՆԽՁԼԽՊ ԱԷ ԨԽՆԼԹՄՄ ԹՆԼ ԥԬ ԨՊՍԽԿԽՊ  ԪԹՄԹՌՀՁՇՆ ԞՅՈՀՁԺՁԹՆ

ԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑԣՁՆԹՄԯԽՈՇՊՌԲՆՈՍԺՄՁՋՀԽԼՋՌՍԼՑՈԽՊԾՇՊՅԽԼԺՑԴՁՄԼՄՁԾԽԦՆՌԽՊՆԹՌՁՇՆԹՄԢԹՋՌՇՆԪԡ

ԩԹԺՇՊԹՌՇՊՑԭՊՇՂԽԻՌԫՍՅԺԽՊԞ԰ՌՍԼՑՋՈՇՆՋՇՊԽԼԺՑԠՀԽՅՁՆՇՎԹԞ԰԰ՌՍԼՑՁՆՁՌՁԹՌԽԼԬԻՌՇԺԽՊ

ԹՆԼՋՍԺՅՁՌՌԽԼԬԻՌՇԺԽՊ

ԫՇՌԽԱՀԽԲ԰ԢԭԞԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊ԰ԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅ
Ԣԡ԰ԭԱՁԽՊՋԻՊԽԽՆՁՆԿԺԹՌՌԽՊՑ ՁՋԻՇՅՈՊՁՋԽԼՇԾ

ԽՄԽՎԽՆՋԻՊԽԽՆՁՆԿԹՋՋԹՑՋՁՆՌԽՆԼԽԼՌՇՁԼԽՆՌՁԾՑԹԻՀԽՅՁԻԹՄ0ՋՄՁՃԽՄՑ ԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՁԽՁՌՋՈՇՌԽՆՌՁԹՄՌՇ

ՁՆՌԽՊԹԻՌՏՁՌՀՌՀԽԽՋՌՊՇԿԽՆԹՆԼՊՇԿԽՆՇՊՌՀՑՊՇՁԼ
ԢԞՇՊԱՈԹՌՀՏԹՑՋԱՀԽՊՇԺՍՋՌՆԽՋՋՇԾՌՀԽԱՁԽՊԺԹՌՌԽՊՑ

ՁՋԺԹՋԽԼՇՆՌՀԽՋՌՊԽՆԿՌՀՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՌՇՁԼԽՆՌՁԾՑՈՇՌԽՆՌՁԹՄ ԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՏՁՌՀ

ԻՇՅՈՄԽՅԽՆՌԹՊՑԽՆԼՈՇՁՆՌՋՏՁՌՀՁՆՌՀԽԹՋՋԹՑՏՀԽՊԽԹՎԹՁՄԹԺՄԽԹՆԼՊԽԼՍՆԼԹՆԻՑԹԻՊՇՋՋՌՀԽԺԹՌՌԽՊՑԱՀՍՋՌՀԽ

ՊԽՋՍՄՌՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՋՀՇՍՄԼՆՇՌԺԽԻՇՆՋՁԼԽՊԽԼՁՆՁՋՇՄԹՌՁՇՆԺՍՌՊԹՌՀԽՊՋՀՇՍՄԼԺԽԻՇՆՋՁԼԽՊԽԼՁՆՌՀԽ

ԻՇՆՌԽՐՌՇԾՇՌՀԽՊԹՋՋԹՑՋՁՆՌՀԽԺԹՌՌԽՊՑԹՋՏԽՄՄԹՋԬՌՀԽՊ԰ԻՁԽՆՌՁԾՁԻԹՄՄՑԯԽՄԽՎԹՆՌԦՆԾՇՊՅԹՌՁՇՆ
Ԭ԰ԯԦԦՆՇՊԼԽՊ

ՌՇԼԽՌԽՊՅՁՆԽՁԾԹԻՀԽՅՁԻԹՄՀԹՋՌՀԽՈՇՌԽՆՌՁԹՄՌՇՁՆՌԽՊԹԻՌՏՁՌՀՌՀԽԢԞՇՊԱՈԹՌՀՏԹՑՋԹԴԽՁԿՀՌՇԾԢՎՁԼԽՆԻԽ


ԴՇԢԽՎԹՄՍԹՌՁՇՆՇԾԱՁԽՊԹՋՋԹՑՊԽՋՍՄՌՋՁՆԻՇՅԺՁՆԹՌՁՇՆՏՁՌՀՌՀԽԾՁՆԼՁՆԿՋՁՆՌՀԽԬ԰ԯԦՋՀՇՍՄԼԺԽՍՆԼԽՊՌԹՃԽՆ


ՊԽԾԽՊՌՇՌՀԽԴՇԢԡՇԻՍՅԽՆՌ

#$%&'($)*+,

ԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋՁՆՌՀՁՋԡԢԯՌԽՅՈՄԹՌԽԹՊԽՇԾԾԽՊԽԼԹՋԹԿԽՆԽՊԹՄՊԽԾԽՊԽՆԻԽՌՇԹՁԼՁՆ

ՈՊԽՈԹՊԹՌՁՇՆՇԾՌՀԽԡԢԯԱՀԽՈՍՊՈՇՋԽՇԾՌՀԽՋԽ ՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋՁՋՆՇՌՌՇՋԽՊՎԽԹՋՋՍԺՋՌՁՌՍՌԽԾՇՊՌՀԽԱԽՋՌ

ԤՍՁԼԽՄՁՆԽՋՆՇՊՌՇՈՊՇՎՁԼԽԹՆՑԿՍՁԼԹՆԻԽՇՆՀՇՏՌՀԽՋՌՍԼՑՋՀՇՍՄԼԺԽԻՇՆԼՍԻՌԽԼ

Page 4 of 294
  	
 
	  	
  	
 	   	 
 	     
 	 	  

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !"#$ %!&&'()

ԱՀԽԼԹՑԹՋՋԹՑՇԾՅԹՄԹՌՀՁՇՆՇՆԹՅՈՀՁԺՁԹՆՅԽՌԹՅՇՊՈՀՇՋՁՋՇԾ԰ՇՍՌՀԞԾՊՁԻԹՆԻՄԹՏԽԼԾՊՇԿՋ
ԵԽՆՇՈՍՋՄԹԽՎՁՋ

ՏԹՋԻՇՆԼՍԻՌԽԼՍՆԼԽՊԾՄՇՏՌՀՊՇՍԿՀԻՇՆԼՁՌՁՇՆՋԞՅՈՀՁԺՁԹՆՄԹՊՎԹԽԹՌԫՁԽՍՏՃՇՇՈԣԹԺԽՊ
ԫԣ԰ՌԹԿԽ
ՈԽՊ

ԻՇՆՌՊՇՄՇՊՌՊԽԹՌՅԽՆՌԿՊՇՍՈՊԽՈՄՁԻԹՌԽՏԽՊԽԽՐՈՇՋԽԼՌՇՅԹՄԹՌՀՁՇՆ
ՈՍՊՁՌՑԹՌՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋ

ՇԾ  
ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ  ԹՆԼ ՅԿ ԹՁԩՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽ


ԩԬԮԻՇՆՌՊՇՄԹՆԼՅԿԹՁԩԱՀԽՌԽՋՌՋՑՋՌԽՅՏԹՋՅԹՁՆՌԹՁՆԽԼԹՌՌՇոԠԹՆԼԹ

ՈԥՇԾՌՇ

ԱՀԽՊԽՏԽՊԽՆՇՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՇՆՋՍՊՎՁՎԹՄՇԾՌԹԼՈՇՄԽՋԼՍՊՁՆԿՌՀԽԼԹՑՌԽՋՌԣՊՇՅԡԹՑՋՌՀՊՇՍԿՀ

ՋՍՊՎՁՎԹՄՏԹՋ  ԹՆԼ  ՁՆՌՀԽ ՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ ԹՆԼ ԹՆԼՅԿ ԹՁԩ

ՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑԣՊՇՅԡԹՑՌՀՊՇՍԿՀՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆՋՍՊՎՁՎԹՄՏԹՋԹՆԼ

ՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑԞՌՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆ

ՌԹՁՄԻՍՊՎԹՌՍՊԽՏԹՋՇԺՋԽՊՎԽԼՁՆՇԾՌԹԼՈՇՄԽՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՆԼՇԾՌԹԼՈՇՄԽՋՁՆ

ՌՀԽ ՄՇՏ ՁՆՌԽՊՅԽԼՁԹՌԽ ԹՆԼ ՀՁԿՀ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԿՊՇՍՈՋ ՊԽՋՈԽԻՌՁՎԽՄՑ ԱՀԽ ՁՆԻՁԼԽՆԻԽ ՇԾ ՌԹՁՄ ԻՍՊՎԹՌՍՊԽ ԼՁԼ ՆՇՌ

ԹՈՈԽԹՊՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼ

ԱՀԽՊԽՏԽՊԽ ՆՇ ՋՁԿՆՁԾՁԻԹՆՌ ԼՁԾԾԽՊԽՆԻԽՋ ՁՆ ԼԽՎԽՄՇՈՅԽՆՌԹՄ ԽՆԼՈՇՁՆՌՋ 
ՁԽԫԣ ԼԽՎԽՄՇՈՅԽՆՌԹՄ ՋՌԹԿԽ ՀՁՆԼ ՄՁՅԺ

ՄԽՆԿՌՀ ՆՇՊՅԹՄՁՒԽԼ ՀՁՆԼ ՄԽԿ ՄԽՆԿՌՀ ՁՆ ԹՆՑ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ՏՀԽՆ ԻՇՅՈԹՊԽԼ ՌՇ ՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ ԫՇ

ԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌՏԹՋՇԺՋԽՊՎԽԼԱՀԽՊԽՏԽՊԽՆՇԼՁԹԿՆՇՋՌՁԻԹԺՆՇՊՅԹՄՁՌՁԽՋՇԺՋԽՊՎԽԼԺՑՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄ

ԽՐԹՅՁՆԹՌՁՇՆՇԾՌՀԽՌՀՑՊՇՁԼՋՇԾՌԹԼՈՇՄԽՋՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈՏՀԽՆԻՇՅՈԹՊԽԼՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՌՌՀԽ

ՌԽՊՅՁՆԹՌՁՇՆՇԾՌՀԽԼԹՑՌԽՋՌ

ԡԹՑՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ
԰ԳԩԹՆԼԺՇԼՑՏԽՁԿՀՌՏԽՊԽՋՁԿՆՁԾՁԻԹՆՌՄՑՁՆԻՊԽԹՋԽԼ
ՈԺՑԹՆԼ

ՊԽՋՈԽԻՌՁՎԽՄՑՁՆՌՀԽՄՇՏԻՇՆԻԽՆՌՊԹՌՁՇՆԿՊՇՍՈՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԥՇՏԽՎԽՊՌՀԽՊԽՏԽՊԽՆՇՋՁԿՆՁԾՁԻԹՆՌ

ԽԾԾԽԻՌՋ ՇՆ ԿՊՇՏՌՀ ՈԹՊԹՅԽՌԽՊՋ ՁՆ ՌՀԽ ՁՆՌԽՊՅԽԼՁԹՌԽ ԹՆԼ ՀՁԿՀ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԿՊՇՍՈՋ ԻՇՅՈԹՊԽԼՌՇՌՀԽ ՆԽԿԹՌՁՎԽ

ԻՇՆՌՊՇՄԣՍՊՌՀԽՊՅՇՊԽ԰ԳԩԹՆԼԺՇԼՑՏԽՁԿՀՌՁՆՌՀԽՌՊԽԹՌԽԼԿՊՇՍՈՋՏԽՊԽԻՇՅՈԹՊԹԺՄԽՌՇՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՋՇՆԡԹՑ


ԽԾԾԽԻՌՋՇՆԺՇԼՑՏԽՁԿՀՌՏԽՊԽՇԺՋԽՊՎԽԼԹՌԹՆԼՅԿԹՁԩՁՆՌՀԽՊԹՆԿԽԾՁՆԼԽՊՋՍԿԿԽՋՌՁՆԿՌՀԽՀՁԿՀ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀՁՋՋՌՍԼՑՏԹՋՋՍԾԾՁԻՁԽՆՌ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

ԩԹՌԽՋՌԹԿԽ 
ԫԣ  ՌԹԼՈՇՄԽՋ ՏԽՊԽ ԽՐԻՄՍԼԽԼ ԾՊՇՅ ԹՆԹՄՑՋԽՋ ՏՁՌՀ ՊԽՋՈԽԻՌ ՌՇ ԺՇԼՑ ՏԽՁԿՀՌ ԰Գԩ ԹՆԼ ԥԩԩ


ՁՆԻՄՍԼՁՆԿ ՆՇՊՅԹՄՁՒԽԼ ԥԩԩ ԺՑ ՌՀԽ ՊԽՎՁԽՏԽՊ ՌՀԽՋԽ ՁՆԻՄՍԼԽԼ  
  
  
 ԹՆԼ  


ՌԹԼՈՇՄԽՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՄՇՏՁՆՌԽՊՅԽԼՁԹՌԽԹՆԼՀՁԿՀՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ

ԱՀԽՋՌՍԼՑՅԽՌԹՄՄՇԾՌՀԽՈԽՊԾՇՊՅԹՆԻԽԹՆԼՎԹՄՁԼՁՌՑԻՊՁՌԽՊՁԹՏՁՌՀՌՀԽԾՇՄՄՇՏՁՆԿԽՐԻԽՈՌՁՇՆՋ

x ԱՀԽԠԳԾՇՊՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՇԾՌՀԽ ՄՇՏՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ 
 ՅԿ ԹՁԩ ՏԹՋ

 ԽՐԻԽԽԼՁՆԿ ՌՀԽ ՈԽՊԾՇՊՅԹՆԻԽ ԻՊՁՌԽՊՁՇՆ ՇԾ ՅԹՁՆՌԹՁՆՁՆԿ Թ ԠԳ ՇԾ  ՇՎԽՊ ՌՀԽ ԼԹՑ ՌԽՋՌ

ԥՇՏԽՎԽՊՁՌՋՀՇՍՄԼԺԽՆՇՌԽԼՌՀԹՌՌՀՁՋԠԳՏԹՋԻԹՄԻՍՄԹՌԽԼԺՑՌՀԽՊԽՎՁԽՏԽՊՍՋՁՆԿԼԹՑՋԹՅՈՄԽՋՏՀՁԻՀ

ՏԽՊԽՄՇՏԹՆԼՊԽՈՇՊՌԽԼՌՇԺԽԼՍԽՌՇՋԹՅՈՄՁՆԿՇՊԹՆԹՄՑՌՁԻԹՄԽՊՊՇՊԦԾՌՀԽԹԼԼՁՌՁՇՆԹՄՄՑԻՇՄՄԽԻՌԽԼԡԹՑ

ՋԹՅՈՄԽՋ
ՌԽՋՌՋՑՋՌԽՅՅԹՁՆՌԹՁՆԽԼՁՆԹԺՋԽՆԻԽՇԾՌԹԼՈՇՄԽՋԹՊԽՍՋԽԼՁՆՄՁԽՍՇԾԡԹՑՋԹՅՈՄԽՋՌՀԽԠԳ

ՇԾԼՇԽՋՆՇՌԽՐԻԽԽԼՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆ

x ԴԹՌԽՊՌԽՅՈԽՊԹՌՍՊԽՏԹՋՅԹՁՆՌԹՁՆԽԼՏՁՌՀՁՆչւԠՏՁՌՀՌՀԽԽՐԻԽՈՌՁՇՆՇԾԡԹՑՏՀԽՆՌԽՅՈԽՊԹՌՍՊԽՋ

ՁՆՋՇՅԽՌԽՋՌՎԽՋՋԽՄՋՏԽՊԽԹՋՄՇՏԹՋւԠԬՆԡԹՑՋԹՆԼՌՀԽԼՁԾԾԽՊԽՆԻԽՁՆՌԽՅՈԽՊԹՌՍՊԽԺԽՌՏԽԽՆ

ՌԽՋՌՎԽՋՋԽՄՋՏԹՋԹՆԼւԠՊԽՋՈԽԻՌՁՎԽՄՑԱՀԽՋԽՄՁՅՁՌՋԽՐԻԽԽԼՌՀԽՌԽՅՈԽՊԹՌՍՊԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆ

ՇԾչւԠՏՁՌՀՁՆՌԽՊՊԽՈՄՁԻԹՌԽՁՆՌԽՊՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋՆՇՌԽՐԻԽԽԼՁՆԿւԠ

ԴՀՁՄԽ ՌՀԽՋԽ ԼԽՈԹՊՌՍՊԽՋ ԾՊՇՅ ԿՍՁԼԽՄՁՆԽ ՈԽՊԾՇՊՅԹՆԻԽ ԻՊՁՌԽՊՁԹ ԹՆԼ ԼԽՎՁԹՌՁՇՆՋ ԾՊՇՅ ԿՍՁԼԽՄՁՆԽ ՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ՏԽՊԽ ՇԺՋԽՊՎԽԼ ՌՀԽՑ ԼՁԼ ՆՇՌ ՋՍԾԾՁԻՁԽՆՌՄՑ ՁՅՈԹԻՌ ՌՀԽ ԹԺՁՄՁՌՑ ՌՇՁՆՌԽՊՈՊԽՌ ՌՀԽ ՊԽՋՍՄՌՋ ՇԾ ՌՀԽ ԹՋՋԹՑ ԱՀԽ ԹՋՋԹՑ

 

ՌՀԽԢԡ԰ԭԱՁԽՊԱԽՋՌԬՊԼԽՊՊԽՉՍՁՊԽՅԽՆՌՋԾՇՊԹՆԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ



   
	   

ԱԽՋՌՇՊԿԹՆՁՋՅԫԣՋՌԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ

ԱԽՋՌՇՊԿԹՆՁՋՅՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՇՈՌՁՇՆԹՄԫՇՌՊԽՈՇՊՌԽԼ

ԱԽՋՌՌՑՈԽԾՄՇՏՌՀՊՇՍԿՀ

Page 6 of 294
  	
 
	  	
  	
 	   	 
 	     
 	 	  

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

      	 	 
 
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 ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ

 ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ

ԞԺԺՊԽՎՁԹՌՁՇՆՋԡՁԾԾԡՁԾԾԽՊԽՆԻԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ

 Ԟ՟ՑԽՋՠՁՆԼՁԻԹՌԽՋԹՋՁԿՆՁԾՁԻԹՆՌԼՁԾԾԽՊԽՆԻԽԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄՍՆՄԽՋՋ

ՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼ

 ԱՀԽԻՊՁՌԽՊՁԹԾՇՊՋՁԿՆՁԾՁԻԹՆԻԽԹՊԽԼԽՋԻՊՁԺԽԼՁՆՌՀԽԯԽՎՁԽՏԽՊ0ՋԞՆԹՄՑՋՁՋԹՆԼ԰ՌԹՌՁՋՌՁԻԹՄԳԽՊՁԾՁԻԹՌՁՇՆՋԽԻՌՁՇՆՋ

ՇԾՌՀԽԡԢԯԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹ

ԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ

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ԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼԾՇՄՄՇՏՁՆԿԿՍՁԼԽՄՁՆԽՋՇՍՌՄՁՆԽԼՁՆԲ԰ԢՆՎՁՊՇՆՅԽՆՌԹՄ

ԭՊՇՌԽԻՌՁՇՆԞԿԽՆԻՑ0ՋԬԠ԰ԭԭԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋ
ԣՊՇԿԹՆԼ

ԬԢԠԡԱԽՋՌԤՍՁԼԽՄՁՆԽԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԡԽՎՁԹՌՁՇՆՋԾՊՇՅ

ԹՊԽ

 ԦՆԾՇՊՅԹՌՁՇՆՇՆՌՀԽՈԹՊԽՆՌԹՄԻԹՊԽԹՆԼՋՈԹՏՆՁՆԿՏԹՋՆՇՌՊԽՈՇՊՌԽԼ

 ԱՀԽ ԾՄՇՏՌՀՊՇՍԿՀ ՊԹՌԽ ՇԾ  ՅԩՅՁՆ ԹՋ ԻԹՄԻՍՄԹՌԽԼ ԺՑ ՌՀԽ ՊԽՎՁԽՏԽՊ ԽՐԻԽԽԼՋ ՌՀԽ ԿՍՁԼԽՄՁՆԽ

ՊԽԻՇՅՅԽՆԼԹՌՁՇՆՇԾՅԩՅՁՆ

 ԱՀԽԠԳԾՇՊՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՇԾՌՀԽՄՇՏՌՊԽԹՌՅԽՆՌԿՊՇՍՈ
ՁԽՅԿԹՁԩ

ՏԹՋԽՐԻԽԽԼՁՆԿՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆՇԾՅԹՁՆՌԹՁՆՁՆԿԹԠԳՇԾ ?ՇՎԽՊՌՀԽԼԹՑ

ՌԽՋՌ ԥՇՏԽՎԽՊ ՁՌ ՋՀՇՍՄԼ ԺԽ ՆՇՌԽԼՌՀԹՌ ՌՀՁՋԠԳՏԹՋ ԻԹՄԻՍՄԹՌԽԼԺՑ ՌՀԽ ՊԽՎՁԽՏԽՊ ՍՋՁՆԿ ԼԹՑ 

ՋԹՅՈՄԽՋՏՀՁԻՀՏԽՊԽՄՇՏԹՆԼՊԽՈՇՊՌԽԼՌՇԺԽԼՍԽՌՇՋԹՅՈՄՁՆԿՇՊԹՆԹՄՑՌՁԻԹՄԽՊՊՇՊԦԾՌՀԽԹԼԼՁՌՁՇՆԹՄՄՑ

ԻՇՄՄԽԻՌԽԼ ԡԹՑ  ՋԹՅՈՄԽՋ 
ՌԽՋՌ ՋՑՋՌԽՅՅԹՁՆՌԹՁՆԽԼ ՁՆ ԹԺՋԽՆԻԽ ՇԾՌԹԼՈՇՄԽՋ ՁՆ ՄՁԽՍ ՇԾ ԡԹՑ 

ՋԹՅՈՄԽՋՌՀԽԠԳՇԾԼՇԽՋՆՇՌԽՐԻԽԽԼՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆ

Page 7 of 294
  	
 
	  	
  	
 	   	 
 	     
 	 	  

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 ԴԹՌԽՊ ՌԽՅՈԽՊԹՌՍՊԽ ՏԹՋ ՅԹՁՆՌԹՁՆԽԼ ՏՁՌՀՁՆ  չ ւԠ ՏՁՌՀ ՌՀԽ ԽՐԻԽՈՌՁՇՆ ՇԾ ԡԹՑ  ՏՀԽՆ

ՌԽՅՈԽՊԹՌՍՊԽՋՁՆՋՇՅԽՌԽՋՌՎԽՋՋԽՄՋՏԽՊԽԹՋՄՇՏԹՋւԠԬՆԡԹՑՋԹՆԼՌՀԽԼՁԾԾԽՊԽՆԻԽՁՆ

ՌԽՅՈԽՊԹՌՍՊԽ ԺԽՌՏԽԽՆ ՌԽՋՌ ՎԽՋՋԽՄՋ ՏԹՋ  ԹՆԼ ւԠ ՊԽՋՈԽԻՌՁՎԽՄՑ  ԱՀԽՋԽ ՄՁՅՁՌՋ ԽՐԻԽԽԼ ՌՀԽ

ՌԽՅՈԽՊԹՌՍՊԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆՇԾչւԠՏՁՌՀՁՆՌԽՊՊԽՈՄՁԻԹՌԽՁՆՌԽՊՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋՆՇՌ

ԽՐԻԽԽԼՁՆԿւԠ

ԴՀՁՄԽՌՀԽՋԽԼԽՈԹՊՌՍՊԽՋԾՊՇՅԿՍՁԼԽՄՁՆԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹԹՆԼԼԽՎՁԹՌՁՇՆՋԾՊՇՅԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ՏԽՊԽՇԺՋԽՊՎԽԼՌՀԽՑԼՁԼՆՇՌՋՍԾԾՁԻՁԽՆՌՄՑՁՅՈԹԻՌՌՀԽԹԺՁՄՁՌՑՌՇՁՆՌԽՊՈՊԽՌՌՀԽՊԽՋՍՄՌՋՇԾՌՀԽԹՋՋԹՑԱՀԽԹՋՋԹՑ

 

ՌՀԽԢԡ԰ԭԱՁԽՊԱԽՋՌԬՊԼԽՊՊԽՉՍՁՊԽՅԽՆՌՋԾՇՊԹՆԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԬԠ԰ԭԭ

ԤՍՁԼԽՄՁՆԽ

	  

 

 ԰ՁԿՆԽԼԹՆԼԼԹՌԽԼԫՇԡԹՌԹԠՇՆԾՁԼԽՆՌՁԹՄՁՌՑԤԩԭԹՆԼԮՍԹՄՁՌՑԞՋՋՍՊԹՆԻԽ

ՋՌԹՌԽՅԽՆՌՋՏԽՊԽՈՊՇՎՁԼԽԼԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼՁՆԻՇՅՈՄՁԹՆԻԽՏՁՌՀՌՀԽ

ԤՇՇԼԩԹԺՇՊԹՌՇՊՑԭՊԹԻՌՁԻԽ԰ՌԹՆԼԹՊԼՋԹՋՋՈԽԻՁԾՁԽԼՁՆԠԣԯՏՁՌՀՌՀԽ

ԾՇՄՄՇՏՁՆԿԽՐԻԽՈՌՁՇՆՋՈԽՊՁՇԼՁԻԹՆԹՄՑՋԽՋՇԾՏԹՌԽՊԾՇՊՈՇՌԽՆՌՁԹՄԻՇՆՌԹՅՁՆԹՆՌՋՏԽՊԽ

ՆՇՌՈԽՊԾՇՊՅԽԼԹԻԻՇՊԼՁՆԿՌՇԤԩԭՋՌԹՆԼԹՊԼՋԺՍՌՏԽՊԽՈԽՊԾՇՊՅԽԼՍՋՁՆԿԹԻԽՊՌՁԾՁԽԼ

ՄԹԺՇՊԹՌՇՊՑԹՆԼՋՌԹՆԼԹՊԼԲ԰ԢԭԞԹՆԹՄՑՌՁԻԹՄՅԽՌՀՇԼՋՈՊԽՄՁՅՁՆԹՊՑԹՆԹՄՑՋԽՋՇԾ

ՏԹՌԽՊՁՇԼՁԼԽԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽՆՇՌՈԽՊԾՇՊՅԽԼԹԻԻՇՊԼՁՆԿՌՇԤԩԭ    

   ԪԹՄԹՌՀՁՇՆԱԽԻՀՆՁԻԹՄ


  	
 

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 ԱՀԽՋՌՍԼՑԹՍՌՀՇՊՋՊԽՈՇՊՌՈՊԽՄՁՅՁՆԹՊՑՌՊՁԹՄՋՋՀՇՏԽԼՌՀԹՌՅԹՄԹՌՀՁՇՆ

ՋՌՇԻՃՋՈՊԽՈԹՊԽԼՁՆԹԻՁԼՁԾՁԽԼԼԽՁՇՆՁՒԽԼՏԹՌԽՊՏԽՊԽՋՌԹԺՄԽԾՇՊԹՌՄԽԹՋՌ

ԼԹՑՋԞՈՊՁՅԹՊՑՋՌՇԻՃՇԾՅԹՄԹՌՀՁՇՆՌԽԻՀՆՁԻԹՄԹՌՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋ

ՇԾՅԿԹՁԩՏԹՋՈՊԽՈԹՊԽԼՁՆԼԽՁՇՆՁՒԽԼՏԹՌԽՊ
ՎՁԹՊԽՎԽՊՋԽ

ՇՋՅՇՋՁՋԽՎԽՊՑՌՇԼԹՑՋԼՍՊՁՆԿՌՀԽՌԽՋՌ

Page 8 of 294
  	
 
	  	
  	
 	   	 
 	     
 	 	  

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ

ՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

	 	 
  	
 	

 
 !

   ԱԽԻՀՆՁԻԹՄՅԹՄԹՌՀՁՇՆՏԹՋՋՌՇՊԽԼՁՆԹՊԽԾՊՁԿԽՊԹՌՇՊԹՌ+ւԠԱՀԽՋՌՇԻՃ

ՋՇՄՍՌՁՇՆՏԹՋՀԽՄԼՍՆԼԽՊԹՅԺՁԽՆՌԻՇՆԼՁՌՁՇՆՋՈՊՇՌԽԻՌԽԼԾՊՇՅՄՁԿՀՌԹՆԼ

ՅՁՐԽԼԻՇՆՌՁՆՍՇՍՋՄՑՍՋՁՆԿԹՌՇՈԼՇՏՆԽՄԽԻՌՊՁԻՅՁՐԽՊ

Page 9 of 294
	

		
	
		
			

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

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԰ՈԽԻՁԽՋԻՇՅՅՇՆՆԹՅԽ ԞԾՊՁԻԹՆԻՄԹՏԽԼԾՊՇԿ ԢԭԞՊԽԻՇՅՅԽՆԼՋԞԾՊՁԻԹՆԻՄԹՏԽԼԾՊՇԿ


ԵԽՆՇՈՍՋՄԹԽՎՁՋԴԽՋՌԽՊՆ#ԞԾՊՁԻԹ%ԻՄԹՏԽԼ

ԾՊՇԿ԰ՁՄՍՊԹՆԹ
ԵԽՆՇՈՍՋՌՊՇՈՁԻԹՄՁՋՅԹՑԺԽ

ՍՋԽԼԹՋԹՆԹՄՌԽՊՆԹՌԽՋՈԽԻՁԽՋՀՇՏԽՎԽՊԹ

ՄՁՋՌՇԾԹՄՄՇԾՌՀԽՆԽԻԽՋՋԹՊՑՈՊՇՌՇԻՇՄ

ԼԽՎՁԹՌՁՇՆՋՌՇԹԻԻՇՅՅՇԼԹՌԽՌՀՁՋՋՈԽԻՁԽՋՁՋ

ՊԽԻՇՅՅԽՆԼԽԼԾՇՊՁՆԻՄՍՋՁՇՆՁՆՌՀԽՋՌՍԼՑ

ՊԽՈՇՊՌԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽ

ՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹՍՋԽԼՌՇՋՍՈՈՇՊՌՌՀԽ

ՊԽՄՁԹԺՁՄՁՌՑՇԾՌՀԽՌԽՋՌԺԽՁԼԽՆՌՁԾՁԽԼ

԰ՈԽԻՁԽՋՋԻՁԽՆՌՁԾՁԻՆԹՅԽ ԵԽՆՇՈՍՋՄԹԽՎՁՋ԰ՈԽԻՁԽՋՋՌՊԹՁՆ
ՁԾՋՌԹՌԽԼ ԫՇՌՋՈԽԻՁԾՁԽԼ

 Բ԰ԢՆՎՁՊՇՆՅԽՆՌԹՄԭՊՇՌԽԻՌՁՇՆԞԿԽՆԻՑ
ԢԭԞ
ԠՇՊՊԽԻՌՁՇՆՋԹՆԼԠՄԹՊՁԾՁԻԹՌՁՇՆՋՇՆԱԽԻՀՆՁԻԹՄԞՋՈԽԻՌՋՇԾՌՀԽԱԽՋՌԤՍՁԼԽՄՁՆԽՋԾՇՊՌՀԽԢՆԼՇԻՊՁՆԽ

ԡՁՋՊՍՈՌՇՊ԰ԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅԱՁԽՊԞՋՋԹՑՋ
ԬԠ԰ԭԭԱԽՋՌԤՍՁԼԽՄՁՆԽ԰ԽՊՁԽՋԪԹՊԻՀԬԾԾՁԻԽՇԾԠՀԽՅՁԻԹՄ԰ԹԾԽՌՑԹՆԼԭՇՄՄՍՌՁՇՆԭՊԽՎԽՆՌՁՇՆ
ԬԠ԰ԭԭԴԹՋՀՁՆԿՌՇՆԡԠ
ՀՌՌՈՏՏՏԽՈԹԿՇՎԽՆԼՇՈՍԺՋԹՋՋԹՑՎԹՄՁԼԹՌՁՇՆԻՄԹՊՁԾՁԻԹՌՁՇՆԼՇԻՈԼԾ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !	"#

	
	


ԴԽՊԽՈԹՊԽՆՌՋՅԹՁՆՌԹՁՆԽԼԹՋՁՆՀՇՍՋԽՋՌՇԻՃ!

ԶԽՋ ԫՇՁՆԾՇՊՅԹՌՁՇՆՁՋՈՊՇՎՁԼԽԼՇՆՈԹՊԽՆՌՋ

ԱՀԽՋՌՍԼՑԹՍՌՀՇՊՋՊԽՈՇՊՌՌՀԹՌՄԹՊՎԹԽՏԽՊԽՇԺՌԹՁՆԽԼԾՊՇՅԻՍՄՌՍՊԽՋՅԹՁՆՌԹՁՆԽԼԺՑԴՁՄԼՄՁԾԽԦՆՌԽՊՆԹՌՁՇՆԹՄԩՌԼԹՆԼՌՀԹՌԹՄՄՌԹԼՈՇՄԽՋՏԽՊԽԼԽՊՁՎԽԼԾՊՇՅԽԿԿՋՋՈԹՏՆԽԼՇՆՌՀԽՋԹՅԽԼԹՑ

ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՄԹՊՎԹԽ ՍՋԽԼ ՁՆ ՌՀԽ

ԹՋՋԹՑԺԽԼԽՊՁՎԽԼԾՊՇՅՁՆՀՇՍՋԽԹԼՍՄՌՋ

ԴԽՊԽՈԹՊԽՆՌԹՄԹԻԻՄՁՅԹՌՁՇՆԻՇՆԼՁՌՁՇՆՋՋԹՅԽԹՋԼԽԾՁՆՁՌՁՎԽՌԽՋՌ!

ԫՇՌՊԽՈՇՊՌԽԼ

ԞԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼԾՇՊՈԹՊԽՆՌԹՄ

ԾՊՇԿՋ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԫՇՌՊԽՈՇՊՌԽԼ

ԡԽՌԹՁՄՋՇՆՈԹՊԽՆՌԹՄԾԽԽԼՁՆԿ ԫՇՌՊԽՈՇՊՌԽԼԡԽՌԹՁՄՋՇՆՈԹՊԽՆՌԹՄՀԽԹՄՌՀ ԫՇՌՊԽՈՇՊՌԽԼ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !"
	
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ԟԽՋՌՋՁՆԿՄԽՋՈԹՏՆ! ԫՇՌՊԽՈՇՊՌԽԼ ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌՌՀԽԺԽՋՌ

-ՁՆԼՁՎՁԼՍԹՄՋՈԹՏՆՋՏՁՌՀԹՅՁՆՁՅՍՅՇԾ

ՄԹՊՎԹԽՋՈԹՏՆԺԽԽՎԹՄՍԹՌԽԼՌՇՁԼԽՆՌՁԾՑ

ՌՀԽԺԽՋՌՋՁՆԿՄԽՋՈԹՏՆԹՆԼՌՀԹՌՌՀԽՄԹՊՎԹԽ

ՋԽՄԽԻՌԽԼԾՇՊՌԽՋՌՁՆԿՇՊՁԿՁՆԹՌԽԾՊՇՅՌՀԽԺԽՋՌ

ՋՁՆԿՄԽՋՈԹՏՆ
ՁԽ ՌՀԽՋՈԹՏՆՋԹՊԽՆՇՌԻՇ

ՅՁՐԽԼ

ԫՍՅԺԽՊՇԾՋՈԹՏՆՋԽՎԹՄՍԹՌԽԼ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԫՇՌՊԽՈՇՊՌԽԼ

ԫՍՅԺԽՊՇԾԽԿԿՋՋԹՅՈՄԽԼՈԽՊՋՈԹՏՆ

ԫՇՌՊԽՈՇՊՌԽԼ

ԫԣՋՌԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽԼԽԾՁՆՁՌՁՎԽՋՌՍԼՑ

ԺԽՁՆՁՌՁԹՌԽԼՏՁՌՀՄԹՊՎԹԽԹՌԫՁԽՍՏՃՇՇՈ-

ԣԹԺԽՊ
ԫԣԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽ
 +

ԼԹՑՋՈՇՋՌԾԽՊՌՁՄՁՒԹՌՁՇՆ

ԞԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ԼԹՑՋՈՇՋՌ

ԾԽՊՌՁՄՁՒԹՌՁՇՆ
ԼՈԾ

ԪԽԹՆՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾՊԽՈՇՊՌԽԼ

ԫՇՌՊԽՈՇՊՌԽԼ

ԯԹՆԿԽՇԾՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾՊԽՈՇՊՌԽԼ

ԫՇՌՊԽՈՇՊՌԽԼ

ԴԹՋՌՀԽՇՈՌՁՇՆԹՄՋՁՒԽՋԽՄԽԻՌՁՇՆՅԽՌՀՇԼՍՋԽԼ!

ԫՇ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !	"#

	
	


ԡԽՌԹՁՄՋՇՆՄԹՊՎԹՄՋԽՄԽԻՌՁՇՆ ԩԹՊՎԹՄՋԽՄԽԻՌՁՇՆՏԹՋ

ՋՁՅՁՄԹՊՌՇԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԡԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽՇԾԽԹԻՀՌԹԼՈՇՄԽՍՋԽԼՁՆՌՀԽՌԽՋՌՏԹՋԼԽՌԽՊՅՁՆԽԼԹՋԹԻԻՍՊԹՌԽՄՑԹՋՈՇՋՋՁԺՄԽՍՋՁՆԿՌՀԽԼՁՋՋԽԻՌՁՇՆՅՁԻՊՇՋԻՇՈԽԱՀԽՈՊՁՅԹՊՑԼԽՎԽՄՇՈՅԽՆՌԹՄԾԽԹՌՍՊԽԾՇՊՋԽՄԽԻՌՁՆԿՋՌԹԿԽՌԹԼՈՇՄԽՋՏԹՋՀՁՆԼՄՁՅԺՅՇՊՈՀՇՄՇԿՑԱԹԼՈՇՄԽՋՌՀԹՌՅԽՌՌՀԽՋՌԹԿԽԻՊՁՌԽՊՁԹԾՇՊՌՀԽՌԽՋՌՏԽՊԽՁՆԼՁՋԻՊՁՅՁՆԹՌԽՄՑՋԽՄԽԻՌԽԼԹՆԼՈՄԹԻԽԼՇՆԽՇՊՌՏՇԹՌԹՌՁՅԽՁՆՌՇՌՊԹՆՋԾԽՊՎԽՋՋԽՄՋՍՆՌՁՄԽԹԻՀՎԽՋՋԽՄԻՇՆՌԹՁՆԽԼՌԹԼՈՇՄԽՋԬՆԽ ՌՊԹՆՋԾԽՊՎԽՋՋԽՄՏԹՋՌՀԽՆՊԹՆԼՇՅՄՑ

ԹՋՋՁԿՆԽԼՌՇԽԹԻՀՌԽՋՌԻՀԹՅԺԽՊԹՆԼՌՀԽՌԹԼՈՇՄԽՋՏԽՊԽԿԽՆՌՄՑՌՊԹՆՋԾԽՊՊԽԼՌՇՌՀԽՌԽՋՌԻՀԹՅԺԽՊՋ

ԩՇԹԼՁՆԿՊԹՌԽ
ՊԽԹՊՁՆԿԼԽՆՋՁՌՑ ԫՇՌՋՈԽԻՁԾՁԽԼ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՊԽԹՊՁՆԿ ԼԽՆՋՁՌՑ


ՄՇԹԼՁՆԿ ՊԹՌԽ ՆՇՌ ԽՐԻԽԽԼ ԹՈՈՊՇՐՁՅԹՌԽՄՑ 

ՄԹՊՎԹԽԩ ԻՍՄՌՍՊՁՆԿ ՋՑՋՌԽՅ ԾՇՊ ԾՄՇՏՌՀՊՇՍԿՀ

ՋՑՋՌԽՅՋ ՇՊ  ՌԹԼՈՇՄԽՋԩ ՁՆ ՋՌԹՌՁԻՊԽՆԽՏԹՄ

ԽՐՈՇՋՍՊԽՋՑՋՌԽՅՋ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !"	#$

	
	


ԱՑՈԽՇԾԾՇՇԼ ԰ԽՊԹԪՁԻՊՇՆն ԢԭԞՊԽԻՇՅՅԽՆԼՋ԰ԽՊԹԪՁԻՊՇՆն ՌՀՊՇՍԿՀՇՍՌ

ՈՊԽԽՐՈՇՋՍՊԽ
ԹԾՌԽՊԫԣՋՌԹԿԽԹՆԼ

ԼՍՊՁՆԿՌՀԽԽՆՌՁՊԽԼԼԽԾՁՆՁՌՁՎԽՋՌՍԼՑԦԾ

ԹՆՇՌՀԽՊԼՁԽՌՁՋՍՋԽԼՌՀԽՋՌՍԼՑՊԽՈՇՊՌՋՀՇՍՄԼ

ՈՊՇՎՁԼԽԹՆԹՄՑՋՁՋՇԾՁՇԼՁԼԽԻՇՆՌԽՆՌԹՆԼ

ՈՇՌԽՆՌՁԹՄԻՇՆՌԹՅՁՆԹՆՌՋԹՆԼՌՀԽԼՁԽՌՋՀՇՍՄԼ

ԼԽՅՇՆՋՌՊԹՌԽԽՉՍԹՄՈԽՊԾՇՊՅԹՆԻԽՌՇ԰ԽՊԹ

ԪՁԻՊՇՆն 

԰ՇՍՊԻԽՇԾԾՇՇԼ ԰ԽՊԹԫՇՊՌՀԞՅԽՊՁԻԹ

ԪՇՆՌԿՇՅԽՊՑՎՁՄՄԽԭԞ

ԦՇԼՁԼԽԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆԼՁԽՌ
ՁԾՃՆՇՏՆ

ԫՇՌՊԽՈՇՊՌԽԼ

ԣՊԽՉՍԽՆԻՑՇԾԾԽԽԼՁՆԿ ՌՁՅԽՋԼԹՑ ԱԹԼՈՇՄԽՋՏԽՊԽԾԽԼՌՏՁԻԽՇՆՌՀԽԾՁՊՋՌԼԹՑ

ԹՆԼՇՆԻԽՇՆՌՀԽՄԹՋՌԼԹՑՇԾՌՀԽՌԽՋՌ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾԽԽԼՁՆԿՇԻԻՍՊԹՌ

ՄԽԹՋՌՌՏՁԻԽՈԽՊԼԹՑ

ԡԽՌԹՁՄՋՇՆԾԽԽԼՁՆԿՊԽԿՁՅԽ ԣԽԽԼՁՆԿՊԽԿՁՅԽՏԹՋ

ՌՀԽՋԹՅԽԹՋԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԣՇՇԼՊԹՌՁՇՆՋԼՍՊՁՆԿՌՀԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼՋՌԹՊՌԽԼԹՌՅԿՄԹՊՎԹԼԹՑԹՆԼՁՆԻՊԽԹՋԽԼԺՑՁՆԻՊԽՅԽՆՌՋՇԾՅԿՍՆՌՁՄՊԽԹԻՀՁՆԿՅԿՄԹՊՎԹԼԹՑԼՍՊՁՆԿՌՀԽԾՁՆԹՄՏԽԽՃՇԾՌԽՋՌՁՆԿ

ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌԾՇՇԼՊԹՌՁՇՆՋԼՍՊՁՆԿ

ՌՀԽՈՊԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼԺԽՁՆԻՊԽԹՋԽԼԹՄՇՆԿ

ՏՁՌՀՄԹՊՎԹՄԿՊՇՏՌՀՌՇԹՈՈՊՇՐՁՅԹՌԽՄՑ

ՅԿՄԹՊՎԹԼԹՑԺՑՌԽՋՌՁՆՁՌՁԹՌՁՇՆԢԭԞԹՆԼ

ԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌԾՇՇԼՊԹՌՁՇՆՋ

ՁՆԻՊԽԹՋԽԾՊՇՅՅԿՄԹՊՎԹԼԹՑԹՌՌԽՋՌ

ՁՆՁՌՁԹՌՁՇՆ
԰ՌՍԼՑԡԹՑՌՇ

ՅԿՄԹՊՎԹԼԹՑՁՆՌՀԽՄԹՋՌՏԽԽՃՇԾ ՌՀԽՌԽՋՌ


԰ՌՍԼՑԡԹՑ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ԱՑՈԽՇԾԽՐՈՇՋՍՊԽ ԣՄՇՏՌՀՊՇՍԿՀ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԹԾՄՇՏ

ՌՀՊՇՍԿՀՋՑՋՌԽՅ

ԱՑՈԽՇԾԾՄՇՏՌՀՊՇՍԿՀԼՁՄՍՌՁՇՆՋՑՋՌԽՅ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԠՇՆՌՁՆՍՇՍՋԾՄՇՏԼՁՄՍՌԽՊ ԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊՋՇՊ

ԻՇՆՌՁՆՍՇՍՋԾՄՇՏՋԽՊՁԹՄԼՁՄՍՌԽՊՋԹՊԽ

ՊԽԻՇՅՅԽՆԼԽԼ

 ԞԼԼՁՌՁՇՆԹՄԿՍՁԼԹՆԻԽԾՇՊԹՉՍԹՌՁԻՌԽՋՌԼԽՋՁԿՆՁՋՄՇԻԹՌԽԼՁՆԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ԰ՈԽԻՁԹՄԠՇՆՋՁԼԽՊԹՌՁՇՆՋԾՇՊԠՇՆԼՍԻՌՁՆԿԞՉՍԹՌՁԻԩԹԺՇՊԹՌՇՊՑ԰ՌՍԼՁԽՋ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !	"#

	
	


ԣՄՇՏՌՀՊՇՍԿՀՊԹՌԽ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԫՇՌՋՈԽԻՁԾՁԽԼԺՑՋՌՍԼՑԹՍՌՀՇՊԯԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼՅԩՅՁՆ

ԱՀԽԾՄՇՏՇԾԼՁՄՍՌՁՇՆՏԹՌԽՊՁՆՌՇԽԹԻՀՅՁՐՁՆԿԻՀԹՅԺԽՊՏԹՋԹԼՂՍՋՌԽԼՌՇՈՊՇՎՁԼԽԹՈՈՊՇՐՁՅԹՌԽՄՑՎՇՄՍՅԽԹԼԼՁՌՁՇՆՋՇԾՌԽՋՌՏԹՌԽՊՁՆԽԹԻՀՌԽՋՌԻՀԹՅԺԽՊՈԽՊԼԹՑԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՏԹՋՈՍՅՈԽԼՁՆՌՇ ՌՀԽԼՁՄՍՌԽՊ

ՅՁՐՁՆԿԻՀԹՅԺԽՊՋԹՌՌԹՊԿԽՌՊԹՌԽՋՇԾ

ԹՆԼՅԩՅՁՆԾՇՊՌՀԽԹՆԼՅԿԹՁԩՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋՊԽՋՈԽԻՌՁՎԽՄՑԦՆՌՀԽՅՁՐՁՆԿԻՀԹՅԺԽՊՋՌՀԽՋՌՇԻՃՏԹՋՅՁՐԽԼՏՁՌՀ ՏԽՄՄՏԹՌԽՊԼԽՄՁՎԽՊԽԼԹՌԹՌԹՊԿԽՌ

ՊԹՌԽՇԾՅԩՅՁՆՌՇԹԻՀՁԽՎԽՌՀԽՆՇՅՁՆԹՄՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ

ԯԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼԾՄՇՏՊԹՌԽՎՇՄՍՅԽՋԼԹՑՐԩԩԼԹՑՅԩԼԹՑՅԩԼԹՑՐԼԹՑՅՁՆՅԩՅՁՆ

ԯԽԻՇՅՅԽՆԼԽԼԾՄՇՏՌՀՊՇՍԿՀՊԹՌԽՁՋ

ՅԩՅՁՆ
ԻՇՅՈՄԽՌԽՎՇՄՍՅԽՊԽՈՄԹԻԽՅԽՆՌ

ԻԹԽՎԽՊՑՀՊՋ

Page 16 of 294
	

		
	
		
			

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

ԡԽՌԹՁՄՋՇՆՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊԾՄՇՏՌՀՊՇՍԿՀՋՑՋՌԽՅՋ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԣՄՍՁԼՅԽՌԽՊՁՆԿՈՍՅՈՋ
ԣՄՍՁԼԪԽՌԽՊՁՆԿԦՆԻ԰ՑՇՋՋԽՌԫԶՏԽՊԽՍՋԽԼՌՇԼԽՄՁՎԽՊՎՇՄՍՅԽՋՇԾԹՋՁՆԿՄԽՌԽՋՌՋՍԺՋՌԹՆԻԽՋՌՇԻՃՋՇՄՍՌՁՇՆՌՇՅՁՐՁՆԿԻՀԹՅԺԽՊՋՁՆԼՁՋԻՊՁՅՁՆԹՌԽՄՑԹՋՋՁԿՆԽԼՌՇԽԹԻՀՌՊԽԹՌՅԽՆՌԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՏԹՋԼՁՄՍՌԽԼՏՁՌՀՏԽՄՄՏԹՌԽՊՁՆՌՀԽՅՁՐՁՆԿԻՀԹՅԺԽՊՋՁՆՇՊԼԽՊՌՇՇԺՌԹՁՆՌՀԽԼԽՋՁՊԽԼՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՈՊՁՇՊՌՇԼԽՄՁՎԽՊՑՌՇՌՀԽՌԽՋՌԻՀԹՅԺԽՊԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՏԹՋԼՁՄՍՌԽԼՏՁՌՀՏԽՄՄՏԹՌԽՊՁՆՌՀԽՅՁՐՁՆԿԻՀԹՅԺԽՊՋՁՆՇՊԼԽՊՌՇՇԺՌԹՁՆՌՀԽԼԽՋՁՊԽԼՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՈՊՁՇՊՌՇԼԽՄՁՎԽՊՑՌՇՌՀԽՌԽՋՌԻՀԹՅԺԽՊՋԞԾՌԽՊՅՁՐՁՆԿՌՀԽՌԽՋՌՏԹՌԽՊՁՆԽԹԻՀՅՁՐՁՆԿԻՀԹՅԺԽՊՏԹՋՈՍՅՈԽԼՁՆՌՇՌՀԽԹՈՈՊՇՈՊՁԹՌԽՊԽՈՄՁԻԹՌԽՌԽՋՌԻՀԹՅԺԽՊՍՋՁՆԿԹՈԽՊՁՋՌԹՄՌՁԻՈՍՅՈ
ԠՇՄԽԭԹՊՅԽՊԦՆՋՌՊՍՅԽՆՌԠՇՅՈԹՆՑԠՀՁԻԹՇԦԩԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆԹՆԼ

ՌՀԽՌԽՋՌՋՇՄՍՌՁՇՆՋՏԽՊԽՋՌՁՊՊԽԼԻՇՆՌՁՆՍՇՍՋՄՑԹՆԼՏԽՊԽՈՊՇՌԽԻՌԽԼԾՊՇՅՄՁԿՀՌԱՀԽՈՊՇՈՇՊՌՁՇՆՇԾ

ՌԽՋՌՏԹՌԽՊՌՀԹՌՏԹՋՈՍՅՈԽԼՁՆՌՇԽԹԻՀՊԽՈՄՁԻԹՌԽՌԽՋՌԻՀԹՅԺԽՊՏԹՋԻՀԽԻՃԽԼՈՊՁՇՊՌՇՌՀԽՌԽՋՌԹՆԼԹՈՈՊՇՐՁՅԹՌԽՄՑՏԽԽՃՄՑԼՍՊՁՆԿՌՀԽՌԽՋՌՌՇ ԽՆՋՍՊԽ

ՌՀԹՌԾՄՇՏՊԹՌԽՋՎԹՊՁԽԼԺՑՆՇՅՇՊԽՌՀԹՆ

չՇԾՌՀԽՅԽԹՆԾՄՇՏՊԹՌԽԾՇՊՌՀԽՊԽՈՄՁԻԹՌԽՋ

ԯԽԻՇՅՅԽՆԼԽԼՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊԾՄՇՏ

ՌՀՊՇՍԿՀՋՑՋՌԽՅՋԪՁՐՁՆԿԻՀԹՅԺԽՊՁՋ

ՊԽԻՇՅՅԽՆԼԽԼԺՍՌՆՇՌՊԽՉՍՁՊԽԼ

ԞԽՊԹՌՁՇՆՁՋՆՇՌՊԽԻՇՅՅԽՆԼԽԼԾՇՊ

ՅՁՐՁՆԿ ԞԼԽՅՇՆՋՌՊԹՌՁՇՆՌՀԹՌՌՀԽՌԽՋՌ

ՋՇՄՍՌՁՇՆՁՋԻՇՅՈՄԽՌԽՄՑՅՁՐԽԼԺԽԾՇՊԽ

ՁՆՌՊՇԼՍԻԽԼՁՆՌՇՌՀԽՌԽՋՌՋՑՋՌԽՅՁՋ

ՊԽԻՇՅՅԽՆԼԽԼ ԱՀԽՊԽԻՇՅՅԽՆԼԽԼԾՄՇՏ

ՋՈՄՁՌՌՁՆԿԹԻԻՍՊԹԻՑՁՋՏՁՌՀՁՆ

Page 17 of 294
	

		
	
		
			

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !	"#

	
	


ԯԽՆԽՏԹՄՈԽՊՁՇԼԾՇՊՋՌԹՌՁԻՊԽՆԽՏԹՄ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԫԞ ԦԾՋՌԹՌՁԻՊԽՆԽՏԹՄՁՋՍՋԽԼԢԭԞ

ՊԽԻՇՅՅԽՆԼՋՀՊՊԽՆԽՏԹՄՊԽՆԽՏԹՄ

ՈԽՊՁՇԼՁՋՊԽԻՇՅՅԽՆԼԽԼՆՇՌՌՇԽՐԻԽԽԼ

ՀՇՍՊՋ

ԞԽՊԹՌՁՇՆ! ԫՇ ԢԭԞՊԽԻՇՅՅԽՆԼՋՅԹՁՆՌԹՁՆՁՆԿԼՁՋՋՇՄՎԽԼ

ՇՐՑԿԽՆԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԹՁՊ

ՋԹՌՍՊԹՌՁՇՆ
 ՅԿԩԞԽՊԹՌՁՇՆՅԹՑԺԽՅԹՁՆՌԹՁՆԽԼՌՀՊՇՍԿՀԺՍԺԺՄԽՊՋԦՌՁՋ

ՊԽԻՇՅՅԽՆԼԽԼՌՇՋԽՌԺՍԺԺՄԽՊՋԹՌՄԽՎԽՄՋ

ՌՀԹՌԼՇՆՇՌԻԹՍՋԽՋՌՊԽՋՋՇՆՌՀԽՌԹԼՈՇՄԽՋ

Page 18 of 294
	

		
	
		
			

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !"	#$

	
	


԰ՇՍՊԻԽՇԾԼՁՄՍՌՁՇՆՏԹՌԽՊ ԴԽՄՄՏԹՌԽՊ ԱՀԽՏԽՄՄՏԹՌԽՊՈԹՋՋԽԼՌՀՊՇՍԿՀԹՋԹՆԼ

ԾՁՄՌԽՊՌՇՊԽՅՇՎԽՈԹՊՌՁԻՄԽՋԿՊԽԹՌԽՊՌՀԹՆԹՈՈՊՇՐՁՅԹՌԽՄՑսՅԹՆԼՈՍՅՈԽԼՁՆՌՇԹԩՋՌՇՊԹԿԽՌԹՆՃՏՀԽՊԽՌՀԽՏԹՌԽՊ

ՏԹՋԹԽՊԹՌԽԼՏՁՌՀՋՈՊԹՑՆՇՒՒՄԽՋ ԭՊՁՇՊՌՇ

ՍՋԽՌՀԽՏԹՌԽՊՏԹՋԾՁՄՌԽՊԽԼՌՇսՅՌՇՊԽՅՇՎԽԾՁՆԽՈԹՊՌՁԻՄԽՋԱՀԽՏԽՄՄՏԹՌԽՊՁՋԻՀԹՊԹԻՌԽՊՁՒԽԼԹՋՅՇԼԽՊԹՌԽՄՑՀԹՊԼՏԹՌԽՊ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՆԹՌՍՊԹՄՇՊՊԽԻՇՆՋՌՁՌՍՌԽԼ

ՏԹՌԽՊՁՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՆԹՌՍՊԹՄ

ՏԹՌԽՊԺԽՋՌԽՊՁՄՁՒԽԼՏՁՌՀԲԳԹՆԼՌԽՋՌԽԼԾՇՊ

ՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊ

ՈՇՋՋՁԺՄԽԻՇՆՌԹՅՁՆԹՆՌՋՁՆԻՄՍԼՁՆԿՃՆՇՏՆ

ՋՍԺՋՌՊԹՌԽՋՇԾՌՀԽՁՇԼՁՆԽՌՊԹՆՋՈՇՊՌԽՊՇԾՌՀԽ

ՌՀՑՊՇՁԼԿՄԹՆԼ
ԽԿԾՄՍՇՊՁԼԽԻՀՄՇՊԹՌԽ

ՈԽՊԻՀՄՇՊԹՌԽԬԢԠԡԹԻԻԽՈՌՋԹՆՑՏԹՌԽՊՁՆ

ՏՀՁԻՀՌՀԽՌԽՋՌՋՈԽԻՁԽՋՋՀՇՏԻՇՆՌՊՇՄ

ՋՍՊՎՁՎԹՄԹՌՄԽԹՋՌԹՋԿՇՇԼԹՋՁՆԼՁԻԹՌԽԼՁՆ

ՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽ

ԴԹՋԼՁՄՍՌՁՇՆՏԹՌԽՊԹՆԹՄՑՒԽԼԾՇՊՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊԻՇՆՌԹՅՁՆԹՆՌՋ!

ԶԽՋ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !"	#$

	
	


ԦՇԼՁԼԽՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՆՏԹՌԽՊ!

ԫՇ ԦԾՊԽԻՇՆՋՌՁՌՍՌԽԼՏԹՌԽՊՁՋՍՋԽԼՇՊՁԾ

ԺԹԻՃԿՊՇՍՆԼՄԽՎԽՄՋՇԾՁՇԼՁԼԽՁՆՆԹՌՍՊԹՄ

ՏԹՌԽՊԹՊԽՄԽՋՋՌՀԹՆսԿԩՁՇԼՁԼԽ

ՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՋՊԽԻՇՅՅԽՆԼԽԼԱՀՁՋ

ՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՋՁՆԹԼԼՁՌՁՇՆՌՇՌՀԽ

ՊԽԻՇՅՅԽՆԼԽԼԼՁԽՌԹՊՑՋՇՍՊԻԽՇԾՁՇԼՁԼԽ


ԽԿՁՆ԰ԽՊԹԪՁԻՊՇՆ

ԱԽՋՌՎԽՋՋԽՄՌՑՈԽՅԹՌԽՊՁԹՄՋ ԩԿՄԹՋՋԹՉՍԹՊՁԹ ԢԭԞ ԹՆԼ ԬԢԠԡ ՊԽԻՇՅՅԽՆԼ ՌՀԹՌ ՏԹՌԽՊ

ԻՇՆՌԹԻՌ ՈՇՊՌՁՇՆՋ ՇԾ ՌՀԽ ՋՑՋՌԽՅ ՆՇՌ

ԻՇՅՈՊՇՅՁՋԽ ՌՀԽ ՋՌՍԼՑ 
ԽԿ ԹՄՄ ԿՄԹՋՋ

ՎԽՋՋԽՄՋՇՊԿՄԹՋՋՎԽՋՋԽՄՋՏՁՌՀՋՌԹՁՆՄԽՋՋՋՌԽԽՄ

ԾՊԹՅԽՋԹՊԽԹԻԻԽՈՌԹԺՄԽԽՐԹՅՈՄԽՋ

ԱԽՋՌՎԽՋՋԽՄՋՁՒԽ ՐՐԻՅ ԱՀԽԼԽՈՌՀՇԾՌՀԽՌԽՋՌՏԹՌԽՊՁՆԹ

ՊԽՈՊԽՋԽՆՌԹՌՁՎԽԻՀԹՅԺԽՊՏԹՋԻՅ

ԣՁՄՄՎՇՄՍՅԽ ԩ

ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋՇՆԽՐՈՇՋՍՊԽՋՑՋՌԽՅ

%&'	()*


'+

Page 20 of 294
	

		
	
		
			

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ



 
!

	
	


ԥԹՊԼՆԽՋՋ
ՅԿԩԹՋԠԹԠԬ   

ԴԽԽՃՄՑ


ԻՇՆՌՊՇՄԹՆԼՌՀԽՀՁԿՀԽՋՌ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՌՊԽԹՌՅԽՆՌԿՊՇՍՈ

ՇՆՄՑ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՀԹՊԼՆԽՋՋՌՇ

ՅԿԩԹՋԠԹԠԬ

Ոԥ    ԴԽԽՃՄՑ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՈԥչՁՆՌԽՊ

ՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋ

ՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼ

ԡՁՋՋՇՄՎԽԼՇՐՑԿԽՆ
ՅԿԩ    ԴԽԽՃՄՑ

ԢԭԞՊԽԻՇՅՅԽՆԼՋԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆ


ԡԬ ՅԿԩ
 ԹՁՊՋԹՌՍՊԹՌՁՇՆ

ԬԢԠԡՊԽԻՇՅՅԽՆԼՋԡԬԻՇՆԻԽՆՌՊԹՌՁՇՆ

 ՅԿԩ 
 ԹՁՊՋԹՌՍՊԹՌՁՇՆ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ



 
!

	
	


ԱԽՅՈԽՊԹՌՍՊԽ
ոԠ   

ԴԽԽՃՄՑ

ԞԼԼՁՌՁՇՆԹՄՌԽՅՈԽՊԹՌՍՊԽ

ՅԽԹՋՍՊԽՅԽՆՌՋՏԽՊԽՌԹՃԽՆ

ԺԽՌՏԽԽՆԡԹՑՋԹՆԼՌՇ

ԽՆՋՍՊԽՌՀԹՌՌԽՅՈԽՊԹՌՍՊԽՋՏԽՊԽ

ՏՁՌՀՁՆՌՀԽՌԹՊԿԽՌՊԹՆԿԽԼՍԽՌՇԹ

ԻՇՆՌՊՇՄՄԽՊՅԹՄԾՍՆԻՌՁՇՆՇՆԡԹՑ

ԱԽՅՈԽՊԹՌՍՊԽԹՄՋՇՏԹՋՅՇՆՁՌՇՊԽԼ

ԻՇՆՌՁՆՍՇՍՋՄՑՁՆՇՆԽՆԽԿԹՌՁՎԽ

ԻՇՆՌՊՇՄՊԽՈՄՁԻԹՌԽԼՍՊՁՆԿՌՀԽԹՋՋԹՑ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌԽՅՈԽՊԹՌՍՊԽչՇԠ

ՁՆՌԽՊՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊՌՊԽԹՌՅԽՆՌ

ԼՁԾԾԽՊԽՆՌՁԹՄՋՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼՇԠ

ԦՇԼՁԼԽ սԿԩ

ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՉՍԹՌՁԻՁՇԼՁԼԽՊԹՆԿԽ

-սԿԩ
ՋՍՈՈՄԽՅԽՆՌԹՄՁՇԼՁԼԽ

ՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼսԿԩ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ



 
!

	
	


ԞՅՅՇՆՁԹ ԫՇՌՊԽՈՇՊՌԽԼ ԤԽՆԽՊԹՄՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋԾՇՊԾՊԽՉՍԽՆԻՑ

ՇԾՅԽԹՋՍՊԽՅԽՆՌՋԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌ

ՏԹՌԽՊՉՍԹՄՁՌՑՈԹՊԹՅԽՌԽՊՋԺԽՅԽԹՋՍՊԽԼՁՆ

ԹԻՇՆՌՊՇՄԹՆԼԹՌՇՆԽՌԽՋՌՁՌԽՅ

ԻՇՆԻԽՆՌՊԹՌՁՇՆԹՌՄԽԹՋՌՏԽԽՃՄՑԦՆՋՌԹՌՁԻ

ՊԽՆԽՏԹՄՋՑՋՌԽՅՋՏԹՌԽՊՉՍԹՄՁՌՑ

ՈԹՊԹՅԽՌԽՊՋՁՆԻՄՍԼՁՆԿԹՅՅՇՆՁԹՋՀՇՍՄԼ

ԺԽՅԽԹՋՍՊԽԼՂՍՋՌՈՊՁՇՊՌՇՊԽՆԽՏԹՄԦՆ

ԹԼԼՁՌՁՇՆԢԭԞՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌԡԬԺԽ

ՅԽԹՋՍՊԽԼԹՌԽԹԻՀԻՇՆԻԽՆՌՊԹՌՁՇՆԹՌՄԽԹՋՌ

ՏԽԽՃՄՑԹՆԼՌՀԹՌՌԽՅՈԽՊԹՌՍՊԽԺԽ

ՅԽԹՋՍՊԽԼԻՇՆՌՁՆՍՇՍՋՄՑԬԢԠԡ

ՊԽԻՇՅՅԽՆԼՋՌՀԹՌ ԡԬԹՆԼՌԽՅՈԽՊԹՌՍՊԽ

ԺԽՅԽԹՋՍՊԽԼԹՌՄԽԹՋՌՏԽԽՃՄՑԹՆԼՌՀԹՌՈԥ

ԹՆԼՀԹՊԼՆԽՋՋԺԽՅԽԹՋՍՊԽԼԹՌՄԽԹՋՌԹՌ

ՌՀԽԺԽԿՁՆՆՁՆԿԹՆԼԽՆԼՇԾՌՀԽՌԽՋՌ

ԣՄՍՇՊՁԼԽ ՅԿԩ
ՏԽՄՄՏԹՌԽՊԭԽՊԻՀՄՇՊԹՌԽ ԫՇՌԯԽՈՇՊՌԽԼԠՀՄՇՊԹՌԽ ԫՇՌԯԽՈՇՊՌԽԼ

ԞՄՃԹՄՁՆՁՌՑ
ՅԿԩԹՋԠԹԠԬ   

ԴԽԽՃՄՑ


ԻՇՆՌՊՇՄԹՆԼՌՀԽՀՁԿՀԽՋՌ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՌՊԽԹՌՅԽՆՌԿՊՇՍՈ

ՇՆՄՑ

ԠՇՆԼՍԻՌՁՎՁՌՑ
ս԰ԻՅ   

 ԬՆԡԹՑԹԻՇՆՌՊՇՄՄԽՊՅԹՄԾՍՆԻՌՁՇՆԽԼԹՆԼՌՀԽՌԽՅՈԽՊԹՌՍՊԽՋՁՆՋԽՎԽՊԹՄՌԽՋՌԻՀԹՅԺԽՊՋՏԽՊԽՋՄՁԿՀՌՄՑԺԽՄՇՏՌՀԽԼԽՋՁՊԽԼՊԹՆԿԽՊԹՆԿՁՆԿԾՊՇՅ

ՌՇոԠԱՀԽԻՇՆՌՊՇՄՄԽՊՏԹՋԻՇՊՊԽԻՌԽԼՇՆՌՀԽՋԹՅԽԼԹՑԹՆԼՌԽՅՈԽՊԹՌՍՊԽՋՏԽՊԽԻՇՆԾՁՊՅԽԼԱՀՁՋՄԽՋՋՌՀԹՆոԠՎԹՊՁԹՆԻԽՀԹԼ ՆՇԹՈՈԹՊԽՆՌԹԼՎԽՊՋԽՁՅՈԹԻՌՇՆՌՀԽՌԹԼՈՇՄԽՋ

 ԯԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼԹՋԹՎԽՊԹԿԽՇԾՅՁՆՁՅՍՅԹՆԼՅԹՐՁՅՍՅՎԹՄՍԽՋ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

  
	

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ԴԹՋԹՊԹՆԿԽԾՁՆԼԽՊԻՇՆԼՍԻՌԽԼ!

ԶԽՋ

ԦԾՑԽՋՏՀԹՌՏԹՋՌՀԽՅԽՌՀՇԼԾՇՊԼԽՌԽՊՅՁՆՁՆԿՌՀԽՀՁԿՀԽՋՌՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽՊԹՆԿԽԾՁՆԼԽՊ!

ԫՇՅՁՆԹՄՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽՋԽՄԽԻՌԽԼԺՑՌՀԽ԰ՈՇՆՋՇՊԺԹՋԽԼՇՆՌՀԽԹԻՍՌԽՀՊԩԠԾՇՊԵԽՆՇՈՍՋՇԾՅԿԹՁԩ

ԫՇՅՁՆԹՄՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՁՆՌՀԽՊԹՆԿԽԾՁՆԼԽՊԹՆԼՅԿԹՁԩ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌ

ՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽ

ՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾՌՀԽՌԽՋՌ

ԻՇՅՈՇՍՆԼՅԿԩՇՊ

ԼԽՅՇՆՋՌՊԹՌԽՋԹԼԽՉՍԹՌԽԽՎՁԼԽՆԻԽ

ՇԾՌՇՐՁԻՁՌՑ
ԽԿՅՇՊՌԹՄՁՌՑ

ՏՀՁԻՀԽՎԽՊԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌ

԰ՈԽԻՁԽՋ ԵԽՆՇՈՍՋՄԹԽՎՁՋԩՁԾԽՋՌԹԿԽ ԫԣՋՌԹԿԽԱԽՋՌԼՍՊԹՌՁՇՆ ԼԹՑՋ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋ ԴԹՌԽՊՌԽՅՈԽՊԹՌՍՊԽՋ

ՏԽՊԽՏՁՌՀՁՆՌՀԽչոԠՊԹՆԿԽԽՋՌԹԺՄՁՋՀԽԼԾՇՊՌՀԽՌԽՋՌԡՁՋՋՇՄՎԽԼՇՐՑԿԽՆԻՇՆԻԽՆՌՊԹՌՁՇՆՋՊԽՅԹՁՆԽԼ ՇԾՋԹՌՍՊԹՌՁՇՆ
ՅԿԩԪԽԹՋՍՊԽՅԽՆՌՋՇԾՈԥՊԹՆԿԽԼԾՊՇՅՌՇԼՍՊՁՆԿՌՀԽՌԽՋՌ

ԪԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽԹՆԼՅԿԹՁԩՏՀՁԻՀՊԽՈՊԽՋԽՆՌԹՆԼՇԾՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋՊԽՋՈԽԻՌՁՎԽՄՑ԰ԹՅՈՄԽՋՇԾՌՀԽՅԿԹՁԩՌԽՋՌՋՇՄՍՌՁՇՆԻՇՄՄԽԻՌԽԼԼՍՊՁՆԿՌՀԽՌԽՋՌՀԹԼՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՌՀԹՌՊԹՆԿԽԼԾՊՇՅՌՇՇԾՌՀԽՆՇՅՁՆԹՄ԰ԹՅՈՄԽՋԻՇՄՄԽԻՌԽԼԼՍՊՁՆԿՌՀԽՌԽՋՌԾՊՇՅՌՀԽԾՁՎԽՀՁԿՀԽՊԻՇՆԻԽՆՌՊԹՌՁՇՆՌԽՋՌՋՇՄՍՌՁՇՆՋՀԹԼՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՌՀԹՌՊԹՆԿԽԼՇԾՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋԦՌՁՋԺԽՄՁԽՎԽԼՌՀԹՌՌՀՁՋՎԹՊՁԹԺՁՄՁՌՑՏԹՋԼՍԽՌՇԹԼՋՇՊՈՌՁՇՆՇԾՌՀԽՅԹՌԽՊՁԹՄՌՇԺՁՇՄՇԿՁԻԹՄՅԹՌԽՊՁԹՄՁՆՌՀԽՌԽՋՌԻՀԹՅԺԽՊԹՌՌՀԽՅԿԹՁԩԻՇՆԻԽՆՌՊԹՌՁՇՆԯԽՈՇՊՌԽԼՎԹՄՍԽՋԾՊՇՅՌՀՁՋՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՏԽՊԽԽՐՌՊԹՈՇՄԹՌԽԼԺԽԻԹՍՋԽՌԽՋՌՅԽԹՋՍՊԽՅԽՆՌՋՏԽՊԽՄԽՋՋՌՀԹՆՌՀԽՄՁՅՁՌՇԾՉՍԹՆՌՁՌԹՌՁՇՆՌՀՊՇՍԿՀՇՍՌՌՀԽՋՌՍԼՑ ԱԹԼՈՇՄԽՋՍՊՎՁՎԹՄՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ

ԿՊՇՍՈՏԹՋԱԹԼՈՇՄԽՋՍՊՎՁՎԹՄՁՆՌՀԽԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԹՋԹՆԼ

ՊԽՋՈԽԻՌՁՎԽՄՑԥՇՏԽՎԽՊՌՀԽՊԽՏԽՊԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼՋՍԺՄԽՌՀԹՄՋՁԿՆՋ

ՇԾՌՇՐՁԻՁՌՑ
ՁԽ ՋՍՊԾԹԻՁՆԿԹՆԼՋՏՁՅՅՁՆԿՁՆԻՁՊԻՄԽՋԹՋՏԽՄՄԹՋՊԽԼՍԻՌՁՇՆՋ

ՁՆԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽՏԽՌՏԽՁԿՀՌՌՇՌԹՄՄԽՆԿՌՀ԰ԳԩԹՆԼԥԩԩԹՅՇՆԿ

ՌԹԼՈՇՄԽՋՁՆՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԴԽՌՏԽՁԿՀՌՏԹՋՌՀԽՅՇՋՌՋԽՆՋՁՌՁՎԽԽՆԼՈՇՁՆՌՅԽԹՋՍՊԽԼՏՁՌՀՊԽԼՍԻՌՁՇՆՋՁՆՏԽՌՏԽՁԿՀՌԹՅՇՆԿՌԹԼՈՇՄԽՋՁՆՌՀԽԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԬՎԽՊԹՄՄ

ՌՀԽՊԽՏԹՋԹՆԹՈՈԹՊԽՆՌՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՇՆԿՊՇՏՌՀԹՆԼ

ԼԽՎԽՄՇՈՅԽՆՌՁՆՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈԦՆՌՀԽԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՅԽԹՆՏԽՌՏԽՁԿՀՌՏԹՋՊԽԼՍԻԽԼԺՑՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽԻՇՆՌՊՇՄԴՀՁՄԽՌՀԽՊԽՏԽՊԽՆՇԻՇՊՊԽՋՈՇՆԼՁՆԿԼԽԻՊԽԹՋԽՋՁՆՌՀԽՄԽՆԿՌՀՈԹՊԹՅԽՌԽՊՋՌՀԽԽԾԾԽԻՌՇՆՏԽՁԿՀՌՁՆՌՀԽԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԹՋԻՇՆՋՁԼԽՊԽԼՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ


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ԱԽՋՌԼՍՊԹՌՁՇՆ ԼԹՑՋ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՌՀԽ ԼՍՊԹՌՁՇՆ ՇԾ ՌՀԽ

ԼԽԾՁՆՁՌՁՎԽՌԽՋՌԺԽԼԹՑՋ

ԪԽՌՀՇԼԾՇՊՋԽՄԽԻՌՁՆԿՌՀԽՀՁԿՀԽՋՌՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ

ԯԽՋՍՄՌՋՇԾՌՀԽՊԹՆԿԽԾՁՆԼԽՊԹՆԼԹԻՍՌԽՀՊ

ԩԠՎԹՄՍԽԾՇՊԵԽՆՇՈՍՋՇԾՅԿԹՁԩ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾ

ՌՀԽՌԽՋՌԻՇՅՈՇՍՆԼՅԿԩՇՊ

ԼԽՅՇՆՋՌՊԹՌԽՋԹԼԽՉՍԹՌԽԽՎՁԼԽՆԻԽՇԾՌՇՐՁԻՁՌՑ


ԽԿՅՇՊՌԹՄՁՌՑՏՀՁԻՀԽՎԽՊ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌ

ԯԽԾԽՊԽՆԻԽՋՌՍԼՑԻՁՌԹՌՁՇՆ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԯԹՆԿԽԾՁՆԼԽՊՋՌՍԼՑԪԯԦԡ

԰ԽՈԹՊԹՌՁՇՆՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ

ԹՆԼ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՌՀԽ ՅԹՐՁՅՍՅ

ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՋԽՈԹՊԹՌՁՇՆ ԺԽ  ԹՆԼ ՌՀԽ

ՅՁՆՁՅՍՅԺԽ

ԫՍՅԺԽՊՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՇԾ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԹՆԼԹԻՇՆՌՊՇՄՈՄՍՋՋՇՄՎԽՆՌ

ԻՇՆՌՊՇՄՁԾԹՈՈՊՇՈՊՁԹՌԽ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !"	#$

	
	


ԞՊԽՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋ

ԹԼՂՍՋՌԽԼԾՇՊՈՍՊՁՌՑ!

ԶԽՋ

ԦՆԼՁԻԹՌԽՌՀԽՌՑՈԽՇԾՎԹՄՍԽՋՈՊԽՋԽՆՌԽԼԾՇՊՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋ

ԪԽԹՆՅԽԹՋՍՊԽԼ

ԩՁՅՁՌՇԾՉՍԹՆՌՁԾՁԻԹՌՁՇՆ
ԩԬԮ ՅԿԹՁԩ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՇՊԻՀԽՅՁԻԹՄՌԽՋՌ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽՄՇՏՌՀԽԩԬԮԹՆԹՄՑՋԽՋԺԽ

ԻՇՆԼՍԻՌԽԼՇՆՌՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋ

ԩԽՎԽՄՇԾԼԽՌԽԻՌՁՇՆ
ԩԬԡ ԫՇՌՊԽՈՇՊՌԽԼ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ԣՊԽՉՍԽՆԻՑՇԾՅԽԹՋՍՊԽՅԽՆՌ ԡԹՑՋ ԡՍՈՄՁԻԹՌԽՏԹՌԽՊՋԹՅՈՄԽՋՏԽՊԽԻՇՄՄԽԻՌԽԼ

ԾՊՇՅԽԹԻՀՊԽՈՄՁԻԹՌԽՌԽՋՌԻՀԹՅԺԽՊԹՌՌՀԽԺԽԿՁՆՆՁՆԿԹՆԼԽՆԼՇԾՌՀԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼ
ԡԹՑՋԹՆԼԹՆԼՇՆԡԹՑՋԹՆԼԡԹՑՋԹՅՈՄԽՋԾՊՇՅՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՏԽՊԽՋՍՋՈԽԻՌ
ՁԽՄՇՏԱՀԽՄՇՏԹՆԹՄՑՌՁԻԹՄ

ՊԽՋՍՄՌՋՏԽՊԽԺԽՄՁԽՎԽԼՌՇԺԽԼՍԽՌՇՋԹՅՈՄՁՆԿՇՊԹՆԹՄՑՌՁԻԹՄԽՊՊՇՊԞԼԼՁՌՁՇՆԹՄ

ՋԹՅՈՄԽՋՏԽՊԽԻՇՄՄԽԻՌԽԼԾՊՇՅՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՌՏՇԼԹՑՋՄԹՌԽՊ
ԡԹՑԹՆԼՏԽՊԽԻՇՆՋՁԼԽՊԽԼՌՇԺԽՊԽՈՊԽՋԽՆՌԹՌՁՎԽՇԾՌՀԽԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽՌԽՋՌԻՀԹՅԺԽՊՋՇՆԡԹՑԹՆԼՍՋԽԼՁՆՌՀԽԻԹՄԻՍՄԹՌՁՇՆՇԾՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺՑՌՀԽՋՌՍԼՑԹՍՌՀՇՊ

ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՌԽՋՌՁՌԽՅ

ԻՇՆԻԽՆՌՊԹՌՁՇՆԺԽՅԽԹՋՍՊԽԼՁՆՇՆԽՌԹՆՃԹՌ

ԽԹԻՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼ

ԽՎԽՊՑՏԽԽՃՌՀԽՊԽԹԾՌԽՊ

ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆԻՇՆՌՊՇՄ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !"	#$

	
	


ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆՋՇՄՎԽՆՌԻՇՆՌՊՇՄ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԫԞ ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԽՍՋԽՇԾԹ

ԻՇՆԻՍՊՊԽՆՌՋՇՄՎԽՆՌԻՇՆՌՊՇՄՏՀԽՆԹ

ՋՇՄՍԺՁՄՁՒՁՆԿԹԿԽՆՌՁՋՍՋԽԼԢԭԞ

ՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ

ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՈԽՊՌԽՋՌՁՌԽՅՌՊԽԹՌՅԽՆՌՄԽՎԽՄ

 ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ

ԫՍՅԺԽՊՇԾՄԹՊՎԹԽՈԽՊՌՊԽԹՌՅԽՆՌԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ

 ԽՉՍԹՄՄՑԼՁՎՁԼԽԼԺԽՌՏԽԽՆՊԽՈՄՁԻԹՌԽՋ

ԴԹՋԹՋՇՄՎԽՆՌՍՋԽԼ! ԫՇ԰ՇՄՎԽՆՌՌՑՈԽ
ՁԾԹՈՈՄՁԻԹԺՄԽ ԫԞԪԹՐՁՅՍՅՋՇՄՎԽՆՌԻՇՆԻԽՆՌՊԹՌՁՇՆ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՋՇՄՎԽՆՌՆՇՌ

ԽՐԻԽԽԼՅՄԩԬԢԠԡՊԽԻՇՅՅԽՆԼՋ

ՌՀԹՌՋՇՄՎԽՆՌՀԹՎԽՆՇԽԾԾԽԻՌՇՆՋՍՊՎՁՎԹՄՆՇՊ

ՈՊՇԼՍԻԽԹՆՑՇՌՀԽՊԹԼՎԽՊՋԽԽԾԾԽԻՌՋԹՆԼՌՀԹՌ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՆՇՌԺԽԿՊԽԹՌԽՊՌՀԹՆՅՄԩ

 ԥՍՌԻՀՁՆՋՇՆԱԥ԰ՀՁՄՄԹԺԽԽՊԫԴՁՆՌԽՊԪԧԭՁԻՃԾՇՊԼԡԟ
ԞԻՍՌԽԹՆԼԻՀՊՇՆՁԻԽԾԾԽԻՌՋՇԾԻԹՊՊՁԽՊՋՇՄՎԽՆՌՋՁՆԹՉՍԹՌՁԻՇՊԿԹՆՁՋՅՋԞԻՊՁՌՁԻԹՄՊԽՎՁԽՏԯԽՎՁԽՏԞՉՍԹՌՁԻ

ԱՇՐՁԻՇՄՇԿՑՈՈ-

 ԬԢԠԡ
ԤՍՁԼԹՆԻԽԡՇԻՍՅԽՆՌՇՆԞՉՍԹՌՁԻԱՇՐՁԻՁՌՑԱԽՋՌՁՆԿՇԾԡՁԾԾՁԻՍՄՌ԰ՍԺՋՌԹՆԻԽՋԹՆԼԪՁՐՌՍՊԽՋԢՆՎՁՊՇՆՅԽՆՌԹՄԥԽԹՄՌՀԹՆԼ԰ԹԾԽՌՑԭՍԺՄՁԻԹՌՁՇՆՋ԰ԽՊՁԽՋՇՆ

ԱԽՋՌՁՆԿԹՆԼԞՋՋԽՋՋՅԽՆՌԫՇԭԹՊՁՋԣՊԹՆԻԽ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !"	#$

	
	


ԴԹՋԹՈՇՋՁՌՁՎԽԻՇՆՌՊՇՄՍՋԽԼ! ԫՇԭՇՋՁՌՁՎԽԻՇՆՌՊՇՄ
ՁԾԹՈՈՄՁԻԹԺՄԽ ԫԞԭՇՋՁՌՁՎԽԻՇՆՌՊՇՄԻՇՆԻԽՆՌՊԹՌՁՇՆ
Ջ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԫԞ

ԭՀՇՌՇՈԽՊՁՇԼ ՀՊՋՄՁԿՀՌ

ՀՊՋԼԹՊՃ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՈՀՇՌՇՈԽՊՁՇԼ


ՄՁԿՀՌԼԹՊՃ

ԩՁԿՀՌՁՆՌԽՆՋՁՌՑԹՌՏԹՌԽՊ0ՋՋՍՊԾԹԻԽ

-ԨՄՍՐ ԢԭԞՊԽԻՇՅՅԽՆԼՋՄՁԿՀՌՁՆՌԽՆՋՁՌՑ-

ԨՄՍՐ
ԹՌՏԹՌԽՊ0ՋՋՍՊԾԹԻԽ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

 !"	#$

	
	


ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋ ԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՏԹՋ

ՀԽՄԼՍՆԼԽՊԹՅԺՁԽՆՌԻՇՆԼՁՌՁՇՆՋՈՊՇՌԽԻՌԽԼԾՊՇՅՄՁԿՀՌՅՁՐԽԼԻՇՆՌՁՆՍՇՍՋՄՑՍՋՁՆԿԹՌՇՈԼՇՏՆԽՄԽԻՌՊՁԻՅՁՐԽՊԹՆԼԹՈՈԽԹՊԽԼԻՄԽԹՊԹՆԼԻՇՄՇՊՄԽՋՋԱՀԽՌԽՋՌՋՇՄՍՌՁՇՆՋԹՋՇԺՋԽՊՎԽԼՁՆՌՀԽՅՁՐՁՆԿԻՀԹՅԺԽՊՋԹՆԼՌԽՋՌԻՀԹՅԺԽՊՋԹՈՈԽԹՊԽԼԻՄԽԹՊԹՆԼԻՇՄՇՊՄԽՋՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԞՌՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆՌՀԽՋՇՄՍՌՁՇՆՋՁՆՌՀԽՅՁՐՁՆԿԻՀԹՅԺԽՊՋՏԽՊԽԻՄԽԹՊԹՆԼԻՇՄՇՊՄԽՋՋ

ԭՊԽՄՁՅՁՆԹՊՑ ՌՊՁԹՄՋ ՋՀՇՏԽԼ ՌՀԹՌ ՅԹՄԹՌՀՁՇՆՋՌՇԻՃՋ ՈՊԽՈԹՊԽԼ ՁՆ ԹԻՁԼՁԾՁԽԼ ԼԽՁՇՆՁՒԽԼՏԹՌԽՊՏԽՊԽՋՌԹԺՄԽԾՇՊԹՌՄԽԹՋՌՌՀՊԽԽԼԹՑՋԞՈՊՁՅԹՊՑՋՌՇԻՃ ՋՇՄՍՌՁՇՆ ՇԾ ՅԹՄԹՌՀՁՇՆ ՌԽԻՀՆՁԻԹՄ ԹՌ ԹՆՇՅՁՆԹՄ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՇԾ  ՅԿ ԹՁԩՏԹՋ

ՈՊԽՈԹՊԽԼ ՁՆ ԼԽՁՇՆՁՒԽԼ ՏԹՌԽՊ 
ՎՁԹ ՊԽՎԽՊՋԽՇՋՅՇՋՁՋԽՎԽՊՑՌՇԼԹՑՋԼՍՊՁՆԿՌՀԽՌԽՋՌԱՀԽ ՋՇՄՍՌՁՇՆ ՏԹՋ ԹԼՂՍՋՌԽԼ ՌՇ  ԹԻՌՁՎԽՁՆԿՊԽԼՁԽՆՌ ԼՍՊՁՆԿ ՈՊԽՈԹՊԹՌՁՇՆ ԺԹՋԽԼ ՇՆ ՌՀԽՌԽՋՌ ՋՍԺՋՌԹՆԻԽ ՈՍՊՁՌՑ 
 ԱՀԽ ԩ

ՋՌՇԻՃՋՇՄՍՌՁՇՆՏԹՋՈՊԽՈԹՊԽԼՁՆԹԩԿՄԹՋՋՌԹՆՃ԰ՁՆԻԽՅԹՄԹՌՀՁՇՆՁՋՋՍԺՂԽԻՌՌՇՋՁԿՆՁԾՁԻԹՆՌՀՑԼՊՇՄՑՋՁՋ ԹՌ Ոԥ % ՌՀԽ ՋՌՇԻՃՋ ՏԽՊԽ

ԹԻՁԼՁԾՁԽԼՌՇԹՌԹՊԿԽՌՈԥՇԾԹՈՈՊՇՐՁՅԹՌԽՄՑՍՋՁՆԿ  ՈՀՇՋՈՀՇՊՁԻ ԹԻՁԼ 
ԥԭԬ ՌՇ

ՅԹՁՆՌԹՁՆ ՋՌԹԺՁՄՁՌՑ ՇՎԽՊ ՌՀԽ ՅԹՐՁՅՍՅ ՋՌՇԻՃՊԽՆԽՏԹՄ ՁՆՌԽՊՎԹՄ ՇԾ ԹՈՈՊՇՐՁՅԹՌԽՄՑ  ՀՇՍՊՋ

ԱՀԽՈԥՇԾՌՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋՏԹՋԻՇՆԾՁՊՅԽԼԹՌ ՌՀԽ ԺԽԿՁՆՆՁՆԿ ԹՆԼ ԽՆԼ ՇԾ ԽԹԻՀ ՋՌՇԻՃՊԽՆԽՏԹՄՈԽՊՁՇԼԹՆԼՊԹՆԿԽԼԾՊՇՅՌՇ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ  ԩԬԮ ԫԞ ԫԞ ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌՌԽՋՌՁՌԽՅ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽՅԹՁՆՌԹՁՆԽԼԹՌԹ

ԻՇԽԾԾՁԻՁԽՆՌՇԾՎԹՊՁԹՌՁՇՆ
ԠԳ 2

ԱՊԽԹՌՅԽՆՌ    ԫԞԱՊԽԹՌՅԽՆՌ    ԫԞԱՊԽԹՌՅԽՆՌ    ԫԞ

ԞԺԺՊԽՎՁԹՌՁՇՆՋԠԳԠՇԽԾԾՁԻՁԽՆՌՇԾՎԹՊՁԹՌՁՇՆ

 ԣՇՊՌՀԽՆՇՅՁՆԹՄՅԿԹՁԩՌՊԽԹՌՅԽՆՌՌՀԽՋՌՍԼՑԹՍՌՀՇՊԻԹՄԻՍՄԹՌԽԼԹՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՇԾՅԿԹՁԩՍՋՁՆԿԡԹՑ

ՋԹՅՈՄԽՋՁՆՄՁԽՍՇԾԡԹՑՋԹՅՈՄԽՋԞԻԻՇՊԼՁՆԿՌՇՌՀԽՋՌՍԼՑԹՍՌՀՇՊԡԹՑՋԹՅՈՄԽՋԾՊՇՅՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՏԽՊԽՋՍՋՈԽԻՌ
ՁԽ

ՄՇՏԹՆԼՌՀԽՊԽԾՇՊԽՆՇՌՁՆԻՄՍԼԽԼՁՆՌՀԽԻԹՄԻՍՄԹՌՁՇՆՇԾՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆԱՀԽՄՇՏԹՆԹՄՑՌՁԻԹՄՊԽՋՍՄՌՋՏԽՊԽԺԽՄՁԽՎԽԼՌՇԺԽԼՍԽՌՇՋԹՅՈՄՁՆԿՇՊԹՆԹՄՑՌՁԻԹՄԽՊՊՇՊԞԼԼՁՌՁՇՆԹՄՋԹՅՈՄԽՋՏԽՊԽԻՇՄՄԽԻՌԽԼԾՊՇՅՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՌՏՇԼԹՑՋՄԹՌԽՊ
ԡԹՑԹՆԼՏԽՊԽ

ԻՇՆՋՁԼԽՊԽԼՌՇԺԽՊԽՈՊԽՋԽՆՌԹՌՁՎԽՇԾՌՀԽԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽՌԽՋՌԻՀԹՅԺԽՊՋՇՆԡԹՑԹՆԼՍՋԽԼՁՆՌՀԽԻԹՄԻՍՄԹՌՁՇՆՇԾՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺՑՌՀԽՋՌՍԼՑԹՍՌՀՇՊ԰ՁՆԻԽԹՆՇՆՋՈԽԻՁԾՁԻՊԹՌՁՇՆԹՄԽՏԹՋԿՁՎԽՆԾՇՊՌՀԽՄՇՏՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾՌՀԽՆՇՅՁՆԹՄՅԿ

ԹՁԩՌՊԽԹՌՅԽՆՌՇՆԡԹՑՌՀԽՊԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼԹՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՇԾՅԿԹՁԩՍՋՁՆԿԡԹՑՋԹՅՈՄԽՋ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ



ՇԾ

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԰ՍՊՎՁՎԹՄԹՆԼԿԽՆԽՊԹՄՇԺՋԽՊՎԹՌՁՇՆՋՌՇՌԹՄՄԽՆԿՌՀՏԽՌՏԽՁԿՀՌՋՆՇՍՌՌՇՎԽՆՌ

ՄԽՆԿՌՀՀՁՆԼՄՁՅԺՄԽՆԿՌՀԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԹՆԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑ԰ՍՊՎՁՎԹՄՏԹՋ

ՅԽԹՋՍՊԽԼՌՀՊՇՍԿՀՇՍՌՌՀԽՋՌՍԼՑՏՀՁՄԽԿՊՇՏՌՀԹՆԼԼԽՎԽՄՇՈՅԽՆՌՅԽԹՋՍՊԽՅԽՆՌՋ

ՏԽՊԽՌԹՃԽՆՇՆԡԹՑԹՆԼԥՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄՅԽԹՋՍՊԽՅԽՆՌՋՏԽՊԽՌԹՃԽՆՇՆ

ԡԹՑՇՆՄՑ

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ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՇԺՋԽՊՎԹՌՁՇՆՋՇԾՅՇՊՌԹՄՁՌՑԹՆԼԻՄՁՆՁԻԹՄՋՁԿՆՋՇԻԻՍՊԼԹՁՄՑԹՌԹՅՁՆՁՅՍՅՇՌՀԽՊ

ՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽՊԽԻՇՅՅԽՆԼԽԼԹՋԾՇՄՄՇՏՋԫԣԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽ
ԼԹՑՋԹՆԼԹՆՑԹՋՑՆԻՀՊՇՆՇՍՋ

ԼԽՎԽՄՇՈՅԽՆՌՁՆԼՁԻԹՌԽԼԺՑՌԹԼՈՇՄԽՋՌՀԹՌԻԹՆՆՇՌԺԽԹՋՋՁԿՆԽԼԹՆԫԣՋՌԹԿԽ
ԼԹՑՋԹՆԼՀՁՆԼՄՁՅԺ

ՄԽՆԿՌՀ
ԼԹՑՋԹՆԼՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ
ԼԹՑՋԹՆԼԺՇԼՑՏԽՁԿՀՌ
ՌԽՋՌՁՆՁՌՁԹՌՁՇՆԾՇՊՇՈՌՁՇՆԹՄ

ՋՁՒԽԺԹՋԽԼՄԹՊՎԹՄՋԽՄԽԻՌՁՇՆԹՆԼՌՀՑՊՇՁԼԿՄԹՆԼԿՊՇՋՋՈԹՌՀՇՄՇԿՑԹՆԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑ
ԼԹՑԫՇՌԽՌՀԽ

ՀՁՋՌՇՈԹՌՀՇՄՇԿՑՋԽԻՌՁՇՆՇԾՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԹՄՋՇՁՆԻՄՍԼԽՋՌՀՑՊՇՁԼԿՊՇՋՋՈԹՌՀՇՄՇԿՑՇԺՋԽՊՎԹՌՁՇՆՋ

Page 33 of 294
  	
 
	  	
  	
 	   	 
 	     
 	 	  

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

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ԣՊՇՅԡԹՑՋՌՀՊՇՍԿՀՋՍՊՎՁՎԹՄՏԹՋԹՆԼՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆ

ՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽԣՊՇՅԡԹՑ

ՌՀՊՇՍԿՀՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆՋՍՊՎՁՎԹՄՏԹՋԹՆԼՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆ

ՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ

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ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ



԰ԹՅՈՄԽՋՁՒԽԹՆԼԻՍՅՍՄԹՌՁՎԽՅՇՊՌԹՄՁՌՑՎԹՄՍԽՋԹՌԡԹՑՈՊՁՇՊՌՇՁՆՌԽՊՁՅՋԹԻՊՁԾՁԻԽ

ԱՀԽՅԽԼՁԹՆԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽՇԾՌՀԽՌԹԼՈՇՄԽՋՇՆԡԹՑՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼ

ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԹՋ
ԱԹԺՄԽԱՀԽՅԽԼՁԹՆԼԽՎԽՄՇՈՅԽՆՌԹՄ

ՋՌԹԿԽՋՇԾՌԹԼՈՇՄԽՋՇՆԡԹՑՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿ

ԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԽՊԽԹՆԼՊԽՋՈԽԻՌՁՎԽՄՑԱՀԽՌՀԹՆԼՌՀՈԽՊԻԽՆՌՁՄԽՋՇԾ

ՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՌԹԼՈՇՄԽՋԹՆԼ ՊԽՋՈԽԻՌՁՎԽՄՑԼՁԼՆՇՌԼՁԾԾԽՊԺՑՅՇՊԽՌՀԹՆԾՇՍՊԫԣՋՌԹԿԽՋԫՇ

ԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌՏԹՋՊԽՈՇՊՌԽԼ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

ԬՆԡԹՑՌՀԽՊԽՏԽՊԽ


ԹՆԼ
ՌԹԼՈՇՄԽՋԿՊԽԹՌԽՊՌՀԹՆՋՌԹԿԽ


ՁԽՋՌԹԿԽՋՌՇՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩ

ՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋ ՊԽՋՈԽԻՌՁՎԽՄՑ ԱՀԽՋԽՌԹԼՈՇՄԽՋՏԽՊԽԽՐԻՄՍԼԽԼԾՊՇՅԹՆԹՄՑՋԽՋ ՏՁՌՀ ՊԽՋՈԽԻՌՌՇԺՇԼՑ

ՏԽՁԿՀՌՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀԹՆԼՀՁՆԼՄԽԿՄԽՆԿՌՀ
ՁՆԻՄՍԼՁՆԿՆՇՊՅԹՄՁՒԽԼՀՁՆԼՄԽԿՄԽՆԿՌՀԺՑՌՀԽՊԽՎՁԽՏԽՊ

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ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ      

      

      

      

ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ

ԪԽԹՆԡԹՑՆՇՊՅԹՄՁՒԽԼՀՁՆԼՄՁՅԺՄԽՆԿՌՀՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼ

ՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԽՊԽԹՆԼՅՅՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽ

ԪԽԹՆԡԹՑՆՇՊՅԹՄՁՒԽԼՀՁՆԼՄՁՅԺՄԽՆԿՌՀՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼ

ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԽՊԽԹՆԼՅՅՊԽՋՈԽԻՌՁՎԽՄՑ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

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 ԰ՍՅՅԹՊՑՊԽՋՍՄՌՋԾՇՊՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ
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ԪԽԹՆ ԡԹՑ  ԰Գԩ ՁՆ ՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ ԹՆԼ ՅԽԹՆՅԽԹՋՍՊԽԼ   ԹՆԼ  ՅԿ ԹՁԩ

ՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԽՊԽԹՆԼՅՅՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽԪԽԹՆ԰ԳԩՇՆԡԹՑ

ՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԽՊԽ

ԹՆԼՅՅՊԽՋՈԽԻՌՁՎԽՄՑ

ԪԽԹՆԡԹՑԺՇԼՑՏԽՁԿՀՌՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿ

ԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԽՊԽԹՆԼԿՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽԪԽԹՆ

ԺՇԼՑՏԽՁԿՀՌՋՇՆԡԹՑՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿ

ԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԽՊԽԹՆԼԿՊԽՋՈԽԻՌՁՎԽՄՑ

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ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼ԰ԡ԰ՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ

 ԞՄՋՇՊԽԾԽՊՊԽԼՌՇԹՋ՟ՏԽՌՏԽՁԿՀՌՠՁՆՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽ

԰ԹՅՈՄՁՆԿԾՊՇՅՌՀԽԹՎԹՁՄԹԺՄԽՌԹԼՈՇՄԽՋԾՇՊՀՁՋՌՇՈԹՌՀՇՄՇԿՑՏԹՋԼՇՆԽՌՇՋԽՄԽԻՌՋՌԹԿԽՅԹՌԻՀԽԼՁՆԼՁՎՁԼՍԹՄՋ

ՏՀԽՆ ՈՇՋՋՁԺՄԽ  ԱՀԽՊԽ ՏԽՊԽ ՆՇ ՊԽՈՄՁԻԹՌԽՋ ՏՁՌՀ ՅՇՊԽ ՌՀԹՆ ԾՁՎԽ ՌԹԼՈՇՄԽՋ ԹՌ ՌՀԽ ԻՇՆՌՊՇՄ ՅԽԼՁԹՆ

ԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԱՀԽՊԽԾՇՊԽԹՄՄՌԹԼՈՇՄԽՋԹՌՌՀԹՌՋՌԹԿԽՏԽՊԽՋԽՄԽԻՌԽԼԾՊՇՅԽԹԻՀՊԽՈՄՁԻԹՌԽԴՀԽՆ

ՌՀԽՊԽՏԽՊԽԾԽՏԽՊՌՀԹՆԾՁՎԽՌԹԼՈՇՄԽՋԹՌՌՀԹՌՋՌԹԿԽՁՆԹ ՊԽՈՄՁԻԹՌԽԹԼԼՁՌՁՇՆԹՄՌԹԼՈՇՄԽՋՏԽՊԽ ՊԹՆԼՇՅՄՑ

ՋԽՄԽԻՌԽԼ
ՍՋՁՆԿԹՊԹՆԼՇՅՆՍՅԺԽՊԿԽՆԽՊԹՌՇՊԾՊՇՅՌՀԽԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽՋՂՍՋՌԹԺՇՎԽԹՆԼՂՍՋՌԺԽՄՇՏ

ՌՀԽՅԽԼՁԹՆԻՇՆՌՊՇՄԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽ
ՁԽԫԣԹՆԼԦՆԹԾԽՏԻԹՋԽՋՋԽԻՌՁՇՆՁՆԿՇԾՌՀԽՀԽԹԼՋ

ՇԾՌՀԽՌԹԼՈՇՄԽՋԼՁԼՆՇՌՈՊՇՎՁԼԽՋՄՁԼԽՋՌՀԹՌԹՄՄՇՏԽԼԾՇՊԹԻԻՍՊԹՌԽԹՋՋԽՋՋՅԽՆՌՇԾՌՀԽՋՁՒԽՇԾՌՀԽՌՀՑՊՇՁԼ

ԿՄԹՆԼՋԦՆՋՍԻՀԻԹՋԽՋՌՀԽՋԽՋՄՁԼԽՋՏԽՊԽՋԽՌԹՋՁԼԽԹՆԼԹԼԼՁՌՁՇՆԹՄՌԹԼՈՇՄԽՋՏԽՊԽՋԽՄԽԻՌԽԼՁՆԹԻԻՇՊԼԹՆԻԽ

ՏՁՌՀ ՌՀԽ ՈՊԽՎՁՇՍՋՄՑ ԼԽՋԻՊՁԺԽԼ ՋԻՀԽՅԽ ԹՆԼ ՌՀԽՋԽ ԹԼԼՁՌՁՇՆԹՄ ՌԹԼՈՇՄԽՋ ՏԽՊԽ ՈՊՇԻԽՋՋԽԼ ՌՇ ՈՊՇՎՁԼԽ

ՊԽՈՄԹԻԽՅԽՆՌՋՄՁԼԽՋ

ԱՀԽՊԽՏԽՊԽՆՇԼՁԹԿՆՇՋՌՁԻԹԺՆՇՊՅԹՄՁՌՁԽՋՇԺՋԽՊՎԽԼԺՑՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄԽՐԹՅՁՆԹՌՁՇՆՇԾՌՀԽՌՀՑՊՇՁԼՋՇԾ

ՌԹԼՈՇՄԽՋՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈՏՀԽՆԻՇՅՈԹՊԽԼՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՌՌՀԽՌԽՊՅՁՆԹՌՁՇՆՇԾՌՀԽ

ԼԹՑՌԽՋՌ



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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

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ՊԽՅԹՊՃԹԺՄԽԪՁՄԼԪՇԼԽՊԹՌԽ԰ԽՎԽՊԽ԰ԽԽԬԢԠԡԫՇԾՇՊՊԽԾԽՊԽՆԻԽ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

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Page 39 of 294
  	
 
	  	
  	
 	   	 
 	     
 	 	  

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

ԱԹԼՈՇՄԽՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԿԽՆԽՊԹՄՄՑԹՈՈԽԹՊԽԼՆՇՊՅԹՄՌՀՊՇՍԿՀՇՍՌՌՀԽՌԽՋՌ

ԬՆԽՌԹԼՈՇՄԽՁՆԽԹԻՀՇԾՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏԹՋՇԺՋԽՊՎԽԼ

ՄՑՁՆԿՇՆՌՀԽԺՇՌՌՇՅՇԾՌՀԽՌԹՆՃՇՆԡԹՑԺՍՌԹՈՈԽԹՊԽԼՆՇՊՅԹՄԺՑԡԹՑԭԽՊՁՇԼՁԻՇԺՋԽՊՎԹՌՁՇՆՋՇԾԹ

ԾԽՏՌԹԼՈՇՄԽՋՌՀԹՌՎՁՋՍԹՄՄՑԹՈՈԽԹՊԽԼՋՅԹՄՄԽՊՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽԻՇՆՌՊՇՄՌԹԼՈՇՄԽՋՏԽՊԽՆՇՌԽԼՁՆՌՀԽ

ՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈԺՍՌՌՀԽՋՁՒԽՏԹՋՆՇՌԻՇՆԾՁՊՅԽԼԺՑՅԽԹՋՍՊԽՅԽՆՌՋՇԾ

ՌՀԽՌԹԼՈՇՄԽՋ ԹՌՌՀԽՌՁՅԽ ՇԾ ՇԺՋԽՊՎԹՌՁՇՆԟԽԿՁՆՆՁՆԿՇՆ ԡԹՑ ՇԾՌՀԽՌԽՋՌ ԹՆ ՁՆԻՊԽԹՋՁՆԿ ՆՍՅԺԽՊ ՇԾ

ՌԹԼՈՇՄԽՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՀԹԼԻՍՊՎԽԼՌԹՁՄՋԥՇՏԽՎԽՊՌՀԽՆՍՅԺԽՊՋՇԾ

ՌԹԼՈՇՄԽՋ ՁՆՌՀԽՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈՋՏՁՌՀ ԻՍՊՎԽԼ ՌԹՁՄՋՏԽՊԽ ԻՇՅՈԹՊԹԺՄԽ ՌՇՌՀԽ ՆՍՅԺԽՊՋ ՁՆՌՀԽ ԻՇՆՌՊՇՄ

ԿՊՇՍՈ  ԱՀԽ ՋՌՍԼՑ ԹՍՌՀՇՊ ՆՇՌԽԼՌՀԹՌ ՁՆԾԽԽԼՁՆԿՌՊՁԹՄՋ ՁՌ ՀԹՋ ԺԽԽՆ ՋՀՇՏՆ ՌՀԹՌԾԽԽԼՁՆԿ ՊԹՌԽՋ ԹՄՇՆԽ

ԻՇՆՌՊՁԺՍՌԽՌՇՌՀԽԹՅՇՍՆՌՇԾԻՍՊՎԹՌՍՊԽՇԺՋԽՊՎԽԼ

ԦՆԻՁԼԽՆՌԼԹՌԹՈՊՇՎՁԼԽԼՁՆԱԹԺՄԽԹՊԽԾՇՊԡԹՑՇԺՋԽՊՎԹՌՁՇՆՋՇՆՄՑԹՋՌՀԽՊԽՁՋՆՇՏԹՑՌՇԼԽՌԽՊՅՁՆԽ

ՌՀԽՌՇՌԹՄՆՍՅԺԽՊՇԾՁՆԻՁԼԽՆԻԽԾՊՇՅՌՀԽԼԹՌԹՈՊՇՎՁԼԽԼ

   

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 ԫՇՌԽՌՀԹՌԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌ
ՍՆԹԺՄԽՌՇՋՌԹԿԽՁՋՊԽՈՇՊՌԽԼՈՊԽՎՁՇՍՋՄՑՁՆ 
 

ԹՆԼՆՇՌ

ՀԽՊԽ

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ԭԹԿԽ



ՇԾ

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԰ՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋԽՋՏԽՊԽՈԽՊԾՇՊՅԽԼՌՇԽՎԹՄՍԹՌԽԼՁԾԾԽՊԽՆԻԽՋԺԽՌՏԽԽՆՌՊԽԹՌՅԽՆՌԹՆԼԻՇՆՌՊՇՄԿՊՇՍՈՋԾՇՊ

ԽԹԻՀՇԾՌՀԽԾՇՄՄՇՏՁՆԿԺՁՇՄՇԿՁԻԹՄԽՆԼՈՇՁՆՌՋՏՀԽՆՈՇՋՋՁԺՄԽՋՍՊՎՁՎԹՄԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԺՇԼՑՏԽՁԿՀՌ

ՏՀՇՄԽԺՇԼՑՄԽՆԿՌՀՋՆՇՍՌՌՇՎԽՆՌՄԽՆԿՌՀՋԹՆԼՀՁՆԼՄՁՅԺՄԽՆԿՌՀ
ՆՇՊՅԹՄՁՒԽԼԺՑԼՁՎՁԼՁՆԿԺՑՌՀԽ԰Գԩ

԰ՁՆԻԽՌՀԽՊԽՏԽՊԽՆՇԹԺՆՇՊՅԹՄՁՌՁԽՋՁՆՌՀԽՌՀՑՊՇՁԼՀՁՋՌՇՄՇԿՑՆՇՋՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋԽՋՏԽՊԽՈԽՊԾՇՊՅԽԼՇՆ

ՀՁՋՌՇՄՇԿՑՊԽՋՍՄՌՋ

ԪՇՋՌԽՆԼՈՇՁՆՌՋՏԽՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԻՇՅՈՄԽՅԽՆՌԹՊՑՋՌԹՌՁՋՌՁԻԹՄԹՈՈՊՇԹԻՀԽՋԦԾՊԽՋՈՇՆՋԽՋԹՈՈԽԹՊԽԼՌՇ

ԺԽՅՇՆՇՌՇՆՁԻ
ՌՊԽՆԼՁՆԿՁՆՇՆԽԼՁՊԽԻՌՁՇՆԹՋՌԽՈԼՇՏՆԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌՏԹՋՍՋԽԼՌՇ

ԽՎԹՄՍԹՌԽՌՊԽՆԼՋՁՆՌՀԽՊԹՆՃՋՇԾՊԽՈՄՁԻԹՌԽՅԽԹՆՋՌՇԼԽՌԽՊՅՁՆԽՈՇՋՋՁԺՄԽԻՇՆԻԽՆՌՊԹՌՁՇՆՊԽՋՈՇՆՋՁՎԽՌՊԽՆԼՋ

ԹՅՇՆԿՌՀԽՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԞՄՄԽՆԼՈՇՁՆՌՋԽՐԻԽՈՌԾՇՊՋՍՊՎՁՎԹՄԹՆԼԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԼԹՌԹՏԽՊԽԹՄՋՇ

ԹՆԹՄՑՒԽԼԺՑՈԽՊԾՇՊՅՁՆԿՈԹՁՊՏՁՋԽԻՇՅՈԹՊՁՋՇՆՋՍՋՁՆԿԡՍՆՆԽՌՌ0ՋՅՍՄՌՁՈՄԽԻՇՅՈԹՊՁՋՇՆՌԽՋՌՌՇԼԽՌԽՊՅՁՆԽ

ՏՀՁԻՀՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԼՁԾԾԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑԾՊՇՅՌՀԽԻՇՆՌՊՇՄԿՊՇՍՈԡԹՌԹԾՇՊ ԽՆԼՈՇՁՆՌՋԹՆԹՄՑՒԽԼԺՑ

ԡՍՆՆԽՌՌ0ՋՌԽՋՌՏԽՊԽԽՎԹՄՍԹՌԽԼԾՇՊՆՇՊՅԹՄՁՌՑՍՋՁՆԿ԰ՀԹՈՁՊՇԴՁՄՃ0ՋՌԽՋՌԹՆԼԾՇՊՀՇՅՇԿԽՆԽՁՌՑՇԾՎԹՊՁԹՆԻԽ

ՍՋՁՆԿԩԽՎԽՆԽ0ՋՌԽՋՌ
ԱՀԽԼԹՌԹԾՇՊՌՀՁՋՋՌՍԼՑՈԹՋՋԽԼՌՀԽԹՋՋՍՅՈՌՁՇՆՋԾՇՊՆՇՊՅԹՄՁՌՑԹՆԼ

ՀՇՅՇԿԽՆԽՁՌՑՇԾՎԹՊՁԹՆԻԽ

԰ՍՊՎՁՎԹՄԹՆԼԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԼԹՌԹՏԽՊԽՆՇՌԹՅԽՆԹԺՄԽՌՇՌՀԽՋՌԹՌՁՋՌՁԻԹՄՅԽՌՀՇԼՋՍՋԽԼԾՇՊԹՆԹՄՑՋՁՋ

ՇԾՌՀԽՇՌՀԽՊԽՆԼՈՇՁՆՌՋ԰ՍՊՎՁՎԹՄՏԹՋԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽՋՌԽՈԼՇՏՆԧՇՆԻՃՀԽԽՊԽԱԽՊՋՌՊԹՌՊԽՆԼՌԽՋՌԹՆԼ

ԣՁՋՀԽՊ0ՋԢՐԹԻՌՌԽՋՌԦՆԹԻԻՇՊԼԹՆԻԽՏՁՌՀՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋՇԾԬԠ԰ԭԭԹՆԹՄՑՋՁՋՇԾՅԽՌԹՅՇՊՈՀՁԻ

ՋՌԹԿԽՏԹՋՈԽՊԾՇՊՅԽԼՍՋՁՆԿՌՀԽՅՍՄՌՁՉՍԹՆՌՁՄԽԹՆԹՄՑՋՁՋԼԽՎԽՄՇՈԽԼԺՑԱ԰ՈՊՁՆԿԽՊԹՆԼԧԤՊԽԽՆԹՆԼ

ՍՋՁՆԿՌՀԽՋՌԽՈԼՇՏՆԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌՇՆՋՌԹԿԽՉՍԹՆՌՁՄԽՋՁՆՌՀԽՊԽՈՄՁԻԹՌԽԻՀԹՅԺԽՊՋ

԰ՌԹՌՁՋՌՁԻԹՄՌԽՋՌՋՍՋԽԼՌՇԽՎԹՄՍԹՌԽՌՊԽԹՌՅԽՆՌԽԾԾԽԻՌՋՏԽՊԽՈԽՊԾՇՊՅԽԼԹՌԹԻՇՆԾՁԼԽՆԻԽՄԽՎԽՄՇԾ


ՍՆՄԽՋՋՇՌՀԽՊՏՁՋԽՆՇՌԽԼՏՁՌՀ԰Ԟ԰ՋՇԾՌՏԹՊԽ

 

ԱՀԽՊԽՏԽՊԽՆՇՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԽԾԾԽԻՌՋՇՆՋՍՊՎՁՎԹՄՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽ

ԻՇՆՌՊՇՄԼՍՊՁՆԿՌՀԽՌԽՋՌ
Ո 

ԞԻԻՇՊԼՁՆԿՌՇՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌՌՀԽՊԽՏԹՋՆՇՋՁԿՆՁԾՁԻԹՆՌՌՊԽՆԼՁՆՌՀԽԡԹՑԺՇԼՑ

ՄԽՆԿՌՀԼԹՌԹ
Ո ԡՍՆՆԽՌՌ0ՋՌԽՋՌՋՀՇՏԽԼՌՀԹՌՌՀԽՁՆԻՊԽԹՋԽՁՆԡԹՑԺՇԼՑՄԽՆԿՌՀՁՆՌՀԽ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ



ՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

ՅԿԹՁԩՆՇՅՁՆԹՄ
ՅԿԹՁԩՅԽԹՆՅԽԹՋՍՊԽԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽ

ՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՏԹՋՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌ
ՈԱՀԽԼՁԾԾԽՊԽՆԻԽԾՊՇՅՌՀԽԻՇՆՌՊՇՄՏԹՋՆՇՌ

ԻՇՆՋՁԼԽՊԽԼՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼՋՁՆԻԽՌՀԽՊԽՏԽՊԽՆՇԼՁԾԾԽՊԽՆԻԽՋՁՆՆՇՊՅԹՄՁՒԽԼՀՁՆԼՄՁՅԺՄԽՆԿՌՀՇՊ

ՋՌԹԿԽ ՇԾ ԼԽՎԽՄՇՈՅԽՆՌ ՇՆ ԡԹՑ  ԦՌ ՁՋ ԹՄՋՇ ԺԹՋԽԼ ՇՆ Թ ՊԽՄԹՌՁՎԽՄՑ ՋՅԹՄՄ ՋԹՅՈՄԽ ՋՁՒԽ ՇԾ  ԾՊՇԿՋ

ԞԼԼՁՌՁՇՆԹՄՄՑՌՀԽԼՁԾԾԽՊԽՆԻԽՏԹՋՆՇՄՇՆԿԽՊԹՈՈԹՊԽՆՌՇՆԡԹՑԱՀԽԡԹՑՁՆԻՊԽԹՋԽՁՆԺՇԼՑՄԽՆԿՌՀ

ՁՆՌՀԽՅԿԹՁԩՆՇՅՁՆԹՄ
ՅԿԹՁԩՅԽԹՆՅԽԹՋՍՊԽԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՊԽՄԹՌՁՎԽՌՇՌՀԽԻՇՆՌՊՇՄ

ՏԹՋՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌ
ՈԾՇՊԺՇՌՀՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌԹՆԼԡՍՆՆԽՌՌ0ՋՌԽՋՌ

ԥՇՏԽՎԽՊՌՀԽՁՆԻՊԽԹՋԽՏԹՋՋՄՁԿՀՌ
ՏԹՋՆՇՌՋՍՈՈՇՊՌԽԼԺՑԹՆՁՆԻՊԽԹՋԽՁՆՇՌՀԽՊԿՊՇՏՌՀՈԹՊԹՅԽՌԽՊՋ

ԹՆԼՌՀԽՊԽԾՇՊԽՏԹՋՆՇՌԻՇՆՋՁԼԽՊԽԼՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼ

ԞԻԻՇՊԼՁՆԿՌՇՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌՌՀԽՊԽՏԹՋՆՇՋՁԿՆՁԾՁԻԹՆՌՌՊԽՆԼՁՆՌՀԽԡԹՑՏԽՌՏԽՁԿՀՌ

ԼԹՌԹ
Ո ԥՇՏԽՎԽՊԡՍՆՆԽՌՌ0ՋՌԽՋՌՁՆԼՁԻԹՌԽԼՌՀԽՊԽՏԹՋԹՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌՁՆԻՊԽԹՋԽՁՆ

ԡԹՑՏԽՌՏԽՁԿՀՌՁՆՌՀԽՅԿԹՁԩՆՇՅՁՆԹՄ
ՅԿԹՁԩՅԽԹՆՅԽԹՋՍՊԽԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈ

ՁՆ ԻՇՅՈԹՊՁՋՇՆ ՌՇՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ 
Ո   ԩՁՃԽՌՇՌԹՄ ՄԽՆԿՌՀՅԽԹՋՍՊԽՅԽՆՌՋ ՇՆԡԹՑ  ՌՀՁՋ

ԼՁԾԾԽՊԽՆԻԽՁՆՏԽՌՏԽՁԿՀՌԾՊՇՅՌՀԽԻՇՆՌՊՇՄՏԹՋՆՇՌԻՇՆՋՁԼԽՊԽԼՌՇԺԽՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼՋՁՆԻԽՌՀԽՊԽՏԽՊԽ

ՆՇԼՁԾԾԽՊԽՆԻԽՋՁՆՆՇՊՅԹՄՁՒԽԼՀՁՆԼՄՁՅԺՄԽՆԿՌՀՇՊՋՌԹԿԽՇԾԼԽՎԽՄՇՈՅԽՆՌՇՆԡԹՑԦՌՁՋԹՄՋՇԺԹՋԽԼՇՆ

ԹՊԽՄԹՌՁՎԽՄՑՋՅԹՄՄՋԹՅՈՄԽՋՁՒԽՇԾԾՊՇԿՋԹՆԼՌՀԽԼՁԾԾԽՊԽՆԻԽՏԹՋՆՇՄՇՆԿԽՊԹՈՈԹՊԽՆՌՇՆԡԹՑԱՀԽՊԽ

ՏԽՊԽՆՇՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԽԾԾԽԻՌՋՇՆԡԹՑՏԽՌՏԽՁԿՀՌՋՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇ

ՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ
Ո 

ԞԻԻՇՊԼՁՆԿՌՇՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌՊԽՆԼՌԽՋՌՌՀԽՊԽՏԹՋՆՇՋՁԿՆՁԾՁԻԹՆՌՌՊԽՆԼՁՆՌՀԽԡԹՑՋՆՇՍՌՌՇ

ՎԽՆՌՄԽՆԿՌՀԼԹՌԹ
Ո ԥՇՏԽՎԽՊԡՍՆՆԽՌՌ0ՋՌԽՋՌՁՆԼՁԻԹՌԽԼՌՀԽՊԽՏԹՋԹՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌ

ՁՆԻՊԽԹՋԽ ՁՆ ԡԹՑ  ՋՆՇՍՌՌՇՎԽՆՌ ՄԽՆԿՌՀ ՁՆ ՌՀԽ  ՅԿ ԹՁԩ ՆՇՅՁՆԹՄ 
 ՅԿ ԹՁԩ ՅԽԹՆ

ՅԽԹՋՍՊԽԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ
ՈԱՀՁՋԼՁԾԾԽՊԽՆԻԽՁՆՋՆՇՍՌ

ՌՇՎԽՆՌՄԽՆԿՌՀՇՆԡԹՑՁՋԻՇՆՋՁՋՌԽՆՌՏՁՌՀՌՀԽԼՁԾԾԽՊԽՆԻԽՋՋԽԽՆՁՆՄԽՆԿՌՀԹՆԼՏԽՌՏԽՁԿՀՌԹՆԼՄՁՃԽ

ՌՀՇՋԽՇՌՀԽՊՈԹՊԹՅԽՌԽՊՋՏԹՋՆՇՌԻՇՆՋՁԼԽՊԽԼՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԱՀԽՊԽՏԽՊԽՆՇՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌ

ԽԾԾԽԻՌՋՇՆԡԹՑՋՆՇՍՌՌՇՎԽՆՌՄԽՆԿՌՀՋՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ


Ո 

ԱՀԽՊԽՏԽՊԽՆՇՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԽԾԾԽԻՌՋՇՆՆՇՊՅԹՄՁՒԽԼՀՁՆԼՄՁՅԺՄԽՆԿՌՀՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈՁՆ

ԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ
Ո ՇՆԡԹՑՇՊ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ



ՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

ԱՀԽՊԽ ՏԽՊԽ ՆՇ ՋՌԹՌՁՋՌՁԻԹՄՄՑ ՋՁԿՆՁԾՁԻԹՆՌ ԽԾԾԽԻՌՋ ՇՆ ԼԽՎԽՄՇՈՅԽՆՌԹՄ ՋՌԹԿԽ ՁՆ ԹՆՑ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ՁՆ

ԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ
Ո ՇՆԡԹՑՋՇՊ

ԱՀԽՊԽՏԽՊԽՆՇՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՇԺՋԽՊՎԽԼԺՑՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄԽՐԹՅՁՆԹՌՁՇՆՇԾՌՀԽՌՀՑՊՇՁԼՋՇԾ

ՌԹԼՈՇՄԽՋՁՆԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈԹՌՌԽՊՅՁՆԹՌՁՇՆՇԾՌՀԽԼԹՑՌԽՋՌ

ԬՎԽՊԹՄՄՌՀԽՊԽՏԽՊԽՆՇԹՈՈԹՊԽՆՌՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՇՆՌԹԼՈՇՄԽԿՊՇՏՌՀԹՆԼԼԽՎԽՄՇՈՅԽՆՌՇԺՋԽՊՎԽԼ

ՁՆՌՀՁՋՌԽՋՌ

  	
 
   
  
   


   
 	 !

ԱՀԽՊԽՎՁԽՏԽՊՎՁՋՍԹՄՄՑԹՆԹՄՑՒԽԼԼԹՌԹԾՇՊՅՇՊՌԹՄՁՌՑԺԽԻԹՍՋԽՌՀԽԼԹՌԹԼՁԼՆՇՌՅԽԽՌ

ՌՀԽԹՋՋՍՅՈՌՁՇՆՋԾՇՊՌՀԽԣՁՋՀԽՊ0ՋԢՐԹԻՌՌԽՋՌԱՀԽ ՊԽՅԹՁՆՁՆԿԽՆԼՈՇՁՆՌՋՏԽՊԽԹՆԹՄՑՒԽԼՁՆԠԢԱԦ԰ԬՆ

ԡԹՑ԰ԳԩԥԩԩՆՇՊՅԹՄՁՒԽԼԥԩԩ
ԥԩԩ԰ԳԩԹՆԼՏԽՌՏԽՁԿՀՌՏԽՊԽՆՇՌԻՇՆՋՁՋՌԽՆՌՏՁՌՀԹՅՇՆՇՌՇՆՁԻ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՊԽՋՈՇՆՋԽԱՀԽՋԽԼԹՌԹՏԽՊԽՆՇՊՅԹՄՄՑԼՁՋՌՊՁԺՍՌԽԼ
԰ՀԹՈՁՊՇԴՁՄՃ0ՋՏՁՌՀԽՉՍԹՄ

ՎԹՊՁԹՆԻԽ
ԩԽՎԽՆԽ0ՋՌԽՋՌ ԹՆԼՌՀԽՊԽԾՇՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԡՍՆՆԽՌՌ0ՋՌԽՋՌԬՆԡԹՑ ԥԩԩԹՆԼ

ՆՇՊՅԹՄՁՒԽԼ ԥԩԩ ԽՐՀՁԺՁՌԽԼ Թ ՅՇՆՇՌՇՆՁԻ ՌՊԽՆԼ ԹՆԼ ՏԽՊԽ ՌՀԽՊԽԾՇՊԽ ԹՆԹՄՑՒԽԼ ՍՋՁՆԿ ՌՀԽ ԧՇՆԻՃՀԽԽՊԽ

ԱԽՊՈՊՋՌՊԹ ՌԽՋՌ ԡԹՌԹ ԾՇՊ ԼԽՎԽՄՇՈՅԽՆՌԹՄ ՋՌԹԿԽ ՏԽՊԽ ԹՆԹՄՑՒԽԼ ՍՋՁՆԿ ՆՇՆՈԹՊԹՅԽՌՊՁԻ ՌԽՋՌՁՆԿ 
ՅԹՆՆ

ՏՀՁՌՆԽՑ ԞՄՄ ՊԽՅԹՁՆՁՆԿ ԡԹՑ  ԽՆԼՈՇՁՆՌՋ ՏԽՊԽ ԹՆԹՄՑՒԽԼ ՍՋՁՆԿ ԡՍՆՆԽՌՌ0Ջ ՌԽՋՌ ԹԾՌԽՊ ՅԽԽՌՁՆԿ ՌՀԽ

ԹՋՋՍՅՈՌՁՇՆՋ ԾՇՊ ՆՇՊՅԹՄՁՌՑ ԹՆԼ ՀՇՅՇԿԽՆԽՁՌՑ ԲՆՄԽՋՋ ՇՌՀԽՊՏՁՋԽ ՁՆԼՁԻԹՌԽԼ ԽԾԾԽԻՌՋ ՏԽՊԽ ԻՇՆՋՁԼԽՊԽԼ

ՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈԱՀԽՊԽՏԽՊԽՇՆՄՑ


ԹՆԼ


ՌԹԼՈՇՄԽՋԿՊԽԹՌԽՊՌՀԹՆՋՌԹԿԽ
ՁԽՋՌԹԿԽՋՌՇՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼ

  ԹՆԼ  ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈՋ ՊԽՋՈԽԻՌՁՎԽՄՑ  ԱՀԽՊԽԾՇՊԽ ՊԽՈՄՁԻԹՌԽ ՅԽԹՆՋ ԹՆԼ

ՅԽԼՁԹՆՋ ԾՇՊ ԺՇԼՑՏԽՁԿՀՌ ԰Գԩ ԹՆԼ ԥԩԩ 
ՁՆԻՄՍԼՁՆԿ ՆՇՊՅԹՄՁՒԽԼ ԥԩԩՏԽՊԽ ԻԹՄԻՍՄԹՌԽԼ ԽՐԻՄՍԼՁՆԿ ԹՄՄ

ՌԹԼՈՇՄԽՋԹՌԫԣՋՌԹԿԽ

	

  	 
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ԱՀԽՊԽՎՁԽՏԽՊ0ՋԹՆԹՄՑՋՁՋԹԿՊԽԽԼՏՁՌՀՌՀԹՌՇԾՌՀԽՋՌՍԼՑԹՍՌՀՇՊԱՀԽՊԽՏԹՋԹՋՁԿՆՁԾՁԻԹՆՌՁՆԻՊԽԹՋԽՁՆԡԹՑ

ՅԽԹՆ԰ԳԩԹՆԼԺՇԼՑՏԽՁԿՀՌՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈԻՇՅՈԹՊԽԼՌՇ

ՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ
ՈՈՊԽՋՈԽԻՌՁՎԽՄՑ԰ՁԿՆՁԾՁԻԹՆՌՁՆԻՊԽԹՋԽՋՁՆՌՀԽՋԽ

ԽՆԼՈՇՁՆՌՋՏԽՊԽՆՇՌՇԺՋԽՊՎԽԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌ

Page 43 of 294
  	
 
	  	
  	
 	   	 
 	     
 	 	  

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

ԿՊՇՍՈՋ ԹՆԼ ՌՀԽՊԽԾՇՊԽ ԹՊԽ ՆՇՌ ԻՇՆՋՁԼԽՊԽԼ ՌՊԽԹՌՅԽՆՌ ՊԽՄԹՌԽԼ  ԱՀԽՊԽ ՏԽՊԽ ՆՇ ՋՁԿՆՁԾՁԻԹՆՌ ԼՁԾԾԽՊԽՆԻԽՋ

ԺԽՌՏԽԽՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԾՇՊԹՆՑՇՌՀԽՊԽՆԼՈՇՁՆՌՋ

Page 44 of 294
	

		
	
		
			

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽ ՇԾ 

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ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ  ԫԞ  ԫԞ  ԫԞ  ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ

         ԫԞ ԫԞ ԫԞ ԫԞ ԫՇ

         ԫԞ ԫԞ ԫԞ ԫԞ ԫՇ

         ԫԞ ԫԞ ԫԞ ԫԞ ԫՇ

԰ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԪԹՆՆԴՀՁՌՆԽՑ ԪԹՆՆԴՀՁՌՆԽՑ ԡՍՆՆԽՌՌ0Ջ ԧՇՆԻՃՀԽԽՊԽ ԫԞ ԫԞ ԫԞ

ԞԺԺՊԽՎՁԹՌՁՇՆՋԡՁԾԾԡՁԾԾԽՊԽՆԻԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ

 ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼԽԾԾԽԻՌՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑ

ՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ

 ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈ ԥՁՆԼՄՁՅԺՄԽՆԿՌՀՁՋՆՇՊՅԹՄՁՒԽԼՌՇՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ
԰Գԩ

Page 45 of 294
  	
 
	  	
  	
 	   	 
 	     
 	 	  

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

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԰ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԡՍՆՆԽՌՌ0Ջ ԡՍՆՆԽՌՌ0Ջ ԡՍՆՆԽՌՌ0Ջ ԡՍՆՆԽՌՌ0Ջ

ԞԺԺՊԽՎՁԹՌՁՇՆՋԡՁԾԾԡՁԾԾԽՊԽՆԻԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ

 ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼԽԾԾԽԻՌՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄ



 ԢԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌ1  2 #2

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x ԦՆԾՇՊՅԹՌՁՇՆՇՆՌՀԽՈԹՊԽՆՌԹՄԻԹՊԽԹՆԼՋՈԹՏՆՁՆԿՏԹՋՆՇՌՊԽՈՇՊՌԽԼ

x ԱՀԽԾՄՇՏՌՀՊՇՍԿՀՊԹՌԽՇԾՅԩՅՁՆԽՐԻԽԽԼՋՌՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՇԾՅԩՅՁՆ

ԱՀԽԾՇՄՄՇՏՁՆԿՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹՏԽՊԽՆՇՌՅԽՌ

x ԱՀԽԠԳԾՇՊՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆ ՇԾՌՀԽՄՇՏՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ 
ՁԽՅԿ

ԹՁԩՏԹՋԽՐԻԽԽԼՁՆԿՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆՇԾՅԹՁՆՌԹՁՆՁՆԿԹԠԳՇԾ 5ՇՎԽՊ

ՌՀԽԼԹՑՌԽՋՌԥՇՏԽՎԽՊՁՌՋՀՇՍՄԼԺԽՆՇՌԽԼՌՀԹՌՌՀՁՋԠԳՏԹՋԻԹՄԻՍՄԹՌԽԼԺՑՌՀԽ ՊԽՎՁԽՏԽՊ

ՍՋՁՆԿԡԹՑՋԹՅՈՄԽՋԦԾԡԹՑՋԹՅՈՄԽՋԹՊԽՍՋԽԼՁՆՄՁԽՍՇԾԡԹՑՋԹՅՈՄԽՋՌՀԽԠԳՇԾ

ԼՇԽՋՆՇՌԽՐԻԽԽԼՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆ

x ԴԹՌԽՊՌԽՅՈԽՊԹՌՍՊԽԼՁԼՆՇՌԼՁԾԾԽՊԺՑՅՇՊԽՌՀԹՆւԠԺԽՌՏԽԽՆՌԽՋՌՎԽՋՋԽՄՋԹՌԹՆՑՇՆԽՌՁՅԽ

ԼՍՊՁՆԿՌՀԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼՏՁՌՀՌՀԽԽՐԻԽՈՌՁՇՆՇԾՅԽԹՋՍՊԽՅԽՆՌՋՌԹՃԽՆՇՆԡԹՑԹՆԼԡԹՑ

ՏՀԽՆՌՀԽԼՁԾԾԽՊԽՆԻԽՏԹՋԹՆԼւԠՊԽՋՈԽԻՌՁՎԽՄՑԴԹՌԽՊՌԽՅՈԽՊԹՌՍՊԽՏԹՋՅԹՁՆՌԹՁՆԽԼ

ՏՁՌՀՁՆչւԠՏՁՌՀՌՀԽԽՐԻԽՈՌՁՇՆՇԾԡԹՑՏՀԽՆՌԽՅՈԽՊԹՌՍՊԽՋՁՆՋՇՅԽՌԽՋՌՎԽՋՋԽՄՋ

ՏԽՊԽԹՋՄՇՏԹՋւԠԱՀԽՋԽՄՁՅՁՌՋԽՐԻԽԽԼՌՀԽՌԽՅՈԽՊԹՌՍՊԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁՇՆՇԾչ

ւԠՏՁՌՀՁՆՌԽՊՊԽՈՄՁԻԹՌԽՁՆՌԽՊՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋՆՇՌԽՐԻԽԽԼՁՆԿւԠԞԻԻՇՊԼՁՆԿՌՇՌՀԽ

Page 46 of 294
  	
 
	  	
  	
 	   	 
 	     
 	 	  

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԡԢԯԱԽՅՈՄԹՌԽԳԽՊՋՁՇՆ԰ԽՈՌԽՅԺԽՊ

ՋՌՍԼՑԹՍՌՀՇՊՌՀԽՋԽՋՄՁԿՀՌԼՁԾԾԽՊԽՆԻԽՋՁՆՌԽՅՈԽՊԹՌՍՊԽՏԽՊԽՇԾՋՀՇՊՌԼՍՊԹՌՁՇՆԹՆԼԼՁԼՆՇՌ

ԹՈՈԽԹՊՌՇԹԼՎԽՊՋԽՄՑՁՅՈԹԻՌՌՀԽՋՌՍԼՑ

ԴՀՁՄԽՌՀԽՋԽԼԽՈԹՊՌՍՊԽՋԾՊՇՅԿՍՁԼԽՄՁՆԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹԹՆԼԼԽՎՁԹՌՁՇՆՋԾՊՇՅԿՍՁԼԽՄՁՆԽ

ՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋՏԽՊԽՇԺՋԽՊՎԽԼՌՀԽՑԼՁԼՆՇՌՋՍԾԾՁԻՁԽՆՌՄՑՁՅՈԹԻՌՌՀԽԹԺՁՄՁՌՑՌՇՁՆՌԽՊՈՊԽՌՌՀԽՊԽՋՍՄՌՋՇԾ

ՌՀԽԹՋՋԹՑԱՀԽԹՋՋԹՑ 

ՌՀԽԢԡ԰ԭԱՁԽՊԱԽՋՌԬՊԼԽՊՊԽՉՍՁՊԽՅԽՆՌՋԾՇՊԹՆԞՅՈՀՁԺՁԹՆ

ԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ

   	



ԣՇՊ ՌՀԽ ՆՇՅՁՆԹՄ  ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ՌՀԽ ՋՌՍԼՑ ԹՍՌՀՇՊ ԻԹՄԻՍՄԹՌԽԼ Թ ՅԽԹՆՅԽԹՋՍՊԽԼ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՇԾՅԿԹՁԩՍՋՁՆԿԡԹՑՋԹՅՈՄԽՋՁՆՄՁԽՍՇԾԡԹՑՋԹՅՈՄԽՋԞԻԻՇՊԼՁՆԿՌՇՌՀԽ

ՋՌՍԼՑ ԹՍՌՀՇՊ ԡԹՑ  ՋԹՅՈՄԽՋ ԾՊՇՅ ՌՀԽ  ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ՏԽՊԽ ՋՍՋՈԽԻՌ 
ՁԽ ՄՇՏ ԹՆԼ

ՌՀԽՊԽԾՇՊԽՆՇՌՁՆԻՄՍԼԽԼՁՆՌՀԽԻԹՄԻՍՄԹՌՁՇՆՇԾՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆԱՀԽՄՇՏԹՆԹՄՑՌՁԻԹՄՊԽՋՍՄՌՋ

ՏԽՊԽԺԽՄՁԽՎԽԼՌՇԺԽԼՍԽՌՇՋԹՅՈՄՁՆԿՇՊԹՆԹՄՑՌՁԻԹՄԽՊՊՇՊԞԼԼՁՌՁՇՆԹՄՋԹՅՈՄԽՋՏԽՊԽԻՇՄՄԽԻՌԽԼԾՊՇՅՌՀԽ

ՅԿԹՁԩՌՊԽԹՌՅԽՆՌՌՏՇԼԹՑՋՄԹՌԽՊ
ԡԹՑԹՆԼՏԽՊԽԻՇՆՋՁԼԽՊԽԼՌՇԺԽՊԽՈՊԽՋԽՆՌԹՌՁՎԽՇԾՌՀԽ

ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՁՆ ՌՀԽ ՌԽՋՌ ԻՀԹՅԺԽՊՋ ՇՆ ԡԹՑ  ԹՆԼ ՍՋԽԼ ՁՆ ՌՀԽ ԻԹՄԻՍՄԹՌՁՇՆ ՇԾ ՅԽԹՆՅԽԹՋՍՊԽԼ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺՑՌՀԽՋՌՍԼՑԹՍՌՀՇՊ԰ՁՆԻԽԹՆՇՆՋՈԽԻՁԾՁԻՊԹՌՁՇՆԹՄԽՏԹՋԿՁՎԽՆԾՇՊՌՀԽՄՇՏՅԽԹՋՍՊԽԼ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾՌՀԽՆՇՅՁՆԹՄՅԿԹՁԩՌՊԽԹՌՅԽՆՌՇՆԡԹՑՌՀԽՊԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼԹՅԽԹՆ

ՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՇԾՅԿԹՁԩՍՋՁՆԿԡԹՑՋԹՅՈՄԽՋ

ԱՀԽՊԽՎՁԽՏԽՊ0ՋՊԽՋՍՄՌՋՏԽՊԽԺԹՋԽԼՇՆՊԽՈՄՁԻԹՌԽՅԽԹՆՋԹՆԼՅԽԼՁԹՆՋԻԹՄԻՍՄԹՌԽԼԺՑԽՐԻՄՍԼՁՆԿՌԹԼՈՇՄԽՋ

ԹՌԫԣՋՌԹԿԽԾՇՊԺՇԼՑՏԽՁԿՀՌՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀԹՆԼՀՁՆԼՄԽԿՄԽՆԿՌՀ
ՁՆԻՄՍԼՁՆԿՆՇՊՅԹՄՁՒԽԼՀՁՆԼ

ՄԽԿՄԽՆԿՌՀԱՀԽՊԽԾՇՊԽՌՀԽՊԽՎՁԽՏԽՊ0ՋՊԽՋՍՄՌՋԹՊԽՊԽՈՇՊՌԽԼՁՆՌՀԽԢՐԽԻՍՌՁՎԽ԰ՍՅՅԹՊՑԹՆԼԠՇՆԻՄՍՋՁՇՆՋ

ՋԽԻՌՁՇՆՋՇԾՌՀՁՋԡԢԯ

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ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

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ԟՇՑԼԠԢԴԹՌԽՊԮՍԹՄՁՌՑՁՆՏԹՊՅՏԹՌԽՊԾՁՋՀՈՇՆԼՋԞԿՊՁԻՍՄՌՍՊԹՄԢՐՈԽՊՁՅԽՆՌ԰ՌԹՌՁՇՆՋԞՍԺՍՊՆ

ԲՆՁՎԽՊՋՁՌՑԞՍԺՍՊՆԞՄԹԺԹՅԹ

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԰ԽՊՁԽՋԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊ԰ԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅԱԽՋՌ

ԤՍՁԼԽՄՁՆԽՋԬԭԭԱ԰ԫՍՅԺԽՊԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋ
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ԬԢԠԡԤՍՁԼԽՄՁՆԽՋԾՇՊԱԽՋՌՁՆԿՇԾ

ԠՀԽՅՁԻԹՄՋԤՍՁԼԽՄՁՆԽԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑԞԼՇՈՌԽԼ԰ԽՈՌԽՅԺԽՊ

ԨԽՆԼԹՄՄԱԷԹՆԼԴԟԫՁՐՇՆԞՆԹՄՑՌՁԻԹՄԪԽՌՀՇԼԳԽՊՁԾՁԻԹՌՁՇՆՋԾՇՊՌՀԽԡԽՌԽՊՅՁՆԹՌՁՇՆՇԾԪԹՄԹՌՀՁՇՆՁՆ

ԣՊԽՋՀՏԹՌԽՊԴՁՄԼՄՁԾԽԦՆՌԽՊՆԹՌՁՇՆԹՄԩՌԼԭՊՇՂԽԻՌԫՍՅԺԽՊԠԲՆՈՍԺՄՁՋՀԽԼՊԽՈՇՊՌՈՊԽՈԹՊԽԼԾՇՊ

ԠՀԽՅՁՆՇՎԹԞ԰ԧԹՆՍԹՊՑ

ԞԭԥԞԞԴԴԞԴԢԣ԰ՌԹՆԼԹՊԼԪԽՌՀՇԼՋԾՇՊՌՀԽԢՐԹՅՁՆԹՌՁՇՆՇԾԴԹՌԽՊԹՆԼԴԹՋՌԽՏԹՌԽՊՌՀ

ԢԼՁՌՁՇՆԞՅԽՊՁԻԹՆԭՍԺՄՁԻՀԽԹՄՌՀԞՋՋՇԻՁԹՌՁՇՆԞՅԽՊՁԻԹՆԴԹՌԽՊԴՇՊՃՋԞՋՋՇԻՁԹՌՁՇՆԴԹՌԽՊԢՆՎՁՊՇՆՅԽՆՌ

ԣԽԼԽՊԹՌՁՇՆԴԹՋՀՁՆԿՌՇՆԡԠ

ԭԽՊՋՇՆԹՄԠՇՅՅՍՆՁԻԹՌՁՇՆԡՊԱՁՅՇՌՀՑ԰ՈՊՁՆԿԽՊԹՆԼԡՊԧՇՀՆԤՊԽԽՆԫՇՎԽՅԺԽՊԡՊԱՁՅՇՌՀՑ

԰ՈՊՁՆԿԽՊԴՁՄԼՄՁԾԽԦՆՌԽՊՆԹՌՁՇՆԹՄԩՌԼԢԹՋՌՇՆԪԡԲ԰ԞԡՊԧՇՀՆԤՊԽԽՆԢԦԡՍՈԬՆՌԼԽԫԽՅՇՍՊՋ

ԹՆԼԠՇՅՈԹՆՑԴՁՄՅՁՆԿՌՇՆԡԢԲ԰Ԟ

ԱՀԽ԰Ԟ԰԰ՑՋՌԽՅԾՇՊԴՁՆԼՇՏՋԳԽՊՋՁՇՆ԰Ԟ԰ԦՆՋՌՁՌՍՌԽԦՆԻԠԹՊՑԫՇՊՌՀԠԹՊՇՄՁՆԹ

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DATA EVALUATION RECORD 

MALATHION 

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

EPA Contract No. EP10H001452 

Task Assignment No. 3-06-2012 

(Revisions to MRID 48617503 to include 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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 61 of 294
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 62 of 294
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 

Page 63 of 294
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 Bmax 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 Kd values was within 

the range reported in the EPA validation program (0.685 to 1.57 nM), and all Bmax values were 

below the recommended range (7 to 16 fmol/100 μg protein). Although the goodness of fit for 

individual runs was acceptable (R2 = 0.880-0.986), there was a large variation in Kd 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 reduced 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, respectively. 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 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. 

The estimated log IC50s for R1881 and the weak positive control (dexamethasone) were −9.3 and 

−4.4 M, respectively. The mean RBA for the weak positive control was 0.0022%. 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 likely due to the concentration differences used for 

R1881 and dexamethasone. The curve for dexamethasone was not fully defined in the first run 

and therefore the calculation for the IC50 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. 

The performance criteria were generally met; however, the bottom percent binding for 

dexamethasone was not achieved in the first run. The concentrations prepared for R1881 and 

dexamethasone 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 (nonradiolabeled) R1881 but with 30X cold R1881, and the resulting curve for dexamethasone fit 

only two of the three criteria (Hill slope and Top plateau). All performance criteria were met for 

the other two runs. 

Based on the results from the three runs, malathion is classified as Equivocal in the Androgen 

Receptor Binding Assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for an Androgen Receptor Binding 

Assay (OCSPP 890.1150). 

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

Page 64 of 294
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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 Operations); 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 

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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 Laboratories (location not specified). 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 received 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 reported), 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 using a commercially available protein kit 

compatible with DTT in the TEDG buffer (Bradford Method; Bio-Rad Dye Reagent 

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

the AR was present in reasonable 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 specific 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 serial dilutions in low-salt 

TEDG+PMSF buffer were prepared to achieve the 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 

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achieve concentrations that were 100-fold greater than each respective radiolabeled 

concentration, resulting in final concentrations in cytosol of 25, 50, 70, 100, 150, 250, 500, 

and 1000 nM. In the absence of cytosol, the 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 [3H]-R1881 was measured. 

For each batch of cytosol, the optimal protein concentration was determined by calculating 

specific binding to differing amounts of protein per tube with 0.25 nM radiolabeled R1881. 

The optimal protein concentration was determined to be 1.86 mg protein/assay tube, which 

resulted in the binding of approximately 25-35% of the total radioactivity added. Cytosolic 

protein used in this assay was thawed fresh for 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, resulting in the 72 samples depicted in 

Table 2. 

TABLE 2. Saturation Binding Experiment Runa,b

Total Binding Non-Specific Binding Radioligand alone 

Tubes 1-24c Tubes 25-48d Tubes 49-72e 

[

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 triamcinolone acetonide and 7.5-45 μL [3

H]-R1881. Samples were dried, 

and 300 μl of prostate cytosol were added. 

d Tubes 25-48 contained 50 μL of triamcinolone acetonide and 7.5-45 μL [3

H]-R1881. R1881 was added in a 

100-fold molar 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 separate 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 supernatant was aspirated 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 

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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 scintillation 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 Experimenta

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 

radioligandb 

Concentration of test substance (as serial dilutions) 10−10 to 10−3 Mc

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 the Guideline is 1 mM PMSF. The PI was identified in the study protocol as 

Calbiochem Protease Inhibitor Cocktail, Set III, EDTA free. It is a mixture of six PIs that includes 4-(2-

aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBSF 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 single 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 binding experiment was provided; however, the 

reviewer was able to calculate from the competitive binding experiment that the amount of 

cytosolic protein used in the assay contained enough receptor to bind 9-10% of the 

[

3

H]-R1881. 

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Dilutions of the test substance, 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 three independent runs on three different days, 

and each run contained three replicates at each concentration plus six 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) Concentrations in Competitive Binding Assay a, b

Solvent Control Reference standard c Weak positive control c Test Chemical None 

DMSO R1881 Dexamethasone Malathion (Hot Mix) 

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 (non-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 preparation, 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 washing 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 experiment, 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 outliers 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 obtained with high receptor concentrations: practical aid for 

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assay, similar methods of nonlinear regression were used to fit a curve (for R1881, the 

positive control, and the test substance) to the Hill equation formula which incorporated log 

IC50 as a parameter to be estimated (XLfitTM Model 204; IDBS, Guildford, UK). The top 

and bottom of the curve, Hill slope, inhibition concentration (IC50), and standard deviations 

were also assessed using XLfitTM. For parameters reported from the saturation binding 

experiment (Kd and Bmax) and competitive binding experiment (log IC50 and RBA), mean 

and standard deviation were calculated for each run and mean and standard error were 

calculated for the composite three runs 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 nonlinear regression model, the test chemical is classified as: 

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

bound. 

Equivocal: The average lowest portion of curves across runs is between 50% and 75% 

radioligand binding (i.e. radioligand displacement is at least 25% but less than 50%), or the 

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

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

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

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

 M and the binding curve does 

not cross 50%, the chemical is judged to be untestable. 

b. Descriptors for receptor binding 

Bmax: maximal binding capacity

Kd: dissociation constant

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

by the competitor

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

Log RBA: Log10(IC50 of R1881 × 100 ÷ IC50 of test substance) 

II. RESULTS 

A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 

are presented in Table 5. The mean Kd for [3H]-R1881 was 1.080±0.690 nM, and the 

estimated Bmax 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 individual Kd values was within the range 

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

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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 (R2 = 0.880-0.986) was acceptable, there 

was a large variation in Kd estimates among runs.

TABLE 5. Saturation Binding Experiment of [3H]-R1881 with Androgen Receptor from Rat Prostate 

Cytosola

Parameter Run 1 Run 2 Run 3 Mean ± SEb

R2 (unweighted) 0.880 0.986 0.893 0.880-0.986 

Bmax (nM) 0.010 0.008 0.011 0.010±0.002 

Bmax (fmol/100 μg protein) 1.087 0.896 1.229 1.071±0.167 

Kd (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 R2

 is reported and the mean ± SE is reported for the other parameters. 

R2

 Goodness of fit for curve calculated for specific binding.

Figure 1 illustrates the non-specific, specific, and total binding curves for [3H]-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 [3H]-R1881 to the androgen receptor. 

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

Binding Experiment.

 

 

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

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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 observed at the highest concentration 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. Malathion 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 malathion was consistently >98% at all concentrations ≤10−4

 M in all three 

runs. Malathion is classified as “equivocal” as the average specific binding for [3H]-R1881 

at the lowest portion of the curve was between 50-75% for the three runs. 

The estimated average log IC50s for R1881 and the weak positive 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 compared to Runs 2 and 3 are likely due to the 

concentration differences used for R1881 and dexamethasone. The curve for 

dexamethasone was not fully defined in the first run and therefore, the calculation for the 

IC50 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 assay from the beginning to the end of the run. 

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

Parameter Run 1 Run 2 Run 3 Mean±SE 

R2 (unweighted) R1881 NR NR NR NA 

 Dexamethasone NR NR NR NA 

 Malathion NR NR NR NA 

Log IC50 (M) R1881 −9.9 −9.0 −9.0 −9.3±0.30b

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

 Malathion NA NA NA NA 

IC50 (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 RBAb 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 

R2 Goodness of fit 

RBA Relative binding affinity

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FIGURE 3. Percentage [3H]-R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, or Malathion (Run 1). 

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FIGURE 4. Percentage [3H]-R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, or Malathion (Run 2). 

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FIGURE 5. Percentage [3H]-R1881 Bound to the Androgen Receptor in the Presence of 

Radioinert R1881, Dexamethasone, or Malathion (Run 3). 

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C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay was 

functioning properly, each run was evaluated using the following criteria (Table 7): 

TABLE 7. Criterion a Tolerance Limit(s) b Value Yes No 

Ligand depletion is minimal. The recommended ratio of 

total binding in the absence of competitor to total amount of 

[

3

H]-R1881 added per assay tube. 

≤15% 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 Kd (nM) 0.685 to 1.57 nM 0.531 to 1.855d

 Xe

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 concentrations for R1881 and dexamethasone were lower than the 

targeted (guideline) levels because of an error 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 dexamethasone fit two of the three criteria (Hill 

slope and Top plateau) but the Bottom plateau was not achieved 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 consistent 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 classified as a “non-binder” in the third valid 

independent run. Thus, malathion has a final classification of “equivocal” for binding to the 

AR.

B. AGENCY COMMENTS: The mean Kd for [3

H]-R1881 was 1.080 nM, and the mean 

estimated Bmax was 1.071 fmol/100 μg protein for the single batch of prostate cytosol that 

was used in the assay. Only one of the three individual Kd values was within the range 

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

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acceptable range. Confidence in these numbers is low based on the large variation in Kd

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 reduced the mean specific binding of the 

ligand, [3H]-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 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 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 IC50 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%. Confidence 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 dexamethasone were lower than the targeted 

(guideline) levels because of an error in the stock preparation. Therefore, non-specific 

binding was not evaluated with 100X of cold (non-radiolabeled) R1881 but with 30X cold 

R1881, and the resulting curve for dexamethasone fit only two of the three criteria (Hill 

slope and Top plateau) in the first run. All performance criteria were met for the other two 

runs. 

The bottom percent binding for dexamethasone was not achieved in the first run, leaving 

only two valid runs instead of the required three. However, as the performance was 

consistent between the other two runs, it is unlikely that information from an additional run 

would change the interpretation 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 achieved in the first run. The 

concentrations prepared for R1881 and dexamethasone 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. 

Page 79 of 294
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 80 of 294
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 81 of 294
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 

Page 82 of 294
Aromatase (Human Recombinant) Assay (2011) / Page 2 of 13 

MALATHION / 057701 OCSPP 890.1200 / OECD None 

averaged 0.75% of the full activity 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 ranges for the top of the 

curve, bottom curve, hill slope, log IC50, and coefficient of variation for replicates of each 

concentration within runs. Also, the coefficients of variations (%CVs) for replicates of each 

concentration of malathion within a run were within the 15% guideline. For 4-OH ASDN, the 

estimated log IC50 averaged –7.16 M, and the slope was –1.01. 

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·mgprotein-1·min−1

 at the highest successfully tested concentration of 10−3.5 M (highest nonprecipitating dose). Precipitation 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 best fit mean inhibition response curve was a straight line (essentially 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. 

Based on the data from the average response curve, malathion is classified as Equivocal for 

aromatase inhibition in this assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for an Aromatase assay (OCSPP 

890.1200). 

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

Assurance statements were provided. 

 

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Aromatase (Human Recombinant) Assay (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 observed 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

CH2CH3

CH2CH3

P O

O

3CH

3CH

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: [1β-3H(N)]-Androst-4-ene-3,17-dione; ([3H]-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 GentestTM (Woburn, MA; Cat. # 456260) 

 Lot # (Expiration Date): 03897 (February 2014) 

Protein Concentration: 7.4 mg/mL 

Cytochrome C Reductase Activity: 290 nmol/min/mg 

Aromatase Activity: 6.0 pmol/min/pmol P450 

Page 84 of 294
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MALATHION / 057701 OCSPP 890.1200 / OECD None 

B. METHODS

1. Assay Components and Preparations: A mixture of non-labeled and radiolabeled 

[

3

H]ASDN was prepared such that the final concentration of ASDN in the assay was 

approximately 0.1 μM, and the amount of tritium added to each incubation tube was 

approximately 0.1 μCi. 

Test chemical(s) were formulated in DMSO such that the total volume 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 specified 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 prepared 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 vials 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. Aromatase 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 determined to be 0.170 nmol/mg protein/min, which 

was greater than the minimum recommended aromatase activity of 0.1 nmol/mgprotein/min. 

3. Aromatase Assay: Each assay run contained 4 tubes 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 

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concentration curve in duplicate for the positive control, and a full concentration curve in 

triplicate for the test substance were established. The aromatase assay was conducted 

generally according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10). 

 The amount of 3H2O 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 concerning demonstration of 

proficiency. However, the report stated that prior to conducting this assay using malathion; 

a full-scale assay consisting of three independent runs was conducted 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 proficiency chemicals in Table 6 of the guideline. 

Data was generated by CeeTox and 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 laboratory) were not reported in regards to the following criteria: 

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

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

 Coefficient of variation (% CV) for replicates within each sample type and 

concentration of 4-OH ASDN was <15%. 

Performance criteria 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 New Technician for Conducting Assay (when 

applicable): Demonstration of proficiency by a new technician, if applicable, was not 

reported. 

TABLE 2. Performance Criteria for the Positive Control 

Parameter Lower Limit Criteria Upper Limit Criteria Actual Lower Limit Actual Upper Limit 

Slope –1.2 –0.8 –1.03 –0.98 

Top (%) 90 110 100.56 106.40 

Bottom (%) –5 +6 0.86 1.25 

Log IC50 (M) –7.3 –7.0 –7.20 –7.10 

a Data were obtained from page 30 of the study report. 

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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 Activity 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 assessed (presence of cloudiness or a 

precipitate). Precipitation 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 lowest 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 componentsa 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 1b 3 All test components plus malathion 1×10–3

Malathion Conc 2b 3 All test components plus malathion 1×10–3.5

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

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

a The complete assay contained buffer, 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 aromatase 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 included in the report (Table 5). 

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TABLE 5. Raw and Calculated Data

Raw/Calculated Data Included (X) 

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

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

Total DPM for each aqueous portion (after extraction) X 

The total DPM present in the assay tube at initiation X 

The percentage of substrate converted to product X 

Total DPM after extraction corrected for background X 

Aromatase activity expressed in nmol/mg protein/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 graphical displays were conducted using 

IDBS XLfit v5.2.0.0 (Guildford, UK). Basic statistical analyses were performed on the 

data, which included means of replicates, standard deviation of the mean, standard error of 

the mean, and coefficient of variation. Analysis of variance (ANOVA) was not performed.

 The response curve was fitted by weighted least 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-Weight statistical analysis for outlier 

analysis. For each run, percent of control activity was plotted versus 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 categories presented in Table 6. 

TABLE 6. Interpretation of Results 

Criteria Interpretation 

Data fit 4-parameter 

nonlinear regression 

model 

Average curve across runs crossed 50% a Inhibitor 

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

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

Data do not fit model --- 

a Ordinarily, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near –1. Unusually steep curves 

may indicate protein denaturing or solubility issues. If the slope of the curve is steeper than –2.0, the result is classified as 

equivocal. 

b If the test compound was not soluble above 10–6 M and the inhibition curve does not cross 50%, the chemical is typically 

determined to be untestable 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 the full activity controls. The response of 

each full activity control within a run was between 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 

Page 88 of 294
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MALATHION / 057701 OCSPP 890.1200 / OECD None 

activity) for each concentration tested across all 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 

IC50, 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 Activity (as Percent of Control) from Independent Runs a

Chemical Conc. Log M # Runs Overall Mean Overall SD Overall SEM Overall %CV 

4-OH ASDN 

(positive control) 

–5 3 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 

 –9b 2 103.15 2.83 1.16 2.7 

 –10b 1 ND ND ND ND 

a Values were calculated by the reviewers based on data 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 

Page 89 of 294
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FIGURE 1. Inhibition Response Curves for 4-OH ASDN. 

Page 90 of 294
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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-precipitating 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 inhibition response curve across all runs is shown in Figure 3. 

Page 91 of 294
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FIGURE 2. Inhibition Response Curves for Malathion. 

Page 92 of 294
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FIGURE 3. Mean Inhibition Response Curve for Malathion. 

The data for malathion were not suitable for modeling. The mean aromatase activity of 

10-3.5 M malathion (highest soluble concentration tested) was 64.58% of control activity, 

and the best fit mean inhibition response curve (Figure 3) was a straight 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 IC50

averaged –7.16 M, and the slope was –1.01. Confidence 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 IC50 (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 Malathion 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. 

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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 recommended ranges for the top of the curve, 

bottom curve, hill slope, log IC50, 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 generally within the 15% guideline. 

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 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 IC50 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 concentration (10–3.5 M) malathion was 64.58% of control activity, and the 

best fit mean inhibition response curve was a straight line (essentially 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 data were not provided. 

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

Page 94 of 294
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 95 of 294
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 96 of 294
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. 

Page 97 of 294
Estrogen Receptor Transcriptional Activation (2011) / Page 2 of 10 

MALATHION / 057701 OCSPP 890.1300/ OECD 455 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions of this assay were not optimal to detect 

very weak activity, malathion responses were similar to those of the negative control 

corticosterone and not comparable to the responses of 17α-methyltestosterone, which was able to 

reach a maximum of 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 cytotoxicity. Because the 

RPCMax < PC10 in both assay runs, malathion was considered 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. 

 

Page 98 of 294
Estrogen Receptor Transcriptional 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

 17β-estradiol (strong estrogen; positive control)

 Supplier: Not reported (NR) 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 50-28-2 

 

 17α-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 

 

 17α-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) 

Page 99 of 294
Estrogen Receptor Transcriptional Activation (2011) / Page 4 of 10 

MALATHION / 057701 OCSPP 890.1300/ OECD 455 

B. METHODS

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

Collection of Research Bioresources Cell Bank and verified to be free of mycoplasma 

infection using a DNA fluorochrome assay. Cells were maintained in Eagles Minimum 

Essential Medium without phenol red, supplemented with 60 mg/L kanamycin and 10% 

dextran-coated charcoal-treated fetal bovine serum (source not reported), in an incubator 

under 5% CO2 at 37 oC. Upon reaching 75-90% confluence, cells were subcultured 5 times 

prior to exposure to test material. Cells used in the range-finding assay were at passage 35. 

New cells at passage 14 were brought out of cryopreservation and used at passages 19 and 

20 for the main assay. 

2. Transcriptional Activation Assays: For each test, cells were plated at a density of 1×104

cells/100 μL medium/well in a 96 well plate and allowed 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 determined 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 Operating Protocol (SOP). A list of reagents was provided, 

but the assay reagent was listed as proprietary information, and the SOP was not provided.

a. Preliminary Test: A preliminary test at concentrations ranging from 10−3 to 10−10 M was 

conducted to determine concentrations resulting in insolubility and/or cytotoxicity. 

b. Proficiency Chemicals: Responsiveness of the test system 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 proficiency testing are 

from the same frozen stock as cells used in the main assay. Cells were tested using the 

following proficiency chemicals, each chemical 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 --- 

17α-Ethynyl estradiol (EE) 57-63-6 10−14 to 10−8 Positive --- 

Hexestrol 84-16-2 10−13 to 10−7 Positive --- 

Genistein 446-72-0 10−12 to 10−5 Positive Cytotoxic at 0.01b, 0.1, and 1 mM 

Estrone 53-16-7 10−12 to 10−6 Positive --- 

Butyl paraben 94-26-8 10−11 to 10−4 Positive Cytotoxic at 0.1b and 1 mM 

1,3,5-Tris(4-

hydroxyphenyl)benzene c

15797-52-1 10−12 to 10−5

 Positive Cytotoxic at 100 μM. PCmax approx. 50% of PC. 

Binds to hERα and has ER antagonistic activity 

Dibutyl phthalate (DBP) 84-74-2 10−11 to 10−4 Negative d Cytotoxic at 1 mM 

Atrazine 1912-24-9 10−11 to 10−4 Negative Cytotoxic at 1 mM b

Corticosterone 50-22-6 10−10 to 10−4 Negative If not cytotoxic at 1 mM, then that should be the 

highest tested concentration 

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

Negative = RPCMax fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs 

b Cytotoxicity is expected to be close to 80% at this concentration. 

c Compound selected to challenge solubility and cytotoxicity. 

d DBP is negative for ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional 

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

therefore unacceptable. 

Page 100 of 294
Estrogen Receptor Transcriptional Activation (2011) / Page 5 of 10 

MALATHION / 057701 OCSPP 890.1300/ OECD 455 

c. Reference Chemicals: To ensure the stability of the response from the cell line, six 

concentrations of each of the following reference chemicals were included in each plate in 

the current assay, along with the test chemical: 

Reference Chemical CAS No. Concentration Range Class 

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

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

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

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

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

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

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

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

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

percentage of the PC. The RPCMax for malathion was calculated, but the software used was 

not reported. The test material was defined as negative for inducing estrogen receptor 

transcriptional activation if the RPCMax < PC10 in at least 2 of 2 (or 2 of 3) runs. Log EC50

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

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

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

4. Definitions 

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

(bottom) and maximum (top) response 

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

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

PC50 = concentration of a test chemical at which the response is 50% of the response 

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

RPCMax = maximum level of response induced by a test chemical, expressed as a percentage 

of the response induced by the positive control (1 nM E2) on the same plate 

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS 

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the 

transcriptional activation assays, 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. 

Page 101 of 294
Estrogen Receptor Transcriptional Activation (2011) / Page 6 of 10 

MALATHION / 057701 OCSPP 890.1300/ OECD 455 

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

Concentration (M) % Viability b Commentsc

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 assays 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 17β-estradiol, 17α-estradiol and 17αmethyltestosterone were tested concurrently with each run of the assay (Table 2b). In the 

first run, the responsiveness of 17β-estradiol indicated decreased sensitivity to strong 

agonists, and the response to 17α-methyltestosterone showed an increased responsiveness to 

very weak agonists; despite the minor deviations this run is considered acceptable. Run 2 

was inadequate as the PC50 could not be calculated for 17α-methyltestosterone indicating a 

decreased sensitivity to very weak agonists. Run 3 was acceptable as 17β-estradiol and 

17α-methyltestosterone performed within the expected range, but the Hill Slope for 

17α-estradiol was higher than expected. The PC-induced fold induction for the three 

reference chemicals was within the Guideline-recommended historical range 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 provided 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 

17α-Ethynyl estradiol Positive 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 Negative Negative Negative NA 

Atrazine Negative Negative NA Negative 

Corticosterone Negative Negative NA Negative 

NA = not applicable. The chemical was not tested at this time. 

Page 102 of 294
Estrogen Receptor Transcriptional Activation (2011) / Page 7 of 10 

MALATHION / 057701 OCSPP 890.1300/ OECD 455 

Table 2b. Performance Criteria for Reference Chemicals in the Proficiency test

Reference Chemical 

Parameter

Acceptable 

Range 

Values Acceptable 

Run 1 Run 2 Run 3 Yes No 

17β-estradiol

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

 Log PC10 <−11 −11.5 −12.5 −12.1 X 

 Log EC50 −11.3 to −10.1 −9.0 −11.3 −10.6 Run 1 

 Hill Slope 0.7 to 1.5 1.2 0.9 0.8 

 Test range (M) 10−14 to 10−8 10−14 to 10−8 10−14 to 10−8 10−14 to 10−8 X 

17α-estradiol

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

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

 Log EC50 −9.6 to −8.4 −8.2 −9.3 −8.9 Run 1 

 Hill Slope 0.9 to 2.0 0.9 0.9 2.9 Run 3 

 Test range (M) 10−12 to 10−6 10−12 to 10−6 10−12 to 10−6 10−12 to 10−6 X 

17α-methyltestosterone

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

 Log PC10 −8.0 to −6.2 −8.1 −6.3 −7.7 Run 1 

 Test range (M) 10−11 to 10−5 10−11 to 10−5 10−11 to 10−5 10−11 to 10−5 X 

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

PC50, log PC10, log EC50, Hill slope) for the concurrently run reference compounds are in 

Table 3. Performance criteria were met for 17β-estradiol and 17α-estradiol; the log EC50 for 

17β-estradiol was -10.0 M for Run 1, which is only just outside the acceptable range. For 

17α-methyltestosterone, PC50 and PC10 values could not be calculated for Run 1, PC50 could 

not be calculated for Run 2, and the PC10 was higher than the expected range, indicating a 

decrease in sensitivity of the assay to very weak agonists. For 17α-methyltestosterone, the 

mean RPCMax was 6.5% for the first run and 37.4% for the second run. 

TABLE 3. Performance Criteria for Reference Chemicalsa

Reference Chemical 

Parameter Acceptable Range Values Acceptable 

Run 1 Run 2 Yes No 

17β-estradiol

 Log PC50 −11.4 to −10.1 −10.0 −10.3 X 

 Log PC10 <−11 −11.1 −11.4 X 

 Log EC50 −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 

17α-estradiol

 Log PC50 −9.6 to −8.1 −8.6 −8.7 X 

 Log PC10 −10.7 to −9.3 −9.6 −9.7 X 

 Log EC50 −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 

17α-methyltestosterone

 Log PC50 −6.0 to −5.1 NC NC Run 1,2 

 Log PC10 −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 

Page 103 of 294
Estrogen Receptor Transcriptional 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 positive controls are included in 

Table 4. The overall mean TA value for the vehicle control, expressed 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 

PC50 (50% of the maximum response) for E2 in this assay was 104881 for the first run and 

77009 for the second run, and the PC10 (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 17β-estradiol (E2) at 1 nM. 

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

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

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

curves depicting fold induction of RTA is presented in Figures 1 and 2. The mean RPCMax

was 3.4% for the first run and 3.7% for the second run, and the associated PCMax was 10−7 M 

for both runs. Because the RPCMax < PC10 in both assay runs, malathion was considered 

negative for estrogen receptor transcriptional activation in this test system. 

TABLE 5. Relative Transcriptional Activation (RTA) of Malathiona

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 

RPCMax 3.4 3.7 

PCMax (M) 10−7 10−7 

PC50 (M) NA NA 

PC10(M) NA NA 

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

* Cytotoxicity observed 

NA = Not Applicable

Page 104 of 294
Estrogen Receptor Transcriptional 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 17β-estradiol and 17αestradiol, although the Log EC50 for 17β-estradiol in Run 1 (-10.0 Log M) was just outside 

the expected range. For 17α-methyltestosterone, PC50 and PC10 values could not be 

calculated for Run 1, the PC50 could not be calculated for Run 2, and the PC10 was higher 

than the expected range, indicating decreased sensitivity to very weak agonists. Six 

replicates of each concentration, including samples of the positive control (PC, 1 nM E2) 

0

5

10

15

20

25

VC PC10 PC ‐11 ‐10 ‐9 ‐8 ‐7 ‐6 ‐5

Fold induction

Log [M]

0

2

4

6

8

10

12

14

16

18

20

VC PC10 PC ‐11 ‐10 ‐9 ‐8 ‐7 ‐6 ‐5

Fold induction

Log [M]

Page 105 of 294
Estrogen Receptor Transcriptional Activation (2011) / Page 10 of 10 

MALATHION / 057701 OCSPP 890.1300/ OECD 455 

and the vehicle control (VC, 0.1% DMSO) were 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 hERα antagonist, 

ICI 182,780 were included; cells responded appropriately to the antagonist (data not shown). 

Cytotoxicity was observed in the first assay at the highest concentrations tested of 

malathion, 17α-estradiol, corticosterone and 17α-methyltestosterone. Cytotoxicity should 

not occur at any of the required concentrations of the reference chemicals and indicate a 

flaw in the system. Additionally, in the second assay, the cells' average response to 10−9 M 

of 17β-estradiol and to 10−7 M or 10−6 M of 17α-estradiol was greater than the positive 

control, 10−9

 M 17β-estradiol. The fold-induction corresponding to the PC10 of the 

concurrent PC was greater than 1+2 standard deviations of the fold-induction value of the 

concurrent VC, and the results 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 10−5M, based on precipitation observed at 

concentrations >10−5

M. Cytotoxicity was observed with malathion at the highest 

concentration tested in the first run. There 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 data. 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 (RPCMax <10%). 

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

solubility, 10−5 M. The mean RPCMax was 3.4% for the first run and 3.7% for the second 

run, and the associated PCMax was 10−7

 M for both runs. Performance criteria were 

generally met for 17β-estradiol and 17α-estradiol, although the EC50 for 17β-estradiol (-

10.0) was just outside the expected range in run 1. For 17α-methyltestosterone, the PC50

and PC10 values could not be calculated for run 1, the PC50 could not be calculated for run 2, 

and the PC10 was higher than the acceptable range, indicating 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 17α-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 cytotoxicity. Because the 

RPCMax < PC10 in both assay runs, malathion was considered 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. 

Page 106 of 294
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Malathion 

EPA MRID Number 48617506 

Page 1Rf93 

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'

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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 estrogen, 

androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and 

redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather 

should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information 

(OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence 

(WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the 

WoE Document). 

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. 

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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 25.3o

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, were 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 (Ļ48%) 

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 

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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 control (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-through 

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

Treatment 

(mg a.i./L)3

[meanmeasured] 

Fecundity 

Fert. 

Success 

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

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

Control No No No No No No No No No No NA NA NA NA0.08 No No No No No No No No No No NA NA NA NA0.22 No No No No No No No No No No NA NA NA NA0.69 Yes No Yes No No Yes Yes Yes No No NA NA NA NA

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

1 A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified. 

2 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may be 

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

3 The mean-measured concentrations were calculated by the reviewer, including the 21-day measured concentrations. 

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I. MATERIALS AND METHODS 

 Guideline Followed: This study was conducted according to the U.S. EPA OCSPP 890.1350: “Fish 

Short-Term Reproductive Assay” and OECD 229 (2009). The following 

deviations were noted: 

1. 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. 

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 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 study was conducted in accordance with 

GLP Standards as published by the U.S. EPA (40 CFR Parts 160), OECD 

Principles of GLP [ENV/MC/CHEM(98)17], with the following exceptions: 

periodic analyses of water for potential contaminants were not performed 

according to GLP standards, but were 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 25qC. 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 diluter 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 

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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. 4qC); analytical standard stored 

frozen (ca. 25qC). 

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B. Test OrganismTable 2: General Information About the Test Species and Acclimation.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead minnow EPA recommends fathead minnow 

(Pimephales promelas). Species scientific name: Pimephales promelas

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

Yes Osage Catfisheries, Inc., Osage Beach, 

Missouri.

EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same as definitive test? 

Yes EPA recommends that fish be acclimated 

under water quality and illumination 

conditions that are similar to the definitive 

test. 

Acclimation period: 2 weeks EPA recommends a minimum two-week 

acclimation period. Note that the 

acclimation period is different from the 

subsequent, in situ pre-exposure phase. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: Pre-exposure mortalities not reported. 

Fish did not receive any treatment for disease during acclimation period. No behavioral abnormalities or clinical signs 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 and 

commercial 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 America

Frequency of feeding: Two times/day 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on feeding: None. 

Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: ca. 5 months EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months 

old.

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

2.7 Based on 10 males used to stock 

aquaria for pre-exposure phase. 

EPA recommends that a subsample 

of fish be weighed before the test to 

estimate the mean weight for each sex. It 

is recommended that the individual weight 

of each fish selected for the test be within 

±20% of the estimated mean for each sex.

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

r 21% 

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

1.2 Based on 10 females used to stock 

aquaria for pre-exposure phase. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

r 21% 

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

Not reported 

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

Not reported 

Duration of pre-exposure phase: 15 days EPA recommends a minimum of 14 days. Were pre-exposure conditions identical to the definitive test? 

Yes EPA recommends that pre-exposure 

conditions, including temperature, 

photoperiod, feeding, etc., be identical to 

definitive test conditions. 

Number of pre-exposure tanks: 32 tanks Extra 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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of males per tank: 2 males/tank Number of females per tank: 4 females/tank Pre-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 days Spawning occurred at least every 3 days. EPA recommends that spawning occur at least twice in the 7 days prior to the 

definitive test. 

Details on pre-exposure: None. 

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Corrections and Clarifications document1 , 

the use of a static renewal system is not 

recommended for this assay.

Type of flow-through dilution system: 

Continuous flow diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: ca. 6 volume 

replacements/day 

Actual 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.

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

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

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

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on toxicant mixing for flow-through systems: 

Flow-splitting chambers were used between 4 replicate test vessels 

Solution from mixing chamber split equally among replicate test vessels. Treatment recoveries prior to test initiation from pretest diluter were 82.0 to 94.1%. Flow rates to each replicate varied by no more 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).

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Source of dilution water: Well water Prior 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 analyzed for pesticides, heavy metals, and other contaminants? 

Yes All potential contaminants were confirmed 

to be below the detection 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).

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Test vessel size: 19 L, 41 x 21 x 25 cm 

high

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 exposure system: 

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

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Temperature (°C) 24.7 25.7 25 ± 1°C Continuous 

EPA recommends temperature 25±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 1oC.

pH 7.9 8.2 Not reported Weekly EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L) 5.7 8.0 Not reported Weekly EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3) 176 184 Not reported Weekly 

EPA recommends total alkalinity > 20 

mg/L as CaCO3.

Hardness [mg/L as CaCO3] 132 144 Not reported Weekly Total 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 reported Not reported 

EPA recommends that unionized 

ammonia in the dilution water be < 1 

Pg/L. 

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Residual chlorine (Pg/L) Not reported Not reported Not reported Not reported 

EPA recommends that residual chlorine 

in dilution water be < 10 Pg/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: NA Not applicable. 

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D. Study Design and Additional Experimental Conditions

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

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? Yes EPA recommends conducting a range-finder if 96-hour 

LC50 data for the fathead minnow are unavailable. 

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

0.20, 0.60, 2.0 mg a.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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional 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 LC50, may be selected as the highest exposure 

concentration in the 21-day test.

Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

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

definitive test be 21 days. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Based on Pilot study results

EPA recommends that the highest test 

concentration is either the solubility limit of 

the test compound, 100 mg/L, or 

demonstrates adequate evidence of toxicity 

(e.g., 1/3 the 96-hour LC50), whichever 

concentration is lowest.

Reference study citation (if applicable):

Not reported. 

Separation of test concentrations:

Three fold EPA suggests that a concentration 

separation of between 0.33 (or three-fold) 

and 0.1 (or ten-fold) is scientifically 

acceptable1 . 

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

concentrations and a control, plus solvent 

control if appropriate.

Are nominal concentrations adjusted for purity? 

No 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Indicate the type of values presented for measured concentrations: 

Mean measured 

Limit of quantification (LOQ): 0.0400 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Was the randomized complete block design used? 

Yes EPA recommends that all fish be randomly 

assigned to tanks during pre-exposure. 

Tanks are then ranked according to preexposure fecundity, and the tanks with the 

highest fecundity are randomly assigned to 

a definitive test treatment and block first. 

Each block contains one replicate of each 

treatment, including controls. 

Number of replicates in control: 4 EPA recommends 4 replicates.Number of replicates in solvent control (if applicable): 

NA EPA recommends the use of a concurrent 

solvent control when a solubilizing agent is 

used. EPA recommends 4 replicates.

Number of replicates per test item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

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

NA EPA recommends that the solvent not 

exceed 0.02 ml/L3. OECD recommends 

that solvent have no effect on survival nor 

produce any other adverse effects and that 

concentration not be greater than 0.1 ml/L4.

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

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

Toxicology, 76, pp.69–92.

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

and Assessment. No. 23. Paris, France.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 16 hrs light 

8 hrs dark 

EPA recommends photoperiod 16:8 

(light:dark).

Light intensity at water’s surface: 

432 to 657 lux EPA recommends light intensity 540 – 

1080 lux (at water’s surface).

Additional details: 

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

Treatment ID 

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration1

(mg a.i./L) 

Mean CV 

(%)1

Measured 

Concentration2

(mg a.i./L) 

Mean CV 

(%)2

Details or 

Remarks 

Guideline 

Recommendations 

Control (dilution water 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 NA 

Treatment 1 0.1 0.08 4% 0.08 8% Treatment 2 0.32 0.25 9% 0.22 26% 

% CV max 

exceeded2

Treatment 3 1.0 0.82 6% 0.69 29% 

% CV max 

exceeded2

Diluter Stock NA NA NA NA NA 

Abbreviations: CV Coefficient of variation. 1 The study authors did not include Day 21 samples in calculations due to suspected handling or analysis error (see Reviewer’s Comment). 2 The reviewer calculated mean measured and %CV including Day 21 samples. 

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E. Observations

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

secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, fatpad 

score, gonadal staging and histopathology, and plasma vitellogenin were evaluated. 

Were raw (individual) data provided? Yes

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical signs occur at least daily. At test 

termination (Day 21), additional observations include body weight and length, nuptial tubercle score, gonadal staging and histopathology, plasma vitellogenin, and 

plasma sex steroids (testosterone and 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 behavior. 

II. RESULTS AND DISCUSSION A. Results

One 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 groups 

exhibited 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 mg 

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a.i/L treatment levels, respectively. Observed clinical signs of toxicity included one fish in the middle treatment group exhibiting a crooked spine, but this 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 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 for the middle treatment group starting at Day 7 and persisting to Day 9. Excess feed was observed in the test chamber in the high treatment group in all replicate tanks from Day 5 to Day 16. 

Table 9: Adult Fish Survival in Pimephales promelas

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

Males Females 

n # Surviving % Survival n # Surviving % Survival 

Control (<LOQ) 8 8 100 16 16 100 0.08 8 8 100 16 16 100 0.22 8 7 88 16 15 94 0.69 8 8 100 16 15 94 

Abbreviations: NA Not applicable. 

LOQ=0.0400 mg a.i./L. 

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Mean 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 the mean-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, and 0.69 mg a.i/L treatment levels, respectively. 

Table 10: Size at Test Termination in Pimephales promelas. 

Treatment 

(mg a.i./L) 

[mean-measured]

Body Weight Length 

Males Females Males Females 

n 

Mean

(g) 

±SD n 

Mean

(g) 

±SD n 

Mean

(mm) 

±SD n 

Mean

(mm) 

±SD 

Control (<LOQ) 4 3.15 0.467 4 1.22 0.092 4 60.6 2.14 4 47.9 1.11 0.08 4 2.88 0.338 4 1.25 0.112 4 59.0 1.68 4 47.3 1.25 0.22 4 2.53 0.427 4 1.24 0.086 4 56.9 2.46 4 47.6 0.55 0.69 4 2.60 0.219 4 1.28 0.071 4 59.6 2.14 4 47.4 1.02 

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

Fecundity1 ±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 GSI1

(%) 

±SD n 

Mean GSI1

(%) 

±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 Stage1 n Median Stage2

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, altered proportion of spermatocytes 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 Observations1

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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Treatment (mg 

a.i./L) 

[mean-measured] 

Diagnostic Observations1

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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Table 15b: Gonadal Histopathology in Male Pimephales promelas. 

Treatment (mg 

a.i./L) 

[mean-measured] 

Diagnostic Observations1

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. 

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Table 16a: Additional Gonadal Histopathology Observations in Male Pimephales promelas. 

Treatment (mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity

Increase 

Spermatogonia 

Efferent Duct 

Diameter Decrease 

Milt Decrease 

Altered Proportions of 

Spermatids 

(Decrease) 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

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

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

0.08 0 8 8 8 8 8 8 8 8 8 7 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 1 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

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Treatment (mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity

Increase 

Spermatogonia 

Efferent Duct 

Diameter Decrease 

Milt Decrease 

Altered Proportions of 

Spermatids 

(Decrease) 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.22 0 8 8 8 8 8 8 8 8 8 7 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 1 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

0.69 0 8 3 8 5 8 4 8 3 8 8 

1 8 0 8 0 8 0 8 0 8 0 

2 8 4 8 2 8 3 8 1 8 0 3 8 1 8 1 8 1 8 2 8 0 4 8 0 8 0 8 0 8 2 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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Table 16b: Additional Gonadal Histopathology Observations in Male Pimephales promelas. 

Treatment (mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity

Primary 

Spermatocytes 

Decrease 

Secondary 

Spermatocytes 

Decrease 

Sertoli CellSpermatocyst 

#/tubule Decrease 

Sertoli CellSpermatocyst Size 

Decrease 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

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

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

0.08 0 8 8 8 8 8 8 8 8 8 7 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 1 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

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Treatment (mg a.i./L) 

[mean-measured] 

Additional Diagnostic Observations1

Severity

Primary 

Spermatocytes 

Decrease 

Secondary 

Spermatocytes 

Decrease 

Sertoli CellSpermatocyst 

#/tubule Decrease 

Sertoli CellSpermatocyst Size 

Decrease 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.22 0 8 8 8 8 8 8 8 8 8 7 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 1 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

0.69 0 8 3 8 5 8 5 8 5 8 8 

1 8 0 8 0 8 0 8 0 8 0 

2 8 2 8 0 8 0 8 0 8 0 3 8 2 8 3 8 3 8 3 8 0 4 8 1 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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Table 17: Gonadal Histopathology in Female Pimephales promelas. 

Treatment (mg a.i./L) 

[mean-measured] 

Diagnostic Observations1

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. 

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Table 18: Additional Gonadal Histopathology Observations in Female Pimephales promelas.

Treatment (mg 

a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

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. 

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Mean male VTG was 970, 270, 390 and 270 and female VTG was 1.1x108, 1.2x108, 6.3x107 and 

1.1x108

 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 1.1x108 9.8x107 

0.08 4 270 291 4 1.2x108

 1.5x108 

0.22 4 390 497 4 6.3x107

 3.1x107 

0.69 4 270 321 4 1.1x108

 1.1x108 

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

LOQ=0.0400 mg a.i./L. 

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Table 20: Plasma Sex Steroids in Pimephales promelas (if measured).

Treatment 

(mg a.i./L) 

[mean-measured]

Plasma Testosterone (T) Plasma 17ȕ-estradiol (E2) 

Males Females Males Females 

n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD 

Control (<LOQ) ND -- -- ND -- -- ND -- -- ND -- -- 0.08 ND -- -- ND -- -- ND -- -- ND -- -- 0.22 ND -- -- ND -- -- ND -- -- ND -- -- 0.69 ND -- -- ND -- -- ND -- -- ND -- -- 

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

LOQ=0.0400 mg a.i./L. 

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One 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 groups showed signs of bruising, erratic swimming, loss of color or no color in tail, lethargy, and crooked spine. All other male and female fish appeared 

normal. 

Table 21: Secondary Sex Characteristics and Clinical Signs in Pimephales promelas.

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs 

Males Females 

Type n Incidence Type n Incidence 

Control (<LOQ) None reported 4 - None reported 4 - 0.08 None reported 4 - None reported 4 - 0.22 None reported 4 -- Crooked spine 4 1 0.69 Bruising, erratic swimming, loss of color or 

no color in tail, lethargy, crooked spine 

4 6 None reported 4 - 

LOQ=0.0400 mg a.i./L. 

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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 methods recommended in the guideline. 

Adult survival (range 91.7-100%) was not significantly 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 score 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, fertilization success, male GSI, and male length. 

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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. 

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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 not 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 not 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. 

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Table 22: Reproductive and HPG Endpoints1,2 for Male Pimephales promelas in the FSTRA with Malathion. 

Treatment 

(mg a.i./L)3

[mean-measured] 

Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

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

Control (<LOQ) 25.5 NA 0 NA NA 0 NA NA NA NA NA 0.08 28.3 0.845 6.1 0.939 No -27.21 0.955 NA NA NA NA 0.22 19.0 0.151 -21.36 0.297 No 5.44 1.00 NA NA NA NA 0.69 10.5 0.006 -2.71 0.994 Yes -25.85 0.961 NA NA NA NA Statistical Test Jonckheere-Terpstra Dunnett’s NA Dunnett’s NA NA 

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

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

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 The mean-measured concentrations were calculated by the reviewer, including the Day 21 measured concentrations 

LOQ=0.0400 mg a.i./L.

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Table 23: Reproductive and HPG Endpoints1,2 for Female Pimephales promelas in the FSTRA with Malathion. 

Treatment 

(mg a.i./L)3

[mean-measured] 

Fecundity Fert. Success Tubercle Score GSI 

Gonadal Staging 

and Histo. 

Plasma VTG Plasma T Plasma E2 

% Diff. p % Diff. p Median p % Diff. p Effect? (Yes/No) % Diff. p % Diff. p % Diff. p 

Control (<LOQ) 0 NA 0 NA 0 NA 0 NA NA NA NA NA NA NA NA 0.08 0.94 0.557 0.35 0.728 0.0 NA -1.12 0.614 No 5.19 0.494 NA NA NA NA 0.22 -10.06 0.416 0.13 0.980 0.0 NA 8.21 0.249 No -43.6 0.889 NA NA NA NA 0.69 -48.25 0.013 - 0.08 0.995 0.0 NA 20.84 0.016 Yes -2.63 1.00 NA NA NA NA Statistical Test 

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

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

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

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

2 Unless otherwise specified, effects are considered statistically significant at p<0.05. 3 The mean-measured concentrations were calculated by the reviewer, including the Day 21 measured concentrations 

LOQ=0.0400 mg a.i./L. 

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Table 24: Growth Endpoints1,2 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 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 

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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 test when parametric assumptions are satisfied. For all 

cases, both tests resulted in the same conclusions. 

Some information was missing from the study report that did not allow the reviewer to confirm if all test 

solution characteristics satisfied the OCSPP 890.1350 guideline requirements. The unionized ammonia 

and residual chlorine was not reported. Guideline 890.1350 recommends that residual chlorine in 

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

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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 guidelines recommended flow-through 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 Carolina 

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION: 

Endpoint Monotonic? Parametric? EDSP EFED Comments Female body weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body weight No; neither the 

mean nor median 

Yes Dunnett’s: 

n.s. p>0.05 Jonckheere: Dose 2 p=0.014 Dose 3 p=0.011 

Dunnett’s: n.s. p>0.05 William’s: Dose 2 p=0.029 Dose 3 p=0.030 

EDSP and EFED same conclusions, no effect Reductions from control were 8, 20, and 17% at the measured 0.08, 0.25, and 0.82 mg a.i/L levels; this does not follow a linear, monotonic pattern. It should be noted, however, that Jonckheere’s and William’s test detected significant 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 a treatment-induced reduction at the 0.25 and 0.82 mg a.i/L levels and observed, but did not quantify, that food consumption was also reduced at affected levels. 

Female body length No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Male body length No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female VTG No No MannWhitney: 

n.s. p>0.05 

MannWhitney: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

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Endpoint Monotonic? Parametric? EDSP EFED Comments Male VTG No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female GSI Yes, medians 

were for increasing trend 

Yes Jonckheere: 

Dose 3 p=0.016 

William’s: Dose 3 p=0.030 

EDSP and EFED same conclusions, significant promotion (p<0.05) of 21% at the top dose. 

Male GSI No No MannWhitney: 

n.s. p>0.05 

MannWhitney: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

Female tubercle score 

NA NA NA NA No score 

Male tubercle score Yes Yes Jonckheere: 

Dose 3 p=0.006 

William’s: Dose 3 p=0.002 

EDSP and EFED same conclusions; significant reduction at Dose 3 (p<0.01; 52% lower than control) 

Fecundity Yes Yes Jonckheere: 

Dose 3 p=0.013 

William’s: Dose 3 p=0.001 

EDSP and EFED same conclusions; significant reduction at Dose 3 (p<0.05; 48% lower than control) 

Fertility No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

EDSP and EFED same conclusions, no effect 

F testosterone NA NA NA NA NA M testosterone NA NA NA NA NA F estradiol NA NA NA NA NA M estradiol 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=0.01 

Levenes 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 

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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 

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PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.24 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 . . . .

.

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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 . .

.

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NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 1.31 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)

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PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.24 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 . .

.

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NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 1.31 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=0.01 

Levenes 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 

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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 

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PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 2.29 0.130 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4

Dose5

 Ctrl 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 . .

.

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***

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 6.16 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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EPA MRID Number 48617506 

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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=0.01 

Levenes 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=0.01 

Levenes 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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EPA MRID Number 48617506 

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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=0.01 

Levenes 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

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Version: 22 September 2011 

 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=0.01 

Levenes 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 

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

***

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Version: 22 September 2011 

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 

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Version: 22 September 2011 

 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 . .

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Version: 22 September 2011 

 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=0.01 

Levenes 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 

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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 

Page 180 of 294
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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=0.01 

Levenes 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

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EPA MRID Number 48617506 

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Version: 22 September 2011 

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

***

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 

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EPA MRID Number 48617506 

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Version: 22 September 2011 

 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

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EPA MRID Number 48617506 

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Version: 22 September 2011 

 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=0.01 

Levenes 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 . . . . . .

.

Page 184 of 294
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EPA MRID Number 48617506 

Page 79Rf93 

Version: 22 September 2011 

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 . . . . . .

.

Page 185 of 294
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EPA MRID Number 48617506 

Page 80Rf93 

Version: 22 September 2011 

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=0.01 

Levenes 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 

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EPA MRID Number 48617506 

Page 81Rf93 

Version: 22 September 2011 

 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 

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EPA MRID Number 48617506 

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Version: 22 September 2011 

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

Page 188 of 294
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EPA MRID Number 48617506 

Page 83Rf93 

Version: 22 September 2011 

 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=0.01 

Levenes 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 

Page 189 of 294
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EPA MRID Number 48617506 

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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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EPA MRID Number 48617506 

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Version: 22 September 2011 

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=0.01 

Levenes 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 

Page 191 of 294
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Malathion 

EPA MRID Number 48617506 

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Version: 22 September 2011 

 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 

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EPA MRID Number 48617506 

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Version: 22 September 2011 

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 

Page 193 of 294
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EPA MRID Number 48617506 

Page 88Rf93 

Version: 22 September 2011 

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=0.01 

Levenes 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 

Page 194 of 294
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EPA MRID Number 48617506 

Page 89Rf93 

Version: 22 September 2011 

 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=0.01 

Levenes 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 . . . . . , .

Page 195 of 294
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Version: 22 September 2011 

 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) ) 

Page 196 of 294
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Malathion 

EPA MRID Number 48617506 

Page 91Rf93 

Version: 22 September 2011 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- 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 

Page 197 of 294
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Malathion 

EPA MRID Number 48617506 

Page 92Rf93 

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=0.01 

Levenes 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 

Page 198 of 294
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Malathion 

EPA MRID Number 48617506 

Page 93Rf93 

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 

Page 199 of 294
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 200 of 294
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 201 of 294
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 

Signat?rf: "7i}-"'-++-~:r=--'---::::tr Date: ~-r'-L-~'-fh~;.;;;.;i...

Signature: ~~--A~::;:::=-­

Date: __ __,~__,_/.,_) ""-r ..... J ...... 1r"--_ 

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. 

Page 202 of 294
In vivo Hershberger Assay (2011) / Page 2 of 18 

MALATHION/ 057701 OCSPP 890.1400/ OECD 441

There were no treatment-related clinical signs of toxicity or gross pathological findings in either 

assay. 

In the androgen agonist assay, there were no treatment-related effects of malathion on accessory 

sex organ weights. At 1000 mg/kg/day, absolute and relative (to body weight) liver weights 

were increased (p≤0.01) by 29% and 32%, respectively, compared 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, 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. 

Percent CVs for the vehicle control and 1000 mg/kg/day group of the androgen agonist assay 

were compared to the performance criteria 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 control; 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). The study 

author noted that the same prosector dissected these organs for every rat in the androgen agonist 

assay. 

In the anti-androgen assay, there were no treatment-related effects for malathion on the weights 

of accessory sex organs. At 1000 mg/kg/day, absolute 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 relative liver weights were increased 

(p≤0.05) by 14% and 10%, respectively, at 100 mg/kg/day. The performance 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 requirements for a Hershberger assay (OCSPP 

890.1400).

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

provided. 

Page 203 of 294
In vivo Hershberger Assay (2011) / 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 Management); M. Vaneman (Director of 

Operations); J. Lech (Senior Manager Technical Operations); J. Yeager 

(Senior Manager Technical Resources); J. Gulbinski (Senior Manager 

Laboratory Sciences); J. Sarsoza (Principal 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 CH3

O O CH3

P

S

O 3CH

O 3CH

Page 204 of 294
In vivo Hershberger Assay (2011) / 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 Laboratories, Inc. (Portage, MI)

 Housing: Rats were housed two/cage in clear plastic nesting boxes with 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 PND 43 and received at the study facility 

at PND 49; the postcastration acclimation 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 potential androgenic 

activity, the test substance was administered daily via oral gavage to castrated male rats. 

Positive androgenic activity is defined as a significant increase in two or more target organ 

weights compared to the vehicle control. To screen for the potential 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 significant decrease in two or more target organ 

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

For both assays, the animals were dosed for 10 consecutive days and necropsied 

approximately 24 hours after the final dose administration for organ weight measurements. 

3. Study Schedule: Sexually mature male rats were castrated on PND 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 administration period was from PND 60 through PND 70. 

Rats were euthanized on PND 70 approximately 24 hours after the last dose and necropsied 

for organ weight measurements. 

4. Animal Assignment: Animals were assigned to the test groups noted in Table 1 using 

computer-generated, weight-ordered randomization procedures. At randomization, the 

weight variation of the rats did not exceed 20% of the mean weight in each group. 

Page 205 of 294
In vivo Hershberger Assay (2011) / Page 5 of 18 

MALATHION/ 057701 OCSPP 890.1400/ OECD 441

TABLE 1. Study Designa

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 controlb 0.4 8 

Anti-Androgen Assay 

Vehicle control (+TPb) 0 8 

Low (+TPb) 100 8 

Mid (+TPb) 300 8 

High (+TPb) 1000 8 

Flutamide + TPb, 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 Dawley 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 times for the anti-androgen assay) by mixing 

appropriate amounts of test substance with corn 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 included 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 storage for 40 hours at 20-25 °C 

 97.2-99.0% following storage for 13 days at 2-8 °C. 

Concentration (% of nominal): 93.1-103.9%

The analytical data indicated that the mixing procedure was adequate 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 given via s.c. injection at 0.5 

Page 206 of 294
In vivo Hershberger Assay (2011) / Page 6 of 18 

MALATHION/ 057701 OCSPP 890.1400/ OECD 441

mL/kg bw, and FT was administered 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 proportional data were analyzed using the 

variance test for homogeneity of the binomial distribution. Continuous data (e.g., body 

weights, body weight gains, food consumption values, and organ weight data) were 

analyzed using Bartlett’s test of homogeneity of variances and the analysis of variance 

(ANOVA), when appropriate [i.e., Bartlett’s test was not significant (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, Fisher’s exact test was used to analyze the data. 

Data from the evaluation for preputial 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 significance of the individual groups.

C. METHODS

1. Clinical Examinations: The rats were checked for viability at least twice daily during the 

study and were observed for general appearance daily during the dosing period. Post-dose 

observations were made daily 1-2 hours following dose administration. 

2. Body Weight: Animals were weighed at randomization, daily throughout 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. 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 anesthetization following by sodium pentobarbital 

injection (rats in the 0, 100, 300, and 1000 mg/kg/day dose groups of the androgen agonist 

assay) or by carbon dioxide asphyxiation (all other rats). The rats were subjected to a gross 

necropsy. The five mandatory androgen-dependent 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 

Page 207 of 294
In vivo Hershberger Assay (2011) / 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 were conducted for rats in the 0, 

100, 300, and 1000 mg/kg/day dose groups of the androgen assay. At study termination, the 

selected rats were anesthetized with isoflurane/oxygen, and blood was collected from the 

inferior vena cava for ChE activity determinations. After collection of blood and 

euthanasia, the brain was collected, weighed, and 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 termination, the death of this rat was not 

considered to be related to malathion dosing. 

2. Clinical signs of toxicity: No treatment-related adverse clinical signs of toxicity were 

observed in animals for any dose groups. 

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain 

data for the androgen agonist assay are presented in Table 2. Body weights and overall 

(Days 1-11) body weight gains in all malathion treatment groups were comparable to 

controls throughout the duration of the assay. 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. 

Page 208 of 294
In vivo Hershberger Assay (2011) / Page 8 of 18 

MALATHION/ 057701 OCSPP 890.1400/ OECD 441

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

Agonist Assaya

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 

Body 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 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. 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 the 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 and Cumulative Body Weight Gains (g) in the 

Anti-Androgen Assaya

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 

Body 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 comparable to the control. Daily mean food 

Page 209 of 294
In vivo Hershberger Assay (2011) / 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 death of one rat when calculating the food 

consumption for the 1000 mg/kg/day group for Days 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 intervals affected by the incorrect calculations. Statistical analyses 

were not conducted by the reviewers. 

Food consumption in the 300 mg/kg/day group was comparable to the controls over the 

duration of the study. In the positive control 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/day) in the Androgen Agonist Assaya

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 8b 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 8b 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 8b 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 8b 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 8b 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 8b 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 not 

account for the death of one rat in this group on Day 6. The mean food consumption values have been recalculated by the 

reviewers and are included in brackets. 

c The study author concluded that this value was significantly 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

Page 210 of 294
In vivo Hershberger Assay (2011) / Page 10 of 18 

MALATHION/ 057701 OCSPP 890.1400/ OECD 441

Selected food consumption data for the anti-androgen agonist assay are presented in Table 

5. 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. 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 positive control group (↓6%). 

TABLE 5. Selected Food Consumption (g/rat/day) in the Anti-Androgen Assaya

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 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 

D. SERUM HORMONE CONCENTRATIONS (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 treatment-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%, respectively, 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 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%). 

Percent CVs for the vehicle control and 1000 mg/kg/day group were compared to the 

performance criteria 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 control; 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). The study author 

Page 211 of 294
In vivo Hershberger Assay (2011) / 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. 

Page 212 of 294
In vivo Hershberger Assay 

MALATHION/ 057701 OCSPP 

TABLE 6. Accessory Sex Organ, Liver, and Kidney Weights from Androgen Agonist Assay in Sprague Dawley Ratsa

Organ 

Dose (mg/kg/day) 

Vehicle Control Positive Control Vehicle + TP (0.4) Malathion (100) Malathion (300) M

N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N Mean SD CV (%) N 

Seminal vesicles (g) 8 0.0615 0.0256 41.6 8 0.6808** (↑1007) 0.1483 21.8 8 0.0656 0.0211 32.2 8 0.0621 0.0306 49.3 7 0Ventral prostate (g) 8 0.0190 0.0096 50.5 8 0.1828** (↑862) 0.0228 12.5 8 0.0166 0.0032 19.3 08 0.0182 0.0036 19.8 7 0LABC (g) 8 0.2232 0.0338 15.1 8 0.6540** (↑193) 0.1254 19.2 8 0.1957 0.0215 11.0 8 0.2235 0.0726 32.5 7 0Cowper’s glands (g) 7 0.0063 0.0026 41.3 8 0.0398** (↑532) 0.0050 12.6 8 0.0072 0.0044 61.1 8 0.0054 0.0020 37.0 7 0Glans penis (g) 8 0.07 0.01 14.3 8 0.10** (↑43) 0.01 10.0 8 0.08 0.01 12.5 8 0.07 0.01 14.3 7 Liver (g) 8 13.6 1.9 14.0 8 14.4 1.9 13.2 8 14.2 1.3 9.2 8 14.8 2.0 13.5 7 1Liver, relative to terminal body weight (%) 

8 4.135 0.136 3.3b 8 4.274 0.258 6.0b 8 4.300 0.178 4.1b 8 4.502** (↑9) 0.190 4.2b 7 5

Kidneys (g) 8 2.0288 0.2618 12.9 8 2.4824** (↑22) 0.3415 13.8 8 2.1092 0.1877 8.9 8 2.1672 0.2746 12.7 7 2Kidney, relative to terminal body weight (%) 

8 0.617 0.042 6.8b 8 0.736** (↑19) 0.060 8.2b 8 0.636 0.031 4.9b 8 0.661* (↑7) 0.027 4.1b 7 0

a Data were obtained from Table 6 on page 64 of the study report. Percent differences from controls, calculated by the reviewers, are included in parentb Calculated by the reviewers. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p≤0.05 

** Significantly different from controls at p≤0.01 
In vivo Hershberger Assay (2011) / Page 13 of 18 

MALATHION/ 057701 OCSPP 890.1400/ OECD 441

Accessory sex organ, liver, and kidney weights for the anti-androgen assay are presented in 

Table 7. In the 100 mg/kg/day group, ventral prostate weights were increased (p≤0.01) 38% 

compared to the control; however, ventral prostate weights in the 300 or 1000 mg/kg/day 

dose groups were similar to controls. There were no malathion treatment-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 kidney 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 relative 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. The %CV values were less than the maximum 

permissible values for all accessory sex organs. 

There were no treatment-related gross pathological findings in either assay. 

Page 214 of 294
In vivo Hershberger Assay 

MALATHION/ 057701 OCSPP 

TABLE 7. Accessory Sex Organ, Liver, and Kidney Weights from Anti-Androgen Agonist Assay in Sprague Dawley Ratsa

Organ

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) Malath

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N 

Seminal vesicles (g) 8 0.7122 0.1333 18.7 8 0.1815** (↓75) 0.0552 30.4 8 0.7233 0.1482 20.5 8 0.7874 0.1108 14.1 8 0Ventral prostate (g) 8 0.1456 0.0251 17.2 8 0.0465** (↓68) 0.0132 28.4 8 0.2013** (↑38) 0.0560 27.8 8 0.1730 0.0337 19.5 8 0LABC (g) 8 0.6304 0.0531 8.4 8 0.3414** (↓46) 0.0588 17.2 8 0.6202 0.0765 12.3 8 0.6543 0.0668 10.2 8 0Cowper’s glands (g) 8 0.0441 0.0106 24.0 7 0.0159** (↓64) 0.0056 35.2 8 0.0498 0.0056 11.2 8 0.0502 0.0089 17.7 8 0Glans penis (g) 8 0.1162 0.0186 16.0 8 0.1008 0.0157 15.6 8 0.1138 0.0132 11.6 8 0.1146 0.0115 10.0 8 0Liver (g) 8 16.0 1.4 8.8 8 15.7 1.4 8.9 8 18.2* (↑14) 1.7 9.3 8 16.8 1.5 8.9 8 Liver, relative to terminal body weight (%) 

8 4.435 0.205 4.6b 8 4.421 0.175 4.0b 8 4.884* (↑10) 0.332 6.8b 8 4.759 0.302 6.3b 8 5

Kidneys (g) 8 2.7033 0.2286 8.5 8 2.6104 0.2359 9.0 8 2.8570 0.2892 10.1 8 2.7188 0.2198 8.1 8 2Kidney, relative to terminal body weight (%) 

8 0.749 0.042 5.6b 8 0.739 0.065 8.8b 8 0.768 0.043 5.6b 8 0.772 0.041 5.3b 8 0

a Data were obtained from Table 14 on page 80 of the study report. Percent differences from controls, calculated by the reviewers, are included in paren

b Calculated by the reviewers. N Number of animals in the group SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p<0.05 ** Significantly different from controls at p<0.01 
In vivo Hershberger Assay (2011) / Page 15 of 18 

MALATHION/ 057701 OCSPP 890.1400/ OECD 441

F. CHOLINESTERASE ACTIVITY: ChE activity data for the 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 Assaya

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 bioassay at dose levels as 

high as 1000 mg/kg bw/day (the highest dose level 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 and kidney weights) and 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 dosing. Because all other rats survived until 

termination, the death of this rat was not considered to be related to malathion dosing. No 

other treatment-related clinical signs of toxicity were observed in animals for any dose 

group. There were no treatment-related gross pathological findings. 

In the androgen agonist assay, 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 controls over the duration of the study. In the 

positive control group, average daily food consumption over Days 1-11 was increased 

(p≤0.01) by 14% compared to the negative control. 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 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) 

in the 300 mg/kg/day group (↑9% and ↑7%). Rats in the positive control group had 

Page 216 of 294
In vivo Hershberger Assay (2011) / 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 criteria 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 control; 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 weights in all malathion treatment groups were comparable 

to controls throughout treatment. Overall 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%, respectively; 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 relative 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 criteria 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 considered to be negative for androgenicity and antiandrogenicity in the Hershberger assay. 

C. STUDY DEFICIENCIES: None 

Page 217 of 294
In vivo Hershberger Assay (2011) / 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 Hershberger 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 receipt at the study facility; rats were 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 kidneys and livers were collected and weighed. Statistical 

analyses, other than calculations of means and standard deviations, were not conducted. 

All animals survived to the scheduled study termination. 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-related 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 consumption values were 

generally comparable across all groups during the study. Slight decreases (↓5-8%) in absolute 

overall food consumption were observed for males (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 and 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 requirements of 40 CFR Part 160. 

Page 218 of 294
In vivo Hershberger Assay (2011) / 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 dose formulations was described, 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 standards and final dilutions of 1 mg/mL and 200 mg/mL fortified 

samples at 15 °C was evaluated by re-analyzing 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 201 mg/mL fortified sample, the percent difference 

between the initial and final result was 24%; a second fortified sample 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 difference is not an indication of instability. 

Stability was evaluated for 1 mg/mL and 200 mg/mL 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 and at 2-8 °C, protected from light, for 13 days. The percent 

difference between the final stability sample 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 and Quality Assurance statements were 

provided. 

Page 219 of 294
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 220 of 294
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 221 of 294
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 

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PNDs 23-25. All other rats survived until scheduled sacrifice. Treatment-related clinical signs 

observed in the high-dose group included effects on gait (decreased motor activity and 

prostration), irregular respiration (dypsnea), nervous system (whole body tremors, 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 low- and mid-dose 

groups, sporadic clinical signs included slight excess salivation, urine stained abdominal fur, 

and/or chromodacryorrhea. 

There were no statistically significant effects on age or weight at VO, mean age at first vaginal 

estrous, mean cycle length, number of females cycling, number of females regularly cycling, or 

cycle status at necropsy. However, there were apparent dose-related trends toward decreased 

percentages in the number of animals cycling and the number of animals regularly cycling in the 

malathion-treated rats. 

Serum T4 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 changes in thyroid weight or histology. Therefore, the biological 

significance of the changes in T4 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 relative kidney weights were increased 

(p≤0.01) by 7% and 11%, respectively. Given that the weight changes in liver and kidneys were 

minor in magnitude and there were no adverse histopathological 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 effects on organ weights, 

including the uterus and ovaries, and there were no 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 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 relevance 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/day) was considered adequate based on the 

mortality, clinical signs and cholinesterase inhibition observed at 1000 mg/kg/day. 

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The assay satisfies the EDSP Tier 1 Test Order requirements 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 CH3

O O CH3

P

S

O 3CH

O 3CH

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 Laboratories, 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 received 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 ensure equal stratification of body weights. 

Littermates were not assigned to the same treatment group. 

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Femalesb 

Control 0 20 + 8 

Low 250 20 + 8 

Mid 500 20 + 8 

High 1000/750c 20 + 8 

a Data were obtained from page 34 of the study report. 

b For the cholinesterase phase of the study, an additional 8 females 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 rats. 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 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 the top, middle and bottom of all dose levels 

on the first day of dosing were collected for concentration and homogeneity analyses. 

Additional samples from the middle of the reduced concentration high dose material 

(150 mg/mL) were collected on the 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. Samples 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 analyses. 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 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 storage 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 adequate 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 analyzed statistically: body weights, body 

weight gains, age and body weight at VO, terminal 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 performed; a Dunnett’s test was 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 Dunn’s Method of Multiple Comparisons. 

The following parameters were analyzed by analysis of covariance (ANCOVA), using 

bodyweight on PND 21 as the covariate: terminal body weights, organ weights (pituitary, 

liver, kidneys, adrenals), initial body weight, final body weight, body weight gain, day of 

first vaginal estrous, cycle length, and age and body weight at VO. If the results of 

ANCOVA were significant, ANOVA was used. A modified Bonferroni correction was used 

for each pairwise comparison. 

Cycling status (cycling vs. not cycling) and percent of animals cycling regularly in the 

control and treatment groups were analyzed using the Variance Test for Homogeneity of the 

Binomial Distribution. 

The RBC and brain ChE data were evaluated as separate variables in one-way ANOVA. A 

Dunnett’s test was conducted 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 Clinical Examinations: All animals were examined twice daily for 

viability and once daily for general appearance prior to dose administration. Post-dose 

clinical examinations were conducted 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 weight 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 termination. 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 observed, vaginal lavages were 

performed daily, and the slides were evaluated microscopically to determine the stage of 

estrus of each female until necropsy. The age of first vaginal estrus after VO was recorded. 

The average cycle length was calculated and reported for complete estrous cycles. At the 

end of the study, the overall pattern of each female was characterized as regularly cycling, 

irregularly cycling, not cycling, or insufficient data. 

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 hours. Immediately following decapitation, trunk 

blood samples were taken for T4, TSH, and serum chemistry analyses. The samples for T4, 

TSH, and serum chemistry were immediately frozen 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 sacrifice. Any tissues with gross lesions were saved 

in 10% neutral-buffered formalin for possible future histopathological examination. 

a. Hormone Analysis: T4 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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) X Albumin globulin ratio (A/G Ratio, by calculation) 

X Alanine aminotransferase (ALT/also SGPT) Bile acids 

X Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase (SDH) 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in female rats based on Guideline 890.1450 

c. Organ Weights and Histopathology: The following CHECKED (X) tissues were 

collected and weighed. The (XX) organs, in addition, were subjected to histological 

examination. 

 UROGENITAL OTHER 

XX Ovaries (paired, without oviducts)*+ XX Thyroid*+ 

XX Uterus*+ X Liver* 

XX Kidneys (paired)*+ X Adrenals (paired)* 

X Cervix X Pituitary* 

* Weights required based on guideline 890.1450 

+ Histopathological examination required based on Guideline 890.1450 

All organs except the thyroid/trachea were weighed prior to fixation. Paired organs 

(kidneys, adrenals, and ovaries) were weighed together. The uterus was weighed “wet” and 

then again following removal of the fluid in the lumen (blotted weight). 

The ovaries (left or right not specified), uterus, and kidneys were fixed in neutral-buffered 

10% formalin, rinsed, and stored in 70% ethanol prior to embedding. The thyroid was fixed 

in neutral-buffered 10% formalin for at least 24 hours. The thyroid gland was dissected 

from the trachea, blotted, weighed, and placed in 70% ethanol until histopathological 

processing. The liver, adrenals, and pituitary were placed in neutral-buffered 10% formalin 

for potential future histopathologic examination. 

After fixation, protocol-specified tissues were shipped to Charles River, Pathology 

Associates, Maryland for processing. Tissue samples from the thyroid, kidney, ovary, and 

uterus were routinely processed into paraffin blocks, sectioned, stained with hematoxylin 

and eosin, and examined microscopically. 

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Thyroid sections were subjectively evaluated for follicular epithelial cell height and colloid 

area, using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest), and any 

abnormalities/lesions were noted. At least two sections of the thyroid were examined. Five 

random sections of an ovary were evaluated for follicular development and any 

abnormalities/lesions (such as atrophy). The ovary sections were evaluated on a five point 

grading scale (1=markedly reduced number or absent; 3=normal; 5=markedly increased 

number) for primordial, primary, secondary, tertiary, and atretic follicles and growth phase, 

mature, and regressive corpora lutea. Uterine histology documented cases of uterine hyperor hypotrophy as characterized by changes in uterine horn diameter and myometrial stromal, 

or endometrial gland development. The stage of the estrous cycle of the female at the time 

of necropsy was taken into account in the final histological assessment, as ovarian and 

uterine cellular changes are dependent upon endocrine status. 

7. Cholinesterase Assay: Animals from the cholinesterase cohort were terminated by sodium 

pentobarbital injection on PND 42, approximately 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 the 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 ChE 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 excess 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 found 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 PNDs 23 and 25 (after 2 to 4 doses). These 

animals displayed various 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 found dead or subsequently recovered (Table 2). These clinical 

signs included effects on gait (decreased motor activity and prostate), irregular respiration 

(gasping), nervous system (whole body tremors, 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 low- and mid-dose groups, sporadic clinical signs 

included slight excess salivation, 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/dayb

# 

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 mortality and clinical observations. 

C. FOOD CONSUMPTION: There were no significant differences 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 attainment of VO are presented in Table 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%) was slightly greater than 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/dayb

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 Table 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 mortality 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 groups, mean kidney weights were increased 

(p≤0.01) by 7% and 12%, respectively, and relative 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 significant 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 performance criteria maximum of 26.97%.

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TABLE 4. Organ Weights at Necropsya

Organ 

Vehicle Control Malathion

250 mg/kg/day 

Malathion 

500 mg/kg/day 

Malathion 

1000/750 mg/kg/dayb

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 mortality 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 presented 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 trends toward decreased percentages in the 

number of animals cycling and regularly cycling. 

Page 233 of 294
Female Pubertal Assay (2012) / Page 13 of 22 

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TABLE 5. Estrous Cyclicitya

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/dayb

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 mortality 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 T4

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 clinical chemistry parameters were concurrent 

with the increased liver weights for all dose groups. 

Total bilirubin levels in the low-, mid-, and high-dose groups were decreased (p≤0.01) by 

17, 31, and 27%, respectively, and triglyceride levels in the mid- and high-dose groups were 

decreased (p≤0.01) by 40 and 53%, respectively. Glucose 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 the following clinical chemistry parameters, 

the values were within the historical control ranges and are not considered to be biologically 

relevant because corresponding changes were 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 aminotransferase, chloride, sodium, and total 

protein treated group levels were similar to those in the control group. 

The Guideline criterion was met for serum T4 level in the control group. 

Page 234 of 294
Female Pubertal Assay (2012) / Page 14 of 22 

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TABLE 6. Hormone Levels and Clinical Chemistrya

Parameter 

Evaluated 

Vehicle Control Malathion

250 mg/kg/day 

Malathion 

500 mg/kg/day 

Malathion 

1000/750 mg/kg/dayb 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N Mean SD 

CV 

(%)

Hormones 

Serum T4, 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 20 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 

Page 235 of 294
Female Pubertal Assay (2012) / Page 15 of 22 

MALATHION/ 057701 OCSPP 890.1450/ OECD None

TABLE 6. Hormone Levels and Clinical Chemistrya

Parameter 

Evaluated 

Vehicle Control Malathion

250 mg/kg/day 

Malathion 

500 mg/kg/day 

Malathion 

1000/750 mg/kg/dayb 

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 treatment-related findings were observed. 

Page 236 of 294
Female Pubertal Assay (2012) / Page 16 of 22 

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TABLE 7. Evaluation of Colloid Area and Follicular Cell Height in the Thyroid Gland a

Treatment 

Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

Gradeb Incidence Gradeb 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/dayc 

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 treatment-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 compared to control (mean of 20 

primary follicles), and the number of atretic follicles in the left ovary were significantly 

decreased (p≤0.05) for the low- and mid-dose groups (means of 3 atretic follicles) compared 

to control (mean of 4.7 atretic follicles). However, these effects are not considered 

treatment-related. 

Page 237 of 294
Female Pubertal Assay (2012) / Page 17 of 22 

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TABLE 8. Incidence of Histpathological Lesions of the Ovaries, Uterus and Kidneya

Findings 

Dose Level (mg/kg/day) 

Vehicle Control Malathion

250 mg/kg/day 

Malathion

500 mg/kg/day 

Malathion

1000/750 

mg/kg/dayb 

# 

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 was 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. 

Page 238 of 294
Female Pubertal Assay (2012) / Page 18 of 22 

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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/dayb 

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 4c

 1.77 0.07 

RBC ChE 

levels 

(U/L) 

42 8 1.27 0.13 8 0.330** 

(↓74.1) 0.085 3c 0.139** 

(↓89.1) 0.057 1c 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 4c 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 observed primarily in the midand high-dose groups. These adverse clinical signs were observed in rats that subsequently 

recovered, were found dead, or were euthanized in extremis prior to scheduled termination. 

The adverse clinical signs included the following: effects on gait, excess salivation, irregular 

respiration, decreased motor 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 cholinesterase cohort and on PNDs 22 to 24 for 

the females in the main study. 

Statistically significantly reductions were observed in RBC and brain ChE activity in all 

malathion-treated female dose groups. 

In the low-, mid- and high-dose groups, statistically significant increases (119%, 115%, and 

124%) were observed in T4 levels; however, the significance of this finding is not clear 

because the increases were not clearly dose dependent, and the T4 values in each dose group 

were within the Testing Facility'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; statistically significant reductions in total 

bilirubin levels at >250 mg/kg/day; and statistically significant decreases in the triglyceride 

levels at >500 mg/kg/day. 

Slight increases or statistically significant increases in absolute and relative kidney and liver 

weights were seen in the mid- and high-dose groups of both sexes, but no histopathological 

Page 239 of 294
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 decrease in the absolute and relative 

pituitary weight was seen in the mid- and high-dose females; however, no histopathological 

findings were observed in the pituitary. No other test article-related organ weight changes 

were noted that were different from their respective controls. There were no correlating 

morphological data, thereby suggesting the kidney and liver weight changes were likely 

adaptive changes to the mid- and 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 the high-dose from the cholinesterase 

cohort were found dead between PNDs 23-25. All other rats survived until scheduled 

sacrifice. The treatment-related clinical signs observed in the high-dose group included 

effects on gait (decreased motor activity and prostate), irregular respiration (dypsnea), 

nervous system (whole body tremors, 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 low- and mid-dose groups, 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 significant effects on mean age and weight at VO, age at first 

vaginal estrous, mean cycle length, number of females cycling, number of females regularly 

cycling, and cycle status at necropsy. Although not statistically significant, there were 

apparent dose-related trends toward decreased percentages in the number of animals cycling 

and the number of animals regularly cycling for the malathion-treated rats. 

 Serum T4 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 affected by treatment, and there were no changes in thyroid weight 

or histology. Therefore, the biological significance of the changes in T4 concentrations is 

unclear. 

Compared to control, absolute and relative liver and kidney weights were significantly 

increased in a dose-dependent manner. Given that the changes were minor in magnitude 

(≤16% increase) and there were no adverse histopathological 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) 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 effects on organ weights; including 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 

Page 240 of 294
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 relevance of these changes is unclear 

as the values were within the historical control ranges for the laboratory and because similar 

changes were not observed in the related 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 ChE 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 the 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%. 

 

Page 241 of 294
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), malathion (96.0%; Batch #9010501) in corn oil 

was administered daily via oral gavage in a dose volume of 5 mL/kg to eight juvenile female 

(PND 22 to 35) and eight male (PND 23 to 36) Sprague Dawley [Crl:CD(SD)] rats/dose group at 

doses of 0, 250, 450, or 600 mg/kg/day. The animals were dosed for 14 consecutive days, and 

necropsied one day after the final dose administration. 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 cholinesterase 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 (concurrently reviewed). 

All rats survived to scheduled termination. Clinical findings included 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 controls. The brain and liver weights (absolute 

and relative to final body weight) were 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. 

Page 242 of 294
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 dose formulations was described, 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 concentration fortified samples. 

The stability of calibration standards and final dilutions of 1 mg/mL and 200 mg/mL fortified 

samples at 15°C was evaluated by re-analyzing the standards and samples at 38 and 37 hours, 

respectively, after 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 fortified sample, the percent difference between the initial and final 

result was 24%; a second fortified sample was prepared and stored at 2-80 °C for 15 days and the 

percent difference between the initial 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/mL dose formulations prepared in corn oil. 

The dose formulations were analyzed prior to and 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. 

Page 243 of 294
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 244 of 294
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 245 of 294
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. 

Page 246 of 294
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 group 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, the observed clinical signs included effects on gait 

(e.g., ataxia, lost righting reflex, and decreased motor activity), irregular 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 chromorhinorrhea). 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 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 lowand mid-dose groups, there was no effect on first day of PPS in the high-dose group. 

Compared to the control group, absolute 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, respectively. The liver and 

kidney weight effects are treatment related and are 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 changes in weights of accessory sex tissues. 

There were no other significant effects on organ weights. 

Serum T4 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 T4 levels were within 

the range of historical control values, and there were no related changes in TSH levels. In 

addition, there were no changes in thyroid weight or any microscopic findings in the thyroids of 

malathion-treated rats. Therefore, the biological relevance of the changes in T4 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 bilirubin 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 were 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 evaluated in the high-dose group. 

Page 247 of 294
Male Pubertal Assay (2012)/ Page 3 of 21

MALATHION/ 057701 OCSPP 890.1500 / OECD None

Based on the ChE data, and the mortality and clinical signs, both the mid- and high-doses (500 

and 750 mg/kg/day) of malathion were clearly excessive for immature male rats; however, the 

low-dose level was adequate. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Male Pubertal Assay (OCSPP 

890.1500). 

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

Quality Assurance statements were provided. 

 

Page 248 of 294
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 CH3

O O CH3

P

S

O 3

CH

O

3

CH

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 Laboratories, 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 

Page 249 of 294
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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 received 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 ensure equal stratification of body weights. 

Littermates were not assigned to the same treatment group. 

TABLE 1. Study Design a

Test group Dose (mg/kg/day) # of Males b 

Control 0 20 + 8 

Low 250 20 + 8 

Mid 500 20 + 8d

High 1000/750 c

 20 + 8e 

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 rats. 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 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 the top, middle and bottom of all dose levels 

on the first day of dosing were collected for concentration and homogeneity analyses. 

Additional samples from the middle of the reduced concentration high dose material 

(150 mg/mL) were collected on the third day of preparation. 

Page 250 of 294
Male Pubertal Assay (2012)/ Page 6 of 21

MALATHION/ 057701 OCSPP 890.1500 / OECD None

A second lot of the bulk test substance (Lot # D2014-OSJ-MLT-01-S) was used beginning 

on October 21, 2011 until test termination. Samples 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 analyses. Additional samples from middle of 

the reduced concentration high dose material (150 mg/mL) were collected 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 storage 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 adequate 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 analyzed statistically: body weights, body 

weight gains, age and body weight at PPS, terminal 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 performed; a Dunnett’s test was 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 Dunn’s Method of Multiple Comparisons. 

The following parameters were analyzed by analysis of covariance (ANCOVA), using 

bodyweight on PND 21 as the covariate: terminal body weights, organ weights (pituitary, 

liver, kidneys, and adrenals), initial body weight, final body weight, body weight gain, and 

age and body weight at PPS. If the results of ANCOVA were significant, ANOVA was 

used. A modified Bonferroni correction was used for each pairwise comparison. 

The RBC and brain ChE data were evaluated as separate variables in one-way ANOVA. A 

Dunnett’s test was conducted if the results of the ANOVA were significant. 

Significance was denoted at p≤0.05 and p≤0.01. 

Page 251 of 294
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 C. METHODS

1. Mortality and Clinical Examinations: All animals were examined twice daily for 

viability and once daily for general appearance prior to dose administration. Post-dose 

clinical examinations were conducted 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 weight 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 termination. 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 hours. Immediately following decapitation, trunk 

blood samples were taken for T4, TSH, testosterone, and serum chemistry analyses. The 

samples for T4, 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 scheduled sacrifice. Any 

tissues with gross lesions were saved in 10% neutral-buffered formalin for possible future 

histopathological examination. 

a. Hormone Analysis: T4, TSH, and testosterone levels were determined with 

radioimmunoassay kits and analyzed with a Wallac 1470 Gamma Counter. 

b. Clinical Chemistry: The following CHECKED (X) parameters were examined. 

Page 252 of 294
Male Pubertal Assay (2012)/ Page 8 of 21

MALATHION/ 057701 OCSPP 890.1500 / OECD None

 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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) X Albumin globulin ratio (A/G Ratio, by calculation) 

X Alanine aminotransferase (ALT/also SGPT) Bile acids 

X Aspartate aminotransferase (AST/also SGOT) 

 Sorbitol dehydrogenase (SDH) 

X Gamma glutamyl transferase (GGT) 

 Glutamate dehydrogenase 

* Recommended for the pubertal assay in male 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 subjected 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 based on guideline 890.1500 

+ Histopathological examination required based on Guideline 890.1500 

All organs except the thyroid/trachea were weighed prior to fixation. Kidneys and adrenals 

were weighed together. Following weighing, the testis and epididymis were fixed in 

Bouin’s solution for no more than 24 hours. The thyroid (with parathyroid) was collected 

with the trachea and fixed in neutral-buffered 10% formalin for at least 24 hrs. Following 

fixation, the thyroid was dissected free of the trachea and weighed. Kidneys were fixed for 

at least 24 hours in neutral-buffered 10% formalin, and then placed in 70% ethanol until 

histological processing. All other 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 blocks, sectioned, stained with 

hematoxylin and eosin, and examined microscopically. 

Page 253 of 294
Male Pubertal Assay (2012)/ Page 9 of 21

MALATHION/ 057701 OCSPP 890.1500 / OECD None

Thyroid sections were subjectively evaluated for follicular epithelial cell height and colloid 

area, using a five point grading scale (1 = shortest/smallest; 5 = tallest/largest), and any 

abnormalities/lesions were noted. At least two sections of the thyroid were examined. 

6. Cholinesterase Assay: Animals from the cholinesterase cohort were terminated by sodium 

pentobarbital injection on PND 53, approximately 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 the 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 ChE 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. Two animals in the mid-dose group were also 

found dead on PNDs 45 and 50. In the cholinesterase 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 other 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 high-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 decreased motor activity), irregular respiration 

(dyspnea), nervous system (e.g., whole body tremors, tremors in the head, slight, moderate, 

and extreme excess salivation and lacrimation), and general toxicity (e.g., urine-stained 

abdominal fur, ungroomed coat, chromodacryorrhea, 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.

Page 254 of 294
Male Pubertal Assay (2012)/ Page 10 of 21

MALATHION/ 057701 OCSPP 890.1500 / OECD None

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/dayb

# 

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 mortality 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 final body weights, overall body weight 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 control, but no effect on first day of PPS 

was observed for the high-dose group.

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%. 

Page 255 of 294
Male Pubertal Assay (2012)/ Page 11 of 21

MALATHION/ 057701 OCSPP 890.1500 / OECD None

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/dayb 

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 22c 69.7 6.2 8.9 22c

 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 19g 236.5 33.0 14.0 15e 225.5 26.0 11.5

A 20 223.7 -- -- 19 235.5 -- -- 19g 236.9 -- -- 15e 224.7 -- -- 

Final body weight 

(g) 

U 20 324.2 20.4 6.3 20 325.0 32.6 10.0 20e 321.1 34.3 10.7 8e 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 20d

42.6**

(↑1.6 

days) 

1.6 3.8 15e 41.5 1.3 3.1 

A 20 41.0 -- -- 20 42.4 -- -- 20 42.5 -- -- 15e 41.6 -- -- 

Age at PPS 

(PND) 

U 20 42.2 1.3 3.1 19f 43.2 1.6 3.7 19g 43.6 2.5 5.7 15e 43.0 1.8 4.2 

A 20 42.1 -- -- 19f 43.2 -- -- 19g 43.6 -- -- 15e 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 mortality 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 euthanized due to clinical observations. 

f Day of maturity could not be determined for one rat. 

g One rat was excluded because testing was discontinued before preputial 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 the 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- and 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 control absolute thyroid weight (0.0326 g) 

was slightly above the performance criteria acceptable range (0.014-0.026 g). 

Page 256 of 294
Male Pubertal Assay (2012)/ Page 12 of 21

MALATHION/ 057701 OCSPP 890.1500 / OECD None

TABLE 4. Organ Weights at Necropsya

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 

(%) Nc Mean SD 

CV 

(%) Nc 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 20 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 20 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 20 575.5 165.0 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 20 280.3 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 20 227.7 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 20 200.3 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 20 236.9 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 Necropsya

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 

(%) Nc Mean SD 

CV 

(%) Nc 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 20 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 mortality and clinical observations. 

c Excludes animals found dead or euthanized due to clinical 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 Table 5. In the low-, mid-, and high-dose groups, 

serum T4 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 increases (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%, respectively, and triglycerides 

were decreased (p≤0.01) by 33, 65, and 68%, respectively. These clinical chemistry effects 

were concurrent with increases in liver weights observed in all dose groups. 

Although significant changes were observed in the following clinical chemistry parameters, 

the values were within the historical control ranges 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%; cholesterol levels were increased 

(p≤0.05) by 11 and 19%; glucose levels 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 protein, urea nitrogen, creatinine, phosphorus, and 

sodium treated group concentrations were similar to control values. 

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The mean control TSH level (2.780) was below the performance criteria acceptable range 

(4.212-24.112). Guideline criteria for serum T4 and testosterone levels were met in the 

control. 

TABLE 5. Hormone Levels and Clinical Chemistrya

Parameter 

Evaluated 

Vehicle Control Malathion

250 mg/kg/day 

Malathion 

500 mg/kg/day 

Malathion 

1000/750 mg/kg/dayb 

N Mean SD 

CV 

(%) N Mean SD 

CV 

(%) N c Mean SD 

CV 

(%) N c Mean SD 

CV 

(%)

Hormones 

Serum T4, 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. 

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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 animals found dead or euthanized due to clinical observations, and includes the rats that were reassigned from the 

cholinesterase phase to the main study. 

d Rats with moderately or severely hemolyzed 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 treatment-related findings in the thyroid gland, epididymides, right 

kidney, liver, testis, or pituitary. 

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TABLE 6. Evaluation of Colloid Area and Follicular Cell Height in the Thyroid Gland a

Treatment 

Groups 

Parameter Evaluated 

Colloid Area Follicular Cell Height 

Gradeb Incidence Gradeb 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/dayc 

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 reductions in RBC and brain ChE 

activity in the low and mid-dose groups (Table 7). No animals from the high-dose group 

were evaluated for RBC and brain ChE activity. 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. 

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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/dayb 

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 6c 2.02 0.050 0c -- -- 

RBC ChE levels 

(U/L) 53 7 1.18 0.145 7 0.245** (↓79.2) 0.061 6c 0.145** (↓87.7) 0.035 0c -- -- 

Brain ChE levels 

(U/L) 53 8 14.2 0.737 8 11.7 

(↓17.4) 0.850 6c 9.25** 

(↓34.6) 1.79 0c -- -- 

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 male rats at both the midand high-doses. Malathion-related adverse clinical 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, irregular respiration, decreased motor activity, 

tremors, as well as additional signs of general toxicity. Due to mortality and adverse 

clinical signs of toxicity 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). Statistically significant 

decreases in mean testosterone levels were observed in all malathion-treated groups. 

Although the mean values for all four dose groups were within the acceptable range for 

control male Sprague-Dawley rats 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-androgenic 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 the decreased testosterone levels are due to alterations in the 

hepatic metabolism of testosterone by the 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 rats with inhibited microsomal cytochrome P450-dependent enzymes 17-hydroxylase and 17, 20-lyase. 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, statistically significant increases were observed in 

mean T4 levels (132-141%); however, the significance of this finding in male rats is not 

clear because the increases were not clearly dose dependent and the T4 values were within 

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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 guidelines. Because malathion has been shown to be an 

inhibitor of mixed function oxidases, the increases seen in this study could be related to 

effects of malathion on the hepatic metabolism of T4, reducing excretion of T4, and/or 

malathion could be overwhelming metabolic processes in the liver at these dose levels and 

altering thyroid hormone metabolism. 

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 significant increase in 

globulin at >250 mg/kg/day; significant reductions in albumin and the A/G ratio at >250 

mg/kg/day; statistically significant reductions in total bilirubin levels in male rats at >250 

mg/kg/day; and significant decreases in the triglyceride levels at >250 mg/kg/day. 

Slight increases or statistically significant increases in absolute and relative kidney and liver 

weights were seen in the mid- and high-dose groups of both sexes, but no histopathological 

findings were associated with these weight changes. These slight changes reflect the 

adaptive, metabolic and execratory responses expected 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 respective controls. There were no correlating 

morphological data, thereby suggesting the kidney and liver weight changes were likely 

adaptive changes to the mid and 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 androgenic or antiandrogenic effects in male 

rats or any effect on pubertal development 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 clinical observations on PND 32. Two animals in the mid-dose 

group were found dead on PNDs 45 and 50. In the cholinesterase 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 recovered. These clinical signs included effects on 

gait, irregular respiration, nervous system, and general toxicity. 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 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, absolute liver weights were increased (p≤0.05) by 21% in 

the mid- and high-dose groups, and the relative (to body) liver weight was increased 

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(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 

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 effects were considered to be treatment related 

and are likely an adaptive response to malathion 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 accessory sex tissues. No other significant effects 

on organ weights were reported. 

Serum T4 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 T4 levels 

were within the range of historical control values, and there were no related changes in TSH 

levels. In addition, there were no changes in weight or microscopic findings in thyroid 

glands of malathion-treated rats. Therefore, the biological relevance of the changes in T4

levels is unclear. 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 bilirubin 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 high-dose groups. The above changes were concurrent with the increases in 

liver weights and likely reflect adaptive changes in liver metabolism. Although significant 

changes were observed in other selected clinical chemistry parameters, the values were 

generally within the historical control ranges and are not considered 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 immature 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 weight (0.0326 g) was slightly above the 

performance criteria acceptable range (0.014-0.026 g). 

 The mean control TSH level (2.780) was below the performance criteria acceptable 

range (4.212-24.112). 

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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), malathion (96.0%; Batch #9010501) in corn oil 

was administered daily via oral gavage in a dose volume of 5 mL/kg to eight juvenile female 

(PND 22 to 35) and eight male (PND 23 to 36) Sprague Dawley [Crl:CD(SD)] rats/dose group at 

doses of 0, 250, 450, or 600 mg/kg/day. The animals were dosed for 14 consecutive days and 

necropsied one day after the final dose administration. 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 cholinesterase 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 (concurrently reviewed). 

All rats survived to scheduled termination. Clinical findings included 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. Mean 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 (absolute 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 cell cholinesterase activity 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 compliance with GLPs. 

 

Page 265 of 294
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 dose formulations was described, 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 standards and final dilutions of 1 mg/mL and 200 mg/mL fortified 

samples at 15 °C was evaluated by re-analyzing 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 fortified sample, the percent difference 

between the initial and final result was 24%; a second fortified sample 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 difference is not an indication of instability. 

Stability was evaluated for 1 mg/mL and 200 mg/mL 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 and at 2-8 °C, protected from light, for 13 days. The percent 

difference between the final stability sample 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 and Quality Assurance statements were 

provided.

Page 266 of 294
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 267 of 294
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 268 of 294
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 

Page 269 of 294
Steroidogenesis Assay (2011) / Page 2 of 14 

MALATHION/057701 OCSPP 890.1550/ OECD None

and an increase in estradiol production at 10 μM of malathion in Run 2 (p<0.05; 1.12-fold 

change). The validity of the observed decrease in testosterone is questionable as precipitation of 

malathion was observed at 100 μM in Runs 2 and 3, and there was no other concentration 

dependent effects noted in testosterone production. Similarly for the observed increase in 

estradiol, the effect was observed at 10 μM in only one of three runs, and no concentration 

dependent changes in estradiol production were observed. 

Based on hormone response in at least three independent runs, malathion treatment did not result 

in statistically significant and reproducible alterations in testosterone or estradiol production. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Steroidogenesis assay 

(OCSPP 890.1550). 

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

provided. 

 

Page 270 of 294
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., Director of Laboratory Operations 

David Blakeman, B.S., Senior Scientist/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., Principal 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 

Page 271 of 294
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 concentration 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 hormone concentrations by supplier 

ITS+ premix: Lot # 05245, 09233 

8. Test Cells: H295R human adrenocortical carcinoma cells (ATCC CLR-2128; lot#7635054) 

at passage 7.5 - 8.5 were incubated in the stock medium at the time of plating, dosing, and 

harvest. Incubation conditions were at 5% CO2 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. 

Page 272 of 294
Steroidogenesis Assay (2011) / Page 5 of 14 

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2. Test Solutions: Malathion was dissolved in DMSO to make a 200 mM stock and then 

serially diluted 1:10 in DMSO. 22R-Hydroxycholesterol (Sigma 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 μM. Malathion was then diluted 

1:2000 in supplemented medium containing 10 μM 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 μM 22R-hydroxycholesterol. When added 

to the cell culture plates, these dilutions yielded final concentrations of 1 μM and 10 μM for 

forskolin, 0.1 μM and 1 μM for prochloraz and 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 μM 

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% CO2 atmosphere. 

4. Exposure: The cells were checked microscopically for good attachment and proper 

morphology, and the medium was removed and 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 total 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 response to forskolin (an inducer of testosterone 

and estradiol production) and prochloraz (an inhibitor of testosterone and estradiol 

production). The QC plate was prepared and dosed in the same manner with either forskolin 

or prochloraz according to the schematic presented in Table 2. 

TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM)a

 1 2 3 4 5 6 

A Blankb

 Blank Blank Backgroundc Background Background 

B DMSO DMSO DMSO DMSO + MeOHd DMSO + MeOH DMSO + MeOH 

C Forskolin 1μM Forskolin 1μM Forskolin 1μM Prochloraz 0.1μM Prochloraz 0.1μM Prochloraz 0.1μM 

D Forskolin 10μM Forskolin 10μM Forskolin 10μM Prochloraz 1μM Prochloraz 1μM Prochloraz 1μM 

a Data were obtained from page 16 of the study report. Dosing was calculated based on a total volume of 1 mL per well. 

b Blank wells received medium containing 22R-hydroxycholesterol. 

c Background wells received medium only. 

d MeOH = 70% methanol was added to these wells for 30 minutes at room temperature following medium removal. 

Page 273 of 294
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 removed, 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 bromide] test immediately after removal of 

the culture medium. Following media removal and/or methanol treatment, 0.5 mL of 0.5 

mg/mL MTT solution in supplemented medium containing 10 μM 22R-hydroxycholesterol 

was added to each well of the test chemical and QC plates. The plates were incubated at 

37±2 °C in a 5% CO2 incubator for 3 hours. Following 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. Following 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 each 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 detection 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 estradiol (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 below maximum response on standard curve 

NA Test compound tested for interference with measurement system 

C. DATA ANALYSIS: Mean values (pg/mL) and standard deviations for testosterone and 

estradiol were calculated for each concentration of the test chemical, reference chemicals, 

and the solvent controls. Relative changes in testosterone and estradiol production were 

calculated using the equation below: 

Relative Change = [Hormone] in each well ÷ [Hormone] of mean solvent (vehicle) control 

For forskolin induction of testosterone, relative change was calculated both with and 

without subtracting the background hormone production from the forskolin-treated wells 

and blank and solvent control wells. This was done because of the high background 

production of testosterone and the small dynamic range for forskolin induction. 

Background hormone production was calculated from three wells with cells on the QC plate 

that received no 22R-hydroxycholesterol at the time of exposure. All concentrations that 

exhibited greater than 20% cytotoxicity in the 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 treatment 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 either the normality or the homogeneity test, a log 

Page 274 of 294
Steroidogenesis Assay (2011) / Page 7 of 14 

MALATHION/057701 OCSPP 890.1550/ OECD None

transformation was performed on the data to attempt 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 performed on the transformed data to evaluate 

statistical significance between the each treatment group and the 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-transformed data set was analyzed using the nonparametric 

Kruskal-Wallis test followed by Dunn’s test to evaluate statistical significance between each 

treatment group and the control group. Data analysis was conducted in Microsoft Excel. 

Statistics were calculated using Unistat 6.0 Light for Excel. Graphs were generated using 

Microsoft Excel. 

II. RESULTS

A. TEST COMPOUND: Precipitation of malathion 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 solvent 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 based 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 malathion-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 significant increase in estradiol production at 10 μM (1.12-fold 

change). There were no other statistically significant changes. 

Page 275 of 294
Steroidogenesis Assay (2011) / Page 8 of 14 

MALATHION/057701 OCSPP 890.1550/ OECD None

TABLE 3. Mean (±SD) Hormone Concentrations Following 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 excluded because precipitation was observed 

*p ≤ 0.05 

Page 276 of 294
Steroidogenesis Assay (2011) / Page 9 of 14 

MALATHION/057701 OCSPP 890.1550/ OECD None

FIGURE 1. Change in Testosterone Production Relative to Malathion Concentration. 

Page 277 of 294
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 presented graphically in Figure 3. Malathion 

was not cytotoxic to the H295R cells at any concentration tested. 

Page 278 of 294
Steroidogenesis Assay (2011) / Page 11 of 14 

MALATHION/057701 OCSPP 890.1550/ OECD None

TABLE 4. Mean (±SD) MTT Cell Viability Results after Treatment with Forskolin, Prochloraz, or 

Malathion for 48 Hours. a 

Compound Concen. 

(μM) 

Cell Viability – Run 2 Cell Viability – 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 5.25 101.6b 6.32 97.1 1.43 

a Data were obtained from pages 23 and 24 of the study report. 

b MTT outlier point removed. Subtracted value for the outlier well was 0.024. Image taken of this well indicates that cells 

are present and adherent. 

SC = Solvent control 

Page 279 of 294
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 exposure to the reference chemicals, SC, 

and blank samples, as well as the fold difference change relative to SC (individual and mean 

± SD) for the three assay runs are presented in Table 5. The 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 production 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 testosterone by 1.80-fold (instead of ≥2-fold as per the 

Page 280 of 294
Steroidogenesis Assay (2011) / Page 13 of 14 

MALATHION/057701 OCSPP 890.1550/ OECD None

guideline) and estradiol by 10.75-fold. However, when the background is subtracted, 10 

μM forskolin induced testosterone an average 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 concentrations in the SC 

were 16% for testosterone and 10% for estradiol, 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 Treatment 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 1.64c 1.31c 1.31c 1.42c 0.18 

Forskolin 10 5898 3054 3596 2.25d 1.61d 1.53d 1.80d 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 values 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 values 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. Precipitate was noted upon observations of the wells under the 

microscope after the 48-hour incubation in the 100 μM wells in runs 2 and 3. The suitable 

highest concentration of malathion that could be tested was 100 μM in Run 4 and 10 μM in 

Runs 2 and 3 because of precipitate observed. A statistically significant reduction of 

testosterone was observed at 100 μM in Run 4. No statistically significant alterations in 

testosterone were observed at any concentration in Run 2 or 3. A statistically significant 

increase in estradiol was observed at 10 μM in Run 2. No statistically significant alterations 

in estradiol were observed at any concentration in Run 3 or 4.

B. AGENCY COMMENTS: Guideline acceptability recommendations and requirements 

were generally met, including lack of cytotoxicity, adequate production of testosterone and 

Page 281 of 294
Steroidogenesis Assay (2011) / Page 14 of 14 

MALATHION/057701 OCSPP 890.1550/ OECD None

estradiol, acceptable reproducibility (low %CV), and appropriate 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 concentration of malathion was 

not be evaluated due to observed precipitation of the test compound. No other significant 

changes for concentration dependent changes were observed in testosterone 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 significant changes were not observed in the other 

two runs at this concentration and no other significant changes were noted at the other 

concentrations tested. 

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

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

 The results of the proficiency test were not reported. 

Page 282 of 294
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 altered by the Health Effects Division subsequent to 

signing by CSS-Dynamac Corporation personnel. 

Page 283 of 294
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

comprised of eleven screening assays intended to identify a chemical’s likely endocrine 

bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) 

pathways. The robustness of the Tier 1 battery is based on the strengths of each individual 

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

individual assay should not be considered in isolation but rather should be considered in 

the context of other assays in the battery as well as Other Scientifically Relevant 

Information (OSRI). In order to determine if a chemical has the potential to interact with 

the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in 

combination with the findings in the OSRI, should be undertaken (refer to the WoE 

Document).

Page 284 of 294
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 

Page 285 of 294
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 duration 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 weights 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 requirements for a uterotrophic assay (OCSPP 

890.1600).

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

provided. 

 

Page 286 of 294
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 Management); M. Vaneman (Director of 

Operations); J. Lech (Senior Manager Technical Operations); J. Yeager 

(Senior Manager Technical Resources); J. Gulbinski (Senior Manager 

Laboratory Sciences); J. Sarsoza (Principal 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: 17α-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 Laboratories, Inc. (Portage, MI)

 Housing: Rats were housed two/cage in clear plastic nesting boxes with 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 CH3

O O CH3

P

S

O 3CH

O 3CH

Page 287 of 294
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 administered the test substance from PND 68 

through 70. Rats were euthanized approximately 24 hours later on PND 71 and necropsied 

for uterine weight measurements. 

3. Animal Assignment: Animals were assigned to the test groups noted in Table 1 using 

computer-generated, weight-ordered randomization 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 Designa

Test Group Dose (mg/kg/day) # of Females 

Estrogen Agonist Assay 

Vehicle Control 0 8 

Low 100 8 

Mid 300 8 

High 1000 8 

17α-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 Sprague 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 includes a limit-dose group of test animals.

5. Dose Preparation and Analysis: Dose formulations were prepared 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 top, 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 included 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%

Page 288 of 294
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 adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: Animals were administered the test 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 volumes were adjusted daily based on the 

concurrent body weight measurement. 

7. Statistics: Clinical observations and other proportional data were analyzed using the 

variance test for homogeneity of the binomial distribution. Continuous data (e.g., body 

weights, body weight gains, and food consumption) were analyzed using Bartlett’s test of 

homogeneity of variances and the analysis of variance (ANOVA), when appropriate [i.e., 

Bartlett’s test was not significant (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, 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 significance of the individual groups.

C. METHODS

1. Clinical Examinations: The rats were checked for viability at least twice daily during the 

study and were observed for general appearance daily during the dosing period. Post-dose 

observations were made 1-2 hours following dose administration. 

2. Body Weight: Animals were weighed at randomization, daily throughout 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. Values 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 collection. Animals were then euthanized with sodium 

pentobarbital (rats in the 0, 100, 300, and 1000 mg/kg/day dose groups) or by carbon 

dioxide asphyxiation (rats in the 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 just below the cervix in order to retain the 

luminal fluid in the uterus. The “wet” uterus (i.e., containing the luminal fluid) was 

weighed. Subsequently, the uterine horns were cut longitudinally and gently blotted with 

Page 289 of 294
In vivo Uterotrophic Assay (2011) / Page 6 of 10 

MALATHION/ 057701 OCSPP 890.1600/ OECD 440

moist filter paper to remove the luminal fluid 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 were 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 cholinesterase 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 initiation of dosing; the evaluations confirmed that all rats were not 

cycling and had been properly ovariectomized. On the day of euthanasia, it was confirmed 

that all rats had achieved vaginal patency. 

B. BODY WEIGHT AND WEIGHT GAIN: Body weight and body weight gain data are 

presented in Table 2. Body weights were comparable to controls in all dose groups over the 

duration of the study. In the 1000 mg/kg/day group, there was a statistically 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 decreased (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%). 

Page 290 of 294
In vivo Uterotrophic Assay (2011) / Page 7 of 10 

MALATHION/ 057701 OCSPP 890.1600/ OECD 440

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 μg/kg/day) 

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 over the duration of the study was 

comparable to the control. 

TABLE 3. Food Consumption (g/rat/day) in the Estrogen Agonist Assaya

Study Day 

#

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 

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 

Page 291 of 294
In vivo Uterotrophic Assay (2011) / Page 8 of 10 

MALATHION/ 057701 OCSPP 890.1600/ OECD 440

D. PATHOLOGY 

1. Uterine and Liver Weights: Uterine and liver weight data are presented in Table 4. 

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 positive controls 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 liver weights were increased (p≤0.05) in the 

300 mg/kg/day group (↑10%). These increases were consistent 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 control 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 ovarian 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 Ratsa

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 rats 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 Assaya

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 uterotrophic bioassay at dose levels as high as 1000 mg/kg bw/day 

(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 weights) and statistically and 

biologically significant RBC and brain ChE inhibition. 

B. AGENCY COMMENTS: 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%. In the positive control group, overall body weight gain was decreased 

(p≤0.01; ↓85%); body weights in this group were comparable 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 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 over the 

duration of the study was comparable to the control. 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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MALATHION/ 057701 OCSPP 890.1600/ OECD 440

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 treatment. 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 

Page 294 of 294