UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460
OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION
MEMORANDUM
Date: 6/29/2015
SUBJECT: Phosmet: Data Evaluation Records (DERs) for EDSP Tier 1 Assays
PC Code: 059201 DP Barcode: D397165, D400672 Decision No.: 459049, 463273 Registration No.: NA Petition No.: NA Regulatory Action: NA Risk Assessment Type: NA Case No.: NA TXR No.: 0056199 CAS No.: 732-11-6 MRID No.: See Table 40 CFR: NA Ver.Apr. 2010
FROM: Greg Akerman, Ph.D. Do- ff--- lmmediate Office Health Effects Division (7509P)
THROUGH: Jess Rowland ~- ~ Deputy Director Health Effects Division
TO: Jolene Trujillo Biologist/Chemical Review Manager Risk Management and Implementation Branch V Pesticide Re-evaluation Division (7505P)
I. ACTION REQUESTED
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 phosmet: Test Order # EDSP-059201-54.
Pagel of2 Page 1 of 260 II. RESPONSE
Attached are the EDSP Tier 1 assay DERs for phosmet.
III. MRID Table
Chemical: Phosmet PC Code: 059201 Guideline Assay MRID 890.1100 Amphibian Metamorphosis Assay (Frog) 48673001 890.1150 Androgen Receptor Binding (Rat Prostate) 48618703 890.1200 Aromatase Assay (Human Recombinant) 48618702 890.1250 Estrogen Receptor Binding 48618704 890.1300 Estrogen Receptor Transcriptional Activation 48618705 (Human Cell Line HeLa-9903) 890.1350 Fish Short-Term Reproduction 48673002 890.1400 Hershberger (Rat) 48618706 890.1450 Female Pubertal (Rat) 48673003 890.1500 Male Pubertal (Rat) 48673003 890.1550 Steroidogenesis (Human Cell Line – H295R) 48618701 890.1600 Uterotrophic (Rat) 48618707
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ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԡԹՌԹԯԽՉՍՁՊԽՅԽՆՌ ԢԭԞԡԭԟԹՊԻՇԼԽ ԬԢԠԡԡԹՌԹԭՇՁՆՌ ԢԭԞԪԯԦԡ ԢԭԞԤՍՁԼԽՄՁՆԽ ԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ ԣՊՇԿ ԱԽՋՌԪԹՌԽՊՁԹՄ ԭՀՇՋՅԽՌԭՍՊՁՌՑ ԠՇՅՅՇՆԫԹՅԽԭՀՇՋՅԽՌ ԠՀԽՅՁԻԹՄԫԹՅԽ ԦԲԭԞԠ ԫՇՌԯԽՈՇՊՌԽԼ ԠԞԫԹՅԽ ԫՇՌԯԽՈՇՊՌԽԼ ԠԞԫՇ ՑՆՇՆՑՅՋ ԦՅՁԼԹՆնԱԽԻՀՆՁԻԹՄ ԢԭԞԭԠԠՇԼԽ
ԭՊՁՅԹՊՑԯԽՎՁԽՏԽՊ ԧՇՀՆԪԹՊՌՇՆՁԿՆԹՌՍՊԽ ՌԹԾԾԻՁԽՆՌՁՋՌԠԹՅԺՊՁԼԿԽԢՆՎՁՊՇՆՅԽՆՌԹՄԦՆԻ ԡԹՌԽ
ԽԻՇՆԼԹՊՑԯԽՎՁԽՏԽՊ ԱԽՊՁԪՑԽՊՋՁԿՆԹՌՍՊԽ ԽՆՁՇՊԻՁԽՆՌՁՋՌԠԹՅԺՊՁԼԿԽԢՆՎՁՊՇՆՅԽՆՌԹՄԦՆԻ ԡԹՌԽ ԽԻՇՆԼԹՊՑԯԽՎՁԽՏԽՊ ԠԹՌՀԽՊՁՆԽԞՍԺԽԽԡԹՌԽ ԽՆՁՇՊԻՁԽՆՌՁՋՌԲԢԭԞԬԭԭԢԣԢԡԢԯԟԦԳ ՁԿՆԹՌՍՊԽ ԡԹՌԽԢՎԹՄՍԹՌՁՇՆԠՇՅՈՄԽՌԽԼ#ԼԼՅՅՅՑՑՑՑ% ԠԦԱԞԱԦԬԫ ԩԽԽԪԯԭՀՇՋՅԽՌԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑՏՁՌՀԞԾՊՁԻԹՆԠՄԹՏԽԼԣՊՇԿ ԵԽՆՇՈՍՋ ՄԹԽՎՁՋ ԣՇՄՄՇՏՁՆԿԬԭԭԱԱԽՋՌԤՍՁԼԽՄՁՆԽԹՆԼԬԢԠԡԱԽՋՌԤՍՁԼԽՄՁՆԽԫՇԲՆՈՍԺՄՁՋՀԽԼՋՌՍԼՑ ՈԽՊԾՇՊՅԽԼԺՑՅՁՌՀԽՊՋԳՁՋԻՁԽՆՌԴԹՊԽՀԹՅԪԹՋՋԹԻՀՍՋԽՌՌՋԩԹԺՇՊԹՌՇՊՑՊԽՈՇՊՌՆՍՅԺԽՊ ՌՍԼՑՋՈՇՆՋՇՊԽԼԺՑԤՇՏԹՆԠՇՅՈԹՆՑԶՍՅԹԞՊՁՒՇՆԹՌՍԼՑԻՇՅՈՄԽՌԽԼԣԽԺՊՍԹՊՑ ԡՁՋԻՄԹՁՅԽՊ ԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋՁՆՌՀՁՋԡԢԯՌԽՅՈՄԹՌԽԹՊԽՇԾԾԽՊԽԼԹՋԹԿԽՆԽՊԹՄՊԽԾԽՊԽՆԻԽՌՇԹՁԼ ՁՆՈՊԽՈԹՊԹՌՁՇՆՇԾՌՀԽԡԢԯԱՀԽՈՍՊՈՇՋԽՇԾՌՀԽՋԽՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋՁՋՆՇՌՌՇՋԽՊՎԽԹՋՋՍԺՋՌՁՌՍՌԽԾՇՊՌՀԽԱԽՋՌ ԤՍՁԼԽՄՁՆԽՋՆՇՊՌՇՈՊՇՎՁԼԽԹՆՑԿՍՁԼԹՆԻԽՇՆՀՇՏՌՀԽՋՌՍԼՑՋՀՇՍՄԼԺԽԻՇՆԼՍԻՌԽԼ
ԭԹԿԽՇԾ
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ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԫՇՌԽ ԱՀԽԲԢԭԞԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅ Ԣԡԭ ԱՁԽՊՋԻՊԽԽՆՁՆԿԺԹՌՌԽՊՑՁՋԻՇՅՈՊՁՋԽԼՇԾԽՄԽՎԽՆ ՋԻՊԽԽՆՁՆԿԹՋՋԹՑՋՁՆՌԽՆԼԽԼՌՇՁԼԽՆՌՁԾՑԹԻՀԽՅՁԻԹՄ0ՋՄՁՃԽՄՑԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՁԽՁՌՋՈՇՌԽՆՌՁԹՄՌՇՁՆՌԽՊԹԻՌՏՁՌՀ ՌՀԽԽՋՌՊՇԿԽՆԹՆԼՊՇԿԽՆՇՊՌՀՑՊՇՁԼ ԢԞՇՊԱ ՈԹՌՀՏԹՑՋԱՀԽՊՇԺՍՋՌՆԽՋՋՇԾՌՀԽԱՁԽՊԺԹՌՌԽՊՑՁՋԺԹՋԽԼՇՆ ՌՀԽՋՌՊԽՆԿՌՀՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՌՇՁԼԽՆՌՁԾՑՈՇՌԽՆՌՁԹՄԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՏՁՌՀԻՇՅՈՄԽՅԽՆՌԹՊՑԽՆԼՈՇՁՆՌՋ ՏՁՌՀՁՆՌՀԽԹՋՋԹՑՏՀԽՊԽԹՎԹՁՄԹԺՄԽԹՆԼՊԽԼՍՆԼԹՆԻՑԹԻՊՇՋՋՌՀԽԺԹՌՌԽՊՑԱՀՍՋՌՀԽՊԽՋՍՄՌՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄ ԹՋՋԹՑՋՀՇՍՄԼՆՇՌԺԽԻՇՆՋՁԼԽՊԽԼՁՆՁՋՇՄԹՌՁՇՆԺՍՌՊԹՌՀԽՊՋՀՇՍՄԼԺԽԻՇՆՋՁԼԽՊԽԼՁՆՌՀԽԻՇՆՌԽՐՌՇԾՇՌՀԽՊԹՋՋԹՑՋ ՁՆՌՀԽԺԹՌՌԽՊՑԹՋՏԽՄՄԹՋԬՌՀԽՊԻՁԽՆՌՁԾՁԻԹՄՄՑԯԽՄԽՎԹՆՌԦՆԾՇՊՅԹՌՁՇՆ ԬԯԦ ԦՆՇՊԼԽՊՌՇԼԽՌԽՊՅՁՆԽՁԾԹԻՀԽՅՁԻԹՄ ՀԹՋՌՀԽՈՇՌԽՆՌՁԹՄՌՇՁՆՌԽՊԹԻՌՏՁՌՀՌՀԽԢԞՇՊԱՈԹՌՀՏԹՑՋԹԴԽՁԿՀՌՇԾԢՎՁԼԽՆԻԽ ԴՇԢ ԽՎԹՄՍԹՌՁՇՆՇԾԱՁԽՊ ԹՋՋԹՑՊԽՋՍՄՌՋՁՆԻՇՅԺՁՆԹՌՁՇՆՏՁՌՀՌՀԽԾՁՆԼՁՆԿՋՁՆՌՀԽԬԯԦՋՀՇՍՄԼԺԽՍՆԼԽՊՌԹՃԽՆ ՊԽԾԽՊՌՇՌՀԽԴՇԢ ԡՇԻՍՅԽՆՌ ԢԵԢԠԲԱԦԳԢԲԪԪԞԯԶ ԱՀԽԼԹՑԹՋՋԹՑՇԾՈՀՇՋՅԽՌՇՆԹՅՈՀՁԺՁԹՆՅԽՌԹՅՇՊՈՀՇՋՁՋՇԾԞԾՊՁԻԹՆԠՄԹՏԽԼԣՊՇԿ ԵԽՆՇՈՍՋՄԹԽՎՁՋ ՏԹՋ ԻՇՆԼՍԻՌԽԼՍՆԼԽՊԾՄՇՏՌՀՊՇՍԿՀԻՇՆԼՁՌՁՇՆՋԞՅՈՀՁԺՁԹՆՄԹՊՎԹԽ ՌԹԼՈՇՄԽՋՌՊԽԹՌՅԽՆՌԿՊՇՍՈՊԽՈՄՁԻԹՌԽ ԹՌ ԫՁԽՍՏՃՇՇՈԣԹԺԽՊ ԫԣ ՋՌԹԿԽՏԽՊԽԽՐՈՇՋԽԼՌՇՈՀՇՋՅԽՌ ՈՍՊՁՌՑ ԹՌՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾ ՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ ԹՆԼՅԿԹՁԩԪԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽ ԩԬԮ ԻՇՆՌՊՇՄ ԹՆԼՅԿԹՁԩԱՀԽՌԽՋՌՋՑՋՌԽՅՏԹՋՅԹՁՆՌԹՁՆԽԼԹՌՌՇոԠԹՆԼԹՈԥՇԾ ՌՇ ԭՀՇՋՅԽՌՋՁԿՆՁԾՁԻԹՆՌՄՑԼԽԻՊԽԹՋԽԼՆՇՊՅԹՄՁՒԽԼՀՁՆԼՄՁՅԺՄԽՆԿՌՀ ԥԩԩ ՁՆՆՇՆՄԹՌԽՋՌԹԿԽՌԹԼՈՇՄԽՋ ԫԣՋՌԹԿԽ ԹՌՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՇՆԡԹՑՋԹՆԼԪԽԼՁԹՆԫՁԽՍՏՃՇՇՈԣԹԺԽՊ ԫԣ ՋՌԹԿԽՏԹՋՍՆԹԾԾԽԻՌԽԼՇՆԡԹՑՌՀՇՍԿՀԹՋՁԿՆՁԾՁԻԹՆՌԼԽՄԹՑՏԹՋՇԺՋԽՊՎԽԼԹՌՇՆԡԹՑԹՌՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԫՇԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌՏԹՋՇԺՋԽՊՎԽԼԪՁՄԼՌՀՑՊՇՁԼԿՄԹՆԼ ՀՑՈԽՊՌՊՇՈՀՑԾՇՄՄՁԻՍՄԹՊԻԽՄՄՀՑՈԽՊՈՄԹՋՁԹԹՆԼԾՇՄՄՁԻՍՄԹՊԻԽՄՄՀՑՈԽՊՌՊՇՈՀՑՏԽՊԽՇԺՋԽՊՎԽԼԹՌԹՄՄՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋ ՁՆԻՄՍԼՁՆԿԻՇՆՌՊՇՄՋ ՌՀԽՊԽՏԹՋՆՇԹՈՈԹՊԽՆՌՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼՈԹՌՌԽՊՆՁՆՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄԾՁՆԼՁՆԿՋԱԹԼՈՇՄԽՋՏՁՌՀ ՋՈՁՆԹՄԻՍՊՎԹՌՍՊԽՏԽՊԽՇԺՋԽՊՎԽԼՁՆՌՇՇԾՌԹԼՈՇՄԽՋՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽ ՊԽՈՇՊՌԽԼԹՄՄԹՆԹՄՑՋԽՋԹՊԽԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ ԣԢԱԞԵ ԻՇՆՌՊՇՄԹՆԼԹՆԹՄՑՋԽՋՇԾԳԩՆՇՊՅԹՄՁՒԽԼ ԥԩԩԹՆԼԺՇԼՑՏԽՁԿՀՌՏԽՊԽՈԽՊԾՇՊՅԽԼԽՐԻՄՍԼՁՆԿՄԹՌԽՋՌԹԿԽ ԫԣՋՌԹԿԽ ՌԹԼՈՇՄԽՋ ԪՁՆՇՊԼԽՎՁԹՌՁՇՆՋԾՊՇՅՌՀԽԿՍՁԼԽՄՁՆԽՏԽՊԽՆՇՌԽԼԺՍՌՌՀԽՑԼՁԼՆՇՌՋՍԺՋՌԹՆՌՁՎԽՄՑԹԾԾԽԻՌՌՀԽՊԽՋՍՄՌՋՇՊՁՆՌԽՊՈՊԽՌԹՌՁՇՆ ՇԾՌՀԽՋՌՍԼՑԱՀԽԹՋՋԹՑՋԹՌՁՋԾՁԽՋՌՀԽԢԡԭԱՁԽՊԱԽՋՌԬՊԼԽՊՊԽՉՍՁՊԽՅԽՆՌԾՇՊԹՆԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԹՋՋԹՑ
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ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԬԠԭԭԤՍՁԼԽՄՁՆԽ ԯԽՋՍՄՌՋՑՆՇՈՋՁՋ ԱԽՋՌՇՊԿԹՆՁՋՅԫԣՋՌԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ԱԽՋՌՇՊԿԹՆՁՋՅՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ՇՈՌՁՇՆԹՄ ԫՇՌԯԽՈՇՊՌԽԼ ԱԽՋՌՌՑՈԽ ԾՄՇՏՌՀՊՇՍԿՀ ԱԹԺՄԽ ՍՅՅԹՊՑ ՇԾ ԡԽՎԽՄՇՈՅԽՆՌԹՄ ԹՆԼ ԱՀՑՊՇՁԼ ԭԹՌՀՇՄՇԿՑԥՁՋՌՇՈԹՌՀՇՄՇԿՑ ԢԾԾԽԻՌՋՁՆՌՀԽԞՅՈՀՁԺՁԹՆ ԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ ԞԪԞ ՏՁՌՀԭՀՇՋՅԽՌ
ԱՀՑՊՇՁԼԤՊՇՋՋ ԫԣԡԽՎԽՄՇՈՅԽՆՌԹՄ ԥՁՆԼԩՁՅԺ ԞՋՑՆԻՀՊՇՆՇՍՋ ԱՊԽԹՌՅԽՆՌ ԹՆԼ ՌԹԿԽ ԩԽՆԿՌՀ ԡԽՎԽՄՇՈՅԽՆՌ ՅԿԹՁԩ ԥՁՋՌՇՈԹՌՀՇՄՇԿՑ
#ՅԽԹՆՅԽԹՋՍՊԽԼ% ԡԹՑ ԡԹՑ ԡԹՑ ԡԹՑ ԡԹՑ ԡԹՑ ԡԹՑ
ԫՇ ԫՇ ԫՇ ԫՇ ԫԞ ԫԞ ԫՇ
ԫՇ ԶԽՋ ԶԽՋ ԶԽՋ ԫԞ ԫԞ ԫՇ
ԫՇ ԶԽՋ ԶԽՋ ԶԽՋ ԫԞ ԫԞ ԫՇ
ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԡՁԾԾԡՁԾԾԽՊԽՆԻԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ
Ԟ՟ՑԽՋՠՁՆԼՁԻԹՌԽՋԹՋՁԿՆՁԾՁԻԹՆՌԼՁԾԾԽՊԽՆԻԽԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ ԻՄԽԹՆՏԹՌԽՊ ԻՇՆՌՊՇՄՍՆՄԽՋՋ ՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼ
ԱՀԽԻՊՁՌԽՊՁԹԾՇՊՋՁԿՆՁԾՁԻԹՆԻԽԹՊԽԼԽՋԻՊՁԺԽԼՁՆՌՀԽԯԽՎՁԽՏԽՊ0ՋԞՆԹՄՑՋՁՋԹՆԼՌԹՌՁՋՌՁԻԹՄԳԽՊՁԾՁԻԹՌՁՇՆՋԽԻՌՁՇՆՋՇԾ ՌՀԽ ԡԢԯ ԠՇՆԻՄՍՋՁՇՆՋ ՊԽԿԹՊԼՁՆԿ ՀՁՋՌՇՈԹՌՀՇՄՇԿՑ ՅԹՑ ԺԽ ՀԽԹՎՁՄՑ ՏԽՁԿՀՌԽԼ ԺՑ ՌՀԽ ԽՐՈԽՊՌ ՇՈՁՆՁՇՆ ՇԾ Թ ԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ
ԥՁՆԼՄՁՅԺՄԽՆԿՌՀՁՋՆՇՊՅԹՄՁՒԽԼՌՇՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ Գԩ
Ԧ ԪԞԱԢԯԦԞԩԞԫԡԪԢԱԥԬԡ ԤՍՁԼԽՄՁՆԽԣՇՄՄՇՏԽԼ ԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼԾՇՄՄՇՏՁՆԿԿՍՁԼԽՄՁՆԽՋՇՍՌՄՁՆԽԼՁՆՌՀԽԲԢՆՎՁՊՇՆՅԽՆՌԹՄ ԭՊՇՌԽԻՌՁՇՆԞԿԽՆԻՑ0ՋԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅԱԽՋՌԤՍՁԼԽՄՁՆԽՋ
ԭԹԿԽՇԾ
Page 5 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋ ԣՊՇԿ ԹՆԼԬԢԠԡԤՍՁԼԽՄՁՆԽԾՇՊԱԽՋՌՁՆԿ ՇԾԠՀԽՅՁԻԹՄՋԫՇԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑԱՀԽԾՇՄՄՇՏՁՆԿ ԼԽՎՁԹՌՁՇՆՋՏԽՊԽՆՇՌԽԼ ԦՌՏԹՋՆՇՌՋՈԽԻՁԾՁԽԼՇՆՏՀՁԻՀԼԹՑՋԽՅԺՊՑՇՋՏԽՊԽԻՀԽԻՃԽԼԾՇՊՎՁԹԺՁՄՁՌՑ ԱՀԽԾՄՇՏՊԹՌԽՊԽՋՍՄՌԽԼՁՆԹՎՇՄՍՅԽՊԽՈՄԹԻԽՅԽՆՌԽՎԽՊՑՀՇՍՊՋԤՍՁԼԹՆԻԽՊԽԻՇՅՅԽՆԼՋԻՇՅՈՄԽՌԽ ՎՇՄՍՅԽՊԽՈՄԹԻԽՅԽՆՌԹՈՈՊՇՐՁՅԹՌԽՄՑԽՎԽՊՑՀՇՍՊՋ ԯԽՋՍՄՌՋԾՊՇՅՌՀԽՈԽՊՁՇԼՁԻՋԻՊԽԽՆՁՆԿԹՆԹՄՑՋՁՋՇԾՌՀԽԼՁՄՍՌՁՇՆՏԹՌԽՊՏԽՊԽՆՇՌՈՊՇՎՁԼԽԼԥՇՏԽՎԽՊՌՀԽ ՋՌՍԼՑԹՍՌՀՇՊՊԽՈՇՊՌԽԼՌՀԹՌՆՇՈԽՋՌՁԻՁԼԽՋԭԠԟՋՇՊՌՇՐՁԻՅԽՌԹՄՋՏԽՊԽԼԽՌԽԻՌԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԻՇՆՋՁԼԽՊԽԼՌՇՐՁԻԹԻԻՇՊԼՁՆԿՌՇԞԱԪՋՌԹՆԼԹՊԼՋ ԱՀԽՄՁԿՀՌՁՆՌԽՆՋՁՌՑ ԨՄՍՐ ՏԹՋՄՇՏԽՊՌՀԹՆՊԽԻՇՅՅԽՆԼԽԼ ԨՄՍՐ
ԴԹՌԽՊՀԹՊԼՆԽՋՋ ՅԿԩԹՋԠԹԠԬ ԽՐԻԽԽԼԽԼՌՀԽՊԽԻՇՅՅԽՆԼԽԼՀԹՊԼՆԽՋՋ ՅԿԩ
ԹՋԠԹԠԬ ԱՀԽՌԽՅՈԽՊԹՌՍՊԽ ոԠ ԾԽՄՄՇՍՌՋՁԼԽՇԾՌՀԽՊԽԻՇՅՅԽՆԼԽԼՊԹՆԿԽ չոԠ ԱՀԽՋԽԼԽՎՁԹՌՁՇՆՋԼՇՆՇՌՋՍԺՋՌԹՆՌՁՎԽՄՑՁՅՈԹԻՌՌՀԽՊԽՋՍՄՌՋՇՊՁՆՌԽՊՈՊԽՌԹՌՁՇՆՇԾՌՀԽՋՌՍԼՑ ԠՇՅՈՄՁԹՆԻԽ ՁԿՆԽԼԹՆԼԼԹՌԽԼԫՇԡԹՌԹԠՇՆԾՁԼԽՆՌՁԹՄՁՌՑԤԩԭԹՆԼԮՍԹՄՁՌՑԞՋՋՍՊԹՆԻԽՋՌԹՌԽՅԽՆՌՋ ՏԽՊԽՈՊՇՎՁԼԽԼԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼՁՆԻՇՅՈՄՁԹՆԻԽՏՁՌՀԹՄՄՈԽՊՌՁՆԽՆՌԬԢԠԡ ԬԢԠԡ ԹՆԼԲԢԭԞ ԠԣԯԭԹՊՌԹՆԼԠԣԯԭԹՊՌ ԤՇՇԼ ԩԹԺՇՊԹՌՇՊՑԭՊԹԻՌՁԻԽՊԽԿՍՄԹՌՁՇՆՋՏՁՌՀՌՀԽԾՇՄՄՇՏՁՆԿԽՐԻԽՈՌՁՇՆ ՊՇՍՌՁՆԽԾՇՇԼԹՆԼ ՏԹՌԽՊԻՇՆՌԹՅՁՆԹՆՌՋԻՊԽԽՆՁՆԿԹՆԹՄՑՋԽՋՏԽՊԽԻՇՆԼՍԻՌԽԼՍՋՁՆԿՋՌԹՆԼԹՊԼԲ ԢԭԞՈՊՇԻԽԼՍՊԽՋԺՑԤԽՇԩԹԺՋԦՆԻԟՊԹՁՆՌՊԽԽԪԹՋՋԹԻՀՍՋԽՌՌԽՋ ԞԱԽՋՌԪԹՌԽՊՁԹՄԭՀՇՋՅԽՌ ԡԽՋԻՊՁՈՌՁՇՆ ՇՄՁԼ ԬԢԠԡՊԽԻՇՅՅԽՆԼՋԼԽՋԻՊՁԺՁՆԿՏԹՌԽՊՋՇՄՍԺՁՄՁՌՑՅԽՄՌՁՆԿԺՇՁՄՁՆԿՈՇՁՆՌՋՌԹԺՁՄՁՌՑՁՆՏԹՌԽՊԹՆԼՄՁԿՀՌՈԨԹԭՇՏՇՊԨՇՏ ՎԹՈՇՊՈՊԽՋՋՍՊԽՇԾՌԽՋՌԻՇՅՈՇՍՆԼԽՐՈՁՊԹՌՁՇՆԼԹՌԽ ԩՇՌԫՇԟԹՌԻՀԫՇ ԤԮԥ
ԭԹԿԽՇԾ
Page 6 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭՍՊՁՌՑ ԦՅՈՍՊՁՌՁԽՋ ԫՇՆԽԯԽՈՇՊՌԽԼ ՌԹԺՁՄՁՌՑՇԾԠՇՅՈՇՍՆԼ ԱՀԽԼԹՑՅԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇՆՄՑՑՁԽՄԼԽԼՊԽԻՇՎԽՊՁԽՋՇԾ ՇԾՆՇՅՁՆԹՄԥՇՏԽՎԽՊԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽՊԽՄԹՌՁՎԽՄՑՋՌԹԺՄԽՏՁՌՀԠԳՋՊԹՆԿՁՆԿ ԾՊՇՅՌՇ ՌՇՊԹԿԽԠՇՆԼՁՌՁՇՆՋՇԾ ԱԽՋՌԠՀԽՅՁԻԹՄՋ ՌՇՊԽԼԹՌՊՇՇՅՌԽՅՈԽՊԹՌՍՊԽՁՆՌՀԽՇՊՁԿՁՆԹՄԻՇՆՌԹՁՆԽՊՁՆԹԼԹՊՃՎԽՆՌՁՄԹՌԽԼ ԻԹԺՁՆԽՌ
ԭԹԿԽՇԾ
Page 7 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԟԱԽՋՌԬՊԿԹՆՁՋՅ ԱԹԺՄԽ ԤԽՆԽՊԹՄԦՆԾՇՊՅԹՌՁՇՆԞԺՇՍՌՌՀԽԱԽՋՌՈԽԻՁԽՋԹՆԼԭԹՊԽՆՌԹՄԠԹՊԽ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ՈԽԻՁԽՋԻՇՅՅՇՆՆԹՅԽ ԞԾՊՁԻԹՆԠՄԹՏԽԼԣՊՇԿ ԢԭԞՊԽԻՇՅՅԽՆԼՋԞԾՊՁԻԹՆԻՄԹՏԽԼԾՊՇԿ ԵԽՆՇՈՍՋՄԹԽՎՁՋ ԴԽՋՌԽՊՆ#ԞԾՊՁԻԹ%ԻՄԹՏԽԼ ՈԽԻՁԽՋՋԻՁԽՆՌՁԾՁԻՆԹՅԽ ԵԽՆՇՈՍՋՄԹԽՎՁՋ ԾՊՇԿՁՄՍՊԹՆԹ ԵԽՆՇՈՍՋ ՌՊՇՈՁԻԹՄՁՋՅԹՑԺԽ ՈԽԻՁԽՋՋՌՊԹՁՆ ՁԾՋՌԹՌԽԼ ԫՇՌԯԽՈՇՊՌԽԼ ՍՋԽԼԹՋԹՆԹՄՌԽՊՆԹՌԽՋՈԽԻՁԽՋ ՀՇՏԽՎԽՊԹՄՁՋՌ ՇԾԹՄՄՇԾՌՀԽՆԽԻԽՋՋԹՊՑՈՊՇՌՇԻՇՄԼԽՎՁԹՌՁՇՆՋՌՇ ԹԻԻՇՅՅՇԼԹՌԽՌՀՁՋՋՈԽԻՁԽՋՁՋՊԽԻՇՅՅԽՆԼԽԼ ԾՇՊՁՆԻՄՍՋՁՇՆՁՆՌՀԽՋՌՍԼՑՊԽՈՇՊՌԱՀԽԿՍՁԼԽՄՁՆԽ ՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹ ՍՋԽԼՌՇՋՍՈՈՇՊՌՌՀԽՊԽՄՁԹԺՁՄՁՌՑՇԾՌՀԽՌԽՋՌԺԽ ՁԼԽՆՌՁԾՁԽԼ
ԲԢՆՎՁՊՇՆՅԽՆՌԹՄԭՊՇՌԽԻՌՁՇՆԞԿԽՆԻՑ ԢԭԞ ԠՇՊՊԽԻՌՁՇՆՋԹՆԼԠՄԹՊՁԾՁԻԹՌՁՇՆՋՇՆԱԽԻՀՆՁԻԹՄԞՋՈԽԻՌՋՇԾՌՀԽԱԽՋՌԤՍՁԼԽՄՁՆԽՋԾՇՊՌՀԽԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊ ԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅԱՁԽՊԞՋՋԹՑՋ ԬԠԭԭԱԽՋՌԤՍՁԼԽՄՁՆԽԽՊՁԽՋ ԪԹՊԻՀԬԾԾՁԻԽՇԾԠՀԽՅՁԻԹՄԹԾԽՌՑԹՆԼԭՇՄՄՍՌՁՇՆԭՊԽՎԽՆՌՁՇՆ ԬԠԭԭ ԴԹՋՀՁՆԿՌՇՆ ԡԠ ՀՌՌՈ ՏՏՏԽՈԹԿՇՎԽՆԼՇՈՍԺՋԹՋՋԹՑՎԹՄՁԼԹՌՁՇՆԻՄԹՊՁԾՁԻԹՌՁՇՆԼՇԻՈԼԾ
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ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԴԽՊԽՈԹՊԽՆՌՋՅԹՁՆՌԹՁՆԽԼԹՋ ԶԽՋ ԦՆՀՇՍՋԽԻՍՄՌՍՊԽՏԹՋՇՊՁԿՁՆԹՄՄՑՇԺՌԹՁՆԽԼ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՄԹՊՎԹԽ ՍՋԽԼ ՁՆ ՌՀԽ ՁՆՀՇՍՋԽՋՌՇԻՃ! ԾՊՇՅԫԹՋԻՇԣՇՊՌԞՌՃՁՆՋՇՆԴՁՋԻՇՆՋՁՆԹՆԼ ԹՋՋԹՑԺԽԼԽՊՁՎԽԼԾՊՇՅՁՆՀՇՍՋԽԹԼՍՄՌՋ ՏԹՋՅԹՁՆՌԹՁՆԽԼԹՋԺՊԽԽԼԽՊՋԾՇՊ ԹՈՈՊՇՐՁՅԹՌԽՄՑՇՆԽՑԽԹՊՈՊՁՇՊՌՇՍՋԽԾՇՊ ԿԽՆԽՊԹՌՁՆԿՌԹԼՈՇՄԽՋՍՋԽԼՁՆՌՀՁՋԽՐՈՇՋՍՊԽ
ԴԽՊԽՈԹՊԽՆՌԹՄԹԻԻՄՁՅԹՌՁՇՆ ԶԽՋ ԻՇՆԼՁՌՁՇՆՋՋԹՅԽԹՋԼԽԾՁՆՁՌՁՎԽ ՌԽՋՌ!
ԞԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼԾՇՊՈԹՊԽՆՌԹՄ ԠՇՆՌՁՆՍՇՍՋ ԾՊՇԿՋ ՁԾԹՈՈՄՁԻԹԺՄԽ
ԡԽՌԹՁՄՋՇՆՈԹՊԽՆՌԹՄԾԽԽԼՁՆԿ ԡՍՊՁՆԿՌՀԽՈՊԽԽՐՈՇՋՍՊԽ ԱԹԼՈՇՄԽՋՏԽՊԽԾԽԼԵԽՆՇՈՍՋԢՐՈՊԽՋՋ ՈԽՊՁՇԼՌԹԼՈՇՄԽՋՏԽՊԽ ԱԹԼՈՇՄԽԣՇՇԼ ԵԽՆՇՈՍՋԢՐՈՊԽՋՋ ԾԽԼՌՏՁԻԽՈԽՊԼԹՑՏՁՌՀ ԟՊՇՇՃՋՎՁՄՄԽԣՄՇՊՁԼԹ ՅԿՌԹԼՈՇՄԽՏՁՌՀ ՁՆԻՊԽԹՋՁՆԿՊԹՌԽՋ ՌՀՊՇՍԿՀՇՍՌՌՀԽ ԽՐՈՇՋՍՊԽ
Page ԭԹԿԽՇԾ 9 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԡԽՌԹՁՄՋՇՆՈԹՊԽՆՌԹՄՀԽԹՄՌՀ ԱՀԽԺՊՇՇԼՋՌՇԻՃԼՁԼՆՇՌ ՋՀՇՏԹՆՑՋՁԿՆՇԾ ՋՁԻՃՆԽՋՋԼՁՋԽԹՋԽ ՁՆՂՍՊՁԽՋՇՊԹԺՆՇՊՅԹՄՁՌՁԽՋ ԾՊՇՅՌՀԽԼԹՑՇԾՊԽԻԽՁՈՌՌՇ ՌՀԽԼԹՑՇԾՈՊԽԽՐՈՇՋՍՊԽ ՁՆՁՌՁԹՌՁՇՆ
ԱԹԺՄԽ ԩԹՊՎԹՄԽՄԽԻՌՁՇՆԹՆԼԠԹՊԽ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԟԽՋՌՋՁՆԿՄԽՋՈԹՏՆ! ԶԽՋ ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌՌՀԽԺԽՋՌ- ՁՆԼՁՎՁԼՍԹՄՋՈԹՏՆՋՏՁՌՀԹՅՁՆՁՅՍՅՇԾ ԫՍՅԺԽՊՇԾՋՈԹՏՆՋԽՎԹՄՍԹՌԽԼ ՁԾ ՄԹՊՎԹԽՋՈԹՏՆԺԽԽՎԹՄՍԹՌԽԼՌՇՁԼԽՆՌՁԾՑՌՀԽ ԹՈՈՄՁԻԹԺՄԽ ԺԽՋՌՋՁՆԿՄԽՋՈԹՏՆԹՆԼՌՀԹՌՌՀԽՄԹՊՎԹԽՋԽՄԽԻՌԽԼ ԫՍՅԺԽՊՇԾԽԿԿՋՋԹՅՈՄԽԼՈԽՊ ԞՌՄԽԹՋՌ ԦՌՏԹՋՆՇՌՋՈԽԻՁԾՁԽԼՇՆՏՀՁԻՀԼԹՑՋ ԾՇՊՌԽՋՌՁՆԿՇՊՁԿՁՆԹՌԽԾՊՇՅՌՀԽԺԽՋՌՋՁՆԿՄԽՋՈԹՏՆ ՋՈԹՏՆ ԽՅԺՊՑՇՋՏԽՊԽԻՀԽԻՃԽԼԾՇՊՎՁԹԺՁՄՁՌՑ ՁԽՌՀԽՋՈԹՏՆՋԹՊԽՆՇՌԻՇՅՁՐԽԼ
ԫԣՋՌԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ԫԣ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽԼԽԾՁՆՁՌՁՎԽՋՌՍԼՑԺԽ
Page ԭԹԿԽՇԾ 10 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԞԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ԼԹՑՋՈՇՋՌԾԽՊՌՁՄՁՒԹՌՁՇՆ ՁՆՁՌՁԹՌԽԼՏՁՌՀՄԹՊՎԹԽԹՌԫՁԽՍՏՃՇՇՈ-ԣԹԺԽՊ ԼՈԾ ԫԣ ԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽ ԼԹՑՋ ՈՇՋՌԾԽՊՌՁՄՁՒԹՌՁՇՆ
ԪԽԹՆՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ԫՇՌՅԽԹՋՍՊԽԼ ՁԾՊԽՈՇՊՌԽԼ
ԯԹՆԿԽՇԾՌՇՌԹՄՄԽՆԿՌՀԹՌՌԽՋՌ ԫՇՌՅԽԹՋՍՊԽԼ ՁՆՁՌՁԹՌՁՇՆ ՁԾՊԽՈՇՊՌԽԼ
ԴԹՋՌՀԽՇՈՌՁՇՆԹՄՋՁՒԽՋԽՄԽԻՌՁՇՆ ԫՇ ՅԽՌՀՇԼՍՋԽԼ!
ԡԽՌԹՁՄՋՇՆՄԹՊՎԹՄՋԽՄԽԻՌՁՇՆ ԫՁՆԽՀՍՆԼՊԽԼՌԹԼՈՇՄԽՋ ԾՊՇՅՌՀԽՀՁԿՀԽՋՌՉՍԹՄՁՌՑ ՋՈԹՏՆՏԽՊԽՌՊԹՆՋԾԽՊՊԽԼ ՌՇՊԽԹՊՁՆԿՎԽՋՋԽՄՋ ՌԹԼՈՇՄԽՋԩ ԬՆԻԽ ՌԹԼՈՇՄԽՋՊԽԹԻՀԽԼԫԣ ՋՌԹԿԽՀԽԹՄՌՀՑ ՌԹԼՈՇՄԽՋՏԽՊԽՊԹՆԼՇՅՄՑ ՋԽՄԽԻՌԽԼԾՇՊՍՋԽՁՆՌՀԽ ԼԽԾՁՆՁՌՁՎԽԽՐՈՇՋՍՊԽ
Page ԭԹԿԽՇԾ 11 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԩՇԹԼՁՆԿՊԹՌԽ ՊԽԹՊՁՆԿԼԽՆՋՁՌՑ +ՄԹՊՎԹԽԩ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՊԽԹՊՁՆԿ ԼԽՆՋՁՌՑ ՄՇԹԼՁՆԿՊԹՌԽ ՆՇՌԽՐԻԽԽԼԹՈՈՊՇՐՁՅԹՌԽՄՑ ՄԹՊՎԹԽԩ ԻՍՄՌՍՊՁՆԿ ՋՑՋՌԽՅ ԾՇՊ ԾՄՇՏՌՀՊՇՍԿՀ ՋՑՋՌԽՅՋ ՇՊ ՌԹԼՈՇՄԽՋԩ ՁՆ ՋՌԹՌՁԻՊԽՆԽՏԹՄ ԽՐՈՇՋՍՊԽՋՑՋՌԽՅՋ
ԱՑՈԽՇԾԾՇՇԼ ԵԽՆՇՈՍՋԢՐՈՊԽՋՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋԽՊԹԪՁԻՊՇՆնՌՀՊՇՍԿՀՇՍՌ ԱԹԼՈՇՄԽԣՇՇԼ ՈՊԽԽՐՈՇՋՍՊԽ ԹԾՌԽՊԫԣՋՌԹԿԽ ԹՆԼ ԼՍՊՁՆԿՌՀԽԽՆՌՁՊԽԼԼԽԾՁՆՁՌՁՎԽՋՌՍԼՑԦԾ ՇՍՊԻԽՇԾԾՇՇԼ ԵԽՆՇՈՍՋԢՐՈՊԽՋՋ ԹՆՇՌՀԽՊԼՁԽՌՁՋՍՋԽԼՌՀԽՋՌՍԼՑՊԽՈՇՊՌՋՀՇՍՄԼ ԟՊՇՇՃՋՎՁՄՄԽԣԩ ՈՊՇՎՁԼԽԹՆԹՄՑՋՁՋՇԾՁՇԼՁԼԽԻՇՆՌԽՆՌԹՆԼՈՇՌԽՆՌՁԹՄ ԦՇԼՁԼԽԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆԼՁԽՌ ՁԾ ԫՇՌՊԽՈՇՊՌԽԼ ԻՇՆՌԹՅՁՆԹՆՌՋԹՆԼՌՀԽԼՁԽՌՋՀՇՍՄԼ ՃՆՇՏՆ ԼԽՅՇՆՋՌՊԹՌԽԽՉՍԹՄՈԽՊԾՇՊՅԹՆԻԽՌՇԽՊԹ ԪՁԻՊՇՆն
ԣՊԽՉՍԽՆԻՑՇԾԾԽԽԼՁՆԿ ՌՁՅԽՋԼԹՑ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾԽԽԼՁՆԿՇԻԻՍՊԹՌՄԽԹՋՌ ՌՏՁԻԽՈԽՊԼԹՑ
Page ԭԹԿԽՇԾ 12 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԡԽՌԹՁՄՋՇՆԾԽԽԼՁՆԿՊԽԿՁՅԽ ԣԽԽԼՁՆԿՊԽԿՁՅԽԾՇՄՄՇՏԽԼ ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌԾՇՇԼՊԹՌՁՇՆՋԼՍՊՁՆԿՌՀԽ ԿՍՁԼԽՄՁՆԽ ՈՊԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼԺԽՁՆԻՊԽԹՋԽԼԹՄՇՆԿՏՁՌՀ ՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ՄԹՊՎԹՄԿՊՇՏՌՀՌՇԹՈՈՊՇՐՁՅԹՌԽՄՑ ՅԿՄԹՊՎԹԼԹՑԺՑՌԽՋՌՁՆՁՌՁԹՌՁՇՆԢԭԞԹՆԼ ԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌԾՇՇԼՊԹՌՁՇՆՋՁՆԻՊԽԹՋԽ ԾՊՇՅՅԿՄԹՊՎԹԼԹՑԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ՌՍԼՑ ԡԹՑ ՌՇՅԿՄԹՊՎԹԼԹՑՁՆՌՀԽՄԹՋՌՏԽԽՃ ՇԾՌՀԽՌԽՋՌ ՌՍԼՑԡԹՑ
Page ԭԹԿԽՇԾ 13 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԠԢՐՈՇՋՍՊԽՑՋՌԽՅ ԱԹԺՄԽ ՍՅՅԹՊՑՇԾԦՆԾՇՊՅԹՌՁՇՆՇՆՌՀԽԢՐՈՇՋՍՊԽՑՋՌԽՅԹՆԼԱԽՋՌԳԽՋՋԽՄԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԱՑՈԽՇԾԽՐՈՇՋՍՊԽ ԣՄՇՏՌՀՊՇՍԿՀ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԹԾՄՇՏՌՀՊՇՍԿՀ ՋՑՋՌԽՅ
ԱՑՈԽՇԾԾՄՇՏՌՀՊՇՍԿՀԼՁՄՍՌՁՇՆ ԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏ ԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊՋՇՊ ՋՑՋՌԽՅ ՁԾԹՈՈՄՁԻԹԺՄԽ ՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊՋ ԻՇՆՌՁՆՍՇՍՋԾՄՇՏՋԽՊՁԹՄԼՁՄՍՌԽՊՋԹՊԽ ՊԽԻՇՅՅԽՆԼԽԼ
ԣՄՇՏՌՀՊՇՍԿՀՊԹՌԽ ՁԾ +ՅԩՅՁՆ ԠԹՄԻՍՄԹՌԽԼԺԹՋԽԼՇՆԩԾՁՄՄՎՇՄՍՅԽԹՆԼ ԯԽԻՇՅՅԽՆԼԽԼԾՄՇՏՌՀՊՇՍԿՀՊԹՌԽՁՋ ԹՈՈՄՁԻԹԺՄԽ ՎՇՄՍՅԽՊԽՈՄԹԻԽՅԽՆՌՋԽՎԽՊՑՀՇՍՊՋԱՀՁՋ ՅԩՅՁՆ ԻՇՅՈՄԽՌԽՎՇՄՍՅԽՊԽՈՄԹԻԽՅԽՆՌԻԹ ՊԽՋՍՄՌԽԼՁՆԹՎՇՄՍՅԽՊԽՈՄԹԻԽՅԽՆՌԽՎԽՊՑ ԽՎԽՊՑՀՊՋ ՀՇՍՊՋ
ԞԼԼՁՌՁՇՆԹՄԿՍՁԼԹՆԻԽԾՇՊԹՉՍԹՌՁԻՌԽՋՌԼԽՋՁԿՆՁՋՄՇԻԹՌԽԼՁՆԬԠԭԭԤՍՁԼԽՄՁՆԽՈԽԻՁԹՄԠՇՆՋՁԼԽՊԹՌՁՇՆՋԾՇՊԠՇՆԼՍԻՌՁՆԿԞՉՍԹՌՁԻԩԹԺՇՊԹՌՇՊՑՌՍԼՁԽՋ
Page ԭԹԿԽՇԾ 14 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԡԽՌԹՁՄՋՇՆՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊ ՌՇԻՃՋՇՄՍՌՁՇՆՋԾՁՄՄԽԼ ԯԽԻՇՅՅԽՆԼԽԼՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊ ԾՄՇՏՌՀՊՇՍԿՀՋՑՋՌԽՅՋ ՁԾ ԻՀԽՅՁԻԹՄԼՁՄՍՌԽՊԻԽՄՄՋ ԾՄՇՏՌՀՊՇՍԿՀՋՑՋՌԽՅՋ ԪՁՐՁՆԿԻՀԹՅԺԽՊՁՋ ԹՈՈՄՁԻԹԺՄԽ ՏՀՁԻՀՌՀԽՆՌՊԹՆՋԾԽՊՊԽԼՌՀԽ ՊԽԻՇՅՅԽՆԼԽԼԺՍՌՆՇՌՊԽՉՍՁՊԽԼ ԞԽՊԹՌՁՇՆ ԼՁՄՍՌԽԼՋՌՇԻՃՋՇՄՍՌՁՇՆՋՌՇ ՁՋՆՇՌՊԽԻՇՅՅԽՆԼԽԼԾՇՊՅՁՐՁՆԿ Ԟ ՌՀԽԹՈՈՊՇՈՊՁԹՌԽՌԽՋՌ ԼԽՅՇՆՋՌՊԹՌՁՇՆՌՀԹՌՌՀԽՌԽՋՌՋՇՄՍՌՁՇՆՁՋ ԻՀԹՅԺԽՊ ԻՇՅՈՄԽՌԽՄՑՅՁՐԽԼԺԽԾՇՊԽՁՆՌՊՇԼՍԻԽԼՁՆՌՇՌՀԽ ՌԽՋՌՋՑՋՌԽՅՁՋՊԽԻՇՅՅԽՆԼԽԼ ԱՀԽ ՊԽԻՇՅՅԽՆԼԽԼԾՄՇՏՋՈՄՁՌՌՁՆԿԹԻԻՍՊԹԻՑՁՋ ՏՁՌՀՁՆ
ԯԽՆԽՏԹՄՈԽՊՁՇԼԾՇՊՋՌԹՌՁԻ ԫԞ ԦԾՋՌԹՌՁԻՊԽՆԽՏԹՄՁՋՍՋԽԼԢԭԞՊԽԻՇՅՅԽՆԼՋ ՊԽՆԽՏԹՄ ՁԾԹՈՈՄՁԻԹԺՄԽ ՀՊՊԽՆԽՏԹՄ ՊԽՆԽՏԹՄՈԽՊՁՇԼՁՋ ՊԽԻՇՅՅԽՆԼԽԼՆՇՌՌՇԽՐԻԽԽԼՀՇՍՊՋ
ԞԽՊԹՌՁՇՆ! ԫՇ ԢԭԞՊԽԻՇՅՅԽՆԼՋՅԹՁՆՌԹՁՆՁՆԿԼՁՋՋՇՄՎԽԼ ՇՐՑԿԽՆԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԹՁՊՋԹՌՍՊԹՌՁՇՆ ՅԿԩ ԞԽՊԹՌՁՇՆՅԹՑԺԽՅԹՁՆՌԹՁՆԽԼ ՌՀՊՇՍԿՀԺՍԺԺՄԽՊՋԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՇՋԽՌ ԺՍԺԺՄԽՊՋԹՌՄԽՎԽՄՋՌՀԹՌԼՇՆՇՌԻԹՍՋԽՋՌՊԽՋՋ ՇՆՌՀԽՌԹԼՈՇՄԽՋ
Page ԭԹԿԽՇԾ 15 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ՇՍՊԻԽՇԾԼՁՄՍՌՁՇՆՏԹՌԽՊ ԩԹԺՇՊԹՌՇՊՑՏԽՄՄՏԹՌԽՊ ԱՀԽՏԹՌԽՊՏԹՋՈՍՅՈԽԼՌՇԹԻՇՆԻՊԽՌԽ ԢԭԞՊԽԻՇՅՅԽՆԼՋՆԹՌՍՊԹՄՇՊՊԽԻՇՆՋՌՁՌՍՌԽԼ ՇԺՌԹՁՆԽԼԾՊՇՅԹՆՇՆՋՁՌԽ ՊԽՋԽՊՎՇՁՊՏՀԽՊԽՁՌՏԹՋՋՍՈՈՄԽՅԽՆՌԽԼՇՆ ՏԹՌԽՊ ՁՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՆԹՌՍՊԹՄՏԹՌԽՊ ՅԽՌԽՊԺԽԼՊՇԻՃՏԽՄՄ ԼԽՅԹՆԼՏՁՌՀԼԽԻՀՄՇՊՁՆԹՌԽԼԱՇՏՆՇԾ ԺԽՋՌԽՊՁՄՁՒԽԼՏՁՌՀԲԳԹՆԼՌԽՋՌԽԼԾՇՊ ՏԹՋԾՇՊՌՁԾՁԽԼՏՁՌՀԣԢԱԞԵ ԴԹՊԽՀԹՅՏԽՄՄՏԹՌԽՊԴԹՌԽՊՏԹՋԹԽՊԹՌԽԼՈՊՁՇՊ ՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊ ՋՇՄՍՌՁՇՆԹԻԻՇՊԼՁՆԿՌՇ ՌՇՍՋԽՌՇԽՆՋՍՊԽՈՊՇՈԽՊԽՉՍՁՄՁԺՊԹՌՁՇՆՇԾ ՈՇՋՋՁԺՄԽԻՇՆՌԹՅՁՆԹՆՌՋՁՆԻՄՍԼՁՆԿՃՆՇՏՆ ԞԱԪԿՍՁԼԽՄՁՆԽՋ ԼՁՋՋՇՄՎԽԼԿԹՋԽՋ ՋՍԺՋՌՊԹՌԽՋՇԾՌՀԽՁՇԼՁՆԽՌՊԹՆՋՈՇՊՌԽՊՇԾՌՀԽ ՌՀՑՊՇՁԼԿՄԹՆԼ ԽԿԾՄՍՇՊՁԼԽԻՀՄՇՊԹՌԽ ՈԽՊԻՀՄՇՊԹՌԽ ԬԢԠԡԹԻԻԽՈՌՋԹՆՑՏԹՌԽՊՁՆ ՏՀՁԻՀՌՀԽՌԽՋՌՋՈԽԻՁԽՋՋՀՇՏԻՇՆՌՊՇՄՋՍՊՎՁՎԹՄ ԹՌՄԽԹՋՌԹՋԿՇՇԼԹՋՁՆԼՁԻԹՌԽԼՁՆՌՀԽՌԽՋՌ ԿՍՁԼԽՄՁՆԽ
ԴԹՋԼՁՄՍՌՁՇՆՏԹՌԽՊԹՆԹՄՑՒԽԼԾՇՊ ԶԽՋ ԫՇՈԽՋՌՁԻՁԼԽՋԭԠԟՋՇՊՌՇՐՁԻՅԽՌԹՄՋՏԽՊԽ ՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼ ԼԽՌԽԻՌԽԼԹՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋԻՇՆՋՁԼԽՊԽԼՌՇՐՁԻ ՇՌՀԽՊԻՇՆՌԹՅՁՆԹՆՌՋ! ՁՆԹՆՑՇԾՌՀԽՏԹՌԽՊՋԹՅՈՄԽՋԹՆԹՄՑՒԽԼՁՆ ԹԿՊԽԽՅԽՆՌՏՁՌՀԞԱԪՋՌԹՆԼԹՊԼՈՊԹԻՌՁԻԽՋ
Page ԭԹԿԽՇԾ 16 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԦՇԼՁԼԽՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՆ ԫՇ ԱՀԽՅՇՋՌՊԽԻԽՆՌԹՆԹՄՑՋՁՋՇԾՌՀԽՏԽՄՄՏԹՌԽՊ ԦԾՊԽԻՇՆՋՌՁՌՍՌԽԼՏԹՌԽՊՁՋՍՋԽԼՇՊՁԾ ՏԹՌԽՊ! ԾՇՍՆԼՁՇԼՁԼԽԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾ+սԿԩ ԺԹԻՃԿՊՇՍՆԼՄԽՎԽՄՋՇԾՁՇԼՁԼԽՁՆՆԹՌՍՊԹՄՏԹՌԽՊ ԹՊԽՄԽՋՋՌՀԹՆսԿԩՁՇԼՁԼԽ ՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՋՊԽԻՇՅՅԽՆԼԽԼԱՀՁՋ ՋՍՈՈՄԽՅԽՆՌԹՌՁՇՆՁՋՁՆԹԼԼՁՌՁՇՆՌՇՌՀԽ ՊԽԻՇՅՅԽՆԼԽԼԼՁԽՌԹՊՑՋՇՍՊԻԽՇԾՁՇԼՁԼԽ ԽԿՁՆԽՊԹԪՁԻՊՇՆ
ԱԽՋՌՎԽՋՋԽՄՌՑՈԽՅԹՌԽՊՁԹՄՋ ԤՄԹՋՋԹՉՍԹՊՁԹ ԢԭԞ ԹՆԼ ԬԢԠԡ ՊԽԻՇՅՅԽՆԼ ՌՀԹՌ ՏԹՌԽՊԻՇՆՌԹԻՌ ՈՇՊՌՁՇՆՋ ՇԾ ՌՀԽ ՋՑՋՌԽՅ ՆՇՌ ԻՇՅՈՊՇՅՁՋԽ ՌՀԽ ՋՌՍԼՑ ԽԿ ԹՄՄ ԿՄԹՋՋ ՎԽՋՋԽՄՋՇՊԿՄԹՋՋՎԽՋՋԽՄՋՏՁՌՀՋՌԹՁՆՄԽՋՋՋՌԽԽՄ ԾՊԹՅԽՋԹՊԽԹԻԻԽՈՌԹԺՄԽԽՐԹՅՈՄԽՋ
ԱԽՋՌՎԽՋՋԽՄՋՁՒԽ ՐՐԻՅ
ԣՁՄՄՎՇՄՍՅԽ +ԩ
ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋՇՆԽՐՈՇՋՍՊԽ ՋՑՋՌԽՅ
ԱԹԺՄԽ ՍՅՅԹՊՑՇԾԴԹՌԽՊԮՍԹՄՁՌՑԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋՁՆՌՀԽԱԽՋՌՑՋՌԽՅ
Page ԭԹԿԽՇԾ 17 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԪԽԹՋՍՊԽՅԽՆՌ ԭԹՊԹՅԽՌԽՊ ԪՁՆՁՅՍՅ ԪԹՐՁՅՍՅ ԪԽԹՆ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԦՆՌԽՊՎԹՄ
ԫԞ ԪԽԹՋՍՊԽԼԹՌՌԽՋՌ ԢԭԞՊԽԻՇՅՅԽՆԼՋՀԹՊԼՆԽՋՋՌՇ
ԥԹՊԼՆԽՋՋ ՅԿԩԹՋԠԹԠԬ ՇՆՄՑՊԹՆԿԽՋ ՁՆՁՌՁԹՌՁՇՆԹՆԼՏԽԽՃՄՑ ՅԿԩԹՋԠԹԠԬ ՈՊՇՎՁԼԽԼ ՌՀԽՊԽԹԾՌԽՊ
ԪԽԹՋՍՊԽԼՁՆԹՄՄ ԢԭԞՊԽԻՇՅՅԽՆԼՋՈԥչ ՊԽՈՄՁԻԹՌԽՌԽՋՌՎԽՋՋԽՄՋ ՁՆՌԽՊՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊՌՊԽԹՌՅԽՆՌ Ոԥ ՇՆԡԹՑԹՆԼՁՆ ԼՁԾԾԽՊԽՆՌՁԹՄՋՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼ ԹՄՌԽՊՆԹՌՁՆԿՊԽՈՄՁԻԹՌԽՋ ԼԹՁՄՑՌՀԽՊԽԹԾՌԽՊ
ԪԽԹՋՍՊԽԼՁՆԹՄՄ ԢԭԞՊԽԻՇՅՅԽՆԼՋԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆ ԡԬ ՊԽՈՄՁԻԹՌԽՌԽՋՌՎԽՋՋԽՄՋ ՅԿԩ ԹՁՊՋԹՌՍՊԹՌՁՇՆ ԬԢԠԡ ԡՁՋՋՇՄՎԽԼՇՐՑԿԽՆ ՅԿԩ ՇՆԡԹՑԹՆԼՁՆ ՊԽԻՇՅՅԽՆԼՋԡԬԻՇՆԻԽՆՌՊԹՌՁՇՆ ՅԿԩ ԹՄՌԽՊՆԹՌՁՆԿՊԽՈՄՁԻԹՌԽՋ ԹՁՊՋԹՌՍՊԹՌՁՇՆ ԼԹՁՄՑՌՀԽՊԽԹԾՌԽՊ
Page ԭԹԿԽՇԾ 18 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԪԽԹՋՍՊԽՅԽՆՌ ԭԹՊԹՅԽՌԽՊ ԪՁՆՁՅՍՅ ԪԹՐՁՅՍՅ ԪԽԹՆ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԦՆՌԽՊՎԹՄ
ԪԽԹՋՍՊԽԼՁՆԹՄՄ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌԽՅՈԽՊԹՌՍՊԽչՇԠ ՊԽՈՄՁԻԹՌԽՌԽՋՌՎԽՋՋԽՄՋ ՁՆՌԽՊՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊՌՊԽԹՌՅԽՆՌ ՇՆԡԹՑԹՆԼՁՆ ԼՁԾԾԽՊԽՆՌՁԹՄՋՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼՇԠ ԹՄՌԽՊՆԹՌՁՆԿՊԽՈՄՁԻԹՌԽՋ ԱԽՅՈԽՊԹՌՍՊԽ ոԠ ԼԹՁՄՑՌՀԽՊԽԹԾՌԽՊԞՄՋՇ ՅԽԹՋՍՊԽԼ ԻՇՆՌՁՆՍՇՍՋՄՑՁՆ ԯԽՈՄՁԻԹՌԽԞՇԾՌՀԽ ՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ
ԟԹՋԽԼՇՆՅՇՋՌ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՉՍԹՌՁԻՁՇԼՁԼԽՊԹՆԿԽ ԦՇԼՁԼԽ սԿԩ ԫԞ ԫԞ ՊԽԻԽՆՌԹՆԹՄՑՋՁՋ -սԿԩ ՋՍՈՈՄԽՅԽՆՌԹՄՁՇԼՁԼԽՋՀՇՍՄԼՆՇՌ ԽՐԻԽԽԼսԿԩ
Page ԭԹԿԽՇԾ 19 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԪԽԹՋՍՊԽՅԽՆՌ ԭԹՊԹՅԽՌԽՊ ԪՁՆՁՅՍՅ ԪԹՐՁՅՍՅ ԪԽԹՆ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԦՆՌԽՊՎԹՄ
ԞՅՅՇՆՁԹ ԫՇՌԯԽՈՇՊՌԽԼ ԤԽՆԽՊԹՄՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋԾՇՊԾՊԽՉՍԽՆԻՑՇԾ ՅԽԹՋՍՊԽՅԽՆՌՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌ ԣՄՍՇՊՁԼԽ ԫՇՌԯԽՈՇՊՌԽԼ ՏԹՌԽՊՉՍԹՄՁՌՑՈԹՊԹՅԽՌԽՊՋԺԽՅԽԹՋՍՊԽԼՁՆԹ ԭԽՊԻՀՄՇՊԹՌԽ ԫՇՌԯԽՈՇՊՌԽԼ ԻՇՆՌՊՇՄԹՆԼԹՌՇՆԽՌԽՋՌՁՌԽՅԻՇՆԻԽՆՌՊԹՌՁՇՆ ԠՀՄՇՊԹՌԽ ԫՇՌԯԽՈՇՊՌԽԼ ԹՌՄԽԹՋՌՏԽԽՃՄՑԦՆՋՌԹՌՁԻՊԽՆԽՏԹՄՋՑՋՌԽՅՋ ՏԹՌԽՊՉՍԹՄՁՌՑՈԹՊԹՅԽՌԽՊՋՁՆԻՄՍԼՁՆԿ ԹՅՅՇՆՁԹՋՀՇՍՄԼԺԽՅԽԹՋՍՊԽԼՂՍՋՌՈՊՁՇՊՌՇ ՊԽՆԽՏԹՄԦՆԹԼԼՁՌՁՇՆԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌ ԡԬԺԽՅԽԹՋՍՊԽԼԹՌԽԹԻՀԻՇՆԻԽՆՌՊԹՌՁՇՆԹՌ ԞՄՃԹՄՁՆՁՌՑ ՅԿԩԹՋԠԹԠԬ ԫԞ ԪԽԹՋՍՊԽԼԹՌՌԽՋՌ ՄԽԹՋՌՏԽԽՃՄՑԹՆԼՌՀԹՌՌԽՅՈԽՊԹՌՍՊԽԺԽ ՇՆՄՑՊԹՆԿԽՋ ՁՆՁՌՁԹՌՁՇՆԹՆԼՏԽԽՃՄՑ ՅԽԹՋՍՊԽԼԻՇՆՌՁՆՍՇՍՋՄՑԬԢԠԡ ԠՇՆԼՍԻՌՁՎՁՌՑ սՅՀՇՋԻՅ ՈՊՇՎՁԼԽԼ ՌՀԽՊԽԹԾՌԽՊ ՊԽԻՇՅՅԽՆԼՋՌՀԹՌԡԬԹՆԼՌԽՅՈԽՊԹՌՍՊԽԺԽ ՅԽԹՋՍՊԽԼԹՌՄԽԹՋՌՏԽԽՃՄՑԹՆԼՌՀԹՌՈԥԹՆԼ ՀԹՊԼՆԽՋՋԺԽՅԽԹՋՍՊԽԼԹՌՄԽԹՋՌԹՌՌՀԽ ԺԽԿՁՆՆՁՆԿԹՆԼԽՆԼՇԾՌՀԽՌԽՋՌ
ԡՌՍԼՑԡԽՋՁԿՆԹՆԼԞԼԼՁՌՁՇՆԹՄԢՐՈԽՊՁՅԽՆՌԹՄԠՇՆԼՁՌՁՇՆՋ
Page ԭԹԿԽՇԾ 20 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԱԹԺՄԽ ԯԹՆԿԽԣՁՆԼՁՆԿՌՍԼՑԠՇՆԼՁՌՁՇՆՋ ՁԾԞՈՈՄՁԻԹԺՄԽ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԴԹՋԹՊԹՆԿԽԾՁՆԼԽՊԻՇՆԼՍԻՌԽԼ! ԶԽՋ
ԦԾՑԽՋՏՀԹՌՏԹՋՌՀԽՅԽՌՀՇԼԾՇՊ ՇՄՍԺՁՄՁՌՑՌՊՁԹՄՋՈՊՁՇՊՌՇ ԱՀԽՋԽԻՇՆԼՊԹՆԿԽԾՁՆԼՁՆԿՋՌՍԼՑՑՁԽՄԼԽԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ
ԼԽՌԽՊՅՁՆՁՆԿՌՀԽՀՁԿՀԽՋՌՌԽՋՌ ՌՀԽՊԹՆԿԽԾՁՆԼԽՊ ԹՆԩԠՇԾՅԿԹՁԩԱՀԽՀՁԿՀԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾՌՀԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽ ՊԽՎԽԹՄԽԼՌՀԹՌՈՀՇՋՅԽՌ ՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ ՌԽՋՌԻՇՅՈՇՍՆԼՅԿԩՇՊԼԽՅՇՆՋՌՊԹՌԽՋ
ՊԹՆԿԽԾՁՆԼԽՊ! ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽ ՏԹՋՋԽՌԹՌԹՈՈՊՇՐՁՅԹՌԽՄՑՇԾՌՀԽԢԠ ԹԼԽՉՍԹՌԽԽՎՁԼԽՆԻԽՇԾՌՇՐՁԻՁՌՑ ԽԿ ՊԽՄԹՌՁՎԽՋՌԹԺՄԽՇՎԽՊ ԹՆԼՏՁՌՀԹԼՁՄՍՌՁՇՆԾԹԻՌՇՊՇԾ ՅՇՊՌԹՄՁՌՑ ՏՀՁԻՀԽՎԽՊԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌ ՀՇՍՊՋՍՋՁՆԿԹԺՍԾԾԽՊԽԼ ԹՉՍԽՇՍՋՋՇՄՍՌՁՇՆՇԾՈԥ ԞՆՁՆՁՌՁԹՄ ՊԹՆԿԽԾՁՆԼՁՆԿՋՌՍԼՑՏԹՋ ԻՇՆԼՍԻՌԽԼՏՁՌՀՆՇՅՁՆԹՄ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՍՈՌՇ ՅԿԩԺՍՌՆՇՌՇՐՁԻ ՊԽՋՈՇՆՋԽՏԹՋՇԺՋԽՊՎԽԼ ԞՋԽԻՇՆԼՌՊՁԹՄՏԹՋ ԻՇՆԼՍԻՌԽԼՏՁՌՀՆՇՅՁՆԹՄ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՍՈՌՇ ՅԿԩ
ՈԽԻՁԽՋ ԵԽՆՇՈՍՋՄԹԽՎՁՋ
Page ԭԹԿԽՇԾ 21 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԩՁԾԽՋՌԹԿԽ ԫԣ
ԱԽՋՌԼՍՊԹՌՁՇՆ ՀՇՍՊՋ
ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋ ԱՀԽՁՆՁՌՁԹՄՊԹՆԿԽԾՁՆԼԽՊ ԱՀԽՀՁԿՀԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽՋԽԻՇՆԼ ՏԹՋԻՇՆԼՍԻՌԽԼՍՆԼԽՊ ՊԹՆԿԽԾՁՆԼԽՊՏԹՋՌՀԽՀՁԿՀԽՋՌԹՌՌԹՁՆԹԺՄԽ ԾՄՇՏՌՀՊՇՍԿՀԻՇՆԼՁՌՁՇՆՋ ԻՇՆԻԽՆՌՊԹՌՁՇՆՏՁՌՀՌՀԽՍՋԽՇԾԹԺՍԾԾԽՊԽԼ ՏՁՌՀՆՇՅՁՆԹՄ ԹՉՍԽՇՍՋՋՇՄՍՌՁՇՆՏՁՌՀՈԥ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾ ԹՆԼՅԿԹՁԩ ԱՀԽՋԽԻՇՆԼՏԹՋ ԻՇՆԼՍԻՌԽԼՍՆԼԽՊ ԾՄՇՏՌՀՊՇՍԿՀԻՇՆԼՁՌՁՇՆՋ ՏՁՌՀՆՇՅՁՆԹՄ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾ ԹՆԼՅԿ ԹՁԩ
ԱԹԺՄԽ ԡԽԾՁՆՁՌՁՎԽՌՍԼՑԠՇՆԼՁՌՁՇՆՋ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
Page ԭԹԿԽՇԾ 22 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԱԽՋՌԼՍՊԹՌՁՇՆ ԼԹՑՋ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՌՀԽ ԼՍՊԹՌՁՇՆ ՇԾ ՌՀԽ ԼԽԾՁՆՁՌՁՎԽՌԽՋՌԺԽԼԹՑՋ
ԪԽՌՀՇԼԾՇՊՋԽՄԽԻՌՁՆԿՌՀԽՀՁԿՀԽՋՌ ԯԹՆԿԽԾՁՆԼԽՊՇԾՌՀԽ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ
ՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽԼԽԾՁՆՁՌՁՎԽ ԢԠՏՁՌՀԹԼՁՄՍՌՁՇՆ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾՌՀԽ ՌԽՋՌ ԾԹԻՌՇՊՇԾԾՇՊՌՀԽՌՏՇ ՌԽՋՌԻՇՅՈՇՍՆԼՅԿԩՇՊԼԽՅՇՆՋՌՊԹՌԽՋ ՄՇՏԽՊԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԹԼԽՉՍԹՌԽԽՎՁԼԽՆԻԽՇԾՌՇՐՁԻՁՌՑ ԽԿ ՅՇՊՌԹՄՁՌՑ ՏՀՁԻՀԽՎԽՊԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌ
ԯԽԾԽՊԽՆԻԽՋՌՍԼՑԻՁՌԹՌՁՇՆ ՁԾ ԫՇՌԹՈՈՄՁԻԹԺՄԽ ԹՈՈՄՁԻԹԺՄԽ
ԽՈԹՊԹՌՁՇՆՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՌՀԽ ՅԹՐՁՅՍՅ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՋԽՈԹՊԹՌՁՇՆ ԺԽ ԹՆԼ ՌՀԽ ՅՁՆՁՅՍՅԺԽ
ԫՍՅԺԽՊՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՇԾ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԹՆԼԹԻՇՆՌՊՇՄՈՄՍՋՋՇՄՎԽՆՌ ԻՇՆՌՊՇՄՁԾԹՈՈՊՇՈՊՁԹՌԽ
ԞՊԽՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԶԽՋ ԹԼՂՍՋՌԽԼԾՇՊՈՍՊՁՌՑ!
Page ԭԹԿԽՇԾ 23 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԦՆԼՁԻԹՌԽՌՀԽՌՑՈԽՇԾՎԹՄՍԽՋ ԪԽԹՆՅԽԹՋՍՊԽԼ ՈՊԽՋԽՆՌԽԼԾՇՊՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ
ԩՁՅՁՌՇԾՉՍԹՆՌՁԾՁԻԹՌՁՇՆ ԩԬԮ ՅԿԹՁԩ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՇՊԻՀԽՅՁԻԹՄՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽՄՇՏՌՀԽԩԬԮԹՆԹՄՑՋԽՋԺԽ ԻՇՆԼՍԻՌԽԼՇՆՌՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋ
ԩԽՎԽՄՇԾԼԽՌԽԻՌՁՇՆ ԩԬԡ ԫՇՌԯԽՈՇՊՌԽԼ
ԣՊԽՉՍԽՆԻՑՇԾՅԽԹՋՍՊԽՅԽՆՌ ԡԹՑՋԹՆԼ ԱՀԽՊԽՋՍՄՌՋԾՊՇՅՌՀԽԡԹՑՋԹՅՈՄՁՆԿ ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՌԽՋՌՁՌԽՅԻՇՆԻԽՆՌՊԹՌՁՇՆ ԼԽՅՇՆՋՌՊԹՌԽԼՈՇՇՊԮԠՊԽԻՇՎԽՊՁԽՋ ԺԽՅԽԹՋՍՊԽԼՁՆՇՆԽՌԹՆՃԹՌԽԹԻՀՌՊԽԹՌՅԽՆՌ ՊԹՆԿՁՆԿԾՊՇՅՌՇՇԾՆՇՅՁՆԹՄ ՄԽՎԽՄԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼԽՎԽՊՑՏԽԽՃ ԾՇՊՌՁԾՁԽԼՄԽՎԽՄՋԡՍԽՌՇՌՀԽՈՇՇՊՊԽԻՇՎԽՊՁԽՋ ՌՀԽՊԽԹԾՌԽՊ ՌՀԽՈՊԽԻՁՋՁՇՆԹՆԼՉՍԹՄՁՌՑԻՇՆՌՊՇՄԾՇՊՌՀՁՋ ԹՆԹՄՑՋՁՋՏԽՊԽԼԽԽՅԽԼՉՍԽՋՌՁՇՆԹԺՄԽ ԱՀԽՊԽԾՇՊԽՌՀԽԽՐՈՇՋՍՊԽՋՑՋՌԽՅՏԹՋ ՊԽՋԹՅՈՄԽԼՇՆԡԹՑԱՀԽՋԽՊԽԻՇՎԽՊՁԽՋ ՏԽՊԽՏՁՌՀՁՆԽՐՈԽԻՌԹՌՁՇՆՋԹՆԼՏԽՊԽՍՋԽԼ ԾՇՊԻԹՄԻՍՄԹՌՁՇՆՇԾՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԡԹՑՎԹՄՍԽՋՏԽՊԽ ԽՐԻՄՍԼԽԼ
Page ԭԹԿԽՇԾ 24 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆԻՇՆՌՊՇՄ ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ
ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆՋՇՄՎԽՆՌ ԫԞ ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԽՍՋԽՇԾԹ ԻՇՆՌՊՇՄ ՁԾԹՈՈՄՁԻԹԺՄԽ ԻՇՆԻՍՊՊԽՆՌՋՇՄՎԽՆՌԻՇՆՌՊՇՄՏՀԽՆԹՋՇՄՍԺՁՄՁՒՁՆԿ ԹԿԽՆՌՁՋՍՋԽԼԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ
ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՈԽՊՌԽՋՌՁՌԽՅ ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ ՌՊԽԹՌՅԽՆՌՄԽՎԽՄ
ԫՍՅԺԽՊՇԾՄԹՊՎԹԽՈԽՊՌՊԽԹՌՅԽՆՌԹՌ ՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ԴԹՋԹՋՇՄՎԽՆՌՍՋԽԼ! ԫՇ
ՇՄՎԽՆՌՌՑՈԽ ՁԾԹՈՈՄՁԻԹԺՄԽ ԫԞ
ԪԹՐՁՅՍՅՋՇՄՎԽՆՌԻՇՆԻԽՆՌՊԹՌՁՇՆ ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՋՇՄՎԽՆՌՆՇՌԽՐԻԽԽԼ ՁԾԹՈՈՄՁԻԹԺՄԽ ՅՄԩԬԢԠԡՊԽԻՇՅՅԽՆԼՋՌՀԹՌՋՇՄՎԽՆՌ ՀԹՎԽՆՇԽԾԾԽԻՌՇՆՋՍՊՎՁՎԹՄՆՇՊՈՊՇԼՍԻԽԹՆՑ ՇՌՀԽՊԹԼՎԽՊՋԽԽԾԾԽԻՌՋԹՆԼՌՀԹՌԻՇՆԻԽՆՌՊԹՌՁՇՆ ՆՇՌԺԽԿՊԽԹՌԽՊՌՀԹՆՅՄԩ
ԥՍՌԻՀՁՆՋՇՆԱԥՀՁՄՄԹԺԽԽՊԫԴՁՆՌԽՊԪԧԭՁԻՃԾՇՊԼԡԟ ԞԻՍՌԽԹՆԼԻՀՊՇՆՁԻԽԾԾԽԻՌՋՇԾԻԹՊՊՁԽՊՋՇՄՎԽՆՌՋՁՆԹՉՍԹՌՁԻՇՊԿԹՆՁՋՅՋ ԞԻՊՁՌՁԻԹՄՊԽՎՁԽՏԯԽՎՁԽՏԞՉՍԹՌՁԻ ԱՇՐՁԻՇՄՇԿՑՈՈ- ԬԢԠԡ ԤՍՁԼԹՆԻԽԡՇԻՍՅԽՆՌՇՆԞՉՍԹՌՁԻԱՇՐՁԻՁՌՑԱԽՋՌՁՆԿՇԾԡՁԾԾՁԻՍՄՌՍԺՋՌԹՆԻԽՋԹՆԼԪՁՐՌՍՊԽՋԢՆՎՁՊՇՆՅԽՆՌԹՄԥԽԹՄՌՀԹՆԼԹԾԽՌՑԭՍԺՄՁԻԹՌՁՇՆՋԽՊՁԽՋՇՆ
Page ԭԹԿԽՇԾ 25 of 260 ԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ
ՋԽԼ! ԫՇ
ԹԺՄԽ ԫԞ
ԹՌՁՇՆ Ջ ԫԞ
ՀՊՋՄՁԿՀՌ ԞՅՁՆՍՌԽՄՇՏՄՁԿՀՌՌՊԹՆՋՁՌՁՇՆՈԽՊՁՇԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՈՀՇՌՇՈԽՊՁՇԼ ՀՊՋԼԹՊՃ ՏԹՋՍՋԽԼԺԽՌՏԽԽՆՌՀԽՄՁԿՀՌԹՆԼԼԹՊՃ ՄՁԿՀՌ ԼԹՊՃ ՈՀԹՋԽՋ
ՋՍՊԾԹԻԽ ԨՄՍՐ ԢԭԞՊԽԻՇՅՅԽՆԼՋՄՁԿՀՌՁՆՌԽՆՋՁՌՑ-ԨՄՍՐ ԹՌՏԹՌԽՊ0ՋՋՍՊԾԹԻԽ
ԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋՏԽՊԽՊԽՈՇՊՌԽԼԹՋԻՄԽԹՊ ԹՆԼԻՇՄՇՊՄԽՋՋ Page 26 of 260
ԭԹՊՁՋԣՊԹՆԻԽ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԱԹԺՄԽ ՍՅՅԹՊՑՇԾԱՊԽԹՌՅԽՆՌԠՇՆԻԽՆՌՊԹՌՁՇՆՋՁՆՌՀԽԞՅՈՀՁԺՁԹՆԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ
ԫՇՅՁՆԹՄ ԪԽԹՋՍՊԽԼ ԱՊԽԹՌՅԽՆՌԦԡ ԠՇՆԻԽՆՌՊԹՌՁՇՆ ԠՇՆԻԽՆՌՊԹՌՁՇՆ ԪԽԹՆԠԳ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ՅԿԹՁԩ ՅԿԹՁԩ
ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ ԩԬԮ ԫԞ ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌՌԽՋՌՁՌԽՅ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽՅԹՁՆՌԹՁՆԽԼԹՌԹԻՇԽԾԾՁԻՁԽՆՌՇԾ ԱՊԽԹՌՅԽՆՌ ՎԹՊՁԹՌՁՇՆ ԠԳ ԱՊԽԹՌՅԽՆՌ
ԱՊԽԹՌՅԽՆՌ
ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԠԳԠՇԽԾԾՁԻՁԽՆՌՇԾՎԹՊՁԹՌՁՇՆ ԢԬԺՋԽՊՎԹՌՁՇՆՋ ԟՁՇՄՇԿՁԻԹՄԢՆԼՈՇՁՆՌՋ ԡԹՑ ԫԣՋՌԹԿԽՏԽՌՏԽՁԿՀՌՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ Գԩ ՀՁՆԼՄՁՅԺՄԽՆԿՌՀ ԥԩԩ ՆՇՊՅԹՄՁՒԽԼԥԩԩ ԡԹՑ ԫԣՋՌԹԿԽՏԽՌՏԽՁԿՀՌՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ Գԩ ՀՁՆԼՄՁՅԺՄԽՆԿՌՀ ԥԩԩ ՆՇՊՅԹՄՁՒԽԼԥԩԩՌՀՑՊՇՁԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑ ԡԹՁՄՑ ՅՇՊՌԹՄՁՌՑՋՍԺՄԽՌՀԹՄԽԾԾԽԻՌՋ ԴԽՊԽՊԹՏ ՁՆԼՁՎՁԼՍԹՄ ԼԹՌԹՈՊՇՎՁԼԽԼ!ԶԽՋ
Page ԭԹԿԽՇԾ 27 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՇԺՋԽՊՎԹՌՁՇՆՋՇԾՅՇՊՌԹՄՁՌՑԹՆԼԻՄՁՆՁԻԹՄՋՁԿՆՋՇԻԻՍՊԼԹՁՄՑԹՌԹՅՁՆՁՅՍՅ ՇՌՀԽՊՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽՊԽԻՇՅՅԽՆԼԽԼԹՋԾՇՄՄՇՏՋ ԫԣ ԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽ ԼԹՑՋԹՆԼ ԹՆՑԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌՁՆԼՁԻԹՌԽԼԺՑՌԹԼՈՇՄԽՋՌՀԹՌԻԹՆՆՇՌԺԽԹՋՋՁԿՆԽԼԹՆԫԣՋՌԹԿԽ ԼԹՑՋԹՆԼ ՀՁՆԼՄՁՅԺՄԽՆԿՌՀ ԼԹՑՋԹՆԼ ՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ ԼԹՑՋԹՆԼ ԺՇԼՑՏԽՁԿՀՌ ՌԽՋՌՁՆՁՌՁԹՌՁՇՆԾՇՊՇՈՌՁՇՆԹՄՋՁՒԽԺԹՋԽԼՄԹՊՎԹՄՋԽՄԽԻՌՁՇՆ ԹՆԼՌՀՑՊՇՁԼ ԿՄԹՆԼԿՊՇՋՋՈԹՌՀՇՄՇԿՑԹՆԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑ ԼԹՑ ԫՇՌԽՌՀԽՀՁՋՌՇՈԹՌՀՇՄՇԿՑՋԽԻՌՁՇՆՇԾՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԹՄՋՇՁՆԻՄՍԼԽՋՌՀՑՊՇՁԼԿՊՇՋՋՈԹՌՀՇՄՇԿՑՇԺՋԽՊՎԹՌՁՇՆՋ
Page ԭԹԿԽՇԾ 28 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԦԦ ԯԢԲԩԱԞԫԡԡԦԠԲԦԬԫ Ԟ ԯԽՋՍՄՌՋ ԡԹՁՄՑՄԹՊՎԹՄՋՍՊՎՁՎԹՄՏԹՋՆՇՌՈՊՇՎՁԼԽԼԯԹՌՀԽՊՌՇՌԹՄՋՍՊՎՁՎԹՄՇՎԽՊՌՀԽԼԹՑԽՐՈՇՋՍՊԽՈԽՊՁՇԼՏԹՋՊԽՈՇՊՌԽԼ ԟՑՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆՋՍՊՎՁՎԹՄԹՎԽՊԹԿԽԼՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՆԼ ՄՇՋՋՇԾ ՌԹԼՈՇՄԽՇՎԽՊԼՍՊԹՌՁՇՆՇԾՌԽՋՌՌՁՅԽՈՇՁՆՌՍՆՃՆՇՏՆ ՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿ ԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ ԱԹԺՄԽ ԱԹԺՄԽ ԩԹՊՎԹՄԪՇՊՌԹՄՁՌՑՁՆԵԽՆՇՈՍՋՄԹԽՎՁՋ
ԩԹՊՎԹՄԪՇՊՌԹՄՁՌՑ ԱՊԽԹՌՅԽՆՌ ՅԿԹՁԩ ԡԹՑ ԡԹՑ #ՅԽԹՆՅԽԹՋՍՊԽԼ%
Ն ԪՇՊՌԹՄՁՌՑ ԪՇՊՌԹՄՁՌՑ Ն ԪՇՊՌԹՄՁՌՑ ԪՇՊՌԹՄՁՌՑ
ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ ԫԯ ԫԯ ԫԯ ԫԯ
ԫԯ ԫԯ ԫԯ ԫԯ
ԫԯ ԫԯ ԫԯ ԫԯ
ԫԯ ԫԯ ԫԯ ԫԯ
ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ ԹՅՈՄԽՋՁՒԽԹՆԼԻՍՅՍՄԹՌՁՎԽՅՇՊՌԹՄՁՌՑՎԹՄՍԽՋԹՌԡԹՑՈՊՁՇՊՌՇՁՆՌԽՊՁՅՋԹԻՊՁԾՁԻԽ ԪԽԼՁԹՆԫԣԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽՏԹՋՇՆԡԹՑՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋ ԱԹԺՄԽ ԟՑՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆՅԽԼՁԹՆԫԣԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽՏԹՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՅԽԹՆՅԽԹՋՍՊԽԼ ՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈԹՆԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈՋ ԱԹԺՄԽ ԱՀԽՌՀԹՆԼՌՀՈԽՊԻԽՆՌՁՄԽՋՇՆԡԹՑԼՁԼՆՇՌԼՁԾԾԽՊԺՑՅՇՊԽՌՀԹՆՋՌԹԿԽՋԫՇ ԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌՏԹՋՊԽՈՇՊՌԽԼ ԱԹԺՄԽ ԩԹՊՎԹՄԡԽՎԽՄՇՈՅԽՆՌՁՆԵԽՆՇՈՍՋՄԹԽՎՁՋ-ԡԽՎԽՄՇՈՅԽՆՌԹՄՌԹԿԽԹՆԼԞՋՑՆԻՀՊՇՆՇՍՋԡԽՎԽՄՇՈՅԽՆՌ
ԱՊԽԹՌՅԽՆՌ ՅԿԹՁԩ ԡԽՎԽՄՇՈՅԽՆՌԹՄՌԹԿԽ
ԭԹԿԽՇԾ
Page 29 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
#ՅԽԹՆՅԽԹՋՍՊԽԼ% ԡԹՑ ԡԹՑ
ԪԽԼՁԹՆ ԪԽԼՁԹՆ Ն ԞՋՑՆԻՀՊՇՆՇՍՋ Ն ԞՋՑՆԻՀՊՇՆՇՍՋ ՌԹԿԽ ՌԹԿԽ
ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ ԪԽԹՆՆՇՊՅԹՄՁՒԽԼԥԩԩՊԹՆԿԽԼԾՊՇՅՌՇՇՆԡԹՑԹՆԼԾՊՇՅՌՇՇՆԡԹՑ ԱԹԺՄԽ ԱԹԺՄԽ ԩԹՊՎԹՄԡԽՎԽՄՇՈՅԽՆՌՁՆԵԽՆՇՈՍՋՄԹԽՎՁՋ-ԥՁՆԼԩՁՅԺԩԽՆԿՌՀ
ԥՁՆԼԩՁՅԺԩԽՆԿՌՀ ԥԩԩ ԱՊԽԹՌՅԽՆՌ ԡԹՑ ԡԹՑ ՅԿԹՁԩ ԪԽԹՆ ԥԩԩ ԪԽԹՆ ԥԩԩ Ն չԡ Ն չԡ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ՅՅ Գԩ ՅՅ Գԩ
ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԡՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ ՍՅՅԹՊՑՊԽՋՍՄՌՋԾՇՊՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ Գԩ ԹՊԽՈՊԽՋԽՆՌԽԼՁՆՌՀԽՆԽՐՌՌԹԺՄԽ ԱԹԺՄԽ ԴՀՇՄԽԺՇԼՑՏԽՌՏԽՁԿՀՌՇՆԡԹՑԹՎԽՊԹԿԽԼԿՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼՊԹՆԿԽԼԾՊՇՅՌՇ ԿՁՆՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋ ԱԹԺՄԽ ԬՆԡԹՑԺՇԼՑՏԽՁԿՀՌԹՎԽՊԹԿԽԼԿՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ
ԭԹԿԽՇԾ
Page 30 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԹՆԼՊԹՆԿԽԼԾՊՇՅՌՇԿՁՆՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋ ԱԹԺՄԽ ԩԹՊՎԹՄԤՊՇՏՌՀՁՆԵԽՆՇՈՍՋՄԹԽՎՁՋ
ԱՊԽԹՌՅԽՆՌ ՆՇՍՌԳԽՆՌԩԽՆԿՌՀ Գԩ ԟՇԼՑԴԽՁԿՀՌ
ՅԿԹՁԩ ԡԹՑ ԡԹՑ ԡԹՑ ԡԹՑ #ՅԽԹՆ ԪԽԹՆ ԪԽԹՆ ԪԽԹՆ ԪԽԹՆ Ն չԡ Ն չԡ Ն չԡ Ն չԡ ՅԽԹՋՍՊԽԼ% ՅՅ ՅՅ Կ Կ
ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼԡՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ ԞՄՋՇՊԽԾԽՊՊԽԼՌՇԹՋ՟ՏԽՌՏԽՁԿՀՌՠՁՆՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽ ԱՀՑՊՇՁԼԿՄԹՆԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑՊԽՎԽԹՄԽԼՌՀՑՊՇՁԼԿՄԹՆԼՀՑՈԽՊՌՊՇՈՀՑԾՇՄՄՁԻՍՄԹՊԻԽՄՄՀՑՈԽՊՈՄԹՋՁԹԹՆԼԾՇՄՄՁԻՍՄԹՊ ԻԽՄՄՀՑՈԽՊՌՊՇՈՀՑԥՇՏԽՎԽՊՌՀԽՋԽԾՁՆԼՁՆԿՋՏԽՊԽԻՇՆՋՁԼԽՊԽԼՍՆՊԽՄԹՌԽԼՌՇՌՊԽԹՌՅԽՆՌԺԽԻԹՍՋԽՌՀԽՑՏԽՊԽ ՇՆՄՑՇԾՅՁՄԼՋԽՎԽՊՁՌՑԹՆԼՇԻԻՍՊՊԽԼՁՆԹՄՄՌՊԽԹՌԽԼԿՊՇՍՈՋԹՆԼԻՇՆՌՊՇՄՋՁՆԹՅԹՆՆԽՊՍՆՊԽՄԹՌԽԼՌՇԻՇՆԻԽՆՌՊԹՌՁՇՆ ԱԹԺՄԽՋԹՆԼ
ԭԹԿԽՇԾ
Page 31 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԱԹԺՄԽ ԤՊՇՋՋԭԹՌՀՇՄՇԿՑԹՆԼԥՁՋՌՇՈԹՌՀՇՄՇԿՑՇԾՌՀԽԱՀՑՊՇՁԼԤՄԹՆԼՁՆԵԽՆՇՈՍՋՄԹԽՎՁՋ
ԱՊԽԹՌՅԽՆՌ ԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ
ՅԿԹՁԩ ԱՀՑՊՇՁԼԤՄԹՆԼ ԱՀՑՊՇՁԼԤՄԹՆԼ ԣՇՄՄՁԻՍՄԹՊԠԽՄՄ ԣՇՄՄՁԻՍՄԹՊԠԽՄՄ
#ՅԽԹՆ ԽՎԽՊՁՌՑ ԥՑՈԽՊՌՊՇՈՀՑ ԞՌՊՇՈՀՑ ԥՑՈԽՊՌՊՇՈՀՑ ԥՑՈԽՊՈՄԹՋՁԹ ՅԽԹՋՍՊԽԼ% Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ
ԫԽԿԹՌՁՎԽ ԠՇՆՌՊՇՄ
ԱՀՑՊՇՁԼԿՄԹՆԼԿՊՇՋՋՈԹՌՀՇՄՇԿՑԹՆԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑԹՊԽԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑ ԫՇՌՊԽՅԹՊՃԹԺՄԽ ԪՁՄԼ ԪՇԼԽՊԹՌԽ ԽՎԽՊԽԽԽԬԢԠԡԫՇԾՇՊՊԽԾԽՊԽՆԻԽ ԪՁՋՋՁՆԿՌՁՋՋՍԽ
ԭԹԿԽՇԾ
Page 32 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԱԹԺՄԽ ԞԼԼՁՌՁՇՆԹՄԱՀՑՊՇՁԼԤՄԹՆԼԥՁՋՌՇՈԹՌՀՇՄՇԿՑԬԺՋԽՊՎԹՌՁՇՆՋՁՆԵԽՆՇՈՍՋՄԹԽՎՁՋ
ԞԼԼՁՌՁՇՆԹՄԮՍԹՄՁՌԹՌՁՎԽԬԺՋԽՊՎԹՌՁՇՆՋ ԱՊԽԹՌՅԽՆՌ ԣՇՄՄՁԻՍՄԹՊԩՍՅԽՆ ԣՇՄՄՁԻՍՄԹՊԩՍՅԽՆ ԣՇՄՄՁԻՍՄԹՊԠԽՄՄ ԣՇՄՄՁԻՍՄԹՊԠԽՄՄԥԽՁԿՀՌ ԣՇՄՄՁԻՍՄԹՊԠԽՄՄ ՅԿԹՁԩ ԽՎԽՊՁՌՑ ԞՊԽԹ ԦՆԻՊԽԹՋԽ ԞՊԽԹ ԡԽԻՊԽԹՋԽ ԥԽՁԿՀՌ ԦՆԻՊԽԹՋԽ ԡԽԻՊԽԹՋԽ ՀԹՈԽ #ՅԽԹՆՅԽԹՋՍՊԽԼ% Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ
ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ
Page ԭԹԿԽՇԾ 33 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԞԼԼՁՌՁՇՆԹՄԮՍԹՄՁՌԹՌՁՎԽԬԺՋԽՊՎԹՌՁՇՆՋ ԱՊԽԹՌՅԽՆՌ ԣՇՄՄՁԻՍՄԹՊԩՍՅԽՆ ԣՇՄՄՁԻՍՄԹՊԩՍՅԽՆ ԣՇՄՄՁԻՍՄԹՊԠԽՄՄ ԣՇՄՄՁԻՍՄԹՊԠԽՄՄԥԽՁԿՀՌ ԣՇՄՄՁԻՍՄԹՊԠԽՄՄ ՅԿԹՁԩ ԽՎԽՊՁՌՑ ԞՊԽԹ ԦՆԻՊԽԹՋԽ ԞՊԽԹ ԡԽԻՊԽԹՋԽ ԥԽՁԿՀՌ ԦՆԻՊԽԹՋԽ ԡԽԻՊԽԹՋԽ ՀԹՈԽ #ՅԽԹՆՅԽԹՋՍՊԽԼ% Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ
ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ
ԱՀՑՊՇՁԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑՁՋԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑ ԫՇՌՊԽՅԹՊՃԹԺՄԽ ԪՁՄԼ ԪՇԼԽՊԹՌԽ ԽՎԽՊԽԽԽԬԢԠԡԫՇԾՇՊՊԽԾԽՊԽՆԻԽ ԱՀԽՊԽՏԹՋՅՁՋՋՁՆԿՌՁՋՋՍԽԾՊՇՅՇՆԽՌԹԼՈՇՄԽ ԟԽԿՁՆՆՁՆԿՇՆԡԹՑԹՆԼԻՇՆՌՁՆՍՁՆԿՌՀՊՇՍԿՀՇՍՌՌՀԽԼՍՊԹՌՁՇՆՇԾՌՀԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼՋՈՁՆԹՄԻՍՊՎԹՌՍՊԽՏԹՋՇԺՋԽՊՎԽԼՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋ ԟՑՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆՁՆԻՁԼԽՆԻԽՇԾՋՈՁՆԹՄԻՍՊՎԹՌՍՊԽԹՎԽՊԹԿԽԼՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՆԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ ԱԹԺՄԽ
Page ԭԹԿԽՇԾ 34 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԱԹԺՄԽ ԠՄՁՆՁԻԹՄՁԿՆՋՁՆԵԽՆՇՈՍՋՄԹԽՎՁՋ ԱՊԽԹՌՅԽՆՌ ԠՄՁՆՁԻԹՄՁԿՆՋ ՅԿԹՁԩ
#ՅԽԹՆ ԱՑՈԽ Ն ԦՆԻՁԼԽՆԻԽ ՅԽԹՋՍՊԽԼ%
ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ ՈՁՆԹՄԻՍՊՎԹՌՍՊԽ
ՈՁՆԹՄԻՍՊՎԹՌՍՊԽ
ՈՁՆԹՄԻՍՊՎԹՌՍՊԽ
ՈՁՆԹՄԻՍՊՎԹՌՍՊԽ
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ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԟ ՌՍԼՑԞՍՌՀՇՊ0ՋԞՆԹՄՑՋՁՋԹՆԼԠՇՆԻՄՍՋՁՇՆՋ ԞՆԹՄՑՒԽԼԽՆԼՈՇՁՆՌՋՁՆԻՄՍԼԽԼԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԳԩԥԩԩԹՆԼՏԽՌՏԽՁԿՀՌԞՄՄՋՌԹՌՁՋՌՁԻԹՄԻՇՆԻՄՍՋՁՇՆՋ ՏԽՊԽՅԹԼԽԹՌՌՀԽՄԽՎԽՄՇԾԻԽՊՌԹՁՆՌՑԽՐԻԽՈՌՁՆՌՀԽԻԹՋԽՇԾՌՀԽԺԹՋՁԻԹՋՋՍՅՈՌՁՇՆՌԽՋՌՋ ՀԹՈՁՊՇԴՁՄՃՋ0 ԱԽՋՌԹՆԼԟԹՊՌՄԽՌՌ0ՋԱԽՋՌ ՁՆՏՀՁԻՀՌՀԽՄԽՎԽՄՇԾԻԽՊՌԹՁՆՌՑՏԹՋԹՈՈՄՁԽԼ ԪՇՊՌԹՄՁՌՑՏԹՋԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽԣՁՋՀԽՊ0ՋԢՐԹԻՌԱԽՋՌՏՁՌՀԟՇՆԾԽՊՊՇՆՁԥՇՄՅԹԼՂՍՋՌՅԽՆՌԡԽՎԽՄՇՈՅԽՆՌԹՄ ՋՌԹԿԽՏԹՋԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՋՌԽՈԼՇՏՆՌԽՋՌԥԩԩԳԩԹՆԼՏԽՌՏԽՁԿՀՌՏԽՊԽ ԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌԽՋՌՏՀԽՆԹՅՇՆՇՌՇՆՁԻՌՊԽՆԼՏԹՋՈՊԽՋԽՆՌԴՀԽՆԹՅՇՆՇՌՇՆՁԻ ՌՊԽՆԼՏԹՋՆՇՌԹՈՈԹՊԽՆՌՌՀԽՋԽԽՆԼՈՇՁՆՌՋՏԽՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽԡՍՆՆԽՌՌ0ՋԪՍՄՌՁՈՄԽԠՇՅՈԹՊՁՋՇՆՌԽՋՌ ՏՀԽՆՌՀԽԹՋՋՍՅՈՌՁՇՆՋՇԾՈԹՊԹՅԽՌՊՁԻՋՌԹՌՁՋՌՁԻՋՏԽՊԽՅԽՌՇՊՌՀԽԪԹՆՆԴՀՁՌՆԽՑՌԽՋՌՏՁՌՀԟՇՆԾԽՊՊՇՆՁԥՇՄՅ ԹԼՂՍՋՌՅԽՆՌ ՏՀԽՆ ՌՀԽ ԼԹՌԹ ԼՁԼ ՆՇՌ ՅԽԽՌ ՌՀԽ ԹՋՋՍՅՈՌՁՇՆՋ ՇԾ ՆՇՊՅԹՄՁՌՑ ԹՆԼ ՀՇՅՇՋՃԽԼԹՋՌՁԻՁՌՑ ԠԢԱԦԠՇՅՈՊԽՀԽՆՋՁՎԽԢՆՎՁՊՇՆՅԽՆՌԹՄԱՇՐՁԻՁՌՑԦՆԾՇՊՅԹՌՁՇՆՑՋՌԽՅԱԪԳԽՊՋՁՇՆՋՇԾՌՏԹՊԽՏԹՋՍՋԽԼ ՌՇՈԽՊԾՇՊՅԹՄՄՋՌԹՌՁՋՌՁԻԹՄԻՇՅՈՍՌԹՌՁՇՆՋ ԟՑՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆՆՇԼՁԾԾԽՊԽՆԻԽՋՏԽՊԽԾՇՍՆԼՁՆՄԹՊՎԹՄՋՍՊՎՁՎԹՄԺԽՌՏԽԽՆՌՀԽԻՇՆՌՊՇՄԹՆԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋ ԬՆԡԹՑՆՇՋՁԿՆՁԾՁԻԹՆՌՁՆՀՁԺՁՌՁՇՆՋՇՊՈՊՇՅՇՌՁՇՆՋՏԽՊԽՇԺՋԽՊՎԽԼԾՇՊԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽՇՊՏԽՌՏԽՁԿՀՌ ԹՌԹՆՑՌՊԽԹՌՅԽՆՌԿՊՇՍՈԳԩՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼԹՌՌՀԽՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՏՀԽՊԽԹՋԺՇՌՀԥԩԩ ԹՆԼՆՇՊՅԹՄՁՒԽԼԥԩԩՏԽՊԽՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼԹՌՌՀԽՅՁԼԼՄԽԹՆԼՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԬՆԡԹՑ ԼԽՎԽՄՇՈՅԽՆՌԹՄՋՌԹԿԽԥԩԩԹՆԼՆՇՊՅԹՄՁՒԽԼԥԩԩՏԽՊԽԹՄՄՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼՁՆՌՀԽՅՁԼԼՄԽԹՆԼՀՁԿՀԽՋՌ ՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԥՇՏԽՎԽՊՇՆԡԹՑՏԽՌՏԽՁԿՀՌԹՆԼԳԩՏԽՊԽ ՍՆԹԾԾԽԻՌԽԼԹՌԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԱՀԽՊԽՏԽՊԽՆՇԹՈՈԹՊԽՆՌՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼՌՊԽՆԼՋՁՆՌՀՑՊՇՁԼԿՄԹՆԼ ՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄՇԺՋԽՊՎԹՌՁՇՆՋ ԦՆՊԽԿԹՊԼՋՌՇՋՈՁՆԹՄԻՍՊՎԹՌՍՊԽՌՀԽՋՌՍԼՑԹՍՌՀՇՊՊԽՈՇՊՌԽԼՌՀԹՌՌՀԽՁՆԻՁԼԽՆԻԽՇԾՋՈՁՆԹՄԼԽԾՇՊՅՁՌՁԽՋՁՆՌՀԽ ԻՍՊՊԽՆՌԹՋՋԹՑՏԽՊԽՍՆՊԽՄԹՌԽԼՌՇՌՊԽԹՌՅԽՆՌՏՁՌՀՈՀՇՋՅԽՌԣՍՊՌՀԽՊՁՌՏԹՋՊԽՈՇՊՌԽԼՌՀԹՌԺԹՋԽԼՇՆՀՁՋՌՇՊՁԻԹՄ ԻՇՆՌՊՇՄԼԹՌԹԻՇՄՄԽԻՌԽԼԹՌՅՁՌՀԽՊՋԳՁՋԻՁԽՆՌՌՀԽՋԽԼԽԾՇՊՅՁՌՁԽՋԼՁԼՆՇՌՀԹՎԽԹՆԽԿԹՌՁՎԽՁՅՈԹԻՌՇՆՌՀԽ ՁՆՌԽՊՈՊԽՌԹՌՁՇՆՇԾՌՀՁՋՋՌՍԼՑԱՀԽՋՌՍԼՑԹՍՌՀՇՊՊԽՈՇՊՌԽԼՌՀԹՌԾՇՊՌՀԽՈՊԽՎՁՇՍՋՄՑԻՇՆԼՍԻՌԽԼՋՌՍԼՁԽՋՌՀԽՊԽՏԹՋ ՆՇԻՇՊՊԽՄԹՌՁՇՆԺԽՌՏԽԽՆՌՀԽՈՊԽՋԽՆԻԽՇԾՋՈՁՆԹՄԼԽԾՇՊՅՁՌՁԽՋԹՆԼՌՀԽՁՆԻՊԽԹՋԽՁՆԠԳԾՇՊՈՇՌԽՆՌՁԹՄՄՑԹԾԾԽԻՌԽԼ ԽՆԼՈՇՁՆՌՋ ԽԿԳԩԥԩԩՏԽՌԺՇԼՑՏԽՁԿՀՌ
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Page 36 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԠԯԽՎՁԽՏԽՊ0ՋԞՆԹՄՑՋՁՋԹՆԼԠՇՆԻՄՍՋՁՇՆՋ ՌԹՌՁՋՌՁԻԹՄԪԽՌՀՇԼՋ ՍՊՎՁՎԹՄՊԹՆԿԽԼԾՊՇՅՌՇԱՀԽՊԽԾՇՊԽՌՀԽՊԽՎՁԽՏԽՊՎՁՋՍԹՄՄՑԼԽՌԽՊՅՁՆԽԼՌՀԹՌ ՌՀԽՌԽՋՌՅԹՌԽՊՁԹՄՀԹԼՆՇԽԾԾԽԻՌՇՆՄԹՊՎԹՄՅՇՊՌԹՄՁՌՑԡԹՑԹՆԼԡԹՑԿՊՇՏՌՀՈԹՊԹՅԽՌԽՊՋ ԳԩԹՆԼԺՇԼՑ ՏԽՁԿՀՌ ԼՁԼՆՇՌԽՐՀՁԺՁՌՅՇՆՇՌՇՆՁԻՌՊԽՆԼՋԺՍՌՅԽՌՌՀԽԹՋՋՍՅՈՌՁՇՆՋՇԾՈԹՊԹՅԽՌՊՁԻՋՌԹՌՁՋՌՁԻՋԹՆԼՏԽՊԽ ՌՀԽՊԽԾՇՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽՌՏՇՋՁԼԽԼԡՍՆՆԽՌՌ0ՋՌԽՋՌԡԹՑԹՆԼԡԹՑԼԽՎԽՄՇՈՅԽՆՌԹՄՈԹՊԹՅԽՌԽՊՋ ԫԣՋՌԹԿԽԹՆԼՆՇՊՅԹՄՁՒԽԼԥԩԩ ԼՁԼՆՇՌԽՐՀՁԺՁՌՋՍԺՋՌԹՆՌՁՎԽԼԽՈԹՊՌՍՊԽՋԾՊՇՅՅՇՆՇՌՇՆՁԻՁՌՑ ՌՀԽՊԽԾՇՊԽ ԻՇՆՋՁՋՌԽՆՌՏՁՌՀՌՀԽԬԠԭԭԿՍՁԼԽՄՁՆԽՌՀԽՋԽԽՆԼՈՇՁՆՌՋՏԽՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽՇՆԽՋՁԼԽԼ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՋՌԽՈԼՇՏՆՌՊԽՆԼՌԽՋՌԞՄՄԹՆԹՄՑՋԽՋՏԽՊԽԻՇՆԼՍԻՌԽԼԺՑԻՇՅՈԹՊՁՆԿՌՊԽԹՌՅԽՆՌԼԹՌԹՌՇ ՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՍՋՁՆԿԹՆĮՇԾՍՆՄԽՋՋՋՈԽԻՁԾՁԽԼՇՌՀԽՊՏՁՋԽԯԽՋՍՄՌՋԾՊՇՅՇՆԽՋՁԼԽԼՌՊԽՆԼՌԽՋՌՋ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ ՏԽՊԽԻՇՅՈԹՊԽԼՌՇԹՋԽԻՇՆԼԹՊՑĮ ՌՇՁՄՄՍՋՌՊԹՌԽՏՀԽՌՀԽՊՌՀԽԽԾԾԽԻՌՏՇՍՄԼՋՌՁՄՄ ԺԽԻՇՆՋՁԼԽՊԽԼՋՁԿՆՁԾՁԻԹՆՌՁԾԹՌՏՇՋՁԼԽԼ ՎԽՊՋՍՋՇՆԽՋՁԼԽԼ ՌՊԽՆԼՌԽՋՌՏԽՊԽՍՋԽԼ ԫՇԹՋՑՆԻՀՊՇՆՇՍՋԼԽՎԽՄՇՈՅԽՆՌՏԹՋՊԽՈՇՊՌԽԼ ԠՇՆԻՄՍՋՁՇՆՋ ԱՀԽՊԽՎՁԽՏԽՊԾՇՍՆԼՆՇՋՁԿՆՁԾՁԻԹՆՌԼՁԾԾԽՊԽՆԻԽՋՁՆԡԹՑՏԽՌՏԽՁԿՀՌՇՊՅԽԼՁԹՆԫԣՋՌԹԿԽ ԡՍՆՆԽՌՌ0ՋՌԽՋՌ Ո ԡԹՑԳԩՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼԹՌՌՀԽՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈ ՊԽԼՍԻՌՁՇՆ ԡՍՆՆԽՌՌ0Ջ Ո ԣՍՊՌՀԽՊԡԹՑԥԩԩՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼ ՁՆՀՁԺՁՌՁՇՆՋ ԹՌԹՄՄՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋ ԧՇՆԻՃՀԽԽՊԽՈ= ՏՀԽՊԽԹՋՆՇՊՅԹՄՁՒԽԼԥԩԩՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼ ԹՌՌՀԽՅՁԼԼՄԽԹՆԼ ՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋ ԧՇՆԻՃՀԽԽՊԽՈ ԡԹՑԼԽՎԽՄՇՈՅԽՆՌԹՄՈԹՊԹՅԽՌԽՊՋ ՁԽԫԣՋՌԹԿԽՆՇՊՅԹՄՁՒԽԼԥԩԩ ՏԽՊԽԺՇՌՀՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼԹՌ ՌՀԽՅՁԼԼՄԽԹՆԼՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ ԧՇՆԻՃՀԽԽՊԽՈ= ԬԾՌՀԽ ՌՀՊԽԽԿՊՇՏՌՀՈԹՊԹՅԽՌԽՊՋՇՆԡԹՑ ՁԽԳԩԥԩԩԹՆԼՏԽՌՏԽՁԿՀՌ ՇՆՄՑԥԩԩՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼ ԹՌՌՀԽՅՁԼԼՄԽԹՆԼՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈ ՊԽԼՍԻՌՁՇՆ ԧՇՆԻՃՀԽԽՊԽՈ ԡԹՑԳԩԹՆԼ ՏԽՌՏԽՁԿՀՌՏԽՊԽՍՆԹԾԾԽԻՌԽԼ ԡՍՆՆԽՌՌ0ՋՈ ԞՄՄԽԾԾԽԻՌՋՁԼԽՆՌՁԾՁԽԼԹՋՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌՍՋՁՆԿՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌԽՋՌՊԽՅԹՁՆՋՁԿՆՁԾՁԻԹՆՌՏՀԽՆ ԻՇՅՈԹՊԽԼՌՇՌՀԽԹԼՂՍՋՌԽԼĮ ՏՀՁԻՀԹԻԻՇՍՆՌՋԾՇՊՌՀԽԻՇՆՋԽՊՎԹՌՁՋՅՇԾՌՀԽՇՆԽՋՁԼԽԼՌՊԽՆԼՌԽՋՌ
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Page 37 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԱԹԺՄԽ ԡԽՎԽՄՇՈՅԽՆՌԹՄ ԹՆԼ ԱՀՑՊՇՁԼ ԤՊՇՋՋ ԭԹՌՀՇՄՇԿՑԥՁՋՌՇՈԹՌՀՇՄՇԿՑ ԢՆԼՈՇՁՆՌՋ ՁՆ ՌՀԽ ԞԪԞ ՏՁՌՀ ԭՀՇՋՅԽՌ
ԱՀՑՊՇՁԼԤՊՇՋՋ ԫԣԡԽՎԽՄՇՈՅԽՆՌԹՄՌԹԿԽ ԥՁՆԼԩՁՅԺԩԽՆԿՌՀ ԹՆԼԥՁՋՌՇ ԱՊԽԹՌՅԽՆՌ ՈԹՌՀՇՄՇԿՑ
ՅԿԹՁԩ ԡԹՑ ԡԹՑ ԡԹՑ ԡԹՑ ԡԹՑ
#ՅԽԹՆ ԱՊԽԹՌՅԽՆՌ ՅԽԹՋՍՊԽԼ% ԯԽՄԹՌԽԼ ԪԽԼՁԹՆ Ո ԪԽԼՁԹՆ Ո ԡՁԾԾ Ո ԡՁԾԾ Ո ԢԾԾԽԻՌՋ! ԶԽՋԫՇ
ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ ԫԞ ԫԞ ԫԞ ԫԞ ԫՇ
ԫՇ
ԫՇ
ԫՇ ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԧՇՆԻՃՀԽԽՊԽ ԧՇՆԻՃՀԽԽՊԽ ԧՇՆԻՃՀԽԽՊԽ ԧՇՆԻՃՀԽԽՊԽ ԫԞ ԱԽՊՈՋՌՊԹ ԱԽՊՈՋՌՊԹ ԱԽՊՈՋՌՊԹ ԱԽՊՈՋՌՊԹ
ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԡՁԾԾԡՁԾԾԽՊԽՆԻԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼԽԾԾԽԻՌՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄԠՇՆԻՄՍՋՁՇՆՋ ՊԽԿԹՊԼՁՆԿՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈ ԥՁՆԼՄՁՅԺՄԽՆԿՌՀՁՋՆՇՊՅԹՄՁՒԽԼՌՇՋՆՇՍՌՎԽՆՌՄԽՆԿՌՀ Գԩ
ԭԹԿԽՇԾ
Page 38 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԱԹԺՄԽ ԤՊՇՏՌՀԢՆԼՈՇՁՆՌՋՁՆՌՀԽԞԪԞՏՁՌՀԭՀՇՋՅԽՌ
ԱՊԽԹՌՅԽՆՌ ՆՇՍՌԳԽՆՌԩԽՆԿՌՀ ԟՇԼՑԴԽՁԿՀՌ
ՅԿԹՁԩ ԡԹՑ ԡԹՑ ԡԹՑ ԡԹՑ
#ՅԽԹՆՅԽԹՋՍՊԽԼ% ԡՁԾԾ Ո ԡՁԾԾ Ո ԡՁԾԾ Ո ԡՁԾԾ Ո
ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ
ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԡՍՆՆԽՌՌ0Ջ ԡՍՆՆԽՌՌ0Ջ ԡՍՆՆԽՌՌ0Ջ ԡՍՆՆԽՌՌ0Ջ
ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԡՁԾԾԡՁԾԾԽՊԽՆԻԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼԽԾԾԽԻՌՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ ԻՄԽԹՆՏԹՌԽՊ ԻՇՆՌՊՇՄ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈ ԢՌՍԼՑԡԽԾՁԻՁԽՆԻՁԽՋ ԱՀԽՌԽՅՈԽՊԹՌՍՊԽՎԹՊՁԹՆԻԽ ոԠ ՏԹՋՋՄՁԿՀՌՄՑՇՍՌՋՁԼԽՌՀԽՊԹՆԿԽՊԽԻՇՅՅԽՆԼԽԼՁՆՌՀԽՈԽՊԾՇՊՅԹՆԻԽ ԻՊՁՌԽՊՁԹ չոԠ ԞՄՌՀՇՍԿՀՌՀԽՊԽՏԽՊԽՇՌՀԽՊԼԽՎՁԹՌՁՇՆՋԾՊՇՅՌՀԽԿՍՁԼԽՄՁՆԽՁՆԻՄՍԼՁՆԿՏԹՌԽՊՀԹՊԼՆԽՋՋՌՀԹՌ ՏԹՋՀՁԿՀԽՊՌՀԹՆՊԽԻՇՅՅԽՆԼԽԼՌՀԽՋՌՍԼՑՅԽՌԹՄՄՊԽՅԹՁՆՁՆԿՎԹՄՁԼՁՌՑԹՆԼՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹ ԣԯԽՎՁԽՏԽՊ0ՋԠՇՅՅԽՆՌՋ ԱՀԽՊԽՎՁԽՏԽՊ0ՋՊԽՋՍՄՌՋՏԽՊԽԻՇՅՈԹՊԹԺՄԽՌՇՌՀՇՋԽՇԾՌՀԽՋՌՍԼՑԹՍՌՀՇՊՏՁՌՀՌՀԽԽՐԻԽՈՌՁՇՆՌՀԹՌՌՀԽՊԽՎՁԽՏԽՊ ԾՇՍՆԼՋՁԿՆՁԾՁԻԹՆՌՊԽԼՍԻՌՁՇՆՋՁՆԡԹՑՆՇՊՅԹՄՁՒԽԼԥԩԩԹՌԹՄՄՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՏՀԽՊԽԹՋՌՀԽՋՌՍԼՑԹՍՌՀՇՊ ՇՆՄՑԾՇՍՆԼՊԽԼՍԻՌՁՇՆՋՁՆՌՀԽՅՁԼԼՄԽԹՆԼՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈԱՀԽՋՌՍԼՑԹՍՌՀՇՊԹՄՋՇՅՁՋՁԼԽՆՌՁԾՁԽԼ ՇՆԽՌԹԼՈՇՄԽՁՆՌՀԽՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԱԹԼՈՇՄԽԦԡԡԫԣՋՌԹԿԽ ԹՋԺԽՁՆԿՄԹՌԽՋՌԹԿԽ ԫԣՋՌԹԿԽ ՀՇՏԽՎԽՊԺԹՋԽԼՇՆՌՀԽԼԽՋԻՊՁՈՌՁՎԽՋՌԹՌՁՋՌՁԻՋՁՆՌՀԽՊԽՈՇՊՌՁՌԼՁԼՆՇՌԹՈՈԽԹՊՌՇԺԽԽՐԻՄՍԼԽԼԾՊՇՅ ԹՆԹՄՑՋՁՋ
ԭԹԿԽՇԾ
Page 39 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԦԦԦ ԯԢԣԢԯԢԫԠԢ
ԦՎԽՋԪՁԻՀԹԽՄԠԢԱԦԠՇՅՈՊԽՀԽՆՋՁՎԽԢՆՎՁՊՇՆՅԽՆՌԹՄԱՇՐՁԻՁՌՑԦՆԾՇՊՅԹՌՁՇՆՑՋՌԽՅԱԪԲՋԽՊ0ՋԤՍՁԼԽ ԱՁԼԽՈՇՇՄԻՁԽՆՌՁԾՁԻՇԾՌՏԹՊԽԪԻԨՁՆՄԽՑՎՁՄՄԽԠԹՄՁԾՇՊՆՁԹ ԩՇՈԽՒԞԥՑԼՊՇՄՑՋՁՋՇԾ#Ԡ%ԭՀՇՋՅԽՌԹՌԳԹՊՁՇՍՋՈԥԩԽՎԽՄՋԹՆԼԱԽՅՈԽՊԹՌՍՊԽՋԭԱԯԩԯԽՈՇՊՌԫՍՅԺԽՊ ԴՈՇՆՋՇՊԽԼԹՆԼՋՍԺՅՁՌՌԽԼԺՑԤՇՏԹՆԠՇՅՈԹՆՑՈՈԪԯԦԡ ԪՇՍՆՌԡԦԹՆԼԴԞԟՊՍՆԿՋԞՋՁՅՈՄՁԾՁԽԼԼՇՋՁՆԿԹՈՈԹՊԹՌՍՋԾՇՊԾՁՋՀՌՇՐՁԻՇՄՇԿՁԻԹՄՋՌՍԼՁԽՋԴԹՌԽՊ ԯԽՋԽԹՊԻՀ ԫՁԽՍՏՃՇՇՈԭԡԹՆԼԣԹԺԽՊԧԫՇՊՅԹՄԱԹԺՄԽՇԾԵԽՆՇՈՍՋՄԹԽՎՁՋԤԹՊՄԹՆԼԭՍԺՄՁՋՀՁՆԿԫԽՏԶՇՊՃ ՈՊԹԿՍԽԧԟԪԽԹՋՍՊԽՅԽՆՌՇԾՈՇՄՄՍՌԹՆՌՌՇՐՁԻՁՌՑՌՇԾՁՋՀԟՁՇԹՋՋԹՑՅԽՌՀՇԼՋԾՇՊԹԻՍՌԽՌՇՐՁԻՁՌՑԴԹՌԽՊ ԯԽՋԽԹՊԻՀ
ԭԹԿԽՇԾ
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ԢԭԞԪԯԦԡԫՍՅԺԽՊ
ԞԭԭԢԫԡԦԵԦԬԲԱԭԲԱԬԣԯԢԳԦԢԴԢԯ0ԱԞԱԦԱԦԠԞԩԳԢԯԦԣԦԠԞԱԦԬԫ test for amphib metamorph screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR01 ( 7-d wet weight (g) )
TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals N/A alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) N/A alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.915 0.142 1.571 0.248 USE PARAMETRIC TESTS
******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.51 0.04 0.02 7.47 0.45, 0.57 Dose1 4 0.45 0.02 0.01 3.54 0.42, 0.47 Dose2 4 0.48 0.05 0.03 10.86 0.40, 0.56 Dose3 4 0.44 0.07 0.04 16.03 0.33, 0.56
Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.52 0.46 0.54 . . Dose1 0.44 0.43 0.47 87.96 12.04 Dose2 0.46 0.44 0.56 94.50 5.50 Dose3 0.42 0.39 0.55 87.13 12.87
******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 1.60 0.241
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 0.51 . 0.51 ...... Dose1 0.45 0.228 0.46 0.130 . . . . . Dose2 0.48 0.758 0.46 0.138 0.771 . . . . Dose3 0.44 0.188 0.44 0.051 0.999 0.702 . . .
test for amphib metamorph screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR02 ( 7-d stage (median) )
TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals N/A alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) N/A alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.768 0.001 1.000 0.426 USE NON-PARAMETRIC TESTS
********************************************************************************
ԭԹԿԽՇԾ
Page 41 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 53.50 0.58 0.29 1.08 52.58, 54.42 Dose1 4 53.50 0.58 0.29 1.08 52.58, 54.42 Dose2 4 53.75 0.50 0.25 0.93 52.95, 54.55 Dose3 4 53.50 0.58 0.29 1.08 52.58, 54.42
Level Median Min Max %of Control(means) %Reduction(means) Ctrl 53.50 53.00 54.00 . . Dose1 53.50 53.00 54.00 100.00 0.00 Dose2 54.00 53.00 54.00 100.47 -0.47 Dose3 53.50 53.00 54.00 100.00 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 0.71 0.870
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 53.50 . . Dose1 53.50 1.000 0.500 Dose2 54.00 0.624 0.754 Dose3 53.50 1.000 0.586
DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences)
test for amphib metamorph screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR03 ( 7-d sn-vent length (mm) )
TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals N/A alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) N/A alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.960 0.653 1.293 0.322 USE PARAMETRIC TESTS
******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 20.25 0.67 0.33 3.29 19.19, 21.31 Dose1 4 19.38 0.28 0.14 1.42 18.94, 19.81 Dose2 4 19.73 0.65 0.32 3.30 18.69, 20.76 Dose3 4 18.90 1.02 0.51 5.41 17.27, 20.53
Level Median Min Max %of Control(means) %Reduction(means) Ctrl 20.45 19.30 20.80 . . Dose1 19.35 19.10 19.70 95.68 4.32 Dose2 19.60 19.10 20.60 97.41 2.59 Dose3 18.50 18.20 20.40 93.33 6.67
********************************************************************************
ԭԹԿԽՇԾ
Page 42 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԱՇՐՁԻՁՌՑՇԾԭՀՇՋՅԽՌՌՇԞՅՈՀՁԺՁԹՆՋԪԽՌԹՅՇՊՈՀՇՋՁՋԞՋՋԹՑ
ԢԭԞԪԯԦԡԫՍՅԺԽՊ
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 2.61 0.100
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 20.25 . 20.25 ...... Dose1 19.38 0.237 19.55 0.110 . . . . . Dose2 19.73 0.603 19.55 0.117 0.894 . . . . Dose3 18.90 0.048 18.90 0.012 0.778 0.387 . . .
********************************************************************************
test for amphib metamorph screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR04 ( 7-d hind-limb length (mm) )
TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals N/A alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) N/A alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.971 0.859 2.971 0.074 USE PARAMETRIC TESTS
******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 2.48 0.16 0.08 6.45 2.22, 2.73 Dose1 4 2.26 0.07 0.04 3.13 2.14, 2.37 Dose2 4 2.13 0.09 0.05 4.32 1.98, 2.28 Dose3 4 2.10 0.23 0.11 10.90 1.73, 2.46
Level Median Min Max %of Control(means) %Reduction(means) Ctrl 2.45 2.31 2.69 . . Dose1 2.23 2.21 2.36 91.11 8.89 Dose2 2.14 2.02 2.22 86.06 13.94 Dose3 2.07 1.87 2.37 84.65 15.35
******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 5.19 0.016
Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC
Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5
Ctrl 2.48 . 2.48 ......
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Dose1 2.26 0.146 2.26 0.036 . . . . . Dose2 2.13 0.019 2.13 0.004 0.655 . . . . Dose3 2.10 0.010 2.10 0.002 0.467 0.987 . . .
******************************************************************************** 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 9.09 0.028
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend
Level Median MannWhit p-value Jonckheere p-value Ctrl 2.45 . . Dose1 2.23 0.103 0.022 Dose2 2.14 0.067 <.001 Dose3 2.07 0.103 <.001
DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose1 Jonckheere Dose1
test for amphib metamorph screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR05 ( 7-d norm hind-limb )
TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals N/A alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) N/A alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.953 0.540 3.268 0.059 USE PARAMETRIC TESTS
******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.12 0.01 0.00 6.75 0.11, 0.14 Dose1 4 0.12 0.00 0.00 3.85 0.11, 0.12 Dose2 4 0.11 0.00 0.00 3.05 0.10, 0.11 Dose3 4 0.11 0.01 0.00 7.20 0.10, 0.12
Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.12 0.11 0.13 . . Dose1 0.12 0.11 0.12 95.31 4.69 Dose2 0.11 0.11 0.11 88.37 11.63 Dose3 0.11 0.10 0.12 90.41 9.59
******************************************************************************** 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 4.00 0.035
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
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Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5
Ctrl 0.12 . 0.12 ...... Dose1 0.12 0.467 0.12 0.135 . . . . . Dose2 0.11 0.021 0.11 0.008 0.285 . . . . Dose3 0.11 0.058 0.11 0.008 0.563 0.944 . . .
******************************************************************************** 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 7.19 0.066
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 0.12 . . Dose1 0.12 0.413 0.155 Dose2 0.11 0.080 0.003 Dose3 0.11 0.156 0.007
DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2
test for amphib metamorph screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR06 ( 21-d stage (median) )
TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals N/A alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) N/A alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.936 0.305 1.220 0.345 USE PARAMETRIC TESTS
******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 59.13 0.85 0.43 1.44 57.77, 60.48 Dose1 4 59.13 0.63 0.31 1.06 58.12, 60.13 Dose2 4 58.13 0.25 0.13 0.43 57.73, 58.52 Dose3 4 57.75 0.50 0.25 0.87 56.95, 58.55
Level Median Min Max %of Control(means) %Reduction(means) Ctrl 59.25 58.00 60.00 . . Dose1 59.00 58.50 60.00 100.00 0.00 Dose2 58.00 58.00 58.50 98.31 1.69 Dose3 58.00 57.00 58.00 97.67 2.33
******************************************************************************** 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 9.65 0.022
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MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend
Level Median MannWhit p-value Jonckheere p-value Ctrl 59.25 . . Dose1 59.00 1.000 0.441 Dose2 58.00 0.168 0.021 Dose3 58.00 0.107 <.001
DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2
test for amphib metamorph screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR07 ( 21-d wet weight (g) )
TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals N/A alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) N/A alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.961 0.682 3.150 0.065 USE PARAMETRIC TESTS
******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.66 0.03 0.02 1.89 1.61, 1.71 Dose1 4 1.72 0.09 0.04 5.24 1.57, 1.86 Dose2 4 1.71 0.04 0.02 2.06 1.65, 1.76 Dose3 4 1.61 0.12 0.06 7.39 1.42, 1.80
Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.67 1.61 1.68 . . Dose1 1.71 1.63 1.81 103.56 -3.56 Dose2 1.70 1.67 1.75 103.11 -3.11 Dose3 1.58 1.50 1.78 97.45 2.55
******************************************************************************** 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 1.47 0.273
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 1.66 . 1.69 ...... Dose1 1.72 0.596 1.69 0.822 . . . . . Dose2 1.71 0.684 1.69 0.851 0.999 . . . . Dose3 1.61 0.789 1.61 0.305 0.308 0.369 . . .
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ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for amphib metamorph screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR08 ( 21-d sn-vent length (mm) )
TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals N/A alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) N/A alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.961 0.689 0.816 0.510 USE PARAMETRIC TESTS
******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 29.18 0.50 0.25 1.71 28.38, 29.97 Dose1 4 29.90 0.63 0.32 2.12 28.89, 30.91 Dose2 4 29.93 0.26 0.13 0.88 29.51, 30.34 Dose3 4 29.65 0.58 0.29 1.96 28.73, 30.57
Level Median Min Max %of Control(means) %Reduction(means) Ctrl 29.30 28.50 29.60 . . Dose1 30.00 29.10 30.50 102.49 -2.49 Dose2 29.90 29.70 30.20 102.57 -2.57 Dose3 29.60 29.00 30.40 101.63 -1.63
******************************************************************************** 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 1.84 0.194
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 29.18 . 29.67 ...... Dose1 29.90 0.162 29.67 0.947 . . . . . Dose2 29.93 0.145 29.67 0.961 1.000 . . . . Dose3 29.65 0.447 29.65 0.964 0.900 0.872 . . .
test for amphib metamorph screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR09 ( 21-d hind-limb length (mm) )
TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals N/A alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) N/A alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.950 0.482 5.729 0.011 USE NON-PARAMETRIC TESTS
******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval
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Ctrl 4 15.93 1.23 0.61 7.70 13.97, 17.88 Dose1 4 15.43 0.95 0.48 6.18 13.91, 16.94 Dose2 4 13.98 0.39 0.20 2.82 13.35, 14.60 Dose3 4 11.95 2.16 1.08 18.11 8.51, 15.39
Level Median Min Max %of Control(means) %Reduction(means) Ctrl 16.35 14.20 16.80 . . Dose1 15.85 14.00 16.00 96.86 3.14 Dose2 14.05 13.50 14.30 87.76 12.24 Dose3 11.80 9.80 14.40 75.04 24.96
******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 7.01 0.006
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 15.93 . 15.93 ...... Dose1 15.43 0.913 15.43 0.363 . . . . . Dose2 13.98 0.149 13.98 0.039 0.455 . . . . Dose3 11.95 0.003 11.95 <.001 0.015 0.200 . . .
******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 3 8.31 0.040
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 16.35 . . Dose1 15.85 0.410 0.153 Dose2 14.05 0.152 0.011 Dose3 11.80 0.101 <.001
DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2
test for amphib metamorph screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR10 ( 21-d norm hind-limb )
TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals N/A alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) N/A alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.929 0.231 3.947 0.036 USE NON-PARAMETRIC TESTS
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******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 0.55 0.05 0.02 8.53 0.48, 0.62 Dose1 4 0.53 0.04 0.02 7.21 0.46, 0.59 Dose2 4 0.47 0.02 0.01 3.21 0.44, 0.49 Dose3 4 0.40 0.07 0.03 17.26 0.29, 0.51
Level Median Min Max %of Control(means) %Reduction(means) Ctrl 0.57 0.48 0.58 . . Dose1 0.54 0.47 0.55 95.45 4.55 Dose2 0.47 0.45 0.48 85.00 15.00 Dose3 0.40 0.33 0.48 73.18 26.82
******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 3 12 7.94 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 0.55 . 0.55 ...... Dose1 0.53 0.791 0.53 0.276 . . . . . Dose2 0.47 0.069 0.47 0.017 0.344 . . . . Dose3 0.40 0.002 0.40 <.001 0.014 0.251 . . .
******************************************************************************** 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 9.98 0.019
MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend
Level Median MannWhit p-value Jonckheere p-value Ctrl 0.57 . . Dose1 0.54 0.230 0.072 Dose2 0.47 0.094 0.004 Dose3 0.40 0.080 <.001
DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2
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Page 49 of 260 DATA EVALUATION RECORD
PHOSMET
Study Type: OCSPP 890.1150, Androgen Receptor Binding (Rat Prostate Cytosol)
EPA Contract No. EP10H001452 Task Assignment No. 2-4-2012 (MRID 48618703)
Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive Arlington, VA 22202
Prepared by Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B Durham, NC 27713
Primary Reviewer Signature: David A. McEwen, B.S. Date: 11/30/2011
Secondary Reviewer Signature: Michelle J. Sharpe-Kass, M.S. Date: 12/01/2011
Program Manager: Signature: Jack D. Early, M.S. Date: 12/05/2011
Quality Assurance: Signature: Jack D. Early, M.S. Date: 12/05/2011
This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel.
Page 50 of 260
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 51 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page I of 14 PHOSMET/059201 OCSPP 890.1150/ OECD None
Primary Reviewer: Jessica Ryman. Ph.D .. D.A.B.T. Health Effects Division Sign"Date:~~ / Secondary Reviewer: __G=---re.,.g"'"'Ak-=--e=rm=an~, "'"""P .....h =D ...... ____ Signature: Health Effects Division Date: G It 2 /,~ Template version 0812011
DATA EVALUATION RECORD
STUDY TYPE: Androgen Receptor Binding (Rat Prostate Cytosol); OCSPP 890.1150
PC CODE: 059201 DP BARCODE: D397 I 65
TXR#: 0056199 CAS No.: 732-11-6
TEST MATERIAL (PURITY): Phosmet (96.8% a.i.)
SYNONYMS: 2-(dimethoxyphosphinothioylsulfanylmethyl)isoindol e-1,3-dione; Imidan technical
CITATION: Willoughby, J.A (2011). Phosmet: Androgen Receptor Binding (Rat Prostate Cytosol). CeeTox, Kalamazoo, Ml. Laboratory Study #: 9142V-l 00348ARB, November 2, 2011. MRID 48618703. Unpublished.
SPONSOR: Gowan, 370 Main Street, Yuma, AZ 85364
TEST ORDER#: EDSP-059201-54
EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRIO 48618703), ventral prostate cytosol from Sprague Dawtey rats was used as the source of the AR. 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 (RI 881).
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 (K-0) for [3H]-Rl 881 was 1.080±0.690 nM, and the estimated Bma.x was l.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/ I 00 µg protein). Although the goodness of fit for individual runs was acceptable (R2 = 0.880-0.986), confidence in these numbers is low based on the large variation in l The competitive binding experiment was conducted to measure the binding of a single concentration of [3H]-Rl 881 (10 nM) in the presence of increasing concentrations of phosmet (96.8% purity; logarithmic increase from 10·10 to 10·3 M). Dimethyl sulfoxide (DMSO) was used as a vehicle at a final assay concentration of approximately 3.2%. The assay included dexamethasone as a weak positive control, and RI 881 as the ligand reference standard. Three Page 52 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None independent runs were conducted with 3 replicates per concentration per run. The estimated log IC50s for R1881 and the weak positive control (dexamethasone) were in the ranges of -9.0 to -9.9 M and -4.3 to -4.6 M, respectively. Phosmet displaced more than 50% of the radiolabeled R1181 from the AR in all three competitive binding runs. The estimated log IC50 was -5.0 M in each run and averaged -5.0 M; therefore, phosmet is classified as an AR “binder”. The mean relative binding affinity (RBA) for the positive control dexamethasone was 0.0013%. The RBA for phosmet was calculated to be 0.0050%, indicating a stronger affinity for the AR than the weak positive control. Confidence in these numbers is high due to the small variation. The solvent control responses indicated no drift in the study assay. All performance criteria were met, with the exception of the bottom (% binding) of dexamethasone in Assay #1, and the bottom plateau level could not be accurately calculated because of a deviation where the concentration range for dexamethasone was prepared slightly different than that specified in the guideline. This was considered a minor deviation. Based on the results of the three competitive binding runs, phosmet is classified as a Binder in the Androgen Receptor Binding Assay. The assay satisfies the EDSP Tier 1 Test Order requirements for an Androgen Receptor Binding assay (OCSPP 890.1150). COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance and Quality Assurance statements were provided. Page 53 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None I. MATERIALS AND METHODS A. MATERIALS 1. Test facility: CeeTox, Inc. Location: 4717 Campus Drive, Kalamazoo, MI 49008 Study Director: J.A. Willoughby, Sr. Other Personnel: D. Blakeman, C. Haines, and S. McColley Study Period: September 10, 2011 to September 17, 2011 2. Test substance: Phosmet Description: White crystalline solid Source: Gowan Company Batch #: GQH9118000 Purity: 96.8% Solubility: Not reported Volatility: Not reported Stability: Not reported Storage conditions: Ambient CAS #: 732-11-6 Molecular weight: 317.32 O Structure: S P N S OCH3 OCH3 O 3. Non-labeled ligand: R1881 Supplier: Sigma Aldrich, St. Louis, MO Catalog #: R0908 Batch #: 060M4638 Purity: 98% CAS #: 965-93-5 4. Radioactive ligand: [3H]-R1881 Supplier: Perkin-Elmer, Boston, MA Catalog and Batch #: NET590; Lot # 653698 Date of production: February 24, 2011 Date of use: September 10, 2011 to September 18, 2011 Radiochemical purity: >97% Specific activity: 85.1 Ci/mmol* *The SAadjusted was 82.6 Ci/mol (10-Sept-2011), 82.5 Ci/mol (13-Sept-2011), and 92.5 Ci/mol (17-Sept-2011). Concentration in stock: 1.0 nM 5. Positive control: Dexamethasone Supplier: Sigma Aldrich, St. Louis, MO Catalog #: D1756 Lot #: 077K1050 Purity: 99% CAS # : 50-02-2 6. Solvent/vehicle control: DMSO Justification for choice of solvent: Not provided Final concentration: 3.2% Page 54 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None B. METHODS 1. Preparation of Rat Ventral Prostate Cytosol: The rat ventral prostate tissue was purchased from Charles River Laboratories (location not reported). Male Sprague Dawley rats (number not reported) were castrated at 90 days of age and were euthanized the following day. The ventral prostate tissues were collected and stored at approximately -80°C until use, and were processed as a batch and used for multiple studies. The cytosol was prepared by adding low-salt TEDG buffer (10 mM Tris, 1 mM sodium molybdate, 1.5 mM EDTA, 10% glycerol and 1 mM DTT, 0.5% protease inhibitor) at pH 7.4 to the ventral prostate tissues at 10 mL/g of tissue. The tissues were minced, homogenized at 4°C, and centrifuged for 30 min at 30,000 × g at 4°C. The supernatant was aliquoted (2-4 mL) in tubes and stored at -80°C until used. Protein concentration of the cytosol prepared for this study was determined to be 6.2 mg/mL using the Bradford method (Bio-Rad). 2. Saturation Radioligand Binding Experiment: A saturation binding experiment measuring total and non-specific binding of [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 Tris 10 mM (pH 7.4) (TEDG) 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 [3H]-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 non- specific binding, solutions of non-labeled R1881 were prepared in a similar manner to 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 [3H]-R1881 and 7.5, 15, or 30 µL of 100 nM [3H]-R1881 was measured. Page 55 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None 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 non- current 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 [3H]-R1881 [3H]-R1881 R1881 [3H]-R1881 [3H]-R1881 Final conc. (nM) Final conc. (nM) Final conc. (nM) Initial conc. (nM) (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 [3H]-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 [3H]-R1881. R1881 was added in a 100-fold molar excess of [3H]-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 [3H]-R1881 or 7.5, 15, or 30 µL of 100 nM [3H]-R1881 without cytosol or other components to determine the total counts added. Following addition of triamcinolone acetonide, [3H]-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 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. Page 56 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None 3. Competitive Binding Experiment: A summary of the assay conditions for the competitive binding experiment is presented in Table 3. TABLE 3. Summary of Conditions for Competitive Binding Experiment a Source of receptor Rat prostate cytosol Concentration of radioligand 1 nM Optimization of receptor concentration Sufficient to bind 10-15% of radioligand b Concentration of test substance (as serial dilutions) 10-10 to 10-3 M Incubation Temperature 4±2°C Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% (v/v) Protease inhibitor 0.5% (v/v) DTT 1 mM Sodium Molybdate 1 mM a Data were obtained from page 15 of the study report. b Obtained in Study Report on page 15. 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 [3H]-R1881 (adjusted specific activity of 85.1 Ci/mmol) to the AR in the presence of increasing concentrations of a test substance. DMSO was used as the vehicle, and the solubility of the test material at the top concentrations of 10-3 and 10-4 M in 100% DMSO was determined by visual observation. Additionally, all test material solutions were examined for precipitation following the overnight incubation period. Due to precipitation being observed at 10-3 M, the suitable top concentration of Phosmet for use in all independent runs was 10-4 M. 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, resulting in a total of 81 samples per run. Page 57 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None TABLE 4. Competitor Final Molar (M) Concentrations in Competitive Binding Assay a b Solvent Control Reference standard c Weak positive control d Test Chemical e None DMSO R1881 Dexamethasone Phosmet Tubes 7-12 Tubes 13-18 and 19-33 f Tubes 37-60 Tubes 61-84 Tubes 1-6 1×10-7.48 1×10-6 1×10-3 1×10-4 1×10-3 1×10-4 1×10-8.48 1×10-7 1×10-4 1×10-5 1×10-4 1×10-5 1×10-9.48 1×10-8 1×10-5 1×10-6 1×10-5 1×10-6 1×10-10.48 1×10-9 1×10-6 1×10-7 1×10-6 1×10-7 1×10-11.48 1×10-10 1×10-7 1×10-8 1×10-7 1×10-8 1×10-12.48 1×10-11 1×10-8 1×10-9 1×10-8 1×10-9 -- 1×10-9 1×10-10 1×10-9 1×10-10 -- 1×10-10 1×10-11 1×10-10 1×10-11 a Data were obtained from pages 53-58 of the study report. b Each concentration of each chemical was run in triplicate for a total of 81 tubes per run. Tubes 7-84 contained 50 µL of triamcinolone acetonide and 30 µL [3H]-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 [3H]-R1881. c The concentrations of R1881 used in the 1st competitive binding assay ranged from 1x10-7.48 to 1x10-12.48, while the standard concentrations of 1x10-6 to 1x10-11 were used in the 2nd and 3rd assays. d The concentrations of dexamethasone used in the 1st competitive binding assay ranged from 1x10-3 to 1x10-10, while the standard concentrations of 1x10-4 to 1x10-11 were used in the 2nd and 3rd assays. e The concentrations of phosmet used in the 1st competitive binding assay ranged from 1x10-3 to 1x10-10, while concentrations of 1x10-4 to 1x10-11 were used in the 2nd and 3rd assays. f Tubes 13-18 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881 4. Data Analysis: The maximal binding capacity (Bmax), dissociation constant (Kd), the top and bottom of the curve, Hill slope, inhibition concentration (IC50), and standard deviations were assessed using GraphPad PRISM (GraphPad Software, Inc., San Diego, CA). 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 (i.e. a Log IC50 was obtained). 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. 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). b. Descriptors for receptor binding Bmax: maximal binding capacity Kd: dissociation constants Page 58 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None 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 II. RESULTS A. SATURATION BINDING EXPERIMENT: Saturation binding experiment parameters 3 are presented in Table 5. The mean Kd for [ H]-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 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, confidence in these numbers is low based on the 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 supplemental information provided by the laboratory (MRID 48843501). 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. Page 59 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None FIGURE 1. Binding of [3H]-R1881 to the Androgen Receptor during the Saturation Binding Experiment. Page 60 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None FIGURE 2. Scatchard Plots of the Binding of [3H]-R1881 to the Androgen Receptor. Page 61 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive binding experiments are summarized in Table 6 and shown graphically in Figures 3 – 5. Non-specific binding values were 8.9%, 10.3%, and 14.0%. Phosmet reduced the radiolabeled R1881 binding to the AR by more than 50% in each of the three runs. The estimated log IC50s for R-1881 and the weak positive control (dexamethasone) were in the ranges of -9.0 to -9.9 M and -4.3 to -4.6 M, respectively. The estimated log IC50 for phosmet was -5.0 M in each run which resulted in the mean log IC50 of -5.0 M for the test material. The mean RBA for the positive control was 0.0013%. Phosmet was classified as a “binder” in all three runs, as the RBA was calculated to be 0.0050%, indicating a stronger affinity for the androgen receptor than the positive control, dexamethasone. Confidence in these numbers is high due to the small variation. The solvent control responses indicated no drift in the study assay. TABLE 6. Competitive Binding Assay of Phosmet with AR from Rat Prostate Cytosol a Parameter Assay 1 Assay 2 Assay 3 Mean r2 (unweighted) R1881 NR NR NR NR Dexamethasone NR NR NR NR Phosmet NR NR NR NR b Log IC50 (M) R1881 -9.9 -9.0 -9.0 -9.3 Dexamethasone -4.3 -4.4 -4.6 -4.4b Phosmet -5.0 -5.0 -5.0 -5.0 b -10 -9 -9 -10 IC50 (M) R1881 1.3 x 10 10 10 1.1 x 10 Dexamethasone 5.0 x 10 -5 4.0 x 10 -5 2.5 x 10 -5 4.0 x 10 -5 Phosmet 10 -5 10 -5 10 -5 10 -5 c RBA (as % IC50) R1881 100 100 100 100 Dexamethasone 0.0003 0.0025 0.0040 0.0013 Phosmet 0.0013 0.0100 0.0100 0.0050 a Data were obtained from page 20 of the study report. b Calculated by reviewer c Calculated by reviewer: [IC50 (in M) Dexamethasone or Phosmet / IC50 (in M) R1881] x 100% NA Not applicable NR Not reported RBA (%) relative binding affinity FIGURE 3. Binding of Phosmet, R1881 and Dexamethasone in Assay 1. Page 62 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None FIGURE 4. Binding of Phosmet, R1881 and Dexamethasone in Assay 2. FIGURE 5. Binding of Phosmet, R1881 and Dexamethasone in Assay 3. C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay was functioning properly, each run was evaluated using the following criteria in Table 7: Tolerance TABLE 7. Criterion a Value Yes No Limit(s) b Ligand depletion is minimal. The recommended ratio of total binding in the absence of competitor to total amount of [3H]- ≤15 10.5 to 12.6 X R1881 added per assay tube (% binding) Test chemical Top (% binding) 80 to 115 102.3 to 107.5 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 -1 to 7 X Hill Slope -1.4 to -0.6 -1.0 to -0.6 X d d Saturation Binding Experiment Kd (nM) 0.685 to 1.57 nM 0.531 to 1.855 X Non-specific binding (%)c 8.1-10.0 <20% X a Data were obtained from pages 23-28 of the study report. b These values represent ranges from the validation study. c MRID 48843501 d One value was below the limit, one value was within the limit, and one value was above the limit. Additionally, the curve for the reference material showed that increasing concentrations of unlabeled R1881 displaced [3H]-R1881 in a manner consistent with one-site binding, as Page 63 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None indicated by a Hill slope of -1.0 to -0.6. Examination across the runs indicated consistency of the Hill slope, placement along the X-axis, and top and bottom plateaus. The percentage of the total specific binding in the solvent controls was approximately 10%. This was within the less than the ≤ 20% recommended in the guideline. Ligand depletion was also minimal. Sufficient optimization of the number of specific binding sites is supported by curves for controls of acceptable steepness, low non-specific binding, and low variability. III. DISCUSSION AND CONCLUSIONS A. INVESTIGATOR’S CONCLUSIONS: All three assays achieved the binder criterion described in the guideline (<50% radioligand binding). There were no apparent alterations in radiolabeled R1881 binding at any of the lower concentrations tested. Phosmet was classified as binding based on the average of the three valid assays with a mean value of “2”. The mean RBA (calculated by dividing the log IC50 of the control/test material by the log IC50 of the positive control R1881) was 0.5 for both dexamethasone and phosmet. B. AGENCY COMMENTS: In the saturation binding experiment, the mean Kd for [3H]- 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 acceptable range. In the competitive binding experiment, the estimated log IC50s for R-1881 and the weak positive control (dexamethasone) were in the ranges of -9.0 to -9.9 M and -4.3 to -4.6 M, respectively. The estimated log IC50 for phosmet was -5.0 M in each run, and the mean log IC50 for phosmet was -5.0 M, classifying it as an AR binder. The mean RBA for the positive control was 0.0013%, and the mean RBA for phosmet was 0.0050%, indicating a stronger affinity for the androgen receptor compared to dexamethasone. Confidence in these numbers is high due to the small variation. The solvent control responses indicated no drift in the study assay. All performance criteria were met, with the exception of the bottom (% binding) of dexamethasone in Assay #1, the bottom plateau level could not be accurately calculated because of a deviation where the concentration range for dexamethasone was prepared slightly different than that specified in the guideline. As this was a minor deviation, the reviewers concluded that the performance criteria were met. Phosmet is classified as a binder in the androgen receptor assay. C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study: • The required r2 value for the competitive binding assay was not reported. • Non-specific binding exceeded the tolerance limits for Assays 2 and 3. This was not considered to have a negative impact on the study because there was still enough of a dynamic range in which to detect changes in binding with concentration for control and test chemicals. Page 64 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 14 PHOSMET /059201 OCSPP 890.1150/ OECD None APPENDIX 1 (from page 49 of the Study Report) Page 65 of 260 DATA EVALUATION RECORD PHOSMET Study Type: OCSPP 890.1200; Aromatase Assay EPA Contract No. EP10H001452 Task Assignment No. 2-4-2012 (MRID 48616302) Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive Arlington, VA 22202 Prepared by Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B Durham, NC 27713 Primary Reviewer Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 12/15/2011 Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 12/15/2011 Program Manager: Signature: Jack D. Early, M.S. Date: 12/15/2011 Quality Assurance: Signature: Jack D. Early, M.S. Date: 12/15/2011 This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel. Page 66 of 260 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 67 of 260 Aromatase (Human Recombinant) Assay (2011) I Page I of 13 PHOSMET I 059201 OCSPP 890.1200 I OECD None /- Primary Reviewer: Vincent Chen, M.S Signature:~ c-- Health Effects Division Date: z_y -sv Al W ...LS Secondary Reviewer: Greg Akerman, Ph.D. Signature: ,4 ~ Health Effects Division Date: Gb.:r /1 ) Template version 08/2011 DATA EV ALU ATI ON RECORD STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200 PC CODE: 059201 DP BARCODE: D397165 TXR: 0056199 CAS: 732-11-6 TEST MATERIAL (PURITY): Phosmet (96.8% a.i.) SYNONYMS: IUPAC: 0,0-dimethyl S-phthalimidomethyl phosphorodithioate CAS: S-[(l ,3-dihydro-l ,3-dioxo-2H-isoindol-2-yl)methyl] 0 ,0-dimethyl phosphorodithioate CITATION: Wilga, P.C. (2011). Phosmet: Human Recombinant Aromatase Assay. CeeTox, Inc (4717 Campus Drive, Kalamazoo, MI 49009, US]. Laboratory Study Number: 9142V-100348AROM. 03 November 2011. MRID 48618702. Unpublished. SPONSOR: Gowan Co., 370 Main Street, Yuma, AZ 85364, US TEST ORDER: EDSP-059201-54 EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRJD 48618702), phosmet (96.8% a.i., batch # GQH9 11 8000) in dimethyl sulfoxide (DMSO) was incubated with human recombinant aromatase and tritiated androstenedione ([ 1P-3H(N)]-androst-4-ene-3,17- 4 1 dione; [3H]-ASDN) at concentrations of 0, 10-3, 10-3.s,10- , 10-s, 10-6, 10- , 10-8, 10-9, or 10-10 M for 15 minutes at 37°C to assess the effect on aromatase activity. Due to slight precipitation that was observed at the high concentration in the first two runs, the high concentration was decreased to l 0·3.s M for the third run. 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 to 10-5 M) using a known inhibitor (4-hydroxyandrostenedione; 4-0H ASDN), and the test chemical series (10- 10 to 10-3) with 3 replicates per concentration. Aromatase activity in the full activity controls ranged from 0.150 to 0.376 nmol·mg protein- 1·min- 1 for the 3 test runs, with a mean and standard deviation of0.268±0.081 nmol·mg protein- 1·min- 1. Activity in the background controls ranged from 0.37% to 0.97% and averaged 0.56% of the full control activity. The response of the full activity controls and background Page 68 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 2 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None controls were acceptable for each run. However, each full activity control was not generally within 10% of the average for the full activity controls, as recommended by the EPA. Run 1 had roughly half the activity of the other runs and the 0.5 mL count for total activity was roughly half that of the other runs. This discrepancy was not explained in the methods of the study report, and applies to both the controls and phosmet in run 1 compared to runs 2 and 3. For the positive control substance (4-OH ASDN), aromatase activity averaged 0.257±0.078 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 results 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. For phosmet, aromatase activity averaged 0.231±0.059 nmol∙mg-protein−1∙min−1 at the lowest tested concentration of 10-10 M and 0.033±0.002 nmol∙mg-protein-1∙min−1 at the highest tested concentration of 10−3 M. The lowest portion of the response curve across runs for phosmet was less than 50% activity, indicating that phosmet is an inhibitor of aromatase activity at the highest soluble concentration. Phosmet decreased aromatase activity to 34% at 10−4 M. The estimated log IC50 ranged from -4.37 to -4.89 M for phosmet and averaged -4.54 M compared to 4OH-ASDN, which averaged -7.21 M. The average slope of the concentration response curve was -1.20 for phosmet and -0.97 for 4OH-ASDN. Confidence in the mean log IC50 for both chemicals and the Hill slope for the positive control is high due to the small variation, but the variation was moderate (22.5% CV) for the slope value of phosmet. At 10-10 M in Run 2, aromatase activity was only 69-73% of control, as compared to 95-101% of control in the other runs; no explanation was provided. Based on the data from the average response curve, phosmet is classified as an Inhibitor of aromatase activity in this assay The assay satisfies the EDSP Tier 1 Test Order requirements for an Aromatase assay (OCSPP 890.1200). COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were provided. Page 69 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 3 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None I. MATERIALS AND METHODS A. MATERIALS: 1. Test Substance: Phosmet Description: Off-white crystals Source: Gowan Batch # (Expiration Date): GQH9118000 (January 31, 2013) Purity: 96.8% a.i. Vapor Pressure at 25°C: 0.065 mPa Storage Conditions: Room temperature Stability: Not reported Solvent: DMSO Solubility (in Test Solvent): Approximately 10−3.5M Highest Concentration Tested: 10−3M Stock Solution Preparation Methodology: Not provided; serial dilutions assumed Molecular Weight: 317.3 CAS #: 732-11-6 Structure: 2. Non-Labeled Substrate: Androstenedione (ASDN) CAS # : 63-05-8 Source: Steraloids, Inc. (Cat. # L1712) Batch # (Expiration Date): L1712 (April 2016) Purity: 99.8% 3. Radiolabeled Substrate: [1β-3H(N)]-Androst-4-ene-3,17-dione; ([3H]-ASDN) Source: Perkin Elmer (Cat. # NET-926) Batch # (Expiration Date): 619344 (10 January 2012) Radiochemical Purity (Supplier): 97% Specific Activity: 26.3 Ci/mmol Radiochemical Purity (In-Lab Determination): Not provided 4. Positive Control: 4-hydroxyandrostenedione (4-OH ASDN) CAS # 566-48-3 Source: Sigma-Aldrich (Cat. # F2552) Batch # (Expiration Date): 081K2133 (March 2015) Purity: 99.6% 5. Solvent (Vehicle Control): Dimethyl sulfoxide (DMSO) Source: Sigma-Aldrich (Cat. # D2650) Lot # (Expiration Date): RNBB7617 (February 2013) Justification for Choice of Solvent: Acceptable vehicle by OCSPP guideline 890.1200 Concentration (% of Total Volume in Assays): 1% 6. Test Microsomes: Human recombinant aromatase (CYP19) microsomes Source: BD Gentest™ (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 70 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 4 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None B. METHODS 1. Assay Components and Preparations: A mixture of non-labeled and radiolabeled [3H]-ASDN was prepared such that the final concentration of ASDN in the assay was approximately 100 nM ASDN, and the amount of tritium added to each incubation tube was approximately 0.1 μCi. Test chemical stock solutions were prepared such that the total volume of each test chemical formulation used per assay was 1% (20 µL in 2 mL) of the total assay volume to minimize the potential for the solvent to inhibit the enzyme. Selection of the solvent, DMSO, was based on the following reasons: i) DMSO was listed as an acceptable vehicle in the OCSPP 890.1200 Guidelines; ii) DMSO was less volatile than ethanol; and iii) DMSO was more accurate to pipette because of its density and viscosity. A stock solution of the positive control substance, 4-OH ASDN, was formulated in DMSO. Fresh dilutions of the stock solution were prepared in the same solvent on the day of use. Dilutions were prepared such that the target concentrations of the positive control substance (0.1–10,000 nM; Table 4) were achieved by the addition of 20 μL of the dilution for a final assay volume of 2 mL. Human recombinant microsomes were purchased from BD Gentest™, and stored at −80±10ºC for no longer than 7 months. Microsomes were portioned into individual vials based on the protein concentration of the batch (approximately 0.004 mg/ml microsomal protein/tube) and stored at −80±10ºC until used to minimize freeze-thaw cycles to no more than one. Other assay components, such as sodium phosphate buffer, propylene glycol, and NADPH, are reported in Table 1. TABLE 1. Assay Components and Conditions Assay Factor Values Sodium phosphate buffer (pH 7.4) 0.1M Microsomal protein 0.004 mg/mL NADPH 0.3 mM [3H]ASDN 100 nM Propylene glycol 5% Temperature 37±2°C Incubation time 15 min 2. Suitability Assessments: The protein concentration of the microsomes 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/pmole P450. The minimum mean aromatase activity in the full activity control samples was determined to be 0.150 nmol/mg-protein/min, which was greater than the minimum recommended aromatase activity of 0.1 nmol/mg-protein/min. 3. Aromatase Assay: Each assay run contained 4 tubes for the full enzyme activity and 4 tubes for background activity controls. Two tubes of each control were run at the beginning of the assay, and two of each control were run at the end of the assay. A full concentration curve in duplicate for the positive control, and a full concentration curve in triplicate for the Page 71 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 5 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None test substance were established. The aromatase assay was generally conducted according to the procedures described in OCSPP 890.1200 (Section h, pp. 9-10). 3 The amount of H2O in the aqueous fraction was quantified for each assay tube by LSC, and aromatase activity was reported in units of nmol∙mg-protein-1∙min-1. 4. Demonstration of Proficiency: It was stated that prior to conducting this assay, a full-scale assay consisting of three independent runs was conducted using the positive control (4-OH ASDN) and the four proficiency chemicals (econazole, fenarimol, nitrofen, and atrazine) listed in the OCSPP 890.1200 guideline. Proficiency was demonstrated when the positive control met the performance criteria stated in the guideline and by the correct classification of the proficiency chemicals. It was stated that the data were generated and maintained at the Performing Laboratory; however, chemical proficiency data were not provided. a. Positive Control: Initial Demonstration of Laboratory Proficiency: Prior to conducting the aromatase activity assay, positive control assays were conducted with three independent runs. These data were not provided. However, positive controls were run concurrently with the test compound in the aromatase activity assay. These positive control data met the following criteria: • Mean aromatase activity in the absence of an inhibitor was at least 0.1 nmol/mg- protein/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 (Table 2) were met, and served as guidance in identifying runs that provided parameters in the preferred ranges. Demonstration of Proficiency of New Technician for Conducting Assay (when applicable): The positive control data met the criteria as listed in section (i) of OCSPP 890.1200. 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 -1.02 -0.94 Top (%) 90 110 85.58 100.53 Bottom (%) -5 +6 -0.28 0.69 Log IC50 (M) -7.3 -7.0 -7.35 -7.09 b. Proficiency Chemicals: Data was not provided. TABLE 3. Proficiency Chemicals Compound CAS# Class Concentrations Econazole 24169-02-6 Inhibitor --- Fenarimol 60168-88-9 Inhibitor --- Nitrofen 1836-75-5 Inhibitor --- Atrazine 1912-24-9 Non-inhibitor --- 5. Determination of Aromatase Activity with Test Chemical: The response of aromatase activity to the presence of eight concentrations of the test chemical per run, in triplicate, was tested during three independent runs. After completion of the first run, the data were Page 72 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 6 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None reviewed and solubility was visually assessed (presence of cloudiness or a precipitate). No data adjustments were necessary. The full enzymatic activity was obtained at the two lowest concentrations of the test chemical in two runs but not the third, 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 Phosmet Conc 1a b 3 All test components plus phosmet 1×10-3 Phosmet Conc 1b c 3 All test components plus phosmet 1×10-3.5 Phosmet Conc 2 3 All test components plus phosmet 1×10-4 Phosmet Conc 3 3 All test components plus phosmet 1×10-5 Phosmet Conc 4 3 All test components plus phosmet 1×10-6 Phosmet Conc 5 3 All test components plus phosmet 1×10-7 Phosmet Conc 6 3 All test components plus phosmet 1×10-8 Phosmet Conc 7 3 All test components plus phosmet 1×10-9 Phosmet Conc 8 3 All test components plus phosmet 1×10-10 a The complete assay contained buffer, propylene glycol, microsomal protein, [3H]ASDN, and NADPH. b The high dose for runs 1 and 2. Slight precipitation was observed at this dose. c The high dose for run 3. No precipitation was observed at this dose. 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). 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 XLfit, v5.2.0.0 (IDBS, 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. Page 73 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 7 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None 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; IDBS; v5.2.0.0; Fit Model 208,) and Tukey’s Bi-Weight statistical analysis for outlier analysis. For each run, the individual percent of control values were plotted versus the logarithm (base 10) of the test chemical concentration. The fitted concentration response curve was superimposed on the plot, with individual plots prepared for each run. The average percent of control values versus the logarithm of test chemical concentration for the individual runs were included on the same graph with their respective fitted response curves. In addition, the average percent of control values for each run versus the logarithm of test chemical concentration were plotted on a separate graph along with the average concentration response curve across runs were superimposed on the same plot. 3. Interpretation of Results: Interpretation of the assay results was based on the average of three runs, using the categories presented in Table 6. TABLE 6. Interpretation of Results Criteria Interpretation Data fit 4-parameter Average curve across runs crossed 50% Inhibitor nonlinear regression Average lowest portion of curves across runs is between 50% and 75% activityb Equivocal model Average lowest portion of curves across runs is greater than 75% activity Non-inhibitor Data do not fit model --- II. RESULTS A. CONTROL ACTIVITY: Aromatase activity in the full activity controls ranged from 0.150 to 0.376 nmol∙mg-protein−1∙min−1 for the 3 test runs, with a mean and standard deviation of 0.268±0.081 nmol∙mg-protein−1∙min−1. Activity in the background controls ranged from 0.37% to 0.97% and averaged 0.56% of the full control activity. The response of the full activity controls and background controls were acceptable within each run. However, Run 1 had roughly half the activity of the other runs, and the 0.5 mL count for total activity was roughly half that of the other runs. This discrepancy was not explained in the methods of the study report, and applies to both the controls and phosmet in run 1 compared to runs 2 and 3. B. POSITIVE CONTROL: For the positive control substance (4-OH ASDN), aromatase activity averaged 0.257±0.078 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 activity) for each concentration tested across all 3 runs is presented in Table 7, along with the overall standard deviation and %CV. The inhibition response curves for the positive control are shown in Figure 1. These results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, log IC50, and coefficient of variation for replicates of each concentration. Page 74 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 8 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None TABLE 7. Effect of Phosmet 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 -5 3 0.82 0.12 0.05 14.14 (positive control) -6 3 6.44 1.07 0.43 16.54 -6.5 3 16.66 2.81 1.15 16.89 -7 3 36.05 3.52 1.44 9.76 -7.5 3 63.50 4.18 1.71 6.59 -8 3 79.85 6.57 2.68 8.23 -9 3 92.27 10.27 4.19 11.13 -10 3 96.78 8.58 3.50 8.87 Phosmet -3 3 14.79 5.23 2.14 35.36 -3.5 3 29.49 1.22 0.50 4.14 -4 3 34.08 5.16 2.11 15.13 -5 3 72.43 10.38 4.24 14.33 -6 3 89.94 9.27 3.79 10.31 -7 3 91.36 9.99 4.08 10.93 -8 3 90.40 9.16 3.74 10.13 -9 3 90.63 11.04 4.51 12.18 -10 3 88.53 12.70 5.18 14.34 a Calculated by the reviewers using the information presented on pages 29-31 of the study report. SD Standard Deviation SEM Standard Error of the Mean CV Coefficient of Variance Page 75 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 9 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None FIGURE 1. Inhibition Response Curves for 4-OH ASDN. C. TEST SUBSTANCE: For phosmet, aromatase activity averaged 0.231±0.059 nmol∙mg-protein−1∙min−1 at the lowest tested concentration of 10-10 M and 0.033±0.002 nmol∙mg-protein-1∙min−1 at the highest tested concentration of 10−3 M. The mean aromatase Page 76 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 10 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None activity of phosmet (expressed as % full control activity) for each concentration tested across all 3 runs is presented in Table 7, along with the overall standard deviation and %CV. Inhibition response curves for phosmet from each run are shown in Figure 2, and the average inhibition response curve across all runs is shown in Figure 3. Page 77 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 11 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None FIGURE 2. Inhibition Response Curves for Phosmet. A slight amount of the compound was observed in the miniscus, near the surface for the highest concentration. A slight amount of the compound was observed in the miniscus, near the surface for the highest concentration. Page 78 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 12 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None FIGURE 3. Mean Inhibition Response Curve for Phosmet A slight amount of the compound was observed in the meniscus, near the surface for the highest concentration. The effect of phosmet on inhibition of aromatase activity is presented in Table 8. The estimated log IC50 ranged from -4.37 to -4.89 M for phosmet and averaged -4.54 M compared to 4OH-ASDN, which averaged -7.21 M. The average slope of the concentration response curve was -1.20 for phosmet and -0.97 for 4OH-ASDN. Confidence in the mean log IC50 for both chemicals and the Hill slope for the positive control is high due to the small variation, but the variation was moderate (22.5% CV) for the slope value of phosmet. TABLE 8. Effect of Phosmet and 4OH-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) Phosmet -4.37 -4.37 -4.89 -4.54 0.30 6.62 4-OH ASDN -7.19 -7.09 -7.35 -7.21 0.13 1.82 Slope Phosmet -1.16 -0.95 -1.48 -1.20 0.27 22.48 4-OH ASDN -0.94 -1.02 -0.95 -0.97 0.04 4.49 a Data for individual control runs were obtained from page 27 of the study report. IC50 and slope values for phosmet were calculated by the reviewers using Graphpad Prism based on data provided on pages 44, 49 and 54. Mean, SD, and %CV for phosmet and control were calculated by the reviewers. SD Standard Deviation CV Coefficient of Variance NA Not applicable At 10-10 M in Run 2, aromatase activity was only 69-73% of control, as compared to 95-101% of control in the other runs. No explanation was provided in the study report concerning this observation. Based on the data from the average response curve and the criteria listed above in Table 6, the results support the conclusion that phosmet inhibits aromatase. Page 79 of 260 Aromatase (Human Recombinant) Assay (2011) / Page 13 of 13 PHOSMET / 059201 OCSPP 890.1200 / OECD None III. DISCUSSION AND CONCLUSIONS A. INVESTIGATORS CONCLUSIONS: A decrease in aromatase activity was identified at the very highest concentration of phosmet tested (e.g., 10−3 M), but solubility issues were observed. Phosmet reduced aromatase to 34.1% of control activity at 10−4 M and thus was classified as an inhibitor, per OPPTS 890.1200 guideline, under the conditions of this assay. Phosmet at lower exposures of 10–10 M to 10–5 M showed essentially no effect (e.g., non-inhibitor). B. AGENCY COMMENTS: Aromatase activity in the full activity controls ranged from 0.150 to 0.376 nmol∙mg-protein−1∙min−1 for the 3 test runs, with a mean and standard deviation of 0.268±0.081 nmol∙mg-protein−1∙min−1. Activity in the background controls ranged from 0.37% to 0.97% and averaged 0.56% of the full control activity. The response of the full activity controls and background controls were acceptable for each run. However, each full activity control was not generally within 10% of the average for the full activity controls as recommended by the EPA. Run 1 had roughly half the activity of the other runs and the 0.5 mL count for total activity was roughly half that of the other runs. This discrepancy was not explained in the methods of the study report, and applies to both the controls and phosmet in run 1 compared to runs 2 and 3. For the positive control substance (4-OH ASDN), aromatase activity averaged 0.257±0.078 nmol∙mg- protein−1∙min−1 at the lowest tested concentration (10−10 M) and 0.002±0.001 nmol∙mg- −1 −1 −5 protein ∙min at the highest tested concentration (10 M). The estimated log IC50 ranged from -4.37 to -4.89 M for phosmet and averaged -4.54 M compared to 4OH-ASDN, which averaged −7.21 M. The average slope of the concentration response curve was -1.20 for phosmet and -0.97 for 4OH-ASDN. Confidence in the mean log IC50 for both chemicals and the Hill slope for the positive control is high due to the small variation, but the variation was moderate (22.5% CV) for the slope value of phosmet. At 10-10 M in Run 2, aromatase activity was only 69-73% of control, as compared to 95-101% of control in the other runs; an explanation was not provided. The lowest portion of the response curve across runs for phosmet was less than 50% activity, indicating that phosmet is an inhibitor of aromatase activity at the highest soluble concentration (phosmet decreased aromatase activity to 34% at 10−4 M). Based on the data from the average response curve, the results indicate the phosmet is an inhibitor of aromatase activity in this assay C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study: • Proficiency chemical data were not provided. • Different counts for total activity in 0.5 mL and full activity controls (nmol/mg/min) were observed in Run 1 compared to Runs 2 and 3, but no explanation was provided. • At 10-10 M in Run 2, aromatase activity was only 69-73% of control, as compared to 95- 101% of control in the other runs. No explanation was provided in the study report concerning this observation. • Information on the stability of the stock solution was not reported. Page 80 of 260 DATA EVALUATION RECORD PHOSMET Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay EPA Contract No. EP10H001452 Task Assignment No. 2-4-2012 (MRID 48618704) Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive Arlington, VA 22202 Prepared by Dynamac Corporations 1910 Sedwick Road, Building 100, Suite B Durham, NC 27713 Primary Reviewer Signature: Michelle J. Sharpe-Kass, M.S. Date: 12/12/2011 Secondary Reviewer Signature: David A. McEwen, B.S. Date: 12/19/2011 Program Manager: Signature: Jack D. Early, M.S. Date: 12/23/2011 Quality Assurance: Signature: Jack D. Early, M.S. Date: 12/23/2011 This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel. Page 81 of 260 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 82 of 260 Androgen Receptor Binding (Rat Prostate Cytosol) (2012) I Page J of 13 PRONAMIDE/ 101701 OCSPP 890.1150/ OECD None Primary Reviewer: Patience Browne, Ph.D. Signatu~ e__ Health Effects Division,. Office of Pesticide Progra.ms l>ate: Secondary Reviewer: Greg Akerman, Ph.D . . Signature: ~~~ Health Effects Division; Office of Pesticide Programs · Date: \:20of 2 Template version 08/2011 DATA EVALUATION RECORD STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC); OCSPP 890.1250 PC CODE: 059201 DP BARCODE: D397 l 65 TXR#: 0056199 CAS No.: 732-11-6 TEST MATERIAL CPURIT\'): Phosmet (96.8%) SYNONYMS: Imidan Technical; 2-(dimethoxyphosphinothioylsulfanylmethyl)isoindole-1 ,3- dione CITATION: Willoughby, J.A. Sr. (2011). Phosmet: Estrogen Receptor Binding (Rat Uterine Cytosol). CeeTox, Kalamazoo, MI. 9142V-100348ERB, November 2, 2011. MRID 48618704 Unpublished. SPONSOR: Gowan Company, 370 Main Street, Yuma, AZ 85364 TEST ORDER#: EDSP-059201-54 EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48618704), uterine cytosol from Sprague-Dawley rats was used as the source of ER to conduct saturation and competitive binding experiments. A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radiolabeled reference estrogen prior to routinely conducting ER competitive binding experiments. The competitive binding experiment was conducted to measure the binding of a single concentration of [3H]-17P-estradiol (1 nM) in the presence of increasing concentrations of phosmet (96.8% purity; logarithmic increase from I 0-10 to 10-3 M). DMSO was used as a vehicle at a final concentration of <4%. The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and l 7P estradiol as the natural ligand reference material. Saturation binding data were not provided in the study report; however, summarized saturation binding data (MRID 48843501) from the perfor,ming laboratory were submitted following a request by the Agency. For the Competitive Binding Experiment, a log ICso could not be calculated for phosmet. The mean log ICso for l 7P-estradiol was -9.0 Mand the average log ICso for 19-norethindrone was - 5.5 M. The mean RBA was 0.03% for 19-norethindrone compared to the positive control (I nM I 7P-estradiol), but a RBA could not be calculated for phosmet. Page 83 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None Based on the top % binding for 19-norethindrone at 49%, Run 2 should not be considered for evaluation of phosmet for binding to the ER. The Hill slope for 17β-estradiol in Run 3 (−1.7) was outside of parameter limits. Precipitation was observed at 10-4M (but not 10-3 M) in Run 1 and at the 10-3 M in Runs 2 and 3. In these instances, data for those concentrations were disregarded by the study author despite indications of potential interaction with the estrogen receptor. The investigators failed to follow assay guidelines that recommend testing solubility of test substances in increasing solvent concentrations (up to 10% DMSO) and including half log concentrations at the limit of solubility. Because of insufficient attempts to resolve issues with solubility of the test substance at concentrations that suggested an ER interaction, there is lack of confidence in the results of the three runs; therefore the assay is considered to be technically inadequate. The assay does not satisfy does not satisfy the EDSP Tier 1 Test Order requirements for an Estrogen Receptor Binding assay (OCSPP 890.1250). This assay is not satisfied due to issues with test chemical solubility. However a repeat of this assay is not required since negative results were seen in the Tier 1 ERTA and OSRI which evaluated potential interaction of phosmet with the ER. For additional information see the EDSP Tier 1 Weight of Evidence Analysis for Phosmet (TXR 0057145). COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided. Page 84 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None I. MATERIALS AND METHODS A. MATERIALS 1. Test Facility: CeeTox Location: Kalamazoo, MI Study Director: Jamin A. Willoughby, Ph.D. Other Personnel: Karen Rutherford, Director of Laboratory Operations David Blakeman, Senior Scientiet/Endocrine Group Leader Cameron Haines, Scientist Steven McColley, Scientist Biranna Wallace, Lead Cell Culture Scientist Colleen Toole, Ph.D., Director of Project Management Study Period: May 25- Nov 2, 2011 2. Test substance: Phosmet Description: White crystalline solid Source: Gowan Company (Yuma, AZ) Lot/Batch #: GQH9118000, Exp Jan 31, 2013 Purity: 96.8% a.i. Solubility: Not reported Volatility: Not reported Stability: Not reported Storage conditions: Ambient CAS #: 732-11-6 Molecular weight: 317.32 g/mol O Structure: S P N S OCH3 OCH3 O 3. Non-labeled ligand: 17β-estradiol Supplier: Sigma-Aldrich (St Louis, MO) Catalog # E8875 Batch #: 110M0138V Purity: 100% a.i. CAS #: 50-28-2 4. Radioactive ligand: [3H]-17β-estradiol Supplier: Perkin-Elmer (Boston, MA) Catalog #: NET517 Batch#: 650702 Radiochemical purity: Not reported Specific activity: 130.2 Ci/mol Concentration of stock: 1 nM 5. Positive control: 19-norethindrone Supplier: Sigma-Aldrich (St Louis, MO) Catalog # N4128 Batch #: 030M1359V Purity: 99% a.i. CAS # : 66-22-4 6. Negative control: Octyltriethoxysilane Page 85 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None Supplier: Sigma-Aldrich (St Louis, MO) Catalog # 440213 Batch #: 2499KK Purity: 99.34% a.i. CAS # : 2943-75-1 7. Solvent/vehicle control: DMSO Justification for choice of solvent: None reported Final Concentration 4% v/v B. METHODS 1. Preparation of Rat Uterine Cytosol (RUC): Rat uteri from female Sprague-Dawley rats (number not reported) aged 12-13 weeks were obtained from Harlan Laboratories. Rats were ovariectomized 7 days prior to being euthanized. Upon receipt, the uterine tissue was inspected for signs of residual ovarian tissue after ovariectomy, and compromised tissue was discarded. The uteri were weighed, placed in ice-cold TEDG (Tris, EDTA, DTT, glycerol) + Protease inhibitor (PI) buffer and used immediately. Uteri were homogenized in buffer, centrifuged for 10 min at 2500 × g at 4ºC. Supernatant was transferred and centrifuged for 60 minutes at 105,000 × g, discarding the resulting pellets. Protein concentration of the cytosol was determined to be 3.22 mg/mL using a BioRad Protein Assay Kit (compatible with DTT in the TEDG buffer). Cytosol was divided into aliquots (1-6 mL) for either immediate use or storage at -80ºC until use. 2. Saturation (radioligand) Binding Experiment: It was stated that proficiency records for this assay are archived and maintained at CeeTox, Inc. Saturation binding data were not provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency (Tables 1 and 2). Page 86 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None 3. Competitive Binding Experiment: A summary of the experiment conditions for the Competitive Binding Experiment is included in Table 3. TABLE 3. Summary of Conditions for Competitive Binding Experiment a Source of receptor Rat Uterine Cytosol Concentration of radioligand 1 nM Concentration of receptor Sufficient to bind 11-12% of radioligand b Concentration of test substance (as serial dilutions) 10-10 to 10-3 mM Temperature 4±2 °C Incubation time 16-20 hours Composition of assay buffer Tris 10 mM (pH 7.4) EDTA 1.5 mM Glycerol 10% v/v Protease Inhibitor 0.5% v/v DTT 1 mM a Data were obtained from page 15 of the study report. b Calculated by reviewer. Data obtained from pages 35, 40 and 45 of the study report. A solubility test was performed to determine the appropriate solvent (DMSO) using visual observation (it was not stated if magnification was used). On the day of the assay, the specific activity of the stock solution [3H]-17β-estradiol was adjusted for decay over time, and diluted in TEDG + PI buffer to achieve a final concentration of 1 nM. No details were provided on the determination of the optimal protein concentration, but the amount used was sufficient to bind 11-12% of the radioligand, based on the data provided for total binding and total activity. The amount of protein used in each tube was 80.0 µg protein per tube. Serial dilutions of the phosmet, positive control (19-norethindrone), negative control (octyltriethoxysilane), and reference material (non-labeled 17β-estradiol) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of three runs, and each Page 87 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 6 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None run contained three replicates at each concentration, plus six tubes to determine total activity (master mix) resulting in a total of 102 tubes/assay run. TABLE 4. Molar (M) concentrations in Competitive Binding Assay Run a b Phosmet Positive control Negative control Reference Chemical 19-Norethindrone Octyltriethoxysilane Non-labeled 17β-estradiol Tubes 79-102 c Tubes 31-54 c Tubes 55-78 c Tubes 7-30 c 10-10 10-8.5 10-10 Solvent control d 10-9 10-7.5 10-9 10-11 10-8 10-7 10-8 10-10 10-7 10-6.5 10-7 10-9.5 10-6 10-6 10-6 10-9 10-5 10-5.5 10-5 10-8.5 10-4 10-4.5 10-4 10-8 10-3 10-4 10-3 10-7 a Data were obtained from pages 35-49 of the study report. b Each tube contains: 10µL of either the test substance, positive control, negative control, solvent control, or non-labeled 17β-estradiol; 390 µL of TEDG + PI buffer with [3H]-17β-estradiol; and 100 µL of uterine cytosol (with ER), for a total of 500 µL. c Each concentration of each chemical was run in triplicate, for a total of 93 tubes per run plus 9 tubes to determine total activity and total binding. d Solvent is DMSO (4%) in Tubes 7-9. Tubes were incubated with gentle vortexing for 16-20 hours at 4°C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and vortexed for approximately 10 seconds at 5 minute intervals for a total of 15 minutes. Subsequently, the contents of each tube were washed three times as follows: TEDG + PMSF buffer was added, vortexed, centrifuged for 10 min at 1000 x g, and the supernatant decanted and discarded. Ethanol was added to the HAP pellet remaining in each tube to extract the [3H]-17β-estradiol, followed by vortexing, and centrifugation for 10 min at 1000 x g. An aliquot of supernatant was radioassayed by scintillation counting. The temperature was maintained at 4ºC throughout the assay prior to extraction with ethanol. C. DATA ANALYSIS: For the saturation binding experiment (data not reported), total binding and non-specific binding data were modeled via non-linear regression using Graph Pad Prism (version not reported; GraphPad Software, Inc., La Jolla, CA) or XLfit, no method was reported for outlier exclusion. It was not reported if receptor binding data plots were corrected for ligand depletion. For the competitive binding assay, similar methods of nonlinear regression were used to fit a curve (for 17β-estradiol, the positive control, and the test substance) to the Hill equation formula which incorporated log IC50 as a parameter to be estimated. 1. Definitions a. Classification of test material: Classification of the test material is based on the average of three runs. Each run was first individually classified as follows: Page 88 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 7 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None Interactive = lowest point on the fitted curve within the range of the data is less than 50% (i.e., >50% of the radiolabeled estradiol has been displaced from the ER). Not interactive = there are usable data points at or above 10-6M and either the lowest point on the fitted response curve within the range of the data is above75% (i.e., <25% of the radiolabeled estradiol has been displaced from the ER) or a binding curve cannot be fitted and the lowest average percent binding among concentration groups in the data is above 75%. Equivocal up to the limit of concentrations tested = there are no data points at or above a test chemical concentration of 10-6M and either a binding curve can be fit but ≤50% of the radiolabeled estradiol has been displaced from the ER or a binding curve cannot be fit and the lowest average percent binding among concentration groups in the data is >50%. Equivocal = A run is classified as equivocal if it does not fall into any of the categories above. The categorical classification of each run was assigned a numerical value as follows: Run Classification Numerical Value Interactive 2 Equivocal 1 Not interactive 0 Equivocal up to the limit of concentrations tested “missing” The values for each run were then averaged across runs and the chemical classified using the following ranges: Test Material Classification Numerical Range Interactive average ≥1.5 Equivocal 0.5≥ average <1.5 Not interactive average <0.5 Equivocal up to the limit of concentrations tested “missing” b. Descriptors for receptor binding: Bmax: maximum specific binding number (fmol ER/100 µg cytosol protein) measures the concentration of active receptor sites Kd: dissociation constant (nM), measures the affinity of the receptor for its natural ligand IC50: concentration of the test substance at which 50% of the radioligand is displaced from the receptor Relative Binding Affinity (RBA %): IC50 of 17β-estradiol ÷ IC50 of test substance × 100) Page 89 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 8 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None II. RESULTS A. SATURATION BINDING EXPERIMENT: Saturation binding data were not provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laboratory were submitted following a request by the Agency. Saturation binding experiment parameters are presented in Table 5. Figures 1-6 show the specific, non-specific, and total binding curves for [3H]-17β-estradiol to the estrogen receptor for saturation binding and the Scatchard plots for runs 1-3. Page 90 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 9 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None Page 91 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 10 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None Page 92 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 11 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None Page 93 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 12 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None B. COMPETITIVE BINDING EXPERIMENT: The results from the three Competitive Binding Assays are summarized in Table 6 and shown graphically in Figures 7, 8 and 9. The estimated mean log IC50 was not calculated for phosmet, as the percent binding did not cross the 50% mark. The estimated mean log IC50s for 17β-estradiol and the positive control were −9.0 M and −5.5M, respectively. The mean RBA was 0.03% for the positive control. TABLE 6. Competitive Binding Assay of Phosmet with Estrogen Receptor from Rat Uterine Cytosol a Parameter Run 1 b Run 2 b Run 3 b Mean ± SE r2 (unweighted), 17β-estradiol NR NR NR NA 19-norethindrone NR NR NR NA Phosmet NR NR NR NA Log IC50 (M), 17β-estradiol -9 -9.1 -9 -9.0 19-norethindrone -5.6 -5.4 -5.5 -5.5 Phosmet NA NA NA NA -9 -10 -9 -10 IC50 (M), 17β-estradiol 1.00 x 10 7.94 x 10 1.00 x 10 9.26 x 10 19-norethindrone 2.51 x 10-6 3.98 x 10-6 3.16 x 10-6 3.16 x 10-6 Phosmet NA NA NA NA Log RBA (%), 19-norethindrone -3.40 -3.70 -3.50 -3.53 Phosmet NA NA NA NA RBA (%), 19-norethindrone 0.04 0.02 0.03 0.03 Phosmet NA NA NA NA a Data were obtained from pages 21-22 of the study report. b The mean and standard deviation are reported for the concurrent replicates within each run. r2 Goodness of fit RBA (%) Relative binding affinity NA Not applicable NR Not reported Page 94 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 13 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None FIGURE 71. Percentage E2 Bound to the Estrogen Receptor in the Presence of Phosmet, Unlabeled EW, 19-norethindrone or Octyltriethoxysilane. Run 1 Page 95 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None FIGURE 8. Percentage E2 Bound to the Estrogen Receptor in the Presence of Phosmet, Unlabeled EW, 19-norethindrone or Octyltriethoxysilane. Run 2 Page 96 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None FIGURE 9. Percentage E2 Bound to the Estrogen Receptor in the Presence of Phosmet, Unlabeled EW, 19-norethindrone or Octyltriethoxysilane. Run 3 Page 97 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 16 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None The classification of the test material is based on the average of three runs. Each run was individually classified as shown in Table 7. TABLE 7. Binding Classification of Phosmet with Estrogen Receptor a Run 1 2 3 Mean Binding Classificationc Classification category value b NA NA NA NA NA a Technical deficiencies noted in all three runs. b Classification category value: Interactive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of concentrations tested (“missing”, i.e., not included in calculation of mean). c Interactive = mean ≥1.5; Equivocal = 0.5≤ mean <1.5; Not interactive = mean <0.5 C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay functioned properly, each run was evaluated using the following criteria: Tolerance TABLE 8. Criterion a Value Yes No Limit(s) 17β-estradiol fitted curve parameters Loge residual SD ≤2.35 1.20 to 1.95 X Top (% binding) b 94 to 111 95 to 109 X Bottom (% binding) -4 to 1 -1 to 5 X -1 Hill Slope (log10(M) ) -1.1 to -0.7 -1.7 to -1 X Weak Positive control (19-norethindrone) fitted curve parameters Loge residual SD` NA 0.91 to 1.59 Top (% binding) b NA 49 to 100 Bottom (% binding) NA -9 to 0 -1 Hill Slope (log10(M) ) -1.1 to -0.7 -2.5 to -0.9 Solvent concentration DMSO ≤10% 4% X Negative control (octyltriethoxysilane) does not displace more than ≤25% <11% X 25% of [3H]-17β-estradiol from the ER on average across all concentrations a Data were obtained from pages 25, 27 and 29 of the study report. b The EPA Guideline does not define a set of tolerance limits for 19-norethindrone. Acceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable as they show 19-norethindrone to be an acceptable weak positive control. NA Not applicable Additionally, the curve for the reference material showed that increasing concentrations of unlabeled 17β-estradiol displaced [3H]-17β-estradiol in a manner consistent with one-site binding, as indicated by a Hill slope of -1.0 for the first run. The second and third runs showed Hill slopes of −1.4 and −1.7. The weak positive control, 19-norethindrone had a Hill Slope of -1.4 in the third assay, and the second assay showed a top plateau level of 49% binding, a bottom plateau level of −9% and a hill slope of −2.5. These data are consistent with a weak positive control and therefore, invalidate the run. Phosmet was tested over a concentration range that fully defined the top of the curve. The maximum percent binding 105.7% was within 25 percentage points of the value for the Page 98 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 17 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None lowest concentration of the estradiol standard 95.1%. Examination across the runs indicated consistency of placement along the X-axis, and a top plateau for phosmet. III. DISCUSSION AND CONCLUSIONS A. INVESTIGATOR’S CONCLUSIONS: In all three independent runs, the mean specific binding was >80% for the negative control octyltriethoxysilane. The data for 17β-estradiol and 17-norethyndrone were within the acceptable ranges, with deviations which were minor and not considered to reflect true deviation from the suggested ranges outlined in the OPPTS guideline. The data for 19-norethindrone were outside the acceptance criteria in Run 1. However, the positive and negative controls both were within acceptance criteria. This suggests that the problem with the 19-norethindrone data in the second run was most likely in the preparation of the compound. The mean RBA (calculated by dividing the log IC50 of the test material by the log IC50 of 17β-estradiol) [sic] was 0.6 for 19-norethindrone. There was no RBA to be calculated for phosmet. Phosmet solubilized in DMSO was classified as "non-interacting" in all three independent runs and thus had a final classification of "non-interacting" under the conditions of this study. B. AGENCY COMMENTS: The saturation binding experiment demonstrated that the ER isolated from rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the radio-labeled reference estrogen prior to routinely 3 conducting ER Competitive Binding Experiments. The mean Kd for [ H]-17β-estradiol was 0.331 ± 0.061 nM and the mean Bmax was 0.549 ± 0.100 nM (74.55 ± 3.03 fmol/100μg) protein for the prepared rat uterine cytosol Precipitation was observed at 10-4 M (but not 10-3 M) in Run 1 and 10-3 M concentrations in Runs 2 and 3. In these instances, data for those concentrations were disregarded by the study author despite indications of potential interaction with the estrogen receptor. The investigators failed to follow assay guidelines that recommend testing solubility of test substances in increasing solvent concentrations (up to 10% DMSO) and including half log concentrations at the limit of solubility. Because of insufficient attempts to resolve solubility of test substance at concentrations where there were indications of potential ER interactions, there is lack of in all the 3 runs; therefore the assay is considered to be technically inadequate. In addition, the intra assay weak positive control (19-norethindrone) did not show an acceptable response for run 2, and the Hill slope for 17β-estradiol in the third run (−1.7) did not fall within parameter limits specified in the guidelines. C. STUDY DEFICIENCIES: The following deficiencies were noted that are considered to have had an adverse impact on the results, interpretation or conclusions of this study: • Hill slopes for 17β-estradiol outside guideline criteria. The weak positive control, 19- norethindrone showed a top plateau level of 49% binding in the second run, a bottom plateau level of −9% and a hill slope of −2.5. The Investigator noted that the weak positive control data were well outside the guidelines for acceptance, however, in the Page 99 of 260 Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 18 of 18 PHOSMET / 059201 OCSPP 890.1250/ OECD None same run the positive and negative controls were within the acceptable criteria. They suggest that the problem was not due to the system but the preparation of the compound. • Higher concentrations (<10%) of DMSO were not tested to resolve solubility issues, nor was EtOH tried as an alternative solvent (as instructed by guidelines). • No logical physicochemical rationale for precipitate at 10-4 M and not at 10-3 M; either precipitate was overlooked or technical differences in treatment of assay tubes at different concentrations. Insufficient attempts to resolve issues with solubility of the test substance at concentrations that suggested an ER interaction. Page 100 of 260 DATA EVALUATION RECORD PHOSMET Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation EPA Contract No. EP10H001452 Task Assignment No. 2-4-2012 (MRID 48618705) Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive Arlington, VA 22202 Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B Durham, NC 27713 Primary Reviewer Signature: Michelle J. Sharpe-Kass, M.S. Date: 12/05/2011 Secondary Reviewer Signature: Rebecca Bryan, B.S. Date: 12/07/2011 Program Manager: Signature: Jack D. Early, M.S. Date: 12/15/2011 Quality Assurance: Signature: Jack D. Early, M.S. Date: 12/15/2011 This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel. Page 101 of 260 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 102 of 260 Estrogen Receptor Transcriptional Activation (2011) I Page 1 of2 PHOSMET /059201 OCSPP 890.1300/ OECD 455 Primary Reviewer: Sheila Healy. Ph.D. Signa{~ . ~~_...... """"...... q.. ___ Health Effects Division Date: -c~~-7':~-:::?- Secondary Reviewer: Minerva Mercado, Ph.D., DABT Health Effects Division Template version 08/2011 DATA EVALU ATI ON RECORD STUDY TYPE: Estrogen Receptor Transcriptional Activation (Human cell Line, HeLa-9903); OCSPP 890.1300; OECD 455. PC CODE: 059201 DP BARCODE: 0397165 TXR#: 0056 199 CAS No.: 732-11-6 TEST MATERIAL (PURITY): Phosmet (96.8% a.i.) SYNONYMS: 2-( dimethoxyphosphinothioylsulfanylmethyl)isoindole-1 ,3 -dione; Imidan technical CITATION: Willoughby, J.A. Sr. (2011). Phosmet: Estrogen Receptor Transcriptional Activation (Human cell Line, HeLa-9903). CeeTox, Kalamazoo, Ml. Laboratory Study#: 9142V-l 00348ERTA, November 2, 2011. MRID 48618705. Unpublished. SPONSOR: Gowan, 370 Main Street, Yuma, AZ 85364 TEST ORDER#: EDSP-059201-54 EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (ERTA; MRID 48618705) conducted by CeeTox, hERa.-HeLa-9903 cells cultured in vitro were exposed to phosmet (96.8% a.i., lot# GQH9 l 800) at concentrations increasing logarithmically from I 0- 11 to 10-4 M mg/mL in DMSO (0. 1%) for approximately 24 hours. The assay was performed twice using 96-well plates and each phosmet concentration was tested in 6 wells/plate in each as·say. Cells were exposed to the phosmet for 24 ± 2 hours to induce reporter (luciferase) gene products. Luciferase expression in response to activation of the estrogen receptor by phosmet was measured using a proprietary CeeTox Standard Operating Protocol (SOP), which was not included in the report. Phosmet was tested up to the limit of cytotoxicity, 10-4 M. The mean RPCMax for phosmet was 1.6-3.9%, the PCMax was 10·7 Mand the PC1 0 could not be calculated as the RPCMax was less than 10%. Cytotoxicity of 20.1 % was observed in the highest concentration in the first run. A similar trend of cytotoxicity was observed in the second run, with the highest concentration having cell viability of only 81. 7%. In the main assays, the responsiveness of the cells to the very weak positive control 17a. methyltestosterone was lower than the expected values, indicating a decreased sensitivity of the assay to very weak agonists. The PC10 for 17a.-methyltestosterone should have been reached by Page 103 of 260 Estrogen Receptor Transcriptional Activation (2011) / Page 2 of 2 PHOSMET /059201 OCSPP 890.1300/ OECD 455 10-6 M but its maximum RPC was 8.8%, at that concentration. A higher concentration (10-5 M) reached 47% RPC but in the presence of cytotoxicity. Cytotoxicity was also observed at the highest concentrations of 17α-estradiol and corticosterone. Although the conditions of this assay were not optimal to detect very weak activity, phosmet 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 6.8-8.8% PC. Phosmet was only able to reach a maximum of 1.6-3.9% PC when tested up to the highest concentration possible based on cytotoxicity. Because the RPCMax < PC10 in both assay runs, phosmet 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 104 of 260 Estrogen Receptor Transcriptional Activation (2011) / Page 3 of 9 PHOSMET /059201 OCSPP 890.1300/ OECD 455 I. MATERIALS AND METHODS A. MATERIALS 1. Test Substance: Phosmet Description: Technical, white crystalline solid, MW 317.32 g/mol Source: Gowan Company (no catalog#) Lot/Batch #: GQH91800; Exp 1/31/2013 Purity: 96.8% Solubility: Not reported (NR) Volatility: NR Stability: NR Storage conditions: Ambient Vapor pressure: NR CAS #: 732-11-6 Structure: 2. Reference substances 17β-estradiol (strong estrogen; positive control) Supplier: 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 3. Vehicle(s) Solvent: DMSO. Supplier, lot and purity not reported Solvent control 0.1% (final concentration): Page 105 of 260 Estrogen Receptor Transcriptional Activation (2011) / Page 4 of 9 PHOSMET /059201 OCSPP 890.1300/ OECD 455 B. METHODS 1. Cell Culture: Stably-transfected hERα-HeLa-9903 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank and were verified to be free of mycoplasma infection using DNA Fluorochrome analysis by Bionique Testing Laboratories. 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 37ºC. Cells used in the range-finding study (7/5/2011) were from Passage 21. For the main study new cells were brought up from cryopreservation and were Passages 19 and 20 for the experimental runs (8/23/2011 and 8/25/2011). Upon reaching 75-90% confluence, cells were subcultured into test plates. 2. Transcriptional Activation Assays: For each run, cells were plated in 96-well culture plates at a density of 1×104 cells/100 µL medium/well and allowed to attach for at least 3 hours. The growth media was replaced with media containing serial log dilutions of phosmet in DMSO. Cells were incubated for 24 ± 2 hours at 37 ± 1°C. All concentrations were tested in replicates of 6/plate. Additionally, for each concentration 2 replicates/plate were prepared with hERα antagonist ICI 182,780 co-treatment, to allow for the identification of non- specific induction of the luciferase gene. The total final concentration of DMSO was 0.1%. Transcriptional activation of the estrogen receptor was determined using a proprietary CeeTox SOP, which was not provided. A list of reagents was provided, but ingredients of the luciferase assay reagent were identified as proprietary information. a. Preliminary Test: A preliminary test evaluating concentrations ranging from10-6.5 to 10-3 M was conducted to determine the appropriate concentration range and to determine concentrations resulting in insolubility and/or cytotoxicity. Cytotoxicity was determined by a two-read propidium iodide (PI) uptake assay. Cells were seeded into black-walled 96-well plate and exposed to phosmet at logarithmic concentrations of 10-6.5 to 10-3 M; digitonin was also used as a positive control for cytotoxicity. Following exposure, growth medium was removed and cells were exposed to PI. Background-corrected fluorescence was calculated for each well. Limit of solubility was determined by visual inspection of the test materials and controls after preparation of the final 1X dosing solutions in culture media. A sample of the 1x dosing solution was placed into wells of a clear 96-well plate and an endoscope was used to assess precipitation. b. Proficiency Chemicals: Prior to testing unknown chemicals, each laboratory must confirm the responsiveness of the test system, at least once for each newly prepared batch of cell stocks taken from the frozen stock by independent testing of the 10 proficiency chemicals listed below. Proficiency records were not provided in the study report; however, proficiency data for the performing laboratory was submitted following a request by the Agency (MRID 48843501). The responsiveness of the test system was evaluated on March 5, 2010 and April 12 and April 28, 2011. Cells used for the proficiency tests were at passage 15, 25 and 28, respectively. It is not clear if the cells used in the proficiency tests are from the same frozen stock as cells used in the current assay. Page 106 of 260 Estrogen Receptor Transcriptional Activation (2011) / Page 5 of 9 PHOSMET /059201 OCSPP 890.1300/ OECD 455 Concentration Expected Compound CAS No. Notes Range (M) Response a 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 −12 −5 1,3,5-Tris(4- 15797-52-1 10 to 10 Positive Cytotoxic at 100 µM. PCmax approx. 50% hydroxyphenyl)benzene c PC. Binds hERα and has ER antagonistic activity Dibutyl phthalate (DBP) 84-74-2 10−11 to 10−4 Negative d Cytotoxic at 1 mM Atrazine 1912-24-9 10−11 to 10−4 Negative Cytotoxic at 1 mM b Corticosterone 50-22-6 10−10 to 10−4 Negative If not cytotoxic at 1 mM, then that should be the highest tested concentration a Positive = RPCMax ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPCMax fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs b Cytotoxicity is expected to be close to 80% at this concentration. c Compound selected to challenge solubility and cytotoxicity d DBP is negative for ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional activation. A positive result would indicate that the system is detecting activity other than that due to pure ERα, and is therefore unacceptable. c. Reference Chemicals: To ensure the stability of the response from the cell line, 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 10-14 to 10-8 Strong estrogen 17α-estradiol 57-91-0 10-12 to 10-6 Weak estrogen Corticosterone 50-22-6 10-10 to 10-4 Negative compound 17α-methyltestosterone 58-18-4 10-11 to 10-5 Very weak agonist 3. Data analysis: To obtain the transcriptional activity relative to 1 nM E2 positive control (PC), luminescence signals from the concurrent plate were analyzed by subtracting the mean vehicle control value from each well value to normalize the data. Normalized values were then divided by the mean normalized PC value and multiplied by 100 to express relative transcriptional activity as a percentage of the PC (RPC) (software used to calculate RPCMax not reported). The test material was defined as negative for inducing estrogen receptor transcriptional activation if the RPCMax < PC10 in 2 of 2 (or 2 of 3) runs. Log EC50 and Hill slop 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 Page 107 of 260 Estrogen Receptor Transcriptional Activation (2011) / Page 6 of 9 PHOSMET /059201 OCSPP 890.1300/ OECD 455 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: Percent cell viability is reported in Table 1. Cytotoxicity was observed with phosmet at 10-3 M in the rangefinder (7/5/2011). Based on these results, logarithmic concentrations of phosmet from 10-11 to 10-4 M were selected for the assay. However, cytotoxicity was observed at 10-4 M in the first run (8/23/2011) only. TABLE 1. Preliminary Test for Solubility, Cytotoxicity, and Concentration-Selection for Phosmeta Concentration (M) % Viability Comments 10-6.5 104 10-6 101 10-5.5 100 10-5 96 10-4.5 98 10-4 86 10-3.5 80 10-3 15 Cytotoxicity of >20% was observed E2 1nM NR VC NR a Data were obtained from page 19 of the study report. VC = Vehicle control B. Positive and Negative Reference Chemicals 1. Proficiency Chemicals: Responsiveness of cells to proficiency chemicals was tested in duplicate on different days. Results are reported in Table 2. In general, the responses demonstrate proficiency; however some deviations from validated ranges were noted. Mean activity of the PC (1 nM 17β-estradiol) was > 4-fold VC activity on each plate, for all runs. The reference compounds 17β-estradiol, 17α-estradiol and 17α-methyltestosterone were within the acceptable ranges with a few exceptions in runs 1 and 3, as noted in Appendix 1. TABLE 2. Proficiency Chemicals a Compound Expected Response Lab Response Diethylstilbestrol Positive Positive 17α-Ethynyl estradiol Positive Positive Hexestrol Positive Positive Genistein Positive Positive Estrone Positive Positive Butyl paraben Positive Positive 1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive Positive Dibutyl phthalate Negative Negative Atrazine Negative Negative Corticosterone Negative Negative Page 108 of 260 Estrogen Receptor Transcriptional Activation (2011) / Page 7 of 9 PHOSMET /059201 OCSPP 890.1300/ OECD 455 2. Reference Chemicals: Values derived from the concentration response curve (e.g., log PC50, log PC10, log EC50, Hill slope) for the four concurrently run reference materials are included in Table 3. The transcriptional responsiveness of 17α-estradiol and 17β-estradiol were within the acceptable range. The log PC50 and log PC 10 for the very weak agonist 17α- methyltestosterone were not reported. The study author indicated that the PC10 was not reached due to cytotoxicity at the highest concentration. Inspection of the raw data by the reviewer indicated that 17α-methyltestosterone did not attain transcriptional activation equal to 10% of the positive control; therefore a PC10 or PC 50 could not be calculated. A dose- responsive trend was observed starting at approximately at 10-8 M. Maximum RPC of 17α- methyltestosterone was 8.8% at 10-6 M, and cytotoxicity occurred at the next higher concentration tested (10-5 M testosterone) in both experimental runs. TABLE 3. Performance Criteria for Reference Chemicalsa Reference Chemical Acceptable Range Values Acceptable Parameter Run 1 Run 2 Yes No 17β-estradiol Log PC50 -11.4 to -10.1 -10.3 -10.4 X Log PC10 <-11 -11.5 -11.3 X Log EC50 -11.3 to -10.1 -10.3 -10.3 X Hill Slope 0.7 to 1.5 0.9 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 -8.5 -8.6 X Log PC10 -10.7 to -9.3 -9.6 -9.6 X Log EC50 -9.6 to -8.4 -8.5 -8.6 X Hill Slope 0.9 to 2.0 1.2 1.4 X Test range 10-12 to 10-6 M 10-13 to 10-6 M 10-13 to 10-6 M X Corticosterone Test range 10-10 to 10-4 M 10-11 to 10-4 M 10-11 to 10-4 M X 17α-methyltestosterone Log PC50 -6.0 to -5.1 NC NC X Log PC10 -8.0 to -6.2 NC NC X Test range 10-11 to 10-5 M 10-12 to 10-5 M 10-12 to 10-5 M X a Data were obtained from pages 11 and 22 of the study report. NC = not calculated C. DEFINITIVE ASSAY 1. Vehicle and Positive Controls: Data for the vehicle and positive controls are included in Table 4. The overall mean response values (Light units) for the vehicle control were 10892 for the first run and 9933 for the second run. The mean normalized value for the positive control was 253542 for the first run and 167033 for the second run. Light units corresponding to the PC50 (50% of the maximum response) for E2 in this assay is 132217 for the first run and 88483 for the second run; the PC10 (10% of the maximum response) is 35157 for the first run and 25643 for the second run. Page 109 of 260 Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 9 PHOSMET /059201 OCSPP 890.1300/ OECD 455 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 10892 2749 253542 142342 23 2 9933 1246 167033 34868 17 a Data were obtained from pages 30 and 31 of the study report. Values were calculated by the reviewer. b Positive control was 17β-estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC) 2. Test Material: Relative (to the PC) transcriptional activation at each concentration of phosmet during the two assay runs is presented in Table 5. The concentration-response curves depicting fold induction of relative transcriptional activation is presented in Figure 1 below. The mean RPCMax for phosmet was 1.6% for the first run and 3.9% for the second -7 run, and the associated PCMax was 10 M. Cytotoxicity of 20.1% was observed in the highest concentration in the first run, and a similar trend of cytotoxicity (18.3%) was observed in the second run at the highest concentration. Due to the failed responsiveness of these cells to the very weak agonist 17α- methyltestosterone, the possibility that phosmet is a very weak estrogen agonist could not be evaluated TABLE 5. Relative Transcriptional Activation (RTA) of Phosmeta Parameter RTA (mean ± SD); % of Positive Control (PC) Run 1 Run 2 Conc. (M) Mean SD Mean SD 10-4 --b --b 2.6 0.9 10-5 0.5 0.8 2.5 1.2 10-6 1.6 0.8 3.0 1.2 10-7 1.6 0.8 3.9 0.8 10-8 -0.1 0.8 1.7 1.0 10-9 0.8 1.0 1.4 1.3 10-10 -0.1 0.7 0.9 1.2 10-11 0.0 1.8 0.8 1.1 Log EC50 NA NA Hill Slope NA NA RPCMax 1.6% 3.9% -6 -7 -7 PCMax 10 - 10 10 PC50 NA NA PC10 NA NA a Data were obtained from pages 20 and 21 of the study report. b Not reported due to cytotoxicity >20% NA= Not Applicable Page 110 of 260 Estrogen Receptor Transcriptional Activation (2011) / Page 9 of 9 PHOSMET /059201 OCSPP 890.1300/ OECD 455 FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of phosmet compared to the Positive Control. VC= Vehicle Control PC= Positive Control (1 nM E2) 3. Performance Criteria: Proficiency chemicals were tested in duplicate on different days, with appropriate responses (Table 2). The transcriptional responsiveness of 17α-estradiol and 17β-estradiol were within the acceptable range. However, 17α-methyltestosterone did not induce transcriptional activation equal to 50% or 10% of the positive control; therefore a PC10 and PC 50 could not be calculated for this very weak agonist. Based on calculations conducted by the reviewer on the raw data provided, the fold-induction corresponding to PC10 of concurrent PC was greater than 1+2 standard deviations of the fold-induction value of the concurrent VC as expected. The CV of the luminescence data was not reported. Page 111 of 260 Estrogen Receptor Transcriptional Activation (2011) / Page 10 of 9 PHOSMET /059201 OCSPP 890.1300/ OECD 455 III. DISCUSSION AND CONCLUSIONS A. INVESTIGATORS’ CONCLUSIONS: The suitable top concentration of phosmet for use in the transcriptional activation assays was 10-4 M. Cytotoxicity was observed in the highest concentration tested (10-3 M) in the range finding test and at 10-4 M in the first valid run. Cytotoxicity was <20% at all of the exposure concentrations tested in the second valid run. Phosmet did not result in in an increase in luciferase activity at any of the viable concentrations tested in either run; phosmet is not an agonist of hERα in the HeLa-9903 model system. B. AGENCY COMMENTS: Phosmet was tested up to the limit of cytotoxicity, 10-4 M. Cytotoxicity of 20.1% was observed in the highest concentration in the first run. A similar trend of cytotoxicity was observed in the second run, with the highest concentration having cell viability of only 81.7%. -7 The mean RPCMax for phosmet was 1.6-3.9%, the PCMax was 10 M and the PC 10 could not be calculated as the RPCMax was less than 10%. Although the conditions of this assay were not optimal to detect very weak activity, phosmet 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 6.8-8.8% PC. Phosmet was only able to reach a maximum of 1.6-3.9% PC when tested up to the highest concentration possible based on cytotoxicity. Because the RPCMax < PC10 in both assay runs, phosmet was considered negative for estrogen receptor transcriptional activation in this test system. C. STUDY DEFICIENCIES: The following deficiencies were noted: • 17α-methyltestosterone did not induced transcriptional activation equal to 50% or 10% of the positive control; therefore a PC10 and PC50 could not be calculated. • Cytotoxicity observed at the highest concentrations of 17α-estradiol, 17α- methyltestosterone and corticosterone. • The source for the fetal bovine serum was not provided. Page 112 of 260 Estrogen Receptor Transcriptional Activation (2011) / Page 11 of 9 PHOSMET /059201 OCSPP 890.1300/ OECD 455 APPENDIX 1. Performance Criteria for Reference Chemicals in Proficiency Test (MRID 48843501) Reference Chemical Values Acceptable Acceptable Range Parameter Run 1 Run 2 Run 3 Yes No PC Fold Induction 21.1-24.6 75.1-84.7 17.9-20.2 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 Run 1 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 X Test range 10−14 to 10−8 M 10-13 to 10-7 10-15 to 10-7 M 10-15 to 10-7 M Run 1 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 10−12 to 10−6 M 10-15 to 10-7 M 10-13 to 10-5 M 10-13 to 10-5 M Run 1 17α-methyltestosterone Log PC50 −6.0 to −5.1 -6.2 NR -5.2 Run 1,2 Log PC10 −8.0 to −6.2 -8.1 -6.3 -7.7 Run 1 Log EC50 NA -6.2 NR -5.1 NA Hill Slope NA 0.7 0.3 0.4 NA Test range 10−11 to 10−5 M 10-12 to 10-4 M 10-12 to 10-4 M 10-12 to 10-4 M X NA = not applicable NR = not reported Page 113 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԡԹՌԹԯԽՉՍՁՊԽՅԽՆՌ ԢԭԞԡԭԟԹՊԻՇԼԽ ԢԭԞԪԯԦԡ ԢԭԞԤՍՁԼԽՄՁՆԽ ԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑ ԱԽՋՌՅԹՌԽՊՁԹՄ ԭՀՇՋՅԽՌ ԭՍՊՁՌՑ ԠՇՅՅՇՆՆԹՅԽ ԠՀԽՅՁԻԹՄՆԹՅԽ ԦԲԭԞԠ ԠԞՆԹՅԽ ԠԞԫՇ ® ՑՆՇՆՑՅՋ ԦՅՁԼԹՆ ԢԭԞԭԠԠՇԼԽ ԭՊՁՅԹՊՑԯԽՎՁԽՏԽՊ ԧՇԹՆԤԹՁԼՇՋՁԿՆԹՌՍՊԽ ԽՆՁՇՊԻՁԽՆՌՁՋՌԠԹՅԺՊՁԼԿԽԢՆՎՁՊՇՆՅԽՆՌԹՄԦՆԻ ԡԹՌԽ ԽԻՇՆԼԹՊՑԯԽՎՁԽՏԽՊ ԱԽՊՁԪՑԽՊՋՁԿՆԹՌՍՊԽ ԽՆՁՇՊԻՁԽՆՌՁՋՌԠԹՅԺՊՁԼԿԽԢՆՎՁՊՇՆՅԽՆՌԹՄԦՆԻԡԹՌԽ ԭՊՁՅԹՊՑԯԽՎՁԽՏԽՊ ԠԹՌՀԽՊՁՆԽԞՍԺԽԽՁԿՆԹՌՍՊԽ ԟՁՇՄՇԿՁՋՌԲԢԭԞԢԣԢԡԢԯԟԦԳԡԹՌԽ Digitally signed by AMY BLANKINSHIP DN: c=US, o=U.S. Government, AMY ou=USEPA, ou=Staff, cn=AMY ԽԻՇՆԼԹՊՑԯԽՎՁԽՏԽՊ Ջ ԞՅՑԟՄԹՆՃՁՆՋՀՁՈՁԿՆԹՌՍՊԽ BLANKINSHIP, dnQualifier=0000040917 BLANKINSHIP Date: 2015.06.15 05:23:09 -04'00' ԽՆՁՇՊԻՁԽՆՌՁՋՌԲԢԭԞԢԣԢԡԢԯԟԳԦԡԹՌԽ ԡԹՌԽԢՎԹՄՍԹՌՁՇՆԠՇՅՈՄԽՌԽԼ#ԼԼՅՅՑՑՑՑ% ԠԦԱԞԱԦԬԫ ԶՇՊՃ ԡԬ ԭՀՇՋՅԽՌ ՀՇՊՌՌԽՊՅ ԯԽՈՊՇԼՍԻՌՁՇՆ ԞՋՋԹՑ ՏՁՌՀ ՌՀԽ ԣԹՌՀԽԹԼ ԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋ ՈՊՇՅԽՄԹՋ ԣՇՄՄՇՏՁՆԿ ԬԭԭԱ ԹՆԼ ԬԢԠԡ ԤՍՁԼԽՄՁՆԽՋ ԲՆՈՍԺՄՁՋՀԽԼ ՋՌՍԼՑ ՈԽՊԾՇՊՅԽԼԺՑՅՁՌՀԽՊՋԳՁՋԻՁԽՆՌԴԹՊԽՀԹՅԪԹՋՋԹԻՀՍՋԽՌՌՋԩԹԺՌՍԼՑԫՇ ՌՍԼՑՋՈՇՆՋՇՊԽԼ ԺՑԤՇՏԹՆԠՇՅՈԹՆՑԶՍՅԹԞՊՁՒՇՆԹՌՍԼՑԻՇՅՈՄԽՌԽԼԣԽԺՊՍԹՊՑ ԭԹԿԽՇԾ Page 114 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԫՇՌԽ ԱՀԽԲԢԭԞԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅ Ԣԡԭ ԱՁԽՊՋԻՊԽԽՆՁՆԿԺԹՌՌԽՊՑՁՋԻՇՅՈՊՁՋԽԼՇԾ ԽՄԽՎԽՆՋԻՊԽԽՆՁՆԿԹՋՋԹՑՋՁՆՌԽՆԼԽԼՌՇՁԼԽՆՌՁԾՑԹԻՀԽՅՁԻԹՄ0ՋՄՁՃԽՄՑԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՁԽՁՌՋՈՇՌԽՆՌՁԹՄՌՇ ՁՆՌԽՊԹԻՌՏՁՌՀՌՀԽԽՋՌՊՇԿԽՆԹՆԼՊՇԿԽՆՇՊՌՀՑՊՇՁԼ ԢԞՇՊԱ ՈԹՌՀՏԹՑՋԱՀԽՊՇԺՍՋՌՆԽՋՋՇԾՌՀԽԱՁԽՊ ԺԹՌՌԽՊՑՁՋԺԹՋԽԼՇՆՌՀԽՋՌՊԽՆԿՌՀՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՌՇՁԼԽՆՌՁԾՑՈՇՌԽՆՌՁԹՄԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՏՁՌՀ ԻՇՅՈՄԽՅԽՆՌԹՊՑԽՆԼՈՇՁՆՌՋՏՁՌՀՁՆՌՀԽԹՋՋԹՑՏՀԽՊԽԹՎԹՁՄԹԺՄԽԹՆԼՊԽԼՍՆԼԹՆԻՑԹԻՊՇՋՋՌՀԽԺԹՌՌԽՊՑԱՀՍՋ ՌՀԽՊԽՋՍՄՌՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՋՀՇՍՄԼՆՇՌԺԽԻՇՆՋՁԼԽՊԽԼՁՆՁՋՇՄԹՌՁՇՆԺՍՌՊԹՌՀԽՊՋՀՇՍՄԼԺԽԻՇՆՋՁԼԽՊԽԼ ՁՆՌՀԽԻՇՆՌԽՐՌՇԾՇՌՀԽՊԹՋՋԹՑՋՁՆՌՀԽԺԹՌՌԽՊՑԹՋՏԽՄՄԹՋԬՌՀԽՊԻՁԽՆՌՁԾՁԻԹՄՄՑԯԽՄԽՎԹՆՌԦՆԾՇՊՅԹՌՁՇՆ ԬԯԦ ԦՆՇՊԼԽՊՌՇԼԽՌԽՊՅՁՆԽՁԾԹԻՀԽՅՁԻԹՄՀԹՋՌՀԽՈՇՌԽՆՌՁԹՄՌՇՁՆՌԽՊԹԻՌՏՁՌՀՌՀԽԢԞՇՊԱՈԹՌՀՏԹՑՋԹԴԽՁԿՀՌՇԾ ԢՎՁԼԽՆԻԽ ԴՇԢ ԽՎԹՄՍԹՌՁՇՆՇԾԱՁԽՊԹՋՋԹՑՊԽՋՍՄՌՋՁՆԻՇՅԺՁՆԹՌՁՇՆՏՁՌՀՌՀԽԾՁՆԼՁՆԿՋՁՆՌՀԽԬԯԦՋՀՇՍՄԼ ԺԽՍՆԼԽՊՌԹՃԽՆ ՊԽԾԽՊՌՇՌՀԽԴՇԢԡՇԻՍՅԽՆՌ ԢԵԢԠԲԱԦԳԢԲԪԪԞԯԶ ԱՀԽ ԼԹՑ ՋՀՇՊՌՌԽՊՅ ՊԽՈՊՇԼՍԻՌՁՇՆ ԹՋՋԹՑ ՇԾ ՈՀՇՋՅԽՌ ՏՁՌՀ ԾԹՌՀԽԹԼ ՅՁՆՆՇՏՋ ԭՁՅԽՈՀԹՄԽՋ ՈՊՇՅԽՄԹՋ ՏԹՋ ԻՇՆԼՍԻՌԽԼՍՆԼԽՊԾՄՇՏՌՀՊՇՍԿՀԻՇՆԼՁՌՁՇՆՋՁՐՌԽԽՆԿՊՇՍՈՋՇԾՅՇՆՌՀՇՄԼԾՁՋՀ ՅԹՄԽՋԹՆԼԾԽՅԹՄԽՋՈԽՊ ԿՊՇՍՈ ԿՊՇՍՈՋ ՈԽՊ ՌՊԽԹՌՅԽՆՌ ՏԽՊԽ ԽՐՈՇՋԽԼ ՌՇ ՈՀՇՋՅԽՌ ՈՍՊՁՌՑ ԹՌ ՆՇՅՁՆԹՄ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ՇԾ ԻՇՆՌՊՇՄ ԹՆԼ ՅԿ ԹՁԩ ՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ՏԽՊԽ ԩԬԮ ԹՆԼՅԿԹՁԩԱՀԽՌԽՋՌՋՑՋՌԽՅՏԹՋՅԹՁՆՌԹՁՆԽԼԹՌՌՇՇԠԹՆԼԹՈԥՇԾՌՇ ՍՊՎՁՎԹՄՏԹՋՄՇՏԹՌՌՀԽՀՁԿՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ՏՁՌՀՇՆՄՑՇՆԽՅԹՄԽԹՆԼՇՆԽԾԽՅԹՄԽՋՍՊՎՁՎՁՆԿՌՇՋՌՍԼՑ ՌԽՊՅՁՆԹՌՁՇՆ ԽՆԼՈՇՁՆՌՋԹՌՌՀՁՋՌՊԽԹՌՅԽՆՌՄԽՎԽՄՏԽՊԽՆՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼՇՊՁՆԻՄՍԼԽԼՁՆՌՀԽՊԽՋՍՄՌՋԞՌՌՀԽ ՁՆՌԽՊՅԽԼՁԹՌԽԻՇՆԻԽՆՌՊԹՌՁՇՆՌՀԽՊԽՏԹՋԹԼԽԻՊԽԹՋԽՁՆՋՍՊՎՁՎԹՄԻՇՅՈԹՊԽԼՌՇԻՇՆՌՊՇՄԹՆԼԿԽՆԽՊԹՄՁՒԽԼՇՎԽՊՌ ՋՁԿՆՋՇԾՌՇՐՁԻՁՌՑՁՆԺՇՌՀՅԹՄԽՋԹՆԼԾԽՅԹՄԽՋՁՆԻՄՍԼՁՆԿՄԽՌՀԹՊԿՑՁՆԹՄՄՋՍՊՎՁՎՁՆԿԾՁՋՀԹՆԼՄՇՋՋՇԾԽՉՍՁՄՁԺՊՁՍՅՁՆ ԾԽՅԹՄԽՋԞՌՌՀԽՄՇՏԻՇՆԻԽՆՌՊԹՌՁՇՆՋՍՊՎՁՎԹՄՏԹՋՊԽԼՍԻԽԼԺՑԻՇՅՈԹՊԽԼՌՇԻՇՆՌՊՇՄԟՇԼՑՏԽՁԿՀՌԹՆԼՄԽՆԿՌՀ ՏԽՊԽՆՇՌՋՁԿՆՁԾՁԻԹՆՌՄՑԼՁԾԾԽՊԽՆՌՁՆՅԹՄԽՇՊԾԽՅԹՄԽԾՁՋՀԹՌԹՆՑՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԦՆՌՀԽԻՇՆՌՊՇՄԿՊՇՍՈՋՈԹՏՆՁՆԿՇԻԻՍՊՊԽԼԹՌՄԽԹՋՌԽՎԽՊՑԼԹՑՋՁՆՌՏՇՇԾՌՀԽԾՇՍՊՊԽՈՄՁԻԹՌԽՋԹՆԼԾԽԻՍՆԼՁՌՑՏԹՋ ԽԿԿՋԾԽՅԹՄԽԼԹՑՊԽՈՄՁԻԹՌԽ ԾԽԻՍՆԼՁՌՑ ՏԹՋ ԽԿԿՋԾԽՅԹՄԽԼԹՑ ԾՇՊ ՌՀՊԽԽ ՊԽՈՄՁԻԹՌԽՋ ԹՆԼ ԽԿԿՋԾԽՅԹՄԽԼԹՑ ԾՇՊ ՇՆԽ ՊԽՈՄՁԻԹՌԽ ԣԽՊՌՁՄՁՒԹՌՁՇՆ ՋՍԻԻԽՋՋ ՁՆ ՌՀԽ ԻՇՆՌՊՇՄ ԿՊՇՍՈ ԹՎԽՊԹԿԽԼ ԞՌ ՌՀԽ ՁՆՌԽՊՅԽԼՁԹՌԽԻՇՆԻԽՆՌՊԹՌՁՇՆՈՀՇՋՅԽՌՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼԾԽՊՌՁՄՁՌՑ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ Ո ԺՑԹՆԼ ԾԽԻՍՆԼՁՌՑ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ Ո ԺՑԻՇՅՈԹՊԽԼՌՇՌՀԽԻՇՆՌՊՇՄ ԭԹԿԽՇԾ Page 115 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԣԽՅԹՄԽ ԿՇՆԹԼՇՋՇՅԹՌՁԻ ՁՆԼԽՐ ԤԦ ՏԹՋ ՋՁԿՆՁԾՁԻԹՆՌՄՑ ՊԽԼՍԻԽԼ ԺՑ ԹՌ ՌՀԽ ՁՆՌԽՊՅԽԼՁԹՌԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ Ո ՊԽՄԹՌՁՎԽ ՌՇ ԻՇՆՌՊՇՄ ՅԹՄԽ ԤԦ ՏԹՋ ՍՆԹԾԾԽԻՌԽԼ ԺՑ ՈՀՇՋՅԽՌ ՌՊԽԹՌՅԽՆՌ Ո ԴՀՁՄԽ ՆՇՌ ՋՌԹՌՁՋՌՁԻԹՄՄՑ ՋՁԿՆՁԾՁԻԹՆՌ ԪԹՆՆԴՀՁՌՆԽՑ Ո ՅԽԼՁԹՆ ՅԹՄԽ ՆՍՈՌՁԹՄ ՌՍԺԽՊԻՄԽ ՋԻՇՊԽՋ ՏԽՊԽՊԽԼՍԻԽԼԺՑԹՌՌՀԽՁՆՌԽՊՅԽԼՁԹՌԽԻՇՆԻԽՆՌՊԹՌՁՇՆԻՇՅՈԹՊԽԼՌՇՌՀԽԻՇՆՌՊՇՄ ՆՇՌՍԺԽՊԻՄԽՋՏԽՊԽՇԺՋԽՊՎԽԼ ԾՇՊ ԾԽՅԹՄԽՋ ԪԹՄԽ ԹՆԼ ԾԽՅԹՄԽ ՎՁՌԽՄՄՇԿԽՆՁՆ ԳԱԤ ՄԽՎԽՄՋ ՏԽՊԽ ՆՇՌ ՋՁԿՆՁԾՁԻԹՆՌՄՑ ԼՁԾԾԽՊԽՆՌ ԾՊՇՅ ՌՀԽ ԻՇՆՌՊՇՄ ԹՄՌՀՇՍԿՀՌՀԽՊԽՏԹՋԹՆԹՈՈԹՊԽՆՌՁՆԻՊԽԹՋԽՁՆՅԹՄԽԳԱԤԹՌՌՀԽՁՆՌԽՊՅԽԼՁԹՌԽԻՇՆԻԽՆՌՊԹՌՁՇՆԼՍԽՋՇՄԽՄՑՌՇ ՀՁԿՀՄԽՎԽՄՋՁՆՌՏՇՅԹՄԽՋՁՆՌՀՁՋԿՊՇՍՈԭՄԹՋՅԹՋԽՐՋՌԽՊՇՁԼՋՏԽՊԽՆՇՌՅԽԹՋՍՊԽԼ ԤՇՆԹԼԹՄՋՌԹԿՁՆԿԽԾԾԽԻՌՋՏԽՊԽՁՆԼՁԻԹՌԽԼԾՇՊԾԽՅԹՄԽԾՁՋՀԹՌՌՀԽՄՇՏՌՊԽԹՌՅԽՆՌԻՇՆԻԽՆՌՊԹՌՁՇՆԹՆԼԾՇՊՅԹՄԽՋԹՆԼ ԾԽՅԹՄԽՋ ԹՌ ՌՀԽ ՁՆՌԽՊՅԽԼՁԹՌԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԢԾԾԽԻՌՋ ՇՆ ԿՇՆԹԼԹՄ ՀՁՋՌՇՈԹՌՀՇՄՇԿՑ ՁՆ ՅԹՄԽ ԾՁՋՀ ՁՆԻՄՍԼԽԼ ԹՆ ՁՆԻՊԽԹՋԽԼՈԽՊԻԽՆՌԹԿԽՇԾՋՈԽՊՅԹՌՇԿՇՆՁԹ ԹՆԼՅՁՄԼԼՁԾԾՍՋԽՌՀՁԻՃԽՆՁՆԿՇԾՌՀԽԿԽՊՅՁՆԹՄԽՈՁՌՀԽՄՁՍՅՄՁՆՁՆԿԺՑ ՋՈԽՊՅԹՌՇԿՇՆՁԹԹՌՌՀԽՁՆՌԽՊՅԽԼՁԹՌԽԻՇՆԻԽՆՌՊԹՌՁՇՆ ՁՆԾԽՅԹՄԽԾՁՋՀՁՆԻՁԼԽՆԻԽՋՇԾԼԽԻՊԽԹՋԽԼՑՇՄՃՋՑՆՌՀԽՋՁՋՏԽՊԽ ՇԺՋԽՊՎԽԼ ԹՌ ԺՇՌՀ ՄՇՏ ԹՆԼ ՁՆՌԽՊՅԽԼՁԹՌԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ՊԽՄԹՌՁՎԽ ՌՇ ՌՀԽ ԻՇՆՌՊՇՄՋ ԬԻԻԹՋՁՇՆԹՄ ԼՁՋՊՍՈՌՁՇՆՋ ՇԾ ԳԱԤ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՏԽՊԽ ՆՇՌԽԼ ՁՆ ՌՀԽ ԾՇՄՄՁԻՄԽՋ ՇԾ ՋՇՅԽ ԾԽՅԹՄԽՋ ԹՆԼ ՅՁՆՁՅԹՄ ՁՆՌԽՊՋՌՁՌՁԹՄԾՁԺՊՇՋՁՋՏԹՋՇԺՋԽՊՎԽԼԹՌՌՀԽՁՆՌԽՊՅԽԼՁԹՌԽԻՇՆԻԽՆՌՊԹՌՁՇՆԦՆԹԼԼՁՌՁՇՆՁՆԻՁԼԽՆԻԽՋՇԾՁՆԻՊԽԹՋԽԼՇՇԻՑՌԽ ԹՌՊԽՋՁԹՏԽՊԽՇԺՋԽՊՎԽԼՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏՁՌՀԹՆԼՇԾՌՀԽԾՁՋՀԽՐՀՁԺՁՌՁՆԿ ՌՀՁՋԾՁՆԼՁՆԿՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼՄՇՏԹՆԼՅՁԼՌՊԽԹՌՅԽՆՌՋՊԽՋՈԽԻՌՁՎԽՄՑԦՆՇԾՌՀԽՋԽԻԹՋԽՋԿՊԹՆՍՄՇՅԹՌՇՍՋ ՁՆԾՄԹՅՅԹՌՁՇՆՏԹՋՇԺՋԽՊՎԽԼ ԽՎՁԼԽՆԻԽՇԾՅՁԻՊՇՋՈՇՊՁԼՁԹՁՆԾԽԻՌՁՇՆՏԹՋԹՄՋՇՇԺՋԽՊՎԽԼ ԦՆՋՍՅՅԹՊՑԹՌՌՀԽՅՁԼԻՇՆԻԽՆՌՊԹՌՁՇՆԾԽԻՍՆԼՁՌՑԾԽՊՌՁՄՁՒԹՌՁՇՆԹՆԼԾԽՅԹՄԽՏԽՊԽԤԦՏԽՊԽԼԽԻՊԽԹՋԽԼԻՇՅՈԹՊԽԼ ՌՇԻՇՆՌՊՇՄՋԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑՏԹՋԹԾԾԽԻՌԽԼԹՋՋՀՇՏՆԺՑԼԽԻՊԽԹՋԽԼՑՇՄՃՋՑՆՌՀԽՋՁՋՁՆՌՀԽՄՇՏԹՆԼՅՁԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԾԽՅԹՄԽՋ ԹՆԼ ՇԻԻԹՋՁՇՆԹՄ ԼՁՋՊՍՈՌՁՇՆՋ ՇԾ ԾՇՄՄՁԻՍՄԹՊ ԳԱԤ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԱՀԽ ՈՊՇՈՇՊՌՁՇՆ ՇԾ ՋՈԽՊՅԹՌՇԿՇՆՁԹ ՁՆ ՌՀԽ ՌԽՋՌԽՋ ՏԹՋ ՁՆԻՊԽԹՋԽԼ ԹՌ ՌՀԽ ՀՁԿՀ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԥՁԿՀ ՅՇՊՌԹՄՁՌՑ ԹՆԼ ԻՄՁՆՁԻԹՄ ՋՁԿՆՋ ՇԾ ՌՇՐՁԻՁՌՑՁՆՌՀՁՋՋՌՍԼՑՅԹՑԻՇՆԾՇՍՆԼՌՀԽՁՆՌԽՊՈՊԽՌԹՌՁՇՆՇԾՌՀԽՋԽՈԹՊԹՅԽՌԽՊՋ ԞՄՄ ՌՀԽ ՈԽՊԾՇՊՅԹՆԻԽ ԹՆԼ ՎԹՄՁԼՁՌՑ ԻՊՁՌԽՊՁԹ ՏԽՊԽ ՅԽՌ ԾՇՊ ՌՀՁՋ ՋՌՍԼՑ ԽՐԻԽՈՌ ԾՇՊ ՇՆԽ ԣՇՊ ՇՆԽ ՊԽՈՄՁԻԹՌԽ ՁՆ ՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ ՌՀԽ ԾԽԻՍՆԼՁՌՑ ՏԹՋ ԽԿԿՋԾԽՅԹՄԽԼԹՑ ԹՆԼ ՁՌ ԼՁԼ ՆՇՌ ՋՈԹՏՆ ԹՌ ՄԽԹՋՌ ԽՎԽՊՑ ԼԹՑՋ ՅԽԽՌՁՆԿ ՇՆԽ ՇԾ ՌՀԽՋԽ ԻՊՁՌԽՊՁԹ ՁՋ ՍՋԽԼ ՌՇ ԼԽՅՇՆՋՌՊԹՌԽ ԹԻՌՁՎԽ ՋՈԹՏՆՁՆԿ ԞՄՌՀՇՍԿՀ ՆՇՌ Թ ՎԹՄՁԼՁՌՑ ԻՊՁՌԽՊՁԹ ՌՀԽ ԽՆԼՈՇՁՆՌՋ ԹՌ ՌՀԽ ՀՁԿՀ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄ ՅԿ ԹՁԩ ՏԽՊԽ ՆՇՌ ՋՌԹՌՁՋՌՁԻԹՄՄՑ ԹՆԹՄՑՒԽԼ ԼՍԽ ՌՇ ՅՇՊՌԹՄՁՌՑ ԱՀԽՊԽԾՇՊԽՇՆՄՑՌԽՋՌՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՏԽՊԽԹՆԹՄՑՒԽԼՁՆՌՀՁՋՋՌՍԼՑԞԼԼՁՌՁՇՆԹՄՄՑՌՀԽՋՍՊՎՁՎԹՄՊԹՌԽՁՆ ՌՀԽ ՄՇՏԽՋՌ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ՏԹՋ ԹՆԼ ՁՆ ՇՆԽ ՊԽՈՄՁԻԹՌԽ ՌՀՊԽԽ ՇԾ ՌՀԽ ԾՇՍՊ ԹՎԹՁՄԹԺՄԽ ԾԽՅԹՄԽՋ ԼՁԽԼ ԭԹԿԽՇԾ Page 116 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԽՋՋԽՆՌՁԹՄՄՑՄԽԹՎՁՆԿՇՆՄՑՌՀՊԽԽՎՁԹԺՄԽՊԽՈՄՁԻԹՌԽՋՁՆՌՀԽՄՇՏԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆԹՎԹՁՄԹԺՄԽԾՇՊԹՆԹՄՑՋՁՋՁՆՌԽՊՈՊԽՌԹՌՁՇՆ ԞՄՌՀՇՍԿՀ ՁՆԼՁՎՁԼՍԹՄՋ ԼՁՋՈՄԹՑՁՆԿ ԻՄՁՆՁԻԹՄ ՋՁԿՆՋ ՇԾ ՌՇՐՁԻՁՌՑ ՏԽՊԽ ՆՇՌ ԽՐԻՄՍԼԽԼ ԾՊՇՅ ՋՌԹՌՁՋՌՁԻԹՄ ԹՆԹՄՑՋԽՋ ՌՀԽ ՈՊԽՋԽՆԻԽՇԾՌՀԽՋԽԻՄՁՆՁԻԹՄՋՁԿՆՋԹՄՇՆԿՏՁՌՀՌՀԽՀՁԿՀՊԹՌԽՇԾՁՆԻՊԽԹՋԽԼՇՇԻՑՌԽԹՌՊԽՋՁԹՁՆՌՀԽԻՇՆՌՊՇՄՋՅԹՃԽՋՁՌ ՅՇՊԽԼՁԾԾՁԻՍՄՌՌՇՁՆՌԽՊՈՊԽՌՌՀԽՊԽՋՍՄՌՋՇԾՌՀԽՋՌՍԼՑ ԞՄՌՀՇՍԿՀՌՀԽՅՇՊՌԹՄՁՌՑՁՆՌՀԽՀՁԿՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄԻՄՁՆՁԻԹՄՋՁԿՆՋՇԾՌՇՐՁԻՁՌՑՁՆՌՀԽՁՆՌԽՊՅԽԼՁԹՌԽՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԹՆԼ ՆՇՌԽՏՇՊՌՀՑ ՊԹՌԽ ՇԾ ՇՇԻՑՌԽ ԹՌՊԽՋՁԹ ՁՆ ԻՇՆՌՊՇՄ ԾԽՅԹՄԽՋ ՅԹՃԽ ՌՀԽ ՊԽՋՍՄՌՋ ԼՁԾԾՁԻՍՄՌ ՌՇ ՁՆՌԽՊՈՊԽՌ ՏՀԽՆ ԻՇՆՋՁԼԽՊԽԼՁՆԻՇՆՌԽՐՌՇԾՇՌՀԽՊԹՎԹՁՄԹԺՄԽԱՁԽՊԢԡԭԼԹՌԹԾՇՊՈՀՇՋՅԽՌՌՀՁՋՋՌՍԼՑՋԹՌՁՋԾՁԽՋՌՀԽԢԡԭԱՁԽՊ ԱԽՋՌԬՊԼԽՊՊԽՉՍՁՊԽՅԽՆՌԾՇՊԹԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑ ԬԠԭԭԤՍՁԼԽՄՁՆԽ ԭԹԿԽՇԾ Page 117 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԯԽՋՍՄՌՋՑՆՇՈՋՁՋ ԱԽՋՌԬՊԿԹՆՁՋՅԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ՅՇՆՌՀՋ ԪԽԹՆԺՇԼՑՏԽՁԿՀՌԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ՁԾՅԽԹՋՍՊԽԼ ԪԹՄԽԿ ԾԽՅԹՄԽԿ ԪԽԹՆՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ՁԾՅԽԹՋՍՊԽԼ ԫՇՌՊԽՈՇՊՌԽԼ ԱԽՋՌԱՑՈԽ ԣՄՇՏՌՀՊՇՍԿՀՌԹՌՁԻՌԹՌՁԻԯԽՆԽՏԹՄ ԣՄՇՏՌՀՊՇՍԿՀ ԱԹԺՄԽ ՍՅՅԹՊՑՇԾԯԽՈՊՇԼՍԻՌՁՎԽԹՆԼԥԭԤԢԾԾԽԻՌՋՁՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑ ԣԱԯԞ ՏՁՌՀԭՀՇՋՅԽՌ ԱՊԽԹՌՅԽՆՌ ԱՍԺԽՊԻՄԽԻՇՊԽ ԤԦ ԤՇՆԹԼԹՄԥՁՋՌՇ ԭՄԹՋՅԹԳԱԤ ՅԿԹՁԩ ԣԽՊՌ ԣԽԻՍՆԼՁՌՑ #ՅԽԹՆ ՍԻԻԽՋՋ Ԫ ԣ Ԫ ԣ Ԫ ԣ Ԫ ԣ ՅԽԹՋՍՊԽԼ% ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԶԽՋ ԫՇ ԫՇ ԶԽՋ ԶԽՋ ԫՇ ԫՇ ԫՇ ԶԽՋ ԶԽՋ ԶԽՋ ԫՇ ԫՇ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԠՇՆԻԠՇՆԻԽՆՌՊԹՌՁՇՆԡՁԾԾԡՁԾԾԽՊԽՆԻԽԢȕԽՋՌՊԹԼՁՇՄԣԣԽՅԹՄԽԣԽՊՌԣԽՊՌՁՄՁՒԹՌՁՇՆԤԦԤՇՆԹԼՇ ՇՅԹՌՁԻԦՆԼԽՐԥՁՋՌՇԥՁՋՌՇՈԹՌՀՇՄՇԿՑ ԪԪԹՄԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԱԱԽՋՌՇՋՌԽՊՇՆԽԳԱԤԳՁՌԽՄՄՇԿԽՆՁՆ Ԟ՟ՑԽՋՠՁՆԼՁԻԹՌԽՋԹՋՁԿՆՁԾՁԻԹՆՌԼՁԾԾԽՊԽՆԻԽԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ ԻՄԽԹՆՏԹՌԽՊ ԻՇՆՌՊՇՄՍՆՄԽՋՋ ՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼ ԱՀԽԻՊՁՌԽՊՁԹԾՇՊՋՁԿՆՁԾՁԻԹՆԻԽԹՊԽԼԽՋԻՊՁԺԽԼՁՆՌՀԽԯԽՎՁԽՏԽՊ0ՋԞՆԹՄՑՋՁՋԹՆԼՌԹՌՁՋՌՁԻԹՄԳԽՊՁԾՁԻԹՌՁՇՆՋԽԻՌՁՇՆՋՇԾ ՌՀԽԡԢԯԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼ ԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՀՁԿՀՅՇՊՌԹՄՁՌՑ ԭԹԿԽՇԾ Page 118 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԦԪԞԱԢԯԦԞԩԞԫԡԪԢԱԥԬԡ ԤԲԦԡԢԩԦԫԢԣԬԩԩԬԴԢԡ ԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼԹԻԻՇՊԼՁՆԿՌՇՌՀԽԲԢԭԞԬԠԭԭ ՟ԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՎԽԞՋՋԹՑՠԹՆԼԬԢԠԡ ԱՀԽԾՇՄՄՇՏՁՆԿԼԽՎՁԹՌՁՇՆՋՏԽՊԽՆՇՌԽԼ ԱՀԽ ԾԽԻՍՆԼՁՌՑ ՁՆ ՇՆԽ ՊԽՈՄՁԻԹՌԽ ՇԾ ՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ ՏԹՋ ԽԿԿՋԾԽՅԹՄԽԼԹՑՆՇՊԼՁԼՌՀԽՊԽՈՄՁԻԹՌԽՋՈԹՏՆԹՌՄԽԹՋՌԽՎԽՊՑԼԹՑՋ ԪԽԽՌՁՆԿ ՇՆԽՇԾՌՀԽՋԽԻՊՁՌԽՊՁԹՁՋՆԽԽԼԽԼՌՇԼԽՅՇՆՋՌՊԹՌԽԹԻՌՁՎԽՋՈԹՏՆՁՆԿ ԱՀԽ ԽՆԼՈՇՁՆՌՋ ԹՌ ՌՀԽ ՀՁԿՀ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄ ՅԿ ԹՁԩ ՏԽՊԽ ՆՇՌ ՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑ ՇՆՄՑՇՆԽՅԹՄԽԹՆԼՇՆԽԾԽՅԹՄԽՋՍՊՎՁՎԽԼ ԹՌ ՋՌՍԼՑ ՌԽՊՅՁՆԹՌՁՇՆ ԞՄՋՇ ԻՄՁՆՁԻԹՄ ՋՁԿՆՋ ՇԾ ՌՇՐՁԻՁՌՑ ԹՌ ՌՀԽ ՅԿ ԹՁԩ ԻՇՆԻԽՆՌՊԹՌՁՇՆՅԹՃԽՊԽՋՍՄՌՋԹՌՌՀՁՋՌՊԽԹՌՅԽՆՌՄԽՎԽՄԼՁԾԾՁԻՍՄՌՌՇՁՆՌԽՊՈՊԽՌ ԱՀԽՈՀՑՋՁԻՇԻՀԽՅՁԻԹՄՈՊՇՈԽՊՌՁԽՋՇԾՌՀԽՌԽՋՌՅԹՌԽՊՁԹՄՏԽՊԽՆՇՌՊԽՈՇՊՌԽԼ ԱՀԽ ՍՆՁՇՆՁՒԽԼ ԹՅՅՇՆՁԹ ԹՆԼ ՊԽՋՁԼՍԹՄ ԻՀՄՇՊՁՆԽ ՁՆ ՌՀԽ ՌԽՋՌ ՏԹՌԽՊ ՏԽՊԽ ՆՇՌ ՊԽՈՇՊՌԽԼԱՀԽԬԠԭԭՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹԽՋՌԹԺՄՁՋՀՅԹՐՁՅՍՅՄԽՎԽՄՋ ԾՇՊ ՌՀԽՋԽ ՎԹՄՍԽՋ ԹՆԼ ՁՌ ՁՋ ՍՆԻՄԽԹՊ ՁԾ ՌՀԽ ՅԹՐՁՅՍՅ ՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ՏԽՊԽ ԽՐԻԽԽԼԽԼ ԱՀԽՀՁԿՀՁՆԻՁԼԽՆԻԽՇԾՅՇՊՌԹՄՁՌՑԹՆԼԻՄՁՆՁԻԹՄՋՁԿՆՋՅԹՃԽՌՀԽՊԽՋՍՄՌՋՇԾՌՀՁՋՋՌՍԼՑԼՁԾԾՁԻՍՄՌՌՇՁՆՌԽՊՈՊԽՌ ԥՇՏԽՎԽՊԾԽՏԽԾԾԽԻՌՋՏԽՊԽՋԽԽՆՁՆՌՀԽԹԺՋԽՆԻԽՇԾՇՎԽՊՌՌՇՐՁԻՁՌՑՁՆՌՀԽԹՎԹՁՄԹԺՄԽՊԽՈՄՁԻԹՌԽՋՇԾՌՀԽՄՇՏ ՌՊԽԹՌՅԽՆՌՄԽՎԽՄԹՆԼՁՌՁՋԻՇՆՋՁԼԽՊԽԼՍՆՄՁՃԽՄՑՌՀԹՌՊԽՈԽՌՁՌՁՇՆՇԾՌՀԽՋՌՍԼՑԹՌՄՇՏԽՊՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՏՇՍՄԼՑՁԽՄԼ ՋՌՊՇՆԿԽՊՊԽՋՍՄՌՋ ԠԬԪԭԩԦԞԫԠԢ ՁԿՆԽԼ ԹՆԼ ԼԹՌԽԼ Ԥԩԭ ԮՍԹՄՁՌՑ ԞՋՋՍՊԹՆԻԽ ԹՆԼ ԡԹՌԹ ԠՇՆԾՁԼԽՆՌՁԹՄՁՌՑ ԻՄԹՁՅՋ ՋՌԹՌԽՅԽՆՌՋՏԽՊԽՈՊՇՎՁԼԽԼԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼՁՆԹԻԻՇՊԼԹՆԻԽՏՁՌՀԤԩԭ ՌԹՆԼԹՊԼՋ ԹՋ ՈՍԺՄՁՋՀԽԼ ԺՑ ՌՀԽ Բ ԢԭԞ Ԡԣԯ ԭԹՊՌՋ ԹՆԼ ԬԢԠԡ ԭՊՁՆԻՁՈՄԽՋՇԾԤԩԭ#ԢԫԳԪԠԠԥԢԪ % ԞԱԢԱԪԞԱԢԯԦԞԩ ԭՀՇՋՅԽՌԠԞԫՇ ԡԽՋԻՊՁՈՌՁՇՆ ԫՇՆԽՊԽՈՇՊՌԽԼ ԢՐՈՁՊԹՌՁՇՆԼԹՌԽԧԹՆՍԹՊՑ ԭԹԿԽՇԾ Page 119 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԬԢԠԡՊԽԻՇՅՅԽՆԼՋԼԽՋԻՊՁԺՁՆԿՏԹՌԽՊՋՇՄՍԺՁՄՁՌՑՅԽՄՌՁՆԿԺՇՁՄՁՆԿՈՇՁՆՌՋՌԹԺՁՄՁՌՑՁՆՏԹՌԽՊԹՆԼՄՁԿՀՌՈԨԹԭՇՏՇՊ ԨՇՏՎԹՈՇՊՈՊԽՋՋՍՊԽՇԾՌԽՋՌԻՇՅՈՇՍՆԼԽՐՈՁՊԹՌՁՇՆԼԹՌԽ ԩՇՌԫՇԟԹՌԻՀԫՇ ԤԮԥ ԭՍՊՁՌՑ ԦՅՈՍՊՁՌՁԽՋ ԫՇՆԽՊԽՈՇՊՌԽԼ ՌԹԺՁՄՁՌՑ ՇԾ ԠՇՅՈՇՍՆԼ ԞՆԹՄՑՌՁԻԹՄ ՎԽՊՁԾՁԻԹՌՁՇՆ ՇԾ ՌՀԽ ՌԽՋՌ ՅԹՌԽՊՁԹՄ ԾՊՇՅ ՌՁՅԽ ԹՆԼ ԼԹՑՋ ՑՁԽՄԼԽԼՊԽԻՇՎԽՊՁԽՋՇԾՌՇՇԾՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋԱԽՋՌՋՇՄՍՌՁՇՆԾՇՊՌՁԾՁԽԼՏՁՌՀՈՀՇՋՅԽՌ ԹՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾԹՆԼՅԿԹՁԩՑՁԽՄԼԽԼՊԽԻՇՎԽՊՁԽՋՇԾՌՇ ՏՁՌՀ ՇՆԽՇՍՌՄՁԽՊՊԽԻՇՎԽՊՑՇԾՈՇՋՋՁԺՄՑԼՍԽՌՇՋՈՁՃՁՆԿՀԹՆԼՄՁՆԿՇՊՁՆՋՌՊՍՅԽՆՌԽՊՊՇՊՋԱՀԽԠԳ0ՋԾՇՊ ՌՀԽՁՆԼՁՎՁԼՍԹՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԹՆԿԽԼԾՊՇՅԾՇՊՌՀԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼԱՀԽՋԽԼԹՌԹՁՆԼՁԻԹՌԽ ՌՀԽՌԽՋՌՅԹՌԽՊՁԹՄՏԹՋՋՌԹԺՄԽՍՆԼԽՊՌՀԽՌԽՋՌԻՇՆԼՁՌՁՇՆՋ ՌՇՊԹԿԽԠՇՆԼՁՌՁՇՆՋՇԾ ԱԽՋՌԠՀԽՅՁԻԹՄՋ ԱՀԽՌԽՋՌՋՍԺՋՌԹՆԻԽՏԹՋՋՌՇՊԽԼԹՌՊՇՇՅՌԽՅՈԽՊԹՌՍՊԽՁՆԼԹՊՃՎԽՆՌՁՄԹՌԽԼ ԻԹԺՁՆԽՌ ԭԹԿԽՇԾ Page 120 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԟԱԽՋՌՇՊԿԹՆՁՋՅ ԱԹԺՄԽ ԤԽՆԽՊԹՄԦՆԾՇՊՅԹՌՁՇՆԞԺՇՍՌՌՀԽԱԽՋՌՈԽԻՁԽՋԹՆԼԞԻԻՄՁՅԹՌՁՇՆ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ՈԽԻՁԽՋԻՇՅՅՇՆՆԹՅԽ ԣԹՌՀԽԹԼԪՁՆՆՇՏ ԢԭԞՊԽԻՇՅՅԽՆԼՋԾԹՌՀԽԹԼՅՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ՈԽԻՁԽՋՋԻՁԽՆՌՁԾՁԻՆԹՅԽ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ՈԽԻՁԽՋՋՌՊԹՁՆ ՁԾՋՌԹՌԽԼ ԫՇՌՊԽՈՇՊՌԽԼ ԴԽՊԽԾՁՋՀՇԺՌԹՁՆԽԼԾՊՇՅԹ ԶԽՋ ՇՍՊԻԽՆՇՌՊԽՈՇՊՌԽԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽԾՊՇՅԹՋՁՆԿՄԽ ՋՁՆԿՄԽՄԹԺՇՊԹՌՇՊՑՋՌՇԻՃ! ՄԹԺՇՊԹՌՇՊՑՋՌՇԻՃ ԴԽՊԽԹԻԻՄՁՅԹՌՁՇՆԻՇՆԼՁՌՁՇՆՋ ԶԽՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽԹԻԻՄՁՅԹՌԽԼ ՋԹՅԽԹՋԼԽԾՁՆՁՌՁՎԽՌԽՋՌ! ՍՆԼԽՊՏԹՌԽՊՉՍԹՄՁՌՑԹՆԼՁՄՄՍՅՁՆԹՌՁՇՆ ԻՇՆԼՁՌՁՇՆՋՌՀԹՌԹՊԽՋՁՅՁՄԹՊՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽ ՌԽՋՌ ԞԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼ ԼԹՑՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՌՏՇՏԽԽՃ ԹԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼԫՇՌԽՌՀԹՌՌՀԽԹԻԻՄՁՅԹՌՁՇՆ ՈԽՊՁՇԼՁՋԼՁԾԾԽՊԽՆՌԾՊՇՅՌՀԽՋՍԺՋԽՉՍԽՆՌՁՆ ՋՁՌՍՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽ Page ԭԹԿԽՇԾ 121 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԡԽՌԹՁՄՋՇՆՀԽԹՄՌՀ ԪՇՊՌԹՄՁՌՁԽՋԼՍՊՁՆԿԼԹՑՋՈՊՁՇՊՌՇՈՊԽ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՅՇՊՌԹՄՁՌՑԼՍՊՁՆԿՌՀԽ ԽՐՈՇՋՍՊԽՈԽՊՁՇԼՏԽՊԽՆՇՌՊԽՈՇՊՌԽԼ ԼԹՑՋՈՊՁՇՊՌՇՌՀԽՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽԺԽ ՄԽՋՋՌՀԹՆՇԾՌՀԽԻՍՄՌՍՊԽՈՇՈՍՄԹՌՁՇՆԦԾ ԣՁՋՀԼՁԼՆՇՌՊԽԻԽՁՎԽԹՆՑՌՊԽԹՌՅԽՆՌԾՇՊ ՅՇՊՌԹՄՁՌՑԼՍՊՁՆԿՌՀԽՋԽԼԹՑՋՁՋԿՊԽԹՌԽՊՌՀԹՆ ԼՁՋԽԹՋԽԼՍՊՁՆԿԹԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽԾՁՋՀԺԽ ՊԽՂԽԻՌԽԼԦԾՅՇՊՌԹՄՁՌՑՁՋԺԽՌՏԽԽՆԢԭԞ ԟԽՀԹՎՁՇՊԹՄԹԺՆՇՊՅԹՄՁՌՁԽՋՇՊԻՄՁՆՁԻԹՄՋՁԿՆՋ ՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽՀԽՄԼԹՆՇՌՀԽՊ ՏԽՊԽՆՇՌՊԽՈՇՊՌԽԼ ԼԹՑՋԦԾՅՇՊՌԹՄՁՌՁԽՋԿՊԽԹՌԽՊՌՀԹՆՇԻԻՍՊ ԼՍՊՁՆԿՌՀՁՋԽՐՌԽՆԼԽԼԹԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽԾՁՋՀՆՇՌԺԽՍՋԽԼ ԱՑՈԽՇԾԾՇՇԼ ԩՁՎԽԺՊՁՆԽՋՀՊՁՅՈ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽԾԽԼԾՊՇՒԽՆ ՆԹՍՈՄՁՁ ԞՊՌԽՅՁԹ ԺՊՁՆԽՋՀՊՁՅՈՌՏՁԻԽՈԽՊԼԹՑՌՇՈՊՇՅՇՌԽ ԹԻՌՁՎԽՊԽՈՊՇԼՍԻՌՁՇՆԹՆԼՅԹՁՆՌԹՁՆԺՇԼՑ ՇՍՊԻԽՇԾԾՇՇԼ ԫՇՌՊԽՈՇՊՌԽԼ ԻՇՆԼՁՌՁՇՆ ԣՊԽՉՍԽՆԻՑՇԾԾԽԽԼՁՆԿ ՌՁՅԽՋԼԹՑ ԡԽՌԹՁՄՋՇՆԾԽԽԼՁՆԿ ԫՇՆԽ Page ԭԹԿԽՇԾ 122 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԣՁՋՀԽՄԽԻՌՁՇՆԹՆԼԭՊԽԢՐՈՇՋՍՊԽԭԽՊԾՇՊՅԹՆԻԽ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԞԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ՅՇՆՌՀՋ ԯԽՈՇՊՌԽԼԹՋՏԽԽՃՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՊՇԼՍԻՌՁՎԽՄՑՅԹՌՍՊԽ ՋԽՐՍԹՄՄՑԼՁՅՇՊՈՀՁԻ ԾՁՋՀՅՇՆՌՀՋ ՇՄԼ ԪԽԹՆՏԽՁԿՀՌՇԾՅԹՄԽՋԹՌՌԽՋՌ Կ ԟԹՋԽԼՇՆՅԹՄԽՋՍՋԽԼՌՇՋՌՇԻՃ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԹՋՍԺՋԹՅՈՄԽ ՁՆՁՌՁԹՌՁՇՆ ՁԾԼԽՌԽՊՅՁՆԽԼ ԹՉՍԹՊՁԹԾՇՊՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽ ՇԾԾՁՋՀԺԽՏԽՁԿՀԽԼԺԽԾՇՊԽՌՀԽՌԽՋՌՌՇ ԽՋՌՁՅԹՌԽՌՀԽՅԽԹՆՏԽՁԿՀՌԾՇՊԽԹԻՀՋԽՐԦՌՁՋ ԯԹՆԿԽՇԾՁՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՋ ՌՇԿ ԦՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՋՏՁՌՀՁՆչՇԾՌՀԽ ՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՌՀԽՁՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՇԾ ՅԹՄԽՋ ԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ՁԾ ԽՋՌՁՅԹՌԽԼՅԽԹՆ ԽԹԻՀԾՁՋՀՋԽՄԽԻՌԽԼԾՇՊՌՀԽՌԽՋՌԺԽՏՁՌՀՁՆ ԼԽՌԽՊՅՁՆԽԼ չՇԾՌՀԽԽՋՌՁՅԹՌԽԼՅԽԹՆԾՇՊԽԹԻՀՋԽՐ ԪԽԹՆՏԽՁԿՀՌՇԾԾԽՅԹՄԽՋԹՌՌԽՋՌ Կ ԟԹՋԽԼՇՆԾԽՅԹՄԽՋՍՋԽԼՌՇՋՌՇԻՃ ՁՆՁՌՁԹՌՁՇՆ ՁԾԼԽՌԽՊՅՁՆԽԼ ԹՉՍԹՊՁԹԾՇՊՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽ ԯԹՆԿԽՇԾՁՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՋ ՌՇԿ ԦՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՋՏՁՌՀՁՆչՇԾՌՀԽ ԾԽՅԹՄԽՋ ԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ՁԾ ԽՋՌՁՅԹՌԽԼՅԽԹՆ ԼԽՌԽՊՅՁՆԽԼ ԪԽԹՆՄԽՆԿՌՀՇԾՅԹՄԽՋԹՌՌԽՋՌ ԫՇՌՊԽՈՇՊՌԽԼ ՁՆՁՌՁԹՌՁՇՆ ՁԾԼԽՌԽՊՅՁՆԽԼ Page ԭԹԿԽՇԾ 123 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԪԽԹՆՄԽՆԿՌՀՇԾԾԽՅԹՄԽՋԹՌՌԽՋՌ ԫՇՌՊԽՈՇՊՌԽԼ ՁՆՁՌՁԹՌՁՇՆ ՁԾԼԽՌԽՊՅՁՆԽԼ ԡՍՊԹՌՁՇՆՇԾՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽ ԼԹՑՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՇԾԼԹՑՋ ԴԽՊԽՈՊԽԽՐՈՇՋՍՊԽԻՇՆԼՁՌՁՇՆՋ ԶԽՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՈՊԽԽՐՈՇՋՍՊԽ ՁԼԽՆՌՁԻԹՄՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ! ԻՇՆԼՁՌՁՇՆՋՁՆԻՄՍԼՁՆԿՌԽՅՈԽՊԹՌՍՊԽ ՈՀՇՌՇՈԽՊՁՇԼԾԽԽԼՁՆԿԽՌԻԺԽՁԼԽՆՌՁԻԹՄՌՇ ԼԽԾՁՆՁՌՁՎԽՌԽՋՌԻՇՆԼՁՌՁՇՆՋ ԫՍՅԺԽՊՇԾՈՊԽԽՐՈՇՋՍՊԽՌԹՆՃՋ ԢՐՌՊԹՊԽՈՄՁԻԹՌԽՋՏԽՊԽԽՋՌԹԺՄՁՋՀԽԼՌՇ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԹԼԼՁՌՁՇՆԹՄՌԹՆՃՋՋԽՌ ԹԻԻՇՍՆՌԾՇՊԹՈՇՌԽՆՌՁԹՄՄԹԻՃՇԾՋՈԹՏՆՁՆԿ ՍՈԹՌՌՀԽԺԽԿՁՆՆՁՆԿՇԾՈՊԽԽՐՈՇՋՍՊԽՏՁՄՄ ՁՆՋՇՅԽԻՀԹՅԺԽՊՋԹՆԼՇՊՅՇՊՌԹՄՁՌՑ ԽՆՋՍՊԽՌՀԹՌՋՍԾԾՁԻՁԽՆՌՊԽՈՄՁԻԹՌԽՋՏՁՌՀՌՀԽ ԼՍՊՁՆԿՌՀՁՋՈՀԹՋԽ ԻՇՊՊԽԻՌՋԽՐՊԹՌՁՇԹՊԽԹՎԹՁՄԹԺՄԽԾՇՊՌՀԽ ԼԽԾՁՆՁՌՁՎԽՌԽՋՌ ԫՍՅԺԽՊՇԾՅԹՄԽՋՈԽՊՌԹՆՃ ԫՍՅԺԽՊՇԾԾԽՅԹՄԽՋՈԽՊՌԹՆՃ Page ԭԹԿԽՇԾ 124 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԭՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑ ԽԿԿՋԾԽՅԹՄԽ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՈՊԽԽՐՈՇՋՍՊԽ ՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑ ԾԽԻՍՆԼՁՌՑՁՆԽԹԻՀՊԽՈՄՁԻԹՌԽ ՌԹՆՃ ՋԽՄԽԻՌԽԼ ՊԽՈՄՁԻԹՌԽ ԾՇՊՍՋԽՁՆՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌԺԽԹՌՄԽԹՋՌ ԽԿԿՋԾԽՅԹՄԽՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑՊԽՈՄՁԻԹՌԽ ԼՍՊՁՆԿՌՀԽԼԹՑՋՈՊՁՇՊՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ ԫՍՅԺԽՊՇԾՋՈԹՏՆՋԼՍՊՁՆԿՈՊԽ ՌՏՇՌՁՅԽՋՁՆԼԹՑՋ ՈԹՏՆՁՆԿՇԻԻՍՊՊԽԼԹՌՄԽԹՋՌԽՎԽՊՑ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՋՈԹՏՆՁՆԿՇԻԻՍՊԹՌ ԽՐՈՇՋՍՊԽ ԼԹՑՋ ՄԽԹՋՌՌՏՁԻԽՁՆՌՀԽԼԹՑՋՈՊՁՇՊՌՇՌՀԽ ԼԽԾՁՆՁՌՁՎԽՌԽՋՌ ԡԽՌԹՁՄՋՇՆՈՊԽԽՐՈՇՋՍՊԽ ԫՇՆԽ Page ԭԹԿԽՇԾ 125 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԠԢՐՈՇՋՍՊԽՑՋՌԽՅ ԱԹԺՄԽ ՍՅՅԹՊՑՇԾԦՆԾՇՊՅԹՌՁՇՆՇՆՌՀԽԢՐՈՇՋՍՊԽՑՋՌԽՅԹՆԼԱԽՋՌԳԽՋՋԽՄԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԱՑՈԽՇԾԽՐՈՇՋՍՊԽ ԣՄՇՏՌՀՊՇՍԿՀ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԹԾՄՇՏ ՌՀՊՇՍԿՀՋՑՋՌԽՅԞՋՆՇՌԽԼՁՆՌՀԽ ԠՇՊՊԽԻՌՁՇՆՋԹՆԼԠՄԹՊՁԾՁԻԹՌՁՇՆՋԼՇԻՍՅԽՆՌ ՌՀԽՍՋԽՇԾԹՋՌԹՌՁԻՊԽՆԽՏԹՄՋՑՋՌԽՅՁՋՆՇՌ ՊԽԻՇՅՅԽՆԼԽԼԾՇՊՌՀՁՋԹՋՋԹՑ ԱՑՈԽՇԾԾՄՇՏՌՀՊՇՍԿՀԼՁՄՍՌՁՇՆ ԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏ ԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊՋՇՊ ՋՑՋՌԽՅ ՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊ ԻՇՆՌՁՆՍՇՍՋԾՄՇՏՋԽՊՁԹՄԼՁՄՍՌԽՊՋԹՊԽ ՊԽԻՇՅՅԽՆԼԽԼ ԣՄՇՏՌՀՊՇՍԿՀՊԹՌԽ ԠԹԩՀՊ ԠԹՄԻՍՄԹՌԽԼԺՑՌՀԽՊԽՎՁԽՏԽՊԺԹՋԽԼՇՆ ԯԽԻՇՅՅԽՆԼԽԼԾՄՇՏՌՀՊՇՍԿՀՊԹՌԽՁՋ ԽՉՍՁՎԹՄԽՆՌՌՇՎՇՄՍՅԽ ԩԻՑԻՄԽԹՌԹՊԹՌԽՇԾԻՑԻՄԽՋՀՊՋ ՅԩՅՁՆ ԩՀՊ ՇՊԹՌՄԽԹՋՌՌՇՌԹՄ ՊԽՈՄԹԻԽՅԽՆՌՋԼԹՑ ՎՇՄՍՅԽԽՐԻՀԹՆԿԽՋՈԽՊԼԹՑ ԲԢՆՎՁՊՇՆՅԽՆՌԹՄԭՊՇՌԽԻՌՁՇՆԞԿԽՆԻՑ ԢԭԞ ԠՇՊՊԽԻՌՁՇՆՋԹՆԼԠՄԹՊՁԾՁԻԹՌՁՇՆՋՇՆԱԽԻՀՆՁԻԹՄԞՋՈԽԻՌՋՇԾՌՀԽԱԽՋՌԤՍՁԼԽՄՁՆԽՋԾՇՊՌՀԽԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊԻՊԽԽՆՁՆԿ ԭՊՇԿՊԹՅԱՁԽՊԞՋՋԹՑՋ ԬԠԭԭԱԽՋՌԤՍՁԼԽՄՁՆԽԽՊՁԽՋ ԪԹՊԻՀԬԾԾՁԻԽՇԾԠՀԽՅՁԻԹՄԹԾԽՌՑԹՆԼԭՇՄՄՍՌՁՇՆԭՊԽՎԽՆՌՁՇՆ ԬԠԭԭ ԴԹՋՀՁՆԿՌՇՆԡԠ ՀՌՌՈ ՏՏՏԽՈԹԿՇՎԽՆԼՇՈՍԺՋԹՋՋԹՑՎԹՄՁԼԹՌՁՇՆԻՄԹՊՁԾՁԻԹՌՁՇՆԼՇԻՈԼԾ ԞԼԼՁՌՁՇՆԹՄԿՍՁԼԹՆԻԽԾՇՊԹՉՍԹՌՁԻՌԽՋՌԼԽՋՁԿՆՁՋՄՇԻԹՌԽԼՁՆԬԠԭԭԤՍՁԼԽՄՁՆԽՈԽԻՁԹՄԠՇՆՋՁԼԽՊԹՌՁՇՆՋԾՇՊԠՇՆԼՍԻՌՁՆԿԞՉՍԹՌՁԻԩԹԺՇՊԹՌՇՊՑՌՍԼՁԽՋ Page ԭԹԿԽՇԾ 126 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԡԽՌԹՁՄՋՇՆՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊ ԣՄՇՏՋՈՄՁՌՌՁՆԿԻՀԹՅԺԽՊՋՏԽՊԽՍՋԽԼ ԯԽԻՇՅՅԽՆԼԽԼՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊԾՄՇՏ ԾՄՇՏՌՀՊՇՍԿՀՋՑՋՌԽՅՋ ԺԽՌՏԽԽՆԼՁՄՍՌԽՊԻԽՄՄՋԹՆԼՌՀԽՊԽՈՄՁԻԹՌԽ ՌՀՊՇՍԿՀՋՑՋՌԽՅՋ ԪՁՐՁՆԿԻՀԹՅԺԽՊՁՋ ՌԽՋՌՎԽՋՋԽՄՋ ՊԽԻՇՅՅԽՆԼԽԼԺՍՌՆՇՌՊԽՉՍՁՊԽԼ ԞԽՊԹՌՁՇՆՁՋՆՇՌՊԽԻՇՅՅԽՆԼԽԼԾՇՊՅՁՐՁՆԿ ԱՊԽԹՌՅԽՆՌՊԽԻՇՎԽՊՁԽՋՏԽՊԽՆՇՌՅԽԹՋՍՊԽԼ ԞԼԽՅՇՆՋՌՊԹՌՁՇՆՌՀԹՌՌՀԽՌԽՋՌՋՇՄՍՌՁՇՆ ՈՊՁՇՊՌՇՌԽՋՌՁՆՁՌՁԹՌՁՇՆՌՇՁՆԼՁԻԹՌԽՁԾԼՁՄՍՌԽՊ ՁՋԻՇՅՈՄԽՌԽՄՑՅՁՐԽԼԺԽԾՇՊԽՁՆՌՊՇԼՍԻԽԼ ՀԹԼՊԽԹԻՀԽԼԽՉՍՁՄՁԺՊՁՍՅ ՁՆՌՇՌՀԽՌԽՋՌՋՑՋՌԽՅՁՋՊԽԻՇՅՅԽՆԼԽԼ ԱՀԽՊԽԻՇՅՅԽՆԼԽԼԾՄՇՏՋՈՄՁՌՌՁՆԿԹԻԻՍՊԹԻՑ ԣՄՇՏՋՈՄՁՌՌՁՆԿԹԻԻՍՊԹԻՑՏԹՋՆՇՌՊԽՈՇՊՌԽԼ ՁՋՏՁՌՀՁՆ ԞԽՊԹՌՁՇՆ! ԫՇ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹԽՊԹՌՁՇՆՁԾԼՁՋՋՇՄՎԽԼ ՇՐՑԿԽՆՊԽԹԻՀԽՋ ՅԿԩ ՋԹՌՍՊԹՌՁՇՆ ՇՍՊԻԽՇԾԼՁՄՍՌՁՇՆՏԹՌԽՊ ԴԽՄՄՏԹՌԽՊ ԡՁՄՍՌՁՇՆՏԹՌԽՊՏԹՋԹԽՊԹՌԽԼՈՊՁՇՊՌՇՍՋԽ ԢԭԞՊԽԻՇՅՅԽՆԼՋՆԹՌՍՊԹՄՇՊՊԽԻՇՆՋՌՁՌՍՌԽԼ ՏԹՌԽՊ ՁՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՆԹՌՍՊԹՄ ՏԹՌԽՊԺԽՋՌԽՊՁՄՁՒԽԼՏՁՌՀԲԳԹՆԼՌԽՋՌԽԼԾՇՊ ՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊ ՈՇՋՋՁԺՄԽԻՇՆՌԹՅՁՆԹՆՌՋԬԢԠԡԹԻԻԽՈՌՋ ԹՆՑՏԹՌԽՊՁՆՏՀՁԻՀՌՀԽՌԽՋՌՋՈԽԻՁԽՋՋՀՇՏ ԻՇՆՌՊՇՄՋՍՊՎՁՎԹՄԹՌՄԽԹՋՌԹՋԿՇՇԼԹՋ ՁՆԼՁԻԹՌԽԼՁՆՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽ Page ԭԹԿԽՇԾ 127 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԴԹՋԼՁՄՍՌՁՇՆՏԹՌԽՊԹՆԹՄՑՒԽԼ ԶԽՋ ԾՇՊՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋ ԹՆԼՇՌՀԽՊԻՇՆՌԹՅՁՆԹՆՌՋ! ԱԽՋՌՎԽՋՋԽՄՌՑՈԽՅԹՌԽՊՁԹՄՋ ԤՄԹՋՋԹՉՍԹՊՁԹՏՁՌՀ ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌՏԹՌԽՊ ՋՁՄՁԻՇՆԽՋԽԹՄԹՆՌԹՆԼ ԻՇՆՌԹԻՌՈՇՊՌՁՇՆՋՇԾՌՀԽՋՑՋՌԽՅՆՇՌ ՆՑՄՇՆ ԻՇՅՈՊՇՅՁՋԽՌՀԽՋՌՍԼՑ ԽԿԹՄՄԿՄԹՋՋ ՎԽՋՋԽՄՋՇՊԿՄԹՋՋՎԽՋՋԽՄՋՏՁՌՀՋՌԹՁՆՄԽՋՋ ՋՌԽԽՄԾՊԹՅԽՋԹՊԽԹԻԻԽՈՌԹԺՄԽԽՐԹՅՈՄԽՋ ԱԽՋՌՎԽՋՋԽՄՋՁՒԽ ԻՅՏՁԼԽՐԻՅ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԩՌԽՋՌ ՄՇՆԿՐԻՅՀՁԿՀ ԻՀԹՅԺԽՊՋ ԽԿՐՐԻՅ ՋՇՄՍՌՁՇՆԼԽՈՌՀԻՅ ԣՁՄՄՎՇՄՍՅԽ ԩ ԢԭԞՊԽԻՇՅՅԽՆԼՋԩՋՇՄՍՌՁՇՆՈԽՊՌԹՆՃ ՈԹՏՆՁՆԿՋՍԺՋՌՊԹՌԽՅԹՌԽՊՁԹՄ ՁՆԻՀԼՁԹՅԽՌԽՊԹԿԽԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԽԹԻՀՌԹՆՃԻՇՆՌԹՁՆ ԭԳԠԻՍՌՁՆՀԹՄԾ ԹՊԻՀԽՋ ՌՀՊԽԽՋԽՅՁԻՁՊԻՍՄԹՊՋՈԹՏՆՁՆԿՋՍԺՋՌՊԹՌԽՋ ԹՆԼՁՆՌՇԻՅՋԽԻՌՁՇՆՋ ԽԿԹԿԽԼԭԳԠՈՁՈԽԻՅՁՆ ԹՆԼՈՇՋՁՌՁՇՆԽԼՁՆԹ ՄԽՆԿՌՀՋՈՄՁՌՄԽՆԿՌՀՏՁՋԽ ՋԻՊԽԽՆԽԼԿՄԹՋՋԻՇՄՄԽԻՌՁՇՆ ՌՊԹՑ Page ԭԹԿԽՇԾ 128 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ՈԹՏՆՁՆԿՋՍԺՋՌՊԹՌԽՋՁՒԽ ԢԹԻՀՋՍԺՋՌՊԹՌԽՈՄԹԻԽԼՇՆՋԻՊԽԽՆԽԼԿՄԹՋՋ ԭԳԠԻՍՌՁՆՀԹՄԾ ԹՊԻՀԽՋ ԻՇՄՄԽԻՌՁՇՆՌՊԹՑ ՐՐԻՅԩՐԴՐ ԹՆԼՁՆՌՇԻՅՋԽԻՌՁՇՆՋ ԥ ԻՇՎԽՊԽԼՏՁՌՀԻՅՋՌԹՁՆՄԽՋՋՋՌԽԽՄ ՅԽՋՀ ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋՇՆԽՐՈՇՋՍՊԽ ԫՇՆԽ ՋՑՋՌԽՅ ԱԹԺՄԽ ՍՅՅԹՊՑՇԾԴԹՌԽՊԮՍԹՄՁՌՑԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋՁՆՌՀԽԱԽՋՌՑՋՌԽՅ ԪԽԹՋՍՊԽՅԽՆՌ ԭԹՊԹՅԽՌԽՊ ԪՁՆՁՅՍՅ ԪԹՐՁՅՍՅ ԪԽԹՆ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԦՆՌԽՊՎԹՄ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌԽՅՈԽՊԹՌՍՊԽչՇԠ ԱԽՅՈԽՊԹՌՍՊԽ ոԠ ԠՇՆՌՁՆՍՇՍՋ ՁՆՌԽՊՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊՌՊԽԹՌՅԽՆՌ ԼՁԾԾԽՊԽՆՌՁԹՄՋՋՀՇՍՄԼՆՇՌԽՐԻԽԽԼՇԠ Ոԥ ԡԹՁՄՑԢԭԞՊԽԻՇՅՅԽՆԼՋՈԥՌՇ ԢԭԞՊԽԻՇՅՅԽՆԼՋԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆ ԡՁՋՋՇՄՎԽԼՇՐՑԿԽՆ ՅԿԩ ԡԹՁՄՑ ԡԬ ՅԿԩ ԹՁՊՋԹՌՍՊԹՌՁՇՆ ԱՇՌԹՄԹՄՃԹՄՁՆՁՌՑ ՅԿԩԹՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՇՌԹՄԹՄՃԹՄՁՆՁՌՑ ԴԽԽՃՄՑ ԠԹԠԬ ՅԿԩԹՋԠԹԠԬ Page ԭԹԿԽՇԾ 129 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԪԽԹՋՍՊԽՅԽՆՌ ԭԹՊԹՅԽՌԽՊ ԪՁՆՁՅՍՅ ԪԹՐՁՅՍՅ ԪԽԹՆ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԦՆՌԽՊՎԹՄ ԥԹՊԼՆԽՋՋ ՅԿԩԹՋԠԹԠԬ ԴԽԽՃՄՑ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՇՌԹՄՇՊԿԹՆՁԻ ԱՇՌԹՄՇՊԿԹՆՁԻԻԹՊԺՇՆ ՅԿԩ ԫԞ ԫՇՎԡԽԻ ԻԹՊԺՇՆՁՆԼՁՄՍՌՁՇՆՏԹՌԽՊԺԽ ՅԿԩ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՍՆՁՇՆՁՒԽԼ μ ԲՆՁՇՆՁՒԽԼԹՅՅՇՆՁԹ Կԩ ԫՇՌՊԽՈՇՊՌԽԼ ԫՇՌՊԽՈՇՊՌԽԼ ԫՇՌՊԽՈՇՊՌԽԼ ԫՇՌՊԽՈՇՊՌԽԼ ԹՅՅՇՆՁԹՁՆՌՀԽԼՁՄՍՌՁՇՆՏԹՌԽՊԺԽ μ Կԩ μ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՊԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽ ԯԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽ Կԩ ԫՇՌՊԽՈՇՊՌԽԼ ԫՇՌՊԽՈՇՊՌԽԼ ԫՇՌՊԽՈՇՊՌԽԼ ԫՇՌՊԽՈՇՊՌԽԼ μ ՁՆԼՁՄՍՌՁՇՆՏԹՌԽՊԺԽ Կԩ ԤԽՆԽՊԹՄՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋԾՇՊԾՊԽՉՍԽՆԻՑ ՇԾՅԽԹՋՍՊԽՅԽՆՌՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌ ՌԽՅՈԽՊԹՌՍՊԽՈԥԹՆԼԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆ ԺԽՅԽԹՋՍՊԽԼՁՆԹՄՄՌԽՋՌՌԹՆՃՋԹՌՄԽԹՋՌ μ ՏԽԽՃՄՑԹՆԼՌՀԹՌՀԹՊԼՆԽՋՋԹՆԼԹՄՃԹՄՁՆՁՌՑ ԠՇՆԼՍԻՌՁՎՁՌՑ ՅՀՇՋԻՅ ԴԽԽՃՄՑ ԺԽՅԽԹՋՍՊԽԼՁՆԻՇՆՌՊՇՄՋԹՆԼՁՆՇՆԽՌԹՆՃ ԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆԹՌՄԽԹՋՌ ՏԽԽՃՄՑԦՆԹԼԼՁՌՁՇՆԻՇՆՌՁՆՍՇՍՋ ՌԽՅՈԽՊԹՌՍՊԽՅՇՆՁՌՇՊՁՆԿՇԾԹՌՄԽԹՋՌՇՆԽ ՌԹՆՃՁՋԽՆԻՇՍՊԹԿԽԼ Page ԭԹԿԽՇԾ 130 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ ԪԽԹՆՋՏԽՊԽԻԹՄԻՍՄԹՌԽԼԺՑՌՀԽՊԽՎՁԽՏԽՊԹՋՌՀԽԹՎԽՊԹԿԽՇԾՌՀԽՅՁՆՁՅԹԹՆԼՅԹՐՁՅԹԾՇՊՌՀԽՊԹՆԿԽՋՈՊՇՎՁԼԽԼԹԻՊՇՋՋԻՇՆՌՊՇՄԹՆԼՌՊԽԹՌԽԼՄԽՎԽՄՋ ԡՌՍԼՑԡԽՋՁԿՆԹՆԼԞԼԼՁՌՁՇՆԹՄԢՐՈԽՊՁՅԽՆՌԹՄԠՇՆԼՁՌՁՇՆՋ ԱԹԺՄԽ ԯԹՆԿԽԣՁՆԼՁՆԿՌՍԼՑԠՇՆԼՁՌՁՇՆՋ ՁԾԞՈՈՄՁԻԹԺՄԽ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԴԹՋԹՊԹՆԿԽԾՁՆԼԽՊԻՇՆԼՍԻՌԽԼ! ԶԽՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋԻՇՆԼՍԻՌՁՆԿԹՊԹՆԿԽԾՁՆԼԽՊ ՁԾՀՇՍՊԩԠԼԹՌԹԾՇՊՌՀԽԾԹՌՀԽԹԼՅՁՆՆՇՏ ԹՊԽՍՆԹՎԹՁՄԹԺՄԽ ԦԾՑԽՋՏՀԹՌՏԹՋՌՀԽՅԽՌՀՇԼԾՇՊ ԟԹՋԽԼՇՆՌՀԽՊԽՋՍՄՌՋՇԾ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ ԼԽՌԽՊՅՁՆՁՆԿՌՀԽՀՁԿՀԽՋՌՌԽՋՌ ՀՊՊԹՆԿԽԾՁՆԼՁՆԿ ԻՇՆԻԽՆՌՊԹՌՁՇՆԺԽՋԽՄԽԻՌԽԼԺԹՋԽԼՇՆՌՇՐՁԻՁՌՑ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽՊԹՆԿԽ ՌԽՋՌԹՆԼՁՆԻՇՆՋՍՄՌԹՌՁՇՆ ԼԹՌԹԾՇՊՇՌՀԽՊԾՁՋՀՋՌՍԼՁԽՋՇՊՋՈԽԻՁԽՋՁԾ ԾՁՆԼԽՊ! ՏՁՌՀՌՀԽՋՌՍԼՑՋՈՇՆՋՇՊ ԹՎԹՁՄԹԺՄԽԬՌՀԽՊՏՁՋԽԽՁՌՀԽՊՌՀԽՋՇՄՍԺՁՄՁՌՑ ՄՁՅՁՌՇԾՌՀԽՌԽՋՌԻՇՅՈՇՍՆԼՇՊՅԿԩ ՏՀՁԻՀԽՎԽՊՁՋՄՇՏԽՊ ՁՋԹՈՈՊՇՈՊՁԹՌԽ ՈԽԻՁԽՋ ԣԹՌՀԽԹԼԪՁՆՆՇՏ Page ԭԹԿԽՇԾ 131 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԩՁԾԽՋՌԹԿԽ ԫՇՌՊԽՈՇՊՌԽԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՊԹՆԿԽԾՁՆԼՁՆԿՌԽՋՌՋ ԺԽՈԽՊԾՇՊՅԽԼՏՁՌՀԾՁՋՀՇԾՋՁՅՁՄԹՊԹԿԽԹՆԼ ՋՁՒԽՌՇՌՀՇՋԽՌՀԹՌՏՇՍՄԼԺԽՍՌՁՄՁՒԽԼՁՆՌՀԽ ՌԽՋՌ ԱԽՋՌԼՍՊԹՌՁՇՆ ՀՊՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՀՇՍՊԽՐՈՇՋՍՊԽ ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋ ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋԻՇՆԼՍԻՌՁՆԿԹՊԹՆԿԽԾՁՆԼԽՊ ՏՁՌՀԾՁՎԽՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՈՄՍՋԹԻՇՆՌՊՇՄ ՋՁՐՌՇՌԹՄՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋ ՏՁՌՀԾՇՍՊ ԾԽՅԹՄԽՋԹՆԼՌՏՇՅԹՄԽՋՈԽՊԽՐՈՇՋՍՊԽՌԹՆՃ ԾՁՋՀՌՇՌԹՄ ԱՀԽՆՍՅԺԽՊՇԾՅՇՊՌԹՄՁՌՁԽՋ ՌՀԹՌՇԻԻՍՊՅԹՑԺԽՍՋԽԼՌՇԼԽՎԽՄՇՈԹ ԻՇՆԻԽՆՌՊԹՌՁՇՆՊԽՋՈՇՆՋԽԻՍՊՎԽ ԟԹՋԽԼՍՈՇՆՌՀԽՊԽՋՍՄՌՋՌՀԽՀՁԿՀԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՌՀԹՌԼՇԽՋՆՇՌՊԽՋՍՄՌՁՆ ՁՆԻՊԽԹՋԽԼՅՇՊՌԹՄՁՌՑՇՊՋՁԿՆՋՇԾՇՎԽՊՌ ՅՇՊԺՁԼՁՌՑԻՇՅՈԹՊԽԼՌՇԻՇՆՌՊՇՄՋՇՊՌՀԽ ԼԽՊՁՎԽԼՀՊԩԠՅԹՑԺԽՋԽՄԽԻՌԽԼԹՋՌՀԽ ՀՁԿՀԽՋՌԽՐՈՇՋՍՊԽԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽ ԼԹՑՌԽՋՌ Page ԭԹԿԽՇԾ 132 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԡԽԾՁՆՁՌՁՎԽՌՍԼՑԠՇՆԼՁՌՁՇՆՋ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԱԽՋՌԼՍՊԹՌՁՇՆ ԼԹՑՋ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ՌՀԽ ԼՍՊԹՌՁՇՆ ՇԾ ՌՀԽ ԼԽԾՁՆՁՌՁՎԽՌԽՋՌԺԽԼԹՑՋ ԪԽՌՀՇԼԾՇՊՋԽՄԽԻՌՁՆԿՌՀԽՀՁԿՀԽՋՌ ԱՀԽԹԻՍՌԽՌՇՐՁԻՁՌՑ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ ՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽ ՀՇՍՊԩԠԾՇՊՈՀՇՋՅԽՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾ ԼԽԾՁՆՁՌՁՎԽՌԽՋՌ ՏՁՌՀԾԹՌՀԽԹԼՅՁՆՆՇՏՁՋ ՌՀԽՌԽՋՌԻՇՅՈՇՍՆԼՅԿԩՇՊ ՅԿԩ ԼԽՅՇՆՋՌՊԹՌԽՋԹԼԽՉՍԹՌԽԽՎՁԼԽՆԻԽՇԾՌՇՐՁԻՁՌՑ ԽԿՌՀԽՀՇՍՊԩԠ ՏՀՁԻՀԽՎԽՊ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌ ԯԽԾԽՊԽՆԻԽՋՌՍԼՑԻՁՌԹՌՁՇՆ ՁԾ ԫԞ ԹՈՈՄՁԻԹԺՄԽ ԽՈԹՊԹՌՁՇՆՇԾՌԽՋՌ ԱԽՆԾՇՄԼ ԢԭԞՋՍԿԿԽՋՌՋՌՀԹՌԹԻՇՆԻԽՆՌՊԹՌՁՇՆ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԻՇՆՌՊՇՄ ՋԽՈԹՊԹՌՁՇՆՇԾԺԽՌՏԽԽՆ ՇՊՌՀՊԽԽԾՇՄԼ ԹՆԼՅԿ ԹՆԼ ՇՊՌԽՆԾՇՄԼ ՁՋՋԻՁԽՆՌՁԾՁԻԹՄՄՑ ԹՁԩ ԹԻԻԽՈՌԹԺՄԽ ԫՍՅԺԽՊՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՇԾ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԹՆԼԹԻՇՆՌՊՇՄՈՄՍՋՋՇՄՎԽՆՌ ԻՇՆՌՊՇՄՁԾԹՈՈՊՇՈՊՁԹՌԽ Page ԭԹԿԽՇԾ 133 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԞՊԽՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԶԽՋ ԹԼՂՍՋՌԽԼԾՇՊՈՍՊՁՌՑ! ԦՆԼՁԻԹՌԽՌՀԽՌՑՈԽՇԾՎԹՄՍԽՋ ԪԽԹՆՅԽԹՋՍՊԽԼ ՈՊԽՋԽՆՌԽԼԾՇՊՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԩՁՅՁՌՇԾՉՍԹՆՌՁԾՁԻԹՌՁՇՆ ԩԬԮ ՅԿԹՁԩ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՇՊԻՀԽՅՁԻԹՄՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽՄՇՏՌՀԽԩԬԮԹՆԹՄՑՋԽՋԺԽ ԻՇՆԼՍԻՌԽԼՇՆՌՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋ ԩԽՎԽՄՇԾԼԽՌԽԻՌՁՇՆ ԩԬԡ ԫՇՌՊԽՈՇՊՌԽԼ ԣՊԽՉՍԽՆԻՑՇԾՅԽԹՋՍՊԽՅԽՆՌ ԹՆԼԼԹՑՋ ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՌԽՋՌՁՌԽՅ ԻՇՆԻԽՆՌՊԹՌՁՇՆԺԽՅԽԹՋՍՊԽԼՈՊՁՇՊՌՇՌՀԽ ԹԼԼՁՌՁՇՆՇԾԾՁՋՀՁՆԹՄՄՌԹՆՃՋԹՆԼԹՌՄԽԹՋՌ ՏԽԽՃՄՑՌՀԽՊԽԹԾՌԽՊՁՆՌՏՇՊԽՈՄՁԻԹՌԽՋՈԽՊ ՌՊԽԹՌՅԽՆՌՄԽՎԽՄ Page ԭԹԿԽՇԾ 134 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԴԹՋՌՀԽՊԹՆԼՇՅՁՒԽԼԻՇՅՈՄԽՌԽ ԶԽՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԹՄՄԾՁՋՀԺԽՊԹՆԼՇՅՄՑ ԺՄՇԻՃԼԽՋՁԿՆՍՋԽԼ! ԹՋՋՁԿՆԽԼՌՇՌԹՆՃՋԼՍՊՁՆԿՈՊԽԽՐՈՇՋՍՊԽ ԱԹՆՃՋԹՊԽՌՀԽՆՊԹՆՃԽԼԹԻԻՇՊԼՁՆԿՌՇՈՊԽ ԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑԹՆԼՌՀԽՌԹՆՃՋՏՁՌՀՌՀԽ ՀՁԿՀԽՋՌԾԽԻՍՆԼՁՌՑԹՊԽՊԹՆԼՇՅՄՑԹՋՋՁԿՆԽԼՌՇ ԹԼԽԾՁՆՁՌՁՎԽՌԽՋՌՌՊԽԹՌՅԽՆՌԹՆԼԺՄՇԻՃԾՁՊՋՌ ԢԹԻՀԺՄՇԻՃԻՇՆՌԹՁՆՋՇՆԽՊԽՈՄՁԻԹՌԽՇԾԽԹԻՀ ՌՊԽԹՌՅԽՆՌՁՆԻՄՍԼՁՆԿԻՇՆՌՊՇՄՋ ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆԻՇՆՌՊՇՄ ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆՋՇՄՎԽՆՌ ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԹԻՇՆԻՍՊՊԽՆՌ ԻՇՆՌՊՇՄ ՁԾԹՈՈՄՁԻԹԺՄԽ ՋՇՄՎԽՆՌԻՇՆՌՊՇՄՏՀԽՆԹՋՇՄՍԺՁՄՁՒՁՆԿԹԿԽՆՌՁՋ ՍՋԽԼԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՈԽՊՌԽՋՌ ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ ՁՌԽՅՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԫՍՅԺԽՊՇԾՅԹՄԽԾՁՋՀՈԽՊ ԢԭԞՊԽԻՇՅՅԽՆԼՋՅԹՄԽՋՈԽՊՊԽՈՄՁԻԹՌԽ ՊԽՈՄՁԻԹՌԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ԫՍՅԺԽՊՇԾԾԽՅԹՄԽԾՁՋՀՈԽՊ ԢԭԞՊԽԻՇՅՅԽՆԼՋԾԽՅԹՄԽՋՈԽՊՊԽՈՄՁԻԹՌԽ ՊԽՈՄՁԻԹՌԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ Page ԭԹԿԽՇԾ 135 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԴԹՋԹՋՇՄՎԽՆՌՍՋԽԼ! ԫՇ ՇՄՎԽՆՌՌՑՈԽ ՁԾԹՈՈՄՁԻԹԺՄԽ ԫԞ ԪԹՐՁՅՍՅՋՇՄՎԽՆՌԻՇՆԻԽՆՌՊԹՌՁՇՆ ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՋՇՄՎԽՆՌՆՇՌ ՁԾԹՈՈՄՁԻԹԺՄԽ ԽՐԻԽԽԼՅՄԩԬԢԠԡՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌՋՇՄՎԽՆՌՀԹՎԽՆՇԽԾԾԽԻՌՇՆՋՍՊՎՁՎԹՄՆՇՊ ՈՊՇԼՍԻԽԹՆՑՇՌՀԽՊԹԼՎԽՊՋԽԽԾԾԽԻՌՋԹՆԼՌՀԹՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՆՇՌԺԽԿՊԽԹՌԽՊՌՀԹՆՅՄԩ ԴԹՋԹՈՇՋՁՌՁՎԽԻՇՆՌՊՇՄՍՋԽԼ! ԫՇ ԭՇՋՁՌՁՎԽԻՇՆՌՊՇՄ ՁԾԹՈՈՄՁԻԹԺՄԽ ԫԞ ԭՇՋՁՌՁՎԽԻՇՆՌՊՇՄԻՇՆԻԽՆՌՊԹՌՁՇՆ Ջ ԫԞ ՁԾԹՈՈՄՁԻԹԺՄԽ ԭՀՇՌՇՈԽՊՁՇԼ ՀՊՋՄՁԿՀՌ ԢԭԞՊԽԻՇՅՅԽՆԼՋՈՀՇՌՇՈԽՊՁՇԼ ՀՊՋԼԹՊՃ ՄՁԿՀՌ ԼԹՊՃ ԥՍՌԻՀՁՆՋՇՆԱԥՀՁՄՄԹԺԽԽՊԫԴՁՆՌԽՊԪԧԭՁԻՃԾՇՊԼԡԟ ԞԻՍՌԽԹՆԼԻՀՊՇՆՁԻԽԾԾԽԻՌՋՇԾԻԹՊՊՁԽՊՋՇՄՎԽՆՌՋՁՆԹՉՍԹՌՁԻՇՊԿԹՆՁՋՅՋ ԞԻՊՁՌՁԻԹՄՊԽՎՁԽՏԯԽՎՁԽՏԞՉՍԹՌՁԻ ԱՇՐՁԻՇՄՇԿՑՈՈ- ԬԢԠԡ ԤՍՁԼԹՆԻԽԡՇԻՍՅԽՆՌՇՆԞՉՍԹՌՁԻԱՇՐՁԻՁՌՑԱԽՋՌՁՆԿՇԾԡՁԾԾՁԻՍՄՌՍԺՋՌԹՆԻԽՋԹՆԼԪՁՐՌՍՊԽՋԢՆՎՁՊՇՆՅԽՆՌԹՄԥԽԹՄՌՀԹՆԼԹԾԽՌՑԭՍԺՄՁԻԹՌՁՇՆՋԽՊՁԽՋՇՆԱԽՋՌՁՆԿԹՆԼ ԞՋՋԽՋՋՅԽՆՌԫՇԭԹՊՁՋԣՊԹՆԻԽ Page ԭԹԿԽՇԾ 136 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ԩՁԿՀՌՁՆՌԽՆՋՁՌՑԹՌՏԹՌԽՊ0Ջ ՄՍՐ ԢԭԞՊԽԻՇՅՅԽՆԼՋՄՁԿՀՌՁՆՌԽՆՋՁՌՑ- ՋՍՊԾԹԻԽ ՄՍՐ ԹՌՏԹՌԽՊ0ՋՋՍՊԾԹԻԽ ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋ ԱԽՋՌՋՇՄՍՌՁՇՆՋՏԽՊԽՊԽՈՇՊՌԽԼՌՇԺԽԻՄԽԹՊ ԹՆԼԻՇՄՇՊՄԽՋՋ Page ԭԹԿԽՇԾ 137 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ՍՅՅԹՊՑՇԾԱՊԽԹՌՅԽՆՌԠՇՆԻԽՆՌՊԹՌՁՇՆՋՁՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԫՇՅՁՆԹՄ ԪԽԹՆԪԽԹՋՍՊԽԼ ԱՊԽԹՌՅԽՆՌԦԡ ԠՇՆԻԽՆՌՊԹՌՁՇՆ ԠՇՆԻԽՆՌՊԹՌՁՇՆ ԪԽԹՆԠԳ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ ՅԿԹՁԩ ՅԿԹՁԩ ԠՇՆՌՊՇՄ ԼՁՄՍՌՁՇՆ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌԽՋՌ ԩԬԮ ԫԞ ՏԹՌԽՊՇՆՄՑ ՁՌԽՅԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽ ՅԹՁՆՌԹՁՆԽԼԹՌԹԻՇԽԾԾՁԻՁԽՆՌՇԾ ՇՄՎԽՆՌԻՇՆՌՊՇՄ ՁԾ ԫԞ ԫԞ ԫԞ ՎԹՊՁԹՌՁՇՆ ԠԳ ԹՈՈՄՁԻԹԺՄԽ ԯԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼ չ ԱՊԽԹՌՅԽՆՌ ԠԳ ԯԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼ չ ԱՊԽԹՌՅԽՆՌ ԠԳ ԯԽՎՁԽՏԽՊԻԹՄԻՍՄԹՌԽԼ չ ԱՊԽԹՌՅԽՆՌ ԠԳ ԡՁՄՍՌԽՊՌՇԻՃ ԫԞ ԫԞ ԫԞ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԠԳԠՇԽԾԾՁԻՁԽՆՌՇԾՎԹՊՁԹՌՁՇՆ ԩԬԮ ՅԿԹՁԩ ԢԬԺՋԽՊՎԹՌՁՇՆՋ Page ԭԹԿԽՇԾ 138 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԟՁՇՄՇԿՁԻԹՄԢՆԼՈՇՁՆՌՋ ՍՊՎՁՎԹՄԾԽԻՍՆԼՁՌՑԾԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋԹՆԼԻՄՁՆՁԻԹՄՋՁԿՆՋՏԽՊԽՇԺՋԽՊՎԽԼԼԹՁՄՑԞՌՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆ ԡԹՑ ՋԽԻՇՆԼԹՊՑ ՋԽՐ ԻՀԹՊԹԻՌԽՊՁՒԹՌՁՇՆ ԺՇԼՑ ԻՇՄՇՊ ՈԹՌՌԽՊՆ ԺՇԼՑ ՋՀԹՈԽ ԺՇԼՑ ՏԽՁԿՀՌ ՄԽՆԿՌՀ ՌՍԺԽՊԻՄԽ ՋԻՇՊԽ ԿՇՆԹԼԹՄ ՋՌԹԿՁՆԿ ԹՆԼ ՀՁՋՌՇՈԹՌՀՇՄՇԿՑԹՆԼՈՄԹՋՅԹՎՁՌԽՄՄՇԿԽՆՁՆՏԽՊԽԽՎԹՄՍԹՌԽԼԭՄԹՋՅԹՋԽՐՋՌԽՊՇՁԼՋՏԽՊԽՆՇՌՅԽԹՋՍՊԽԼ ԴԽՊԽՊԹՏ ՁՆԼՁՎՁԼՍԹՄ ԼԹՌԹՈՊՇՎՁԼԽԼ!ԶԽՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՇԺՋԽՊՎԹՌՁՇՆՋՇԾՋՍՊՎՁՎԹՄԾԽԻՍՆԼՁՌՑԾԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋՋԽԻՇՆԼԹՊՑՋԽՐԻՀԹՊԹԻՌԽՊՁՋՌՁԻՋԹՆԼՇՌՀԽՊԻՄՁՆՁԻԹՄՋՁԿՆՋՇԻԻՍՊԹՌՄԽԹՋՌԼԹՁՄՑԞՌ ՌԽՋՌ ՌԽՊՅՁՆԹՌՁՇՆ ԼԹՑ ԹԼԼՁՌՁՇՆԹՄ ՇԺՋԽՊՎԹՌՁՇՆՋ ՁՆԻՄՍԼԽ ԺՇԼՑ ՏԽՁԿՀՌ ԹՆԼ ՄԽՆԿՌՀ ՆՍՈՌՁԹՄ ՌՍԺԽՊԻՄԽ ՋԻՇՊԽ ԿՇՆԹԼԹՄ ՋՌԹԿՁՆԿ ԹՆԼ ՀՁՋՌՇՈԹՌՀՇՄՇԿՑ ՈՄԹՋՅԹ ՎՁՌԽՄՄՇԿԽՆՁՆԹՆԼՈՄԹՋՅԹՋԽՐՋՌԽՊՇՁԼՋ ՌԽՋՌՇՋՌԽՊՇՆԽԹՆԼȕԽՋՌՊԹԼՁՇՄՁԾՅԽԹՋՍՊԽԼ ԤՇՆԹԼՇՋՇՅԹՌՁԻՁՆԼԽՐ ԤԦ ՁՋԻԹՄԻՍՄԹՌԽԼՍՋՁՆԿԹՊԹՌՁՇՇԾԿՇՆԹԼՏԽՁԿՀՌ ՌՇԺՇԼՑՏԽՁԿՀՌ ԿՇՆԹԼՏԽՁԿՀՌՌՇՌՀԽՆԽԹՊԽՋՌՅԿԺՇԼՑՏԽՁԿՀՌՁՆՅԿՐ ԹՌՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆ ԠՄՁՆՁԻԹՄՋՁԿՆՋՇԾՇՎԽՊՌՌՇՐՁԻՁՌՑՅԹՑՁՆԻՄՍԼԽ ԺՍՌԹՊԽՆՇՌՄՁՅՁՌԽԼՌՇ ՀԽՅՇՊՊՀԹԿԽԻԽՋՋԹՌՁՇՆՇԾԾԽԽԼՁՆԿԹՆԼՇՌՀԽՊԹԺՆՇՊՅԹՄԺԽՀԹՎՁՇՊ ԦԦԯԢԲԩԱԞԫԡԡԦԠԲԦԬԫ ԣՇՊ ԹՄՄ ՋՌՍԼՑ ԹՍՌՀՇՊ ՊԽՋՍՄՌՋ ՌՀԽ ԽՆԼՈՇՁՆՌՋ ԹՌ ՌՀԽ ՀՁԿՀ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄ ՅԿ ԹՁԩ ՏԽՊԽ ՆՇՌ ՋՌԹՌՁՋՌՁԻԹՄՄՑ ԹՆԹՄՑՒԽԼ ԼՍԽ ՌՇ ՅՇՊՌԹՄՁՌՑ ԞԼԼՁՌՁՇՆԹՄՄՑՌՀԽՋՌՍԼՑԹՍՌՀՇՊՊԽՈՇՊՌԽԼՌՀԹՌՇԺՋԽՊՎԹՌՁՇՆՋԹՌՌՀԽՅԿԹՁԩԻՇՆԻԽՆՌՊԹՌՁՇՆՅԽՌՌՀԽԿՍՁԼԽՄՁՆԽԼԽԾՁՆՁՌՁՇՆՇԾՇՎԽՊՌՅՇՊԺՁԼՁՌՑ ԥՇՏԽՎԽՊՁՆԼՁՎՁԼՍԹՄՋԾՊՇՅՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՏԽՊԽՆՇՌԽՐԻՄՍԼԽԼԾՊՇՅՌՀԽՋՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋՁՋ Page ԭԹԿԽՇԾ 139 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ Ԟ ԯԽՋՍՄՌՋ ԱՀԽՊԽՏԹՋՋՍՊՎՁՎԹՄՁՆՌՀԽԻՇՆՌՊՇՄԿՊՇՍՈԞՌՌՀԽՄՇՏԻՇՆԻԽՆՌՊԹՌՁՇՆԾՇՍՊԾԽՅԹՄԽՋԼՁԽԼԹՆԼՌՀՊԽԽՇԾՌՀԽՋԽԾԽՅԹՄԽՋՏԽՊԽԾՊՇՅՇՆԽՊԽՈՄՁԻԹՌԽԞՌ ՌՀԽՁՆՌԽՊՅԽԼՁԹՌԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆՌՏՇՅԹՄԽՋԹՆԼԾՇՍՊԾԽՅԹՄԽՋԼՁԽԼԼՍՊՁՆԿՌՀԽՋՌՍԼՑԞՄՄԺՍՌՇՆԽՅԹՄԽԹՆԼԾԽՅԹՄԽԼՁԽԼԹՌՌՀԽՀՁԿՀԻՇՆԻԽՆՌՊԹՌՁՇՆ ԬՎԽՊԹՄՄՅԽԹՆՋՍՊՎՁՎԹՄՎԹՄՍԽՋՏԽՊԽԹՆԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆՌՊՇՄ ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋ ՊԽՋՈԽԻՌՁՎԽՄՑ ԱԹԺՄԽ ԞԼՍՄՌԣՁՋՀՍՊՎՁՎԹՄՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԱՊԽԹՌՅԽՆՌ ԪԹՄԽՋ ԣԽՅԹՄԽՋ ՅԿԹՁԩ #ՅԽԹՆՅԽԹՋՍՊԽԼ% Ն ՍՊՎՁՎՁՆԿ ՍՊՎՁՎԹՄ Ն ՍՊՎՁՎՁՆԿ ՍՊՎՁՎԹՄ ԠՇՆՌՊՇՄ ԩԬԮ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ ԦՆՌՀՁՋՌԹԺՄԽ՟ՆՠՊԽԾԽՊՋՌՇՌՀԽՌՇՌԹՄՆՍՅԺԽՊՇԾԾՁՋՀՈԽՊՌՊԽԹՌՅԽՆՌՄԽՎԽՄԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ԩԬԮ ՅԿԹՁԩ Page ԭԹԿԽՇԾ 140 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԪԽԹՆՅԹՄԽԺՇԼՑՏԽՁԿՀՌՎԹՄՍԽՋՏԽՊԽԹՆԼԿԹՆԼԾԽՅԹՄԽԺՇԼՑՏԽՁԿՀՌՎԹՄՍԽՋՏԽՊԽԹՆԼԿՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆՌՊՇՄ ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՊԽՋՈԽԻՌՁՎԽՄՑ ԱԹԺՄԽ ԱԹԺՄԽ ՁՒԽԹՌԱԽՋՌԱԽՊՅՁՆԹՌՁՇՆՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԟՇԼՑԴԽՁԿՀՌ ԩԽՆԿՌՀ ԱՊԽԹՌՅԽՆՌ ԪԹՄԽՋ ԣԽՅԹՄԽՋ ԪԹՄԽՋ ԣԽՅԹՄԽՋ ՅԿԹՁԩ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ԪԽԹՆ ԪԽԹՆ ԪԽԹՆ ԪԽԹՆ Ն չԡ Ն չԡ Ն չԡ Ն չԡ Կ Կ ՅՅ ՅՅ ԠՇՆՌՊՇՄ ԩԬԮ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼԡՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ ԦՆՌՀՁՋՌԹԺՄԽ՟ՆՠՊԽԾԽՊՋՌՇՌՀԽՆՍՅԺԽՊՇԾՁՆԼԽՈԽՆԼԽՆՌՊԽՈՄՁԻԹՌԽՋ ՌԹՆՃՋ ՈԽՊՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՀՁԿՀՅՇՊՌԹՄՁՌՑ ԩԬԮ ՅԿԹՁԩ Page ԭԹԿԽՇԾ 141 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԪԽԹՆԾԽԻՍՆԼՁՌՑՎԹՄՍԽՋՏԽՊԽԹՆԼԹՆԼԾԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋՏԹՋԹՆԼՁՆՌՀԽՅԽԹՆ ՅԽԹՋՍՊԽԼ ԻՇՆՌՊՇՄ ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՊԽՋՈԽԻՌՁՎԽՄՑ ԱԹԺՄԽ ԱԹԺՄԽ ԣԽԻՍՆԼՁՌՑԹՆԼԣԽՊՌՁՄՁՒԹՌՁՇՆՍԻԻԽՋՋՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԱՊԽԹՌՅԽՆՌ ԣԽԻՍՆԼՁՌՑ ԣԽՊՌՁՄՁՒԹՌՁՇՆՍԻԻԽՋՋ ՅԿԹՁԩ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ԪԽԹՆ չԡ ԪԽԹՆ չԡ ԠՇՆՌՊՇՄ ԩԬԮ ԫԞ ԫԞ ԫԞ ԫԞ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼ ԣԽԻՍՆԼՁՌՑՁՋԻԹՄԻՍՄԹՌԽԼԹՋՌՀԽՆՍՅԺԽՊՇԾԽԿԿՋՈԽՊՋՍՊՎՁՎՁՆԿԾԽՅԹՄԽՈԽՊՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑՈԽՊՊԽՈՄՁԻԹՌԽ ԣԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋ ՁՋԻԹՄԻՍՄԹՌԽԼԹՋՌՀԽՆՍՅԺԽՊՇԾԽՅԺՊՑՇՋԼՁՎՁԼԽԼԺՑՌՀԽՆՍՅԺԽՊՇԾԽԿԿՋՅՍՄՌՁՈՄՁԽԼ ԺՑ ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑ ԩԬԮ ՅԿԹՁԩ ԪԽԼՁԹՆՅԹՄԽՌՍԺԽՊԻՄԽՋԻՇՊԽՋՏԽՊԽԹՆԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆՌՊՇՄ ԹՆԼ ՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՊԽՋՈԽԻՌՁՎԽՄՑ ԱԹԺՄԽ ԫՇՆԽՇԾՌՀԽՋՍՊՎՁՎՁՆԿԾԽՅԹՄԽՋՏԽՊԽԾՇՍՆԼՌՇՀԹՎԽ ՌՍԺԽՊԻՄԽՋ ԭԹԿԽՇԾ Page 142 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԫՍՈՌՁԹՄԱՍԺԽՊԻՄԽԻՇՊԽՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԱՊԽԹՌՅԽՆՌ ԪԹՄԽՋ ԣԽՅԹՄԽՋ ՅԿԹՁԩ ԪԽԼՁԹՆ ԪԽԼՁԹՆ Ն Ն #ՅԽԹՆՅԽԹՋՍՊԽԼ% ԱՍԺԽՊԻՄԽԻՇՊԽ ԱՍԺԽՊԻՄԽԻՇՊԽ ԠՇՆՌՊՇՄ ԩԬԮ ԫԞ ԫԞ ԫԞ ԫԞ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼԡՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ ԦՆՌՀՁՋՌԹԺՄԽ՟ՆՠՊԽԾԽՊՋՌՇՌՀԽՆՍՅԺԽՊՇԾՁՆԼԽՈԽՆԼԽՆՌՊԽՈՄՁԻԹՌԽՋ ՌԹՆՃՋ ՈԽՊՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑ ԩԬԮ ՅԿԹՁԩ ԪԽԹՆՅԹՄԽԤԦ ՏԹՋԹՆԼԹՆԼՅԽԹՆԾԽՅԹՄԽԤԦ ՏԹՋԹՆԼՁՆՌՀԽՅԽԹՆ ՅԽԹՋՍՊԽԼ ԻՇՆՌՊՇՄ ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՊԽՋՈԽԻՌՁՎԽՄՑ ԱԹԺՄԽ ԭԹԿԽՇԾ Page 143 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԤՇՆԹԼՇՇՅԹՌՁԻԦՆԼԽՐ ԤԦ ՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԱՊԽԹՌՅԽՆՌ ԪԹՄԽՋ ԣԽՅԹՄԽՋ ՅԿԹՁԩ ԪԽԹՆԤԦ ԪԽԹՆԤԦ Ն չԡ Ն չԡ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ԠՇՆՌՊՇՄ ԩԬԮ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ ԤՇՆԹԼՇՋՇՅԹՌՁԻՁՆԼԽՐ ՁՋԻԹՄԻՍՄԹՌԽԼԹՋԿՇՆԹԼՏԽՁԿՀՌ ՌՇՌՀԽՆԽԹՊԽՋՌՅԿ ԺՇԼՑՏԽՁԿՀՌ ՅԿ Ր ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑ ԩԬԮ ՅԿԹՁԩ ԪԽԼՁԹՆՅԹՄԽԿՇՆԹԼԹՄՋՌԹԿԽՏԹՋԹՆԼԹՆԼՅԽԼՁԹՆԾԽՅԹՄԽԿՇՆԹԼԹՄՋՌԹԿԽՏԹՋԹՆԼՁՆ ՌՀԽ ՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆՌՊՇՄ ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՊԽՋՈԽԻՌՁՎԽՄՑ ԱԹԺՄԽ ԭԹԿԽՇԾ Page 144 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԤՇՆԹԼԹՄՌԹԿՁՆԿՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԱՊԽԹՌՅԽՆՌ ԪԹՄԽՋ ԣԽՅԹՄԽՋ ՅԿԹՁԩ Ն ԪԽԼՁԹՆՌԹԿԽ Ն ԪԽԼՁԹՆՌԹԿԽ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ԠՇՆՌՊՇՄ ԩԬԮ ԫԞ ԫԞ ԫԞ ԫԞ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԧԧՍՎԽՆՁՄԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼԲԱԲՆԹԺՄԽՌՇՋՌԹԿԽ ԦՆՌՀՁՋՌԹԺՄԽ՟ՆՠՊԽԾԽՊՋՌՇՌՀԽՌՇՌԹՄՆՍՅԺԽՊՇԾՁՆԼՁՎՁԼՍԹՄՋՈԽՊՌՊԽԹՌՅԽՆՌՄԽՎԽՄՍՈՇՆՏՀՁԻՀՇԺՋԽՊՎԹՌՁՇՆՋՏԽՊԽՅԹԼԽԹՌ ՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆ ԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼՋՌՀԽԾՇՄՄՇՏՁՆԿԿՇՆԹԼԹՄՋՌԹԿՁՆԿՋԻԹՄԽԾՇՊՅԹՄԽԾԹՌՀԽԹԼՅՁՆՆՇՏ ՍՆԼԽՎԽՄՇՈԽԼ ԽԹՊՄՑ ՋՈԽՊՅԹՌՇԿԽՆՁԻ ՅՁԼՋՈԽՊՅԹՌՇԿԽՆՁԻ ՄԹՌԽՋՈԽՊՅԹՌՇԿԽՆՁԻ ՋՈԽՆՌ ԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼՋՌՀԽԾՇՄՄՇՏՁՆԿԿՇՆԹԼԹՄՋՌԹԿՁՆԿՋԻԹՄԽԾՇՊԾԽՅԹՄԽԾԹՌՀԽԹԼՅՁՆՆՇՏ ՍՆԼԽՎԽՄՇՈԽԼ ԽԹՊՄՑ ԼԽՎԽՄՇՈՅԽՆՌ ՅՁԼԼԽՎԽՄՇՈՅԽՆՌ ՄԹՌԽԼԽՎԽՄՇՈՅԽՆՌ ՄԹՌԽԼԽՎԽՄՇՈՅԽՆՌՀՑԼՊԹՌԽԼ ՈՇՋՌՇՎՍՄԹՌՇՊՑ ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑ ԩԬԮ ՅԿԹՁԩ ԞԻԻՇՊԼՁՆԿՌՇՌՀԽՋՌՍԼՑՊԽՈՇՊՌՁՆՅԹՄԽԾՁՋՀՌՀԽՊԽՏԽՊԽԹՆՁՆԻՊԽԹՋԽԼՈԽՊԻԽՆՌԹԿԽՇԾՋՈԽՊՅԹՌՇԿՇՆՁԹ ԱԹԺՄԽ ՁՆՌՀԽՅԿԹՁԩԿՊՇՍՈՏՁՌՀԾՇՍՊՅՁՆՁՅԹՄՌՇՅՁՄԼՁՆԻՁԼԽՆԻԽՋՇՍՌՇԾՁՆԼՁՎՁԼՍԹՄՋԱՏՇՅԹՄԽՋԹՄՋՇ ՀԹԼՅՁՄԼԼՁԾԾՍՋԽՌՀՁԻՃԽՆՁՆԿՇԾՌՀԽԿԽՊՅՁՆԹՄԽՈՁՌՀԽՄՁՍՅՄՁՆՁՆԿԺՑՋՈԽՊՅԹՌՇԿՇՆՁԹԱՀԽՊԽՏԽՊԽՌՏՇՁՆԻՁԼԽՆԻԽՋՇԾ ՅՁՆՁՅԹՄՁՆԻՊԽԹՋԽԼՌԽՋՌՁԻՍՄԹՊԼԽԿԽՆԽՊԹՌՁՇՆՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՋՏՁՌՀԻՇՆԻՍՊՊԽՆՌ ՅՁՆՁՅԹՄՁՆՌԽՊՋՌՁՌՁԹՄԻԽՄՄՀՑՈԽՊՈՄԹՋՁԹՁՆՌՀԽԻՇՆՌՊՇՄՅԹՄԽԹՆԼՅՁՆՁՅԹՄՄՑՁՆԻՊԽԹՋԽԼՈՊՇՈՇՊՌՁՇՆՇԾՋՈԽՊՅԹՌՇԿՇՆՁԹՁՆ ՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌԱՀԽՊԽՏԽՊԽՌՀՊԽԽՁՆԻՁԼԽՆԻԽՋՇԾՅՁՆՁՅԹՄՁՆՌԽՊՋՌՁՌՁԹՄԻԽՄՄՀՑՈԽՊՈՄԹՋՁԹՇՆԽՁՆՌՀԽ ԻՇՆՌՊՇՄԹՆԼՌՏՇՁՆՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌԱՀԽՊԽՏԽՊԽԹՄՋՇՇՆԽԻՇՆՌՊՇՄԹՆԼՇՆԽՅԹՄԽԾՊՇՅՌՀԽ ՅԿԹՁԩՌՊԽԹՌՅԽՆՌՏՁՌՀՅՁՆՁՅԹՄԹՋՑՆԻՀՊՇՆՇՍՋԿՇՆԹԼԼԽՎԽՄՇՈՅԽՆՌ ԱԹԺՄԽ ԞՄՋՇԹԻԻՇՊԼՁՆԿՌՇՌՀԽՋՌՍԼՑՊԽՈՇՊՌՁՆԾԽՅԹՄԽՋՌՀԽՊԽՏԽՊԽՅՁՄԼԹՆԼՋԽՎԽՊԽՁՆԻՁԼԽՆԻԽՋՇԾԼԽԻՊԽԹՋԽԼՑՇՄՃ ՋՑՆՌՀԽՋՁՋՁՆՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈԹՆԼՅՁՆՁՅԹՄՅՁՄԼՅՇԼԽՊԹՌԽԹՆԼՋԽՎԽՊԽՁՆԻՁԼԽՆԻԽ ՁՆՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈ ԱԹԺՄԽ ԱՀԽՇՆԽԾԽՅԹՄԽՅՇԼԽՊԹՌԽՄՑԹԾԾԽԻՌԽԼԹՄՋՇՀԹԼԹԺՍՆԼԹՆՌ ԭԹԿԽՇԾ Page 145 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԳԱԤՁՆՇՆՄՑԹԾԽՏԾՇՄՄՁԻՄԽՋՏՁՌՀՌՏՇԾԽՅԹՄԽՋՋԽՎԽՊԽՄՑԹԾԾԽԻՌԽԼ ՇՆԽԽԹԻՀՁՆՌՀԽԹՆԼՅԿԹՁԩ ԿՊՇՍՈՋ ՏՁՌՀ ՄՁՌՌՄԽ ԳԱԤ ԾՇՊՅԹՌՁՇՆ ԱՀԽ ԾԽՅԹՄԽ ՏՁՌՀ ՌՀԽ ՋԽՎԽՊԽ ԼԽԻՊԽԹՋԽԼ ՑՇՄՃ ՋՑՆՌՀԽՋՁՋ ՁՆ ՌՀԽ ՅԿ ԹՁԩ ԿՊՇՍՈ ԹՄՋՇ ՀԹԼ ՋԽՎԽՊԽ ԿՊԹՆՍՄՇՅԹՌՇՍՋ ՁՆԾՄԹՅՅԹՌՁՇՆ ՏՁՌՀՁՆ ՌՀԽ ՇՎԹՊՑ ԱԹԺՄԽ ԤՊԹՆՍՄՇՅԹՌՇՍՋ ՁՆԾՄԹՅՅԹՌՁՇՆ ՏԹՋ ՆՇՌ ՈՊԽՋԽՆՌ ՁՆ ՌՀԽ ՋԽՎԽՊԽՄՑ ԹԾԾԽԻՌԽԼ ԾԽՅԹՄԽ ԾՊՇՅ ՌՀԽ ՅԿ ԹՁԩ ԿՊՇՍՈ ԺՍՌ ՏԹՋ ԻՇՅՅՇՆՏՁՌՀՋԽՎԽՆԾԽՅԹՄԽՋՁՆՌՀԽԻՇՆՌՊՇՄՁՆՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌԹՆԼՁՆՌՀԽՅԿ ԹՁԩ ՅՁՆՁՅԹՄ ՌՇ ՅՁՄԼ ԹՆԼ ԹՋՋՇԻՁԹՌԽԼ ՏՁՌՀ ՁՆԻՊԽԹՋԽԼ ՇՇԻՑՌԽ ԹՌՊԽՋՁԹ ԽՁԿՀՌ ԻԹՋԽՋ ՀԹԼ ՈՊՇՌՇՒՇԹՄ ՋՈՇՊԽՋ ՏՁՌՀՁՆԿՊԹՆՍՄՇՅԹՋԞՆՁՆԻՊԽԹՋԽՁՆՇՇԻՑՌԽԹՌՊԽՋՁԹՏԹՋՇԺՋԽՊՎԽԼՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՏՁՌՀ ԹՆԼՇԾՌՀԽԾՁՋՀԽՐՀՁԺՁՌՁՆԿՌՀՁՋԾՁՆԼՁՆԿՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼՄՇՏԹՆԼՅՁԼՌՊԽԹՌՅԽՆՌՋՊԽՋՈԽԻՌՁՎԽՄՑԱՀԽՊԽ ՏԹՋՇՆԽՁՆԻՁԼԽՆԻԽՇԾՄՑՅՈՀՇԻՑՌՁԻՁՆԾՁՄՌՊԹՌԽՋՏՁՌՀՁՆՌՀԽՇՎՁԼՍԻՌՁՆՌՀԽՅԿԹՁԩԿՊՇՍՈԹՆԼՁՆՌՀԽ ՅԿ ԹՁԩ ԱՀԽՊԽ ՏԹՋ ՅՁՆՁՅԹՄ ՁՆՌԽՊՋՌՁՌՁԹՄ ԾՁԺՊՇՋՁՋ ՁՆ ՌՀԽ ՅԿ ԹՁԩ ԿՊՇՍՈ ԭԹԿԽՇԾ Page 146 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑՁՆԪԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ ԱՊԽԹՌՅԽՆՌ ԦՆՌԽՊՋՌՁՌՁԹՄԠԽՄՄ ԦՆԻՊԽԹՋԽԼ ԦՆԻՊԽԹՋԽԼ ԤԽՊՅՁՆԹՄ ՅԿԹՁԩ ԭՊԽՋԽՆԻԽՇԾ ԩԽՑԼՁԿ ԭՊՇՈՇՊՌՁՇՆՇԾ ԱԽՋՌՁԻՍՄԹՊ ԢՈՁՌՀԽՄՁՍՅ #ՅԽԹՆ ԽՎԽՊՁՌՑ ԱԽՋՌՁՋԬՎԹ ՀՑՈԽՊՌՊՇՈՀՑ ՈԽՊՅԹՌՇԿՇՆՁԹ ԡԽԿԽՆԽՊԹՌՁՇՆ ԞՌՊՇՈՀՑ ՅԽԹՋՍՊԽԼ% ՀՑՈԽՊՈՄԹՋՁԹ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ ԠՇՆՌՊՇՄ ԫԞ ԫԞ ԩԬԮ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑԼՁԹԿՆՇՋՌՁԻՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑ ԫՇՌՊԽՅԹՊՃԹԺՄԽ ԪՁՆՁՅԹՄ ԪՁՄԼ ԪՇԼԽՊԹՌԽ ԽՎԽՊԽԽԽԞՈՈԽՆԼՁՐԢՇԾՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑ ԩԬԮ ՅԿԹՁԩ ԭԹԿԽՇԾ Page 147 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԞԼԼՁՌՁՇՆԹՄԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑԬԺՋԽՊՎԹՌՁՇՆՋՁՆԪԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ ԱՊԽԹՌՅԽՆՌ ԡԽԻՊԽԹՋԽԼ ԦՆԻՊԽԹՋԽԼԳԹՋԻՍՄԹՊ ԞՋՑՆԻՀՊՇՆՇՍՋ ԞՄՌԽՊԽԼԭՊՇՈՇՊՌՁՇՆՋ ԤՊԹՆՍՄՇՅԹՌՇՍՋ ՅԿԹՁԩ ԭՊՇՈՇՊՌՁՇՆՇԾ ՇՊԦՆՌԽՊՋՌՁՌՁԹՄ ԤՇՆԹԼ ՇԾՈԽՊՅԹՌՇԻՑՌԽՋ ԽՎԽՊՁՌՑ ԦՆԾՄԹՅՅԹՌՁՇՆ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ՈԽՊՅԹՌՇԿՇՆՁԹ ԭՊՇՌԽՁՆԹԻԽՇՍՋԣՄՍՁԼ ԡԽՎԽՄՇՈՅԽՆՌ ՇՊՈԽՊՅԹՌՁԼՋ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ ԠՇՆՌՊՇՄ ԩԬԮ Page ԭԹԿԽՇԾ 148 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ ԱՊԽԹՌՅԽՆՌ ԡԽԻՊԽԹՋԽԼ ԦՆԻՊԽԹՋԽԼԳԹՋԻՍՄԹՊ ԞՋՑՆԻՀՊՇՆՇՍՋ ԞՄՌԽՊԽԼԭՊՇՈՇՊՌՁՇՆՋ ԤՊԹՆՍՄՇՅԹՌՇՍՋ ՅԿԹՁԩ ԭՊՇՈՇՊՌՁՇՆՇԾ ՇՊԦՆՌԽՊՋՌՁՌՁԹՄ ԤՇՆԹԼ ՇԾՈԽՊՅԹՌՇԻՑՌԽՋ ԽՎԽՊՁՌՑ ԦՆԾՄԹՅՅԹՌՁՇՆ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ՈԽՊՅԹՌՇԿՇՆՁԹ ԭՊՇՌԽՁՆԹԻԽՇՍՋԣՄՍՁԼ ԡԽՎԽՄՇՈՅԽՆՌ ՇՊՈԽՊՅԹՌՁԼՋ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑԼՁԹԿՆՇՋՌՁԻՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑ ԫՇՌՊԽՅԹՊՃԹԺՄԽ ԪՁՆՁՅԹՄ ԪՁՄԼ ԪՇԼԽՊԹՌԽ ԽՎԽՊԽԽԽԞՈՈԽՆԼՁՐԢՇԾ ՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑ ԩԬԮ ՅԿԹՁԩ Page ԭԹԿԽՇԾ 149 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑՁՆԣԽՅԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ ԱՊԽԹՌՅԽՆՌ ԭԽՊՁԾՇՄՄՁԻՍՄԹՊԠԽՄՄ ՅԿԹՁԩ ԦՆԻՊԽԹՋԽԼԬՇԻՑՌԽ ԡԽԻՊԽԹՋԽԼԶՇՄՃ ԦՆԾԽԻՌՁՇՆ ԥՑՈԽՊՈՄԹՋՁԹ #ՅԽԹՆ ԽՎԽՊՁՌՑ ԞՌՊԽՋՁԹ ԣՇՊՅԹՌՁՇՆ ԪՁԻՊՇՋՈՇՊՁԼՁԹ ԥՑՈԽՊՌՊՇՈՀՑ ՅԽԹՋՍՊԽԼ% Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ ԠՇՆՌՊՇՄ ԩԬԮ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԭԹԿԽՇԾ Page 150 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ ԱՊԽԹՌՅԽՆՌ ԭԽՊՁԾՇՄՄՁԻՍՄԹՊԠԽՄՄ ՅԿԹՁԩ ԦՆԻՊԽԹՋԽԼԬՇԻՑՌԽ ԡԽԻՊԽԹՋԽԼԶՇՄՃ ԦՆԾԽԻՌՁՇՆ ԥՑՈԽՊՈՄԹՋՁԹ #ՅԽԹՆ ԽՎԽՊՁՌՑ ԞՌՊԽՋՁԹ ԣՇՊՅԹՌՁՇՆ ԪՁԻՊՇՋՈՇՊՁԼՁԹ ԥՑՈԽՊՌՊՇՈՀՑ ՅԽԹՋՍՊԽԼ% Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑԼՁԹԿՆՇՋՌՁԻՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑ ԫՇՌՊԽՅԹՊՃԹԺՄԽ ԪՁՆՁՅԹՄ ԪՁՄԼ ԪՇԼԽՊԹՌԽ ԽՎԽՊԽԽԽԞՈՈԽՆԼՁՐԢՇԾՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑ ԩԬԮ ՅԿԹՁԩ ԭԹԿԽՇԾ Page 151 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԞԼԼՁՌՁՇՆԹՄԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑԬԺՋԽՊՎԹՌՁՇՆՋՁՆԣԽՅԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋ ՈՊՇՅԽՄԹՋ ԱՊԽԹՌՅԽՆՌ ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ ՅԿԹՁԩ ԢԿԿԡԽԺՊՁՋՁՆ ԤՊԹՆՍՄՇՅԹՌՇՍՋ ԡԽԻՊԽԹՋԽԼԭՇՋՌ ԦՆՌԽՊՋՌՁՌՁԹՄԣՁԺՊՇՋՁՋ #ՅԽԹՆ ԽՎԽՊՁՌՑ ԬՎՁԼՍԻՌ ԦՆԾՄԹՅՅԹՌՁՇՆ ԬՎՍՄԹՌՇՊՑԣՇՄՄՁԻՄԽՋ ՅԽԹՋՍՊԽԼ% Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ ԠՇՆՌՊՇՄ ԩԬԮ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑԼՁԹԿՆՇՋՌՁԻՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑ ԫՇՌՊԽՅԹՊՃԹԺՄԽ ԪՁՆՁՅԹՄ ԪՁՄԼ ԪՇԼԽՊԹՌԽ ԽՎԽՊԽԽԽԞՈՈԽՆԼՁՐԢՇԾՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑ ԩԬԮ ՅԿԹՁԩ ԭԹԿԽՇԾ Page 152 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԪԽԹՆՅԹՄԽԳԱԤՏԹՋՐՐԹՆԼՐԹՆԼԾԽՅԹՄԽԳԱԤՏԹՋՐՐԹՆԼՐ ՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆՌՊՇՄ ԹՆԼՅԿԹՁՅԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՊԽՋՈԽԻՌՁՎԽՄՑ ԱԹԺՄԽ ԱԹԺՄԽ ԭՄԹՋՅԹԳՁՌԽՄՄՇԿԽՆՁՆՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԭՄԹՋՅԹԳՁՌԽՄՄՇԿԽՆՁՆ ԳԱԤ ԱՊԽԹՌՅԽՆՌ ԪԹՄԽՋ ԣԽՅԹՄԽՋ ՅԿԹՁԩ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ԪԽԹՆ ԪԽԹՆ Ն չԡ Ն չԡ ՆԿՅԩՈՄԹՋՅԹ ՆԿՅԩՈՄԹՋՅԹ ԠՇՆՌՊՇՄ ԩԬԮ Ր Ր Ր Ր Ր Ր Ր Ր Ր Ր Ր Ր ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼԡՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ ՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆՋԻԹՄԻՍՄԹՌԽԼԺՑՌՀԽՊԽՎՁԽՏԽՊ ԫՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑ ԩԬԮ ՅԿԹՁԩ ԱԹԺՄԽ ԭՄԹՋՅԹԽՐՌԽՊՇՁԼՋՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԫՇՌՅԽԹՋՍՊԽԼ ԱՀՁՋՌԹԺՄԽՁՋՇՅՁՌՌԽԼԺԽԻԹՍՋԽՋԽՐՋՌԽՊՇՁԼՋՏԽՊԽՆՇՌՅԽԹՋՍՊԽԼ ԭԹԿԽՇԾ Page 153 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱՀԽՊԽՏԽՊԽՆՇՆՇՌԹԺՄԽՇԺՋԽՊՎԹՌՁՇՆՋՏՁՌՀՊԽԿԹՊԼՋՌՇԻՇՄՇՊԹՌՁՇՆԺԹՆԼՁՆԿԻՀԹՆԿԽՋՁՆՇՎՁՈՇՋՁՌՇՊԹՈՈԽԹՊԹՆԻԽՇՊՋՁՒԽՇԾԼՇՊՋԹՄՆԹՈԽՈԹԼԱՀԽՈՊԽՋԽՆԻԽ ՇԾԻՇՄՇՊԽԼԺԹՊՋ ԺԹՆԼՁՆԿ ՁՆՅԹՄԽԾՁՋՀԺՍՌՆՇՌԾԽՅԹՄԽԾՁՋՀ ԱԹԺՄԽ ՁՋԻՇՆՋՁԼԽՊԽԼՆՇՊՅԹՄՋԽՐՍԹՄԼՁՅՇՊՈՀՁՋՅԾՇՊՌՀՁՋՋՈԽԻՁԽՋԥՇՏԽՎԽՊՋՁԿՆՋՇԾ ՅՇՊԺՁԼՁՌՑՁՆԻՄՍԼՁՆԿՄԽՌՀԹՊԿՑԹՆԼՄՇՋՋՇԾԽՉՍՁՄՁԺՊՁՍՅՏԽՊԽՇԺՋԽՊՎԽԼՁՆԾՁՋՀԹՌՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԱԹԺՄԽ ԱԹԺՄԽ ԽԻՇՆԼԹՊՑԽՐԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋԹՆԼԠՄՁՆՁԻԹՄՁԿՆՋՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԹՌԱԽՋՌԱԽՊՅՁՆԹՌՁՇՆ ԽԻՇՆԼԹՊՑԽՐԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋԹՆԼԠՄՁՆՁԻԹՄՁԿՆՋ ԱՊԽԹՌՅԽՆՌ ՅԿԹՁԩ ԪԹՄԽՋ ԣԽՅԹՄԽՋ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ԱՑՈԽ Ն ԦՆԻՁԼԽՆԻԽ ԱՑՈԽ Ն ԦՆԻՁԼԽՆԻԽ ԠՇՆՌՊՇՄ ԩԬԮ ԠՇՄՇՊԺԹՊՋ ԫՇՆԽ ԠՇՄՇՊԺԹՊՋ ԫՇՆԽ ԡԽԾՇՊՅԽԼՄԽՌՀԹՊԿՁԻ ԩԽՌՀԹՊԿՁԻՄՇՋՋՇԾԽՉՍՁՄՁԺՊՁՍՅ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫՇՌԽՌՀԹՌՆՍՈՌՁԹՄՌՍԺԽՊԻՄԽՋԻՇՊԽՁՋՊԽՈՇՊՌԽԼՈՊԽՎՁՇՍՋՄՑՁՆԱԹԺՄԽ ԦՆՌՀՁՋՌԹԺՄԽ՟ՆՠՊԽԾԽՊՋՌՇՌՀԽՆՍՅԺԽՊՇԾՁՆԼՁՎՁԼՍԹՄՋՍՈՇՆՏՀՁԻՀՇԺՋԽՊՎԹՌՁՇՆՋՏԽՊԽՅԹԼԽԹՌՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆ ԩԬԮ ՅԿԹՁԩ Page ԭԹԿԽՇԾ 154 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԟ ՌՍԼՑԞՍՌՀՇՊ0ՋԞՆԹՄՑՋՁՋԹՆԼԠՇՆԻՄՍՋՁՇՆՋ ԱՀԽՋՌՍԼՑԹՍՌՀՇՊԹՆԹՄՑՒԽԼՋՍՊՎՁՎԹՄՏԽՁԿՀՌՄԽՆԿՌՀՌՍԺԽՊԻՄԽՋԻՇՊԽԤԦԾԽՊՌՁՄՁՌՑԾԽԻՍՆԼՁՌՑԹՆԼԳԱԤ ԡԹՌԹՏԽՊԽԿԽՆԼԽՊՋՈԽԻՁԾՁԻԹՆԼԹՆԹՄՑՒԽԼՁՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՋ ԡԽՋԻՊՁՈՌՁՎԽՋՌԹՌՁՋՌՁԻՋ ՅԽԹՆՋՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆԽՌԻ ՏԽՊԽԼԽՌԽՊՅՁՆԽԼԾՇՊԽԹԻՀԽՆԼՈՇՁՆՌՁԿՆՁԾՁԻԹՆՌ ԽԾԾԽԻՌՋՏԽՊԽԼԽՌԽԻՌԽԼ ԾՇՊՈ ՏՁՌՀ ՌՀԽԽՐԻԽՈՌՁՇՆՇԾՀԹՈՁՊՇԴՁՄՃ0ՋԹՆԼԟԹՊՌՄԽՌՌ0ՋԱԽՋՌՋՏՀՁԻՀ ՏԽՊԽԺԹՋԽԼՇՆՈ ԠԢԱԦԳԽՊՋՁՇՆ ՍՊՎՁՎԹՄԼԹՌԹՏԽՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԹԣՁՋՀԽՊ0ՋԽՐԹԻՌ ՌԽՋՌ ՏՁՌՀԟՇՆԾԽՊՊՇՆՁԥՇՄՅԹԼՂՍՋՌՅԽՆՌ ԪԹՄԽԤԦԾԽԻՍՆԼՁՌՑԹՆԼԳԱԤՏԽՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԞԫԬԳԞ ԹՆԼ Թ ՇՆԽՌԹՁՄԽԼ ԡՍՆՆԽՌՌ0Ջ ՌԽՋՌ ԣԽՊՌՁՄՁՌՑ ՅԹՄԽ ՌՍԺԽՊԻՄԽ ՋԻՇՊԽ ՅԹՄԽ ՏԽՁԿՀՌ ԹՆԼ ԾԽՅԹՄԽ ԤԦ ԼԹՌԹ ՏԽՊԽԹՆԹՄՑՒԽԼԺՑԞԫԬԳԞԹՆԼԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌԽՋՌԭՊՁՇՊՌՇԡՍՆՆԽՌՌ0ՋԼԹՌԹՏԽՊԽԹՆԹՄՑՒԽԼԺՑ ՀԹՈՁՊՇԴՁՄՃ0ՋՌԽՋՌԹՆԼԩԽՎԽՆԽ0ՋՌՇՌԽՋՌԾՇՊՆՇՊՅԹՄՁՌՑԹՆԼՀՇՅՇԿԽՆԽՁՌՑՇԾՎԹՊՁԹՆԻԽՋՊԽՋՈԽԻՌՁՎԽՄՑԦԾ ՆՇՊՅԹՄՁՌՑ ԹՆԼ ՀՇՅՇԿԽՆԽՁՌՑ ՌԽՋՌՋ ՏԽՊԽ ՈԹՋՋԽԼ Ո Թ ՈԹՊԹՅԽՌՊՁԻ ԹՆԹՄՑՋՁՋ ՏԹՋ ՈԽՊԾՇՊՅԽԼ ԦԾ ՆՇՆՆՇՊՅԹՄՁՌՑ ՇՊ ՍՆԽՉՍԹՄ ՎԹՊՁԹՆԻԽ ՏԽՊԽ ՁՆԼՁԻԹՌԽԼ Ո Թ ՆՇՆՈԹՊԹՅԽՌՊՁԻ ԹՆԹՄՑՋՁՋ ՏԹՋ ՈԽՊԾՇՊՅԽԼ ՇՆ ՌՀԽ ՊԹՆՃՋ ՇԾ ՌՀԽ ԼԹՌԹ ԱՀԽՋԽ ՅԽՌՀՇԼՋ ԹՈՈԽԹՊ ՌՇ ԺԽ ԻՇՆՋՁՋՌԽՆՌ ՏՁՌՀ ՌՀԽ ՅԽՌՀՇԼՋ ՊԽԻՇՅՅԽՆԼԽԼՁՆՌՀԽԿՍՁԼԽՄՁՆԽ ԣՇՊԹՄՄՋՌՍԼՑԹՍՌՀՇՊՊԽՋՍՄՌՋՌՀԽԽՆԼՈՇՁՆՌՋԹՌՌՀԽՀՁԿՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ՅԿԹՁԩ ՏԽՊԽՆՇՌ ՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼԼՍԽՌՇՅՇՊՌԹՄՁՌՑԱՀԽՋՌՍԼՑԹՍՌՀՇՊՊԽՈՇՊՌԽԼՌՀԹՌՇԺՋԽՊՎԹՌՁՇՆՋԹՌՌՀԽՅԿ ԹՁԩԻՇՆԻԽՆՌՊԹՌՁՇՆՅԽՌՌՀԽԿՍՁԼԽՄՁՆԽԼԽԾՁՆՁՌՁՇՆՇԾՇՎԽՊՌՅՇՊԺՁԼՁՌՑՌՀԽՊԽԾՇՊԽՊԽԼՍԻՌՁՇՆՋԾՇՊԽՆԼՈՇՁՆՌՋ ԹՌՌՀՁՋԻՇՆԻԽՆՌՊԹՌՁՇՆԻԹՆԺԽ՟ԹՌՌՊՁԺՍՌԽԼՌՇԿԽՆԽՊԹՄՌՇՐՁԻՁՌՑԹՋՇՈՈՇՋԽԼՌՇԽԾԾԽԻՌՋՊԽՄԹՌԽԼՌՇԽՆԼՇԻՊՁՆԽ ԹԻՌՁՎՁՌՑՠ ԞԼՍՄՌ ՋՍՊՎՁՎԹՄ ԻՇՅԺՁՆԽԼ ՋԽՐԽՋ ԹՌ ՌՀԽ ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄ ՏԹՋ ՋՁԿՆՁԾՁԻԹՆՌՄՑԼՁԾԾԽՊԽՆՌԻՇՅՈԹՊԽԼՌՇՌՀԽԻՇՆՌՊՇՄՋ Ո ԣՁՋՀԽՊԢՐԹԻՌԟՇՆԾԽՊՊՇՆՁԥՇՄՅՌԽՋՌ ԱԹԺՄԽ ԱՀԽՊԽՏԹՋԹՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԼԽԻՊԽԹՋԽՁՆԾԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋԹՌՅԿԹՁԩ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄՋԻՇՅՈԹՊԽԼՌՇՌՀԽԻՇՆՌՊՇՄ Ո ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌԽՋՌ ԱԹԺՄԽ ԴՀՁՄԽՆՇՌ ՋՌԹՌՁՋՌՁԻԹՄՄՑԼՁԾԾԽՊԽՆՌԾՊՇՅՌՀԽԻՇՆՌՊՇՄՌՀԽՋՍՈՈՊԽՋՋՁՇՆՇԾԾԽԻՍՆԼՁՌՑՁՆՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌ ՏԹՋԻՇՆՋՁԼԽՊԽԼԺՁՇՄՇԿՁԻԹՄՄՑՊԽՄԽՎԹՆՌ Ո ԡՍՆՆԽՌՌ0Ջ ԣՇՊՅԹՄԽՌՍԺԽՊԻՄԽՋԻՇՊԽԹՆԼԺՇԼՑՏԽՁԿՀՌ ՌՀԽՋՌՍԼՑԹՍՌՀՇՊՆՇՌԽԼՊԽԼՍԻՌՁՇՆՋՁՆԽՆԼՈՇՁՆՌՋՇԺՋԽՊՎԽԼԹՌՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈ ՌՀԹՌՅԽՌԿՍՁԼԽՄՁՆԽԼԽԾՁՆՁՌՁՇՆՇԾՇՎԽՊՌՅՇՊԺՁԼՁՌՑՌՀԽՊԽԾՇՊԽՌՀԽՋԽՊԽԼՍԻՌՁՇՆՋՏԽՊԽԹՌՌՊՁԺՍՌԽԼՌՇԿԽՆԽՊԹՄ ՌՇՐՁԻՁՌՑ ԹՋ ՇՈՈՇՋԽԼ ՌՇ ԽԾԾԽԻՌՋ ՊԽՄԹՌԽԼ ՌՇ ԽՆԼՇԻՊՁՆԽ ԹԻՌՁՎՁՌՑ ԱԹԺՄԽՋ ԹՆԼ ԱՀԽՊԽ ՏԹՋ Թ ՋՌԹՌՁՋՌՁԻԹՄՄՑ ՋՁԿՆՁԾՁԻԹՆՌ ԼԽԻՊԽԹՋԽ ՁՆ ՅԽԼՁԹՆ ԾԽՅԹՄԽ ԤԦ ՁՆ ՌՀԽ ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ԻՇՅՈԹՊԽԼՌՇՌՀԽԻՇՆՌՊՇՄ Ո ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌԽՋՌ ԱԹԺՄԽ ԭԹԿԽՇԾ Page 155 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԞՄՌՀՇՍԿՀՆՇՌԹՆԹՄՑՒԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՌՀԽՊԽՏԽՊԽԿԽՆԽՊԹՄՁՒԽԼՇՎԽՊՌՋՁԿՆՋՇԾՌՇՐՁԻՁՌՑՁՆԺՇՌՀՅԹՄԽՋԹՆԼ ԾԽՅԹՄԽՋՁՆԻՄՍԼՁՆԿԹՄՄՋՍՊՎՁՎՁՆԿԾՁՋՀԹՌՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՏՀՁԻՀՏԽՊԽՇԺՋԽՊՎԽԼՌՇԺԽ ՄԽՌՀԹՊԿՁԻ ԱԹԺՄԽ ԦՆՅԹՄԽԾՁՋՀՌՀԽՊԽՏԹՋԹՄՋՇԹՆՁՆԻՊԽԹՋԽԼՈԽՊԻԽՆՌԹԿԽՇԾՋՈԽՊՅԹՌՇԿՇՆՁԹՁՆՌՀԽ ՅԿԹՁԩԿՊՇՍՈԹՆԼՁՆԾԽՅԹՄԽԾՁՋՀԼԽԻՊԽԹՋԽԼՑՇՄՃՋՑՆՌՀԽՋՁՋՁՆԺՇՌՀՌՀԽԹՆԼ ՅԿԹՁԩՌՊԽԹՌՅԽՆՌՋՊԽՄԹՌՁՎԽՌՇՌՀԽԻՇՆՌՊՇՄՋ ԱԹԺՄԽՋԹՆԼ ԠԯԽՎՁԽՏԽՊ0ՋԞՆԹՄՑՋՁՋԹՆԼԠՇՆԻՄՍՋՁՇՆՋ ՌԹՌՁՋՌՁԻԹՄ ԪԽՌՀՇԼՋ ԱՀԽ ՊԽՎՁԽՏԽՊ ԹՆԹՄՑՒԽԼ ՋՍՊՎՁՎԹՄ ՅՇՊՌԹՄՁՌՑ ԼԹՌԹ ԾՇՊ ՅԹՄԽ ԹՆԼ ԾԽՅԹՄԽ ԾՁՋՀ ԻՇՅԺՁՆԽԼՍՋՁՆԿԣՁՋՀԽՊ0ՋԢՐԹԻՌՌԽՋՌՎՁԹԱՇՐՋՌԹՌԣԽՅԹՄԽԺՇԼՑՏԽՁԿՀՌԹՆԼՄԽՆԿՌՀՅԹՄԽՄԽՆԿՌՀ ՅԹՄԽ ԿՇՆԹԼՇՋՇՅԹՌՁԻ ՁՆԼԽՐ ԤԦ ԹՆԼ ԾԽՅԹՄԽ ՎՁՌԽՄՄՇԿԽՆՁՆ ԳԱԤ ՏԽՊԽ ՆՇՌ ԻՇՆՋՁՋՌԽՆՌ ՏՁՌՀ Թ ՅՇՆՇՌՇՆՁԻԻՇՆԻԽՆՌՊԹՌՁՇՆՊԽՋՈՇՆՋԽԞՄՄԼԹՌԹՏԽՊԽՌԽՋՌԽԼԾՇՊՆՇՊՅԹՄՁՌՑՍՋՁՆԿՀԹՈՁՊՇԴՁՄՃՋՌԽՋՌԹՆԼ ԾՇՊՀՇՅՇԿԽՆԽՁՌՑՇԾՎԹՊՁԹՆԻԽՍՋՁՆԿԩԽՎԽՆԽ0ՋՌԽՋՌԱՀԽՋԽԼԹՌԹՅԽՌՌՀԽԹՋՋՍՅՈՌՁՇՆՋՇԾՆՇՊՅԹՄՁՌՑԹՆԼ ՀՇՅՇԿԽՆԽՁՌՑՇԾՎԹՊՁԹՆԻԽԹՆԼՏԽՊԽՌՀԽՆԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽՈԹՊԹՅԽՌՊՁԻՌՏՇՋՁԼԽԼԡՍՆՆԽՌՌ0ՋՌԽՋՌ ԪԹՄԽՅԽԼՁԹՆՌՍԺԽՊԻՄԽՋԻՇՊԽԼՁԼՆՇՌԽՐՀՁԺՁՌԹԻՄԽԹՊՅՇՆՇՌՇՆՁԻՊԽՋՈՇՆՋԽԹՆԼՌՀԽՊԽԾՇՊԽՏԹՋԹՆԹՄՑՒԽԼ ՍՋՁՆԿՌՀԽՌՏՇՋՁԼԽԪԹՆՆԴՀՁՌՆԽՑՌԽՋՌ ԣԽԻՍՆԼՁՌՑԾԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋՅԹՄԽՈՄԹՋՅԹԳԱԤԹՆԼՅԹՄԽԺՇԼՑՏԽՁԿՀՌԽԹԻՀԽՐՀՁԺՁՌԽԼԹՆԹՈՈԹՊԽՆՌ ՅՇՆՇՌՇՆՁԻԹՄՄՑ ԼԽԻՊԽԹՋՁՆԿ ՌՊԽՆԼ ՏՀԽՆ ԻՇՅՈԹՊԽԼ ՌՇ ՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ ՌՀԽՋԽ ԽՆԼՈՇՁՆՌՋ ՏԽՊԽ ԹՆԹՄՑՒԽԼԾՇՊՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԼՁԾԾԽՊԽՆԻԽՋՍՋՁՆԿՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՋՌԽՈԼՇՏՆՌԽՋՌ ԲՆՄԽՋՋ ՇՌՀԽՊՏՁՋԽ ՁՆԼՁԻԹՌԽԼ ԽԾԾԽԻՌՋ ՏԽՊԽ ԻՇՆՋՁԼԽՊԽԼ ՋՌԹՌՁՋՌՁԻԹՄՄՑ ՋՁԿՆՁԾՁԻԹՆՌ ԹՌ Ո ԴՁՌՀ ՌՀԽ ԽՐԻԽՈՌՁՇՆՇԾԣՁՋՀԽՊ0Ջ ԢՐԹԻՌԱԽՋՌԹՄՄԹՆԹՄՑՋԽՋՏԽՊԽԻՇՆԼՍԻՌԽԼՍՋՁՆԿՌՀԽԣԦԥՈՊՇԻԽԼՍՊԽՁՆԞն ԞԦՆՋՌՁՌՍՌԽԠԹՊՑԫԠ ՎԽՊՋՁՇՆ ԫՇՆԽՇԾՌՀԽՋՍՊՎՁՎՁՆԿԾԽՅԹՄԽՋՏԽՊԽԾՇՍՆԼՌՇՀԹՎԽՌՍԺԽՊԻՄԽՋԱՀԽՊԽԾՇՊԽԾԽՅԹՄԽՌՍԺԽՊԻՄԽՋԻՇՊԽՏԹՋ ՆՇՌ ՋՌԹՌՁՋՌՁԻԹՄՄՑ ԹՆԹՄՑՒԽԼ ԥՁՋՌՇՈԹՌՀՇՄՇԿՑ ՇԺՋԽՊՎԹՌՁՇՆՋ ՏԽՊԽ ԽՎԹՄՍԹՌԽԼ ՁՆ ԻՇՆՂՍՆԻՌՁՇՆ ՏՁՌՀ ՌՀԽ ՈԹՌՀՇՄՇԿՁՋՌ0ՋՊԽՈՇՊՌԺՍՌՋԽՎԽՊՁՌՑԹՆԼՁՆԻՁԼԽՆԻԽԼԹՌԹՏԽՊԽՆՇՌՋՍԺՂԽԻՌԽԼՌՇԾՇՊՅԹՄՋՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋԽՋ ԭԹԿԽՇԾ Page 156 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԠՇՆԻՄՍՋՁՇՆՋ ՍՊՎՁՎԹՄՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼ Ո ՌՇՇԾՌՀԽԻՇՆՌՊՇՄԹՌՌՀԽՁՆՌԽՊՅԽԼՁԹՌԽՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԱԹԺՄԽ ԹՆԼ ԾՍՊՌՀԽՊ ՊԽԼՍԻԽԼ ՌՇ ՌՀՇՍԿՀ ՆՇՌ ՋՌԹՌՁՋՌՁԻԹՄՄՑ ԹՆԹՄՑՒԽԼ ԹՌ ՌՀԽ ՀՁԿՀԽՋՌ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄՅԿԹՁԩՏՀՁԻՀՏԹՋԽՐԻՄՍԼԽԼԾՊՇՅԹՆԹՄՑՋԽՋԣՁՋՀՁՆՌՀԽՁՆՌԽՊՅԽԼՁԹՌԽՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԼՁՋՈՄԹՑԽԼ ՋՁԿՆՋ ՇԾ ՅՇՊԺՁԼՁՌՑ ՁՆԻՄՍԼՁՆԿ ՄԽՌՀԹՊԿՑ ԹՆԼ ՄՇՋՋ ՇԾ ԽՉՍՁՄՁԺՊՁՍՅ ԞՄՌՀՇՍԿՀ ՌՀԽ ՁՆՌԽՊՅԽԼՁԹՌԽ ՌՊԽԹՌՅԽՆՌՄԽՎԽՄՏԹՋՆՇՌԽՐԻՄՍԼԽԼԾՊՇՅՋՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋԽՋՌՀԽՈՊԽՋԽՆԻԽՇԾԻՄՁՆՁԻԹՄՋՁԿՆՋՇԾՌՇՐՁԻՁՌՑԹՌ ՌՀՁՋ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄ ՋՀՇՍՄԼ ԺԽ ԻՇՆՋՁԼԽՊԽԼ ՏՀԽՆ ՁՆՌԽՊՈՊԽՌՁՆԿ ՌՀԽ ՊԽՋՍՄՌՋ ԞԼԼՁՌՁՇՆԹՄՄՑ ՌՀԽ ՇՎԽՊԹՄՄ ՋՍՊՎՁՎԹՄՁՆՌՀԽՄՇՏԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈ ՅԿԹՁԩ ՏԹՋՏՁՌՀՌՀՊԽԽՇԾՌՀԽԾՇՍՊԾԽՅԹՄԽՋՁՆ ՇՆԽ ՊԽՈՄՁԻԹՌԽ ԼԽԹԼ ԹԿԹՁՆ ՌՀՁՋ ՊԽՈՄՁԻԹՌԽ ՏԹՋ ՆՇՌ ԽՐԻՄՍԼԽԼ ԾՊՇՅ ՌՀԽ ԹՆԹՄՑՋԽՋ ԺՍՌ ՋՀՇՍՄԼ ԺԽ ԻՇՆՋՁԼԽՊԽԼՏՀԽՆՁՆՌԽՊՈՊԽՌՁՆԿՌՀԽՊԽՋՍՄՌՋ ԣԽԻՍՆԼՁՌՑ Ո ԹՆԼ ԾԽՊՌՁՄՁՌՑ Ո ԹՌ ՌՀԽ ՁՆՌԽՊՅԽԼՁԹՌԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՏԽՊԽ ՋՁԿՆՁԾՁԻԹՆՌՄՑ ԼԽԻՊԽԹՋԽԼ ՏՀԽՆ ԻՇՅՈԹՊԽԼ ՌՇ ՌՀԽ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ ԢԾԾԽԻՌՋ ՇՆ ԾԽՅԹՄԽ ԿՇՆԹԼԹՄ ՀՁՋՌՇՈԹՌՀՇՄՇԿՑ ՁՆԻՄՍԼՁՆԿ ԼԽԻՊԽԹՋԽԼ ՑՇՄՃ ՋՑՆՌՀԽՋՁՋ ՏԽՊԽ ՇԺՋԽՊՎԽԼ ԹՌ ՌՀԽ ՄՇՏ ԹՆԼ ՁՆՌԽՊՅԽԼՁԹՌԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԣԽՅԹՄԽ ԤԦ Ո ՏԹՋ ՋՁԿՆՁԾՁԻԹՆՌՄՑ ԼԽԻՊԽԹՋԽԼ ԹՌ ՁՆՌԽՊՅԽԼՁԹՌԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ԴՀՁՄԽ ՆՇՌ ՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌՅԹՄԽՌՍԺԽՊԻՄԽՋԻՇՊԽՏԽՊԽՊԽԼՍԻԽԼԺՑԹՌՌՀԽՁՆՌԽՊՅԽԼՁԹՌԽԻՇՆԻԽՆՌՊԹՌՁՇՆ ԪԹՆՆԴՀՁՌՆԽՑ Ո ԦՆՅԹՄԽԾՁՋՀՌՀԽՊԽՏԹՋԹՄՋՇԹՆՁՆԻՊԽԹՋԽԼՈԽՊԻԽՆՌԹԿԽՇԾՋՈԽՊՅԹՌՇԿՇՆՁԹԹՌ ՌՀՁՋՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԴՀՁՄԽ ՆՇՌ ՋՁԿՆՁԾՁԻԹՆՌՄՑ ԼՁԾԾԽՊԽՆՌ ՌՀԽՊԽ ՏԹՋ Թ ՁՆԻՊԽԹՋԽ ՁՆ ՅԹՄԽ ԳԱԤ ԹՌ ՌՀԽ ՁՆՌԽՊՅԽԼՁԹՌԽ ԻՇՆԻԽՆՌՊԹՌՁՇՆԿՊՇՍՈԻՇՅՈԹՊԽԼՌՇՌՀԽԻՇՆՌՊՇՄ ՌՀՁՋԼՁԾԾԽՊԽՆԻԽՁՋԼՍԽՌՇՌՏՇՅԹՄԽՋՁՆՌՀՁՋԿՊՇՍՈՏՁՌՀ ՀՁԿՀԽՊԳԱԤՄԽՎԽՄՋԻՇՅՈԹՊԽԼՌՇՌՀԽԻՇՆՌՊՇՄ ԟՇԼՑՏԽՁԿՀՌԹՆԼՄԽՆԿՌՀՏԽՊԽՆՇՌՋՁԿՆՁԾՁԻԹՆՌՄՑԼՁԾԾԽՊԽՆՌՁՆՅԹՄԽՇՊԾԽՅԹՄԽԾՁՋՀԹՌԹՆՑՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԭԹԿԽՇԾ Page 157 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԯԽՈՊՇԼՍԻՌՁՎԽԹՆԼԥԭԤԢՆԼՈՇՁՆՌՋԾՇՊԪԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ՁՆՌՀԽԣԱԯԞՏՁՌՀԭՀՇՋՅԽՌ ԱՊԽԹՌՅԽՆՌ ԤՇՆԹԼԹՄՌԹԿՁՆԿ ԱՍԺԽՊԻՄԽԻՇՊԽ ԤԦ ԭՄԹՋՅԹԳԱԤ ՅԿԹՁԩ ԹՆԼԥՁՋՌՇ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ԪԽԼՁԹՆ Ո ԡՁԾԾ Ո ԢԾԾԽԻՌ! ԶԽՋԫՇ ԡՁԾԾ ԭ ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ ԩԬԮ ԫԞ ԫԞ ԫՇ ԫԞ ԫՇ ԶԽՋ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԪԹՆՆԴՀՁՌՆԽՑ ԡՍՆՆԽՌՌ0Ջ ԫԞ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԠՇՆԻԠՇՆԻԽՆՌՊԹՌՁՇՆԡՁԾԾԡՁԾԾԽՊԽՆԻԽԢ ԽՋՌՊԹԼՁՇՄԤԦԤՇՆԹԼՇՇՅԹՌՁԻԦՆԼԽՐԥՁՋՌՇԥՁՋՌՇՈԹՌՀՇՄՇԿՑԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԱԱԽՋՌՇՋՌԽՊՇՆԽԳԱԤԳՁՌԽՄՄՇԿԽՆՁՆ ԲՆՄԽՋՋ ՇՌՀԽՊՏՁՋԽ ՁՆԼՁԻԹՌԽԼ ԽԾԾԽԻՌՋ ԹՆԼ ՈԽՊԻԽՆՌ ԼՁԾԾԽՊԽՆԻԽՋ ԹՊԽ ՊԽՈՇՊՌԽԼ ԺԹՋԽԼ ՇՆ ԻՇՅՈԹՊՁՋՇՆ ՌՇ ՌՀԽ ՆԽԿԹՌՁՎԽ ԻՄԽԹՆ ՏԹՌԽՊ ԻՇՆՌՊՇՄ ԠՇՆԻՄՍՋՁՇՆՋ ՊԽԿԹՊԼՁՆԿ ՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈ ԱՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՋՌԽՈԼՇՏՆՌԽՋՌՏԹՋՍՋԽԼՌՇԹՆԹՄՑՒԽՅԹՄԽՎՁՌԽՄՄՇԿԽՆՁՆ ԳԱԤ ՊԽՋՈՇՆՋԽԼԹՌԹԺԽԻԹՍՋԽՌՀԽՊԽՎՁԽՏԽՊՎՁՋՍԹՄՄՑԼԽՌԽՊՅՁՆԽԼԹՅՇՆՇՌՇՆՁԻԹՄՄՑԼԽԻՊԽԹՋՁՆԿ ՌՊԽՆԼՁՆՌՀԽՌՊԽԹՌՅԽՆՌՅԽԼՁԹՆՎԹՄՍԽՋ ԹՆԼՆԿՅԩՈՄԹՋՅԹԹՌՌՀԽԹՆԼՅԿԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՊԽՋՈԽԻՌՁՎԽՄՑ ՏՀԽՆԻՇՅՈԹՊԽԼՌՇՌՀԽՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ ՆԿՅԩՈՄԹՋՅԹ ԥՇՏԽՎԽՊՌՀԽՇՎԽՊԹՄՄՅԽԹՆԾՇՊՌՀՁՋՌՊԽԹՌՅԽՆՌՏԹՋԿՊԽԹՌԽՊՌՀԹՆՌՀԽԻՇՆՌՊՇՄԹՆԼՌՀՁՋՏԹՋԼՍԽՌՇՌՏՇՅԹՄԽԾՁՋՀՁՆՌՀՁՋՌՊԽԹՌՅԽՆՌՏՁՌՀՊԽՈՇՊՌԽԼ ԳԱԤՄԽՎԽՄՋՌՀԹՌՏԽՊԽՋՍԺՋՌԹՆՌՁԹՄՄՑԿՊԽԹՌԽՊՌՀԹՆՌՀԽԻՇՆՌՊՇՄՋԭՄԹՋՅԹՋԽՐՋՌԽՊՇՁԼՋՏԽՊԽՆՇՌՅԽԹՋՍՊԽԼ ԩԬԮ ՅԿԹՁԩ Page ԭԹԿԽՇԾ 158 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԯԽՈՊՇԼՍԻՌՁՎԽԹՆԼԥԭԤԢՆԼՈՇՁՆՌՋԾՇՊԣԽՅԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ՁՆՌՀԽԣԱԯԞՏՁՌՀԭՀՇՋՅԽՌ ԤՇՆԹԼԹՄ ԱՊԽԹՌՅԽՆՌ ԣԽԻՍՆԼՁՌՑ ԣԽՊՌՍԻԻԽՋՋ ԱՍԺԽՊԻՄԽԻՇՊԽ ԤԦ ՌԹԿՁՆԿԹՆԼ ԭՄԹՋՅԹԳԱԤ ՅԿԹՁԩ ԥՁՋՌՇ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ԢԾԾԽԻՌ! ԡՁԾԾ Ո ԡՁԾԾ ԭ ԪԽԼՁԹՆ Ո ԡՁԾԾ Ո ԡՁԾԾ Ո ԶԽՋԫՇ ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ ԫԞ ԫԞ ԫԞ ԫԞ ԫՇ ԫԞ ԩԬԮ ԩԬԮ ԫԞ ԶԽՋ ԫԞ ԶԽՋ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ ԫԞ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ ԫԞ ԡՍՆՆԽՌՌ0Ջ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԠՇՆԻԠՇՆԻԽՆՌՊԹՌՁՇՆԡՁԾԾԡՁԾԾԽՊԽՆԻԽԢ ԽՋՌՊԹԼՁՇՄԣԽՊՌԣԽՊՌՁՄՁՒԹՌՁՇՆԤԦԤՇՆԹԼՇՇՅԹՌՁԻԦՆԼԽՐԥՁՋՌՇԥՁՋՌՇՈԹՌՀՇՄՇԿՑԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԱԱԽՋՌՇՋՌԽՊՇՆԽ ԳԱԤԳՁՌԽՄՄՇԿԽՆՁՆ ԲՆՄԽՋՋ ՇՌՀԽՊՏՁՋԽ ՁՆԼՁԻԹՌԽԼ ԽԾԾԽԻՌՋ ԹՆԼ ՈԽՊԻԽՆՌ ԼՁԾԾԽՊԽՆԻԽՋ ԹՊԽ ՊԽՈՇՊՌԽԼ ԺԹՋԽԼ ՇՆ ԻՇՅՈԹՊՁՋՇՆ ՌՇ ՌՀԽ ՆԽԿԹՌՁՎԽ ԻՄԽԹՆ ՏԹՌԽՊ ԻՇՆՌՊՇՄ ԠՇՆԻՄՍՋՁՇՆՋ ՊԽԿԹՊԼՁՆԿ ՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈ ԱՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՋՌԽՈԼՇՏՆՌԽՋՌՏԹՋՍՋԽԼՌՇԹՆԹՄՑՒԽԾԽԻՍՆԼՁՌՑՊԽՋՈՇՆՋԽԼԹՌԹԺԽԻԹՍՋԽՌՀԽՊԽՎՁԽՏԽՊՎՁՋՍԹՄՄՑԼԽՌԽՊՅՁՆԽԼԹՅՇՆՇՌՇՆՁԻԹՄՄՑԼԽԻՊԽԹՋՁՆԿՌՊԽՆԼՁՆՌՀԽ ՌՊԽԹՌՅԽՆՌՅԽԼՁԹՆՎԹՄՍԽՋ ԹՆԼԽԿԿՋԾԽՅԹՄԽՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑԹՌՌՀԽԹՆԼՅԿԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՊԽՋՈԽԻՌՁՎԽՄՑ ՏՀԽՆԻՇՅՈԹՊԽԼՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ ԽԿԿՋԾԽՅԹՄԽՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑ ԩԬԮ ՅԿԹՁԩ Page ԭԹԿԽՇԾ 159 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱԹԺՄԽ ԤՊՇՏՌՀԢՆԼՈՇՁՆՌՋՁՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑ ԣԱԯԞ ՏՁՌՀԭՀՇՋՅԽՌ ԱՊԽԹՌՅԽՆՌ ԟՇԼՑԴԽՁԿՀՌ ԩԽՆԿՌՀ ՅԿԹՁԩ ԪԹՄԽՋ ԣԽՅԹՄԽՋ ԪԹՄԽՋ ԣԽՅԹՄԽՋ #ՅԽԹՆՅԽԹՋՍՊԽԼ% ԡՁԾԾ Ո ԡՁԾԾ Ո ԡՁԾԾ Ո ԡՁԾԾ Ո ԠՇՆՌՊՇՄ ԩԬԮ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԫԞ ԧՇՆԻՃՀԽԽՊԽ ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԡՍՆՆԽՌՌ0Ջ ԡՍՆՆԽՌՌ0Ջ ԡՍՆՆԽՌՌ0Ջ ԱԽՊՈՋՌՊԹ ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԡՁԾԾԡՁԾԾԽՊԽՆԻԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼՈԽՊԻԽՆՌ ԼՁԾԾԽՊԽՆԻԽՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ ԻՄԽԹՆՏԹՌԽՊ ԻՇՆՌՊՇՄ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈ ԩԬԮ ՅԿԹՁԩ ԢՌՍԼՑԡԽԾՁԻՁԽՆԻՁԽՋ ԱՀԽՊԽՏԽՊԽՅՁՆՇՊԼԽՎՁԹՌՁՇՆՋԼԽՋԻՊՁԺԽԼՁՆԽԻՌՁՇՆԦԪԹՌԽՊՁԹՄՋԹՆԼԪԽՌՀՇԼՋՇԾՌՀՁՋԡԢԯՊԽԿԹՊԼՁՆԿԾԹՁՄՍՊԽՌՇ ՊԽՈՇՊՌ ԻԽՊՌԹՁՆ ՏԹՌԽՊ ԻՀԹՊԹԻՌԽՊՁՋՌՁԻՋ ԪՇՊԽ ՁՅՈՇՊՌԹՆՌՄՑ ՌՀԽ ՋՌՍԼՑ ԼՁԼ ՆՇՌ ՅԽԽՌ ՌՀԽ ՅՁՆՁՅՍՅ ՇԾ ՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋՌՀԹՌԼՁԼՆՇՌԽՐՀՁԺՁՌՌՇՐՁԻՁՌՑԹՋՊԽԻՇՅՅԽՆԼԽԼԺՑՌՀԽԬԠԭԭԿՍՁԼԽՄՁՆԽ ԭԹԿԽՇԾ Page 160 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱՀԽԾԽԻՍՆԼՁՌՑՁՆՇՆԽՊԽՈՄՁԻԹՌԽՇԾՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՏԹՋ ԽԿԿՋԾԽՅԹՄԽԼԹՑՆՇՊԼՁԼ ՌՀԽ ՊԽՈՄՁԻԹՌԽ ՋՈԹՏՆ ԹՌ ՄԽԹՋՌ ԽՎԽՊՑ ԼԹՑՋ ԪԽԽՌՁՆԿ ՇՆԽ ՇԾ ՌՀԽՋԽ ԻՊՁՌԽՊՁԹ ՁՆ ՆԽԽԼԽԼ ՌՇ ԼԽՅՇՆՋՌՊԹՌԽԹԻՌՁՎԽՋՈԹՏՆՁՆԿ ԱՀԽԽՆԼՈՇՁՆՌՋԹՌՌՀԽՀՁԿՀՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ՅԿԹՁԩ ՏԽՊԽՆՇՌՋՌԹՌՁՋՌՁԻԹՄՄՑԹՆԹՄՑՒԽԼ ԼՍԽ ՌՇ ՅՇՊՌԹՄՁՌՑ ՇՆՄՑ ՇՆԽ ՅԹՄԽ ԹՆԼ ՇՆԽ ԾԽՅԹՄԽ ՋՍՊՎՁՎԽԼ ԹՌ ՋՌՍԼՑ ՌԽՊՅՁՆԹՌՁՇՆ ԱՀԽՊԽԾՇՊԽ ՇՆՄՑ ՌԽՋՌ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄՋ ՏԽՊԽ ԹՆԹՄՑՒԽԼ ՁՆ ՌՀՁՋ ՋՌՍԼՑ ԬԠԭԭ ԤՍՁԼԽՄՁՆԽ ՊԽԻՇՅՅԽՆԼՋ Թ ՅՁՆՁՅՍՅ ՇԾ ՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՈՄՍՋ Թ ՆԽԿԹՌՁՎԽ ԻՇՆՌՊՇՄ ԞՄՋՇ ՌՀԽ ՋՌՍԼՑ ԹՍՌՀՇՊՊԽՈՇՊՌԽԼՌՀԹՌՇԺՋԽՊՎԹՌՁՇՆՋԹՌՌՀԽՅԿԹՁԩԻՇՆԻԽՆՌՊԹՌՁՇՆՅԽՌՌՀԽԿՍՁԼԽՄՁՆԽ ԼԽԾՁՆՁՌՁՇՆՇԾՇՎԽՊՌՅՇՊԺՁԼՁՌՑՅԹՃՁՆԿՊԽՋՍՄՌՋԾՇՊՌՀՁՋՌՊԽԹՌՅԽՆՌՄԽՎԽՄԼՁԾԾՁԻՍՄՌՌՇՁՆՌԽՊՈՊԽՌ ԱՀԽՌՇՌԹՄՇՊԿԹՆՁԻԻԹՊԺՇՆՍՆՁՇՆՁՒԽԼԹՅՅՇՆՁԹԹՆԼՊԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽՁՆՌՀԽՌԽՋՌՏԹՌԽՊՏԽՊԽ ՆՇՌ ՊԽՈՇՊՌԽԼ ԱՀԽ ԬԠԭԭ ՈԽՊԾՇՊՅԹՆԻԽ ԻՊՁՌԽՊՁԹ ԽՋՌԹԺՄՁՋՀ ՅԹՐՁՅՍՅ ՄԽՎԽՄՋ ԾՇՊ ՌՀԽՋԽՎԹՄՍԽՋԹՆԼՁՌՁՋՍՆԻՄԽԹՊՁԾՌՀԽՅԹՐՁՅՍՅՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋՏԽՊԽԽՐԻԽԽԼԽԼ ԱՀԽՊԽՅԹՁՆՁՆԿՎԹՄՁԼՁՌՑԹՆԼՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹԾՇՊԬԠԭԭՏԽՊԽՅԽՌ ԣԯԽՎՁԽՏԽՊ0ՋԠՇՅՅԽՆՌՋ ԱՀԽ ՊԽՎՁԽՏԽՊ0Ջ ԹՆԼ ՌՀԽ ՋՌՍԼՑ ԹՍՌՀՇՊՋ0 ՊԽՋՍՄՌՋ ՏԽՊԽ ՁՆ ԹԿՊԽԽՅԽՆՌ ՊԽԿԹՊԼՁՆԿ ԽԾԾԽԻՌՋ ՇՆ ՋՍՊՎՁՎԹՄ ԾԽՊՌՁՄՁՒԹՌՁՇՆԹՆԼԾԽՅԹՄԽԤԦԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿԾԽԻՍՆԼՁՌՑՏԽՊԽԹՄՋՇՋՁՅՁՄԹՊՁՆՌՀԹՌՌՀԽՋՍՈՈՊԽՋՋՁՇՆԹՌ ՌՀԽՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄՏԹՋԻՇՆՋՁԼԽՊԽԼԺՁՇՄՇԿՁԻԹՄՄՑՊԽՄԽՎԹՆՌԟՇՌՀՌՀԽՋՌՍԼՑԹՍՌՀՇՊ0ՋԹՆԼ ՊԽՎՁԽՏԽՊ0ՋԹՆԹՄՑՋՁՋԼԽՌԽԻՌԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌՊԽԼՍԻՌՁՇՆՋ Ո ՁՆԾԽՊՌՁՄՁՌՑԹՆԼԾԽՅԹՄԽԤԦԹՌՌՀԽ ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄ ԻՇՆՋՁՋՌԽՆՌ ՏՁՌՀ ՌՀԽ ԻՇՆԻՄՍՋՁՇՆՋ ԾՇՄՄՇՏՁՆԿ ՌՀԽ ԬԠԭԭ ՅԽՌՀՇԼՇՄՇԿՑ ՍՋՁՆԿՌՀԽՊԽՋՍՄՌՋՇԾԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹՌԽՋՌ ԦԾՌՀԽՊԽՎՁԽՏԽՊԾՇՄՄՇՏՋՌՀԽՋՍԿԿԽՋՌՁՇՆՇԾՌՀԽ ՌՊԹԼՁՌՁՇՆԹՄ ԢԣԢԡ ՅԽՌՀՇԼՇՄՇԿՑ ՏՀՁԻՀ ՊԽԻՇՅՅԽՆԼՋ ԴՁՄՄՁԹՅ0Ջ ՌԽՋՌ ԾՇՊ ԻՇՆԻԽՆՌՊԹՌՁՇՆԼԽՈԽՆԼԽՆՌ ՈԹՌՌԽՊՆՋ ՋԹՌՁՋԾՑՁՆԿ ՈԹՊԹՅԽՌՊՁԻ ԹՋՋՍՅՈՌՁՇՆՋ ՋՁԿՆՁԾՁԻԹՆՌ ՊԽԼՍԻՌՁՇՆՋ ՏԽՊԽ ԹՄՋՇ ԼԽՌԽԻՌԽԼ ԹՌ ՌՀԽ ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ՄԽՎԽՄ Ո ԱՀԽ ՇԺՋԽՊՎԹՌՁՇՆՋ ՇԾ ԻՄՁՆՁԻԹՄ ՋՁԿՆՋ ԹՌ ՅԿ ԹՁԩ ՅԽՌ ՌՀԽ ԿՍՁԼԽՄՁՆԽ ԼԽԾՁՆՁՌՁՇՆՇԾՇՎԽՊՌՅՇՊԺՁԼՁՌՑՏՀՁԻՀՄԽՆԼՋՍՆԻԽՊՌԹՁՆՌՑՌՇՌՀԽՁՆՌԽՊՈՊԽՌԹՌՁՇՆՇԾՊԽՋՍՄՌՋԹՌՌՀՁՋՌՊԽԹՌՅԽՆՌՄԽՎԽՄ ԱՀԽ ՊԽՎՁԽՏԽՊ0Ջ ԻՇՆԻՄՍՋՁՇՆՋ ԺԹՋԽԼ ՇՆ ՌՀԽ ԬԠԭԭ ԾՄՇՏԻՀԹՊՌ ԹՊԽ ՈՊԽՋԽՆՌԽԼ ՁՆ ՌՀԽ ԢՐԽԻՍՌՁՎԽ ՍՅՅԹՊՑԹՆԼԠՇՆԻՄՍՋՁՇՆՋՋԽԻՌՁՇՆՋՇԾՌՀՁՋԡԢԯ ԭԹԿԽՇԾ Page 161 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԱՀԽՋՌՍԼՑԹՍՌՀՇՊՋՊԽՈՇՊՌԽԼՅԽԹՆՅԽԹՋՍՊԽԼՊԽԻՇՎԽՊՁԽՋՇԾՈՀՇՋՅԽՌԹՋՅԿԹՁԩԺԹՋԽԼՇՆՆՇՅՁՆԹՄ ԻՇՆԻԽՆՌՊԹՌՁՇՆ ՇԾ ՅԿ ԹՁԩ ՏԹՋ ՁՆԻՇՊՊԽԻՌ ՌՀԽ ՊԽՎՁԽՏԽՊ ՅԽԹՆ ՅԽԹՋՍՊԽԼ ՏԹՋ ՅԿ ԹՁԩ ԺԹՋԽԼՇՆՌՀԽԼԹՌԹՈՊՇՎՁԼԽԼԱՀԽՅԽՌՀՇԼՊԽԻՇՎԽՊՁԽՋՇԾՈՀՇՋՅԽՌՁՆԣՊՇԿԢՅԺՊՑՇԱԽՊԹՌՇԿԽՆԽՋՁՋԞՋՋԹՑ ԵԽՆՇՈՍՋ ԣԢԱԞԵ ՋՇՄՍՌՁՇՆԾՇՊՌՁԾՁԽԼԹՌԹՆԼՅԿԹՁԩՏԽՊԽչ Ն ՍՋՁՆԿԹՆԞԿՁՄԽՆՌԩԻՇՄՍՅՆ ՀՇՏԽՎԽՊԣԢԱԞԵՋՇՄՍՌՁՇՆՏԹՋՆՇՌՍՋԽԼԹՋՌՀԽԼՁՄՍՌՁՇՆՏԹՌԽՊՋՇՍՊԻԽ ՁՆՌՀՁՋԹՋՋԹՑ ԦԦ ԯԢԣԢԯԢԫԠԢ ԞՆՃՄԽՑԤԱԧԽՆՋԽՆԨԪԨԹՀՄԪԡԨՇՊՌԽԧԧԹՆԼԪԹՃՑՆԽՆԢԞԡԽՋԻՊՁՈՌՁՇՆԹՆԼԽՎԹՄՍԹՌՁՇՆ ՇԾԹՋՀՇՊՌՌԽՊՅՊԽՈՊՇԼՍԻՌՁՇՆՌԽՋՌՏՁՌՀՌՀԽԾԹՌՀԽԹԼՅՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԢՆՎՁՊՇՆԱՇՐՁԻՇՄԠՀԽՅ ԞԭԥԞ ԞԴԴԞ ԴԭԠԣ ՌԹՆԼԹՊԼ ԪԽՌՀՇԼՋ ԾՇՊ ՌՀԽ ԢՐԹՅՁՆԹՌՁՇՆ ՇԾ ԴԹՌԽՊ ԹՆԼ ԴԹՋՌԽՏԹՌԽՊ ՌՀ ԢԼՁՌՁՇՆԴԹՋՀՁՆԿՌՇՆԡԠ ԞԱԪ ԠՇՅՅՁՌՌԽԽ Ԣ ՇՆ ԟՁՇՄՇԿՁԻԹՄ ԢԾԾԽԻՌՋ ԹՆԼ ԢՆՎՁՊՇՆՅԽՆՌԹՄ ԣԹՌԽ ՌԹՆԼԹՊԼ ԤՍՁԼԽ ԾՇՊ ԠՇՆԼՍԻՌՁՆԿԢԹՊՄՑԩՁԾԽՌԹԿԽԱՇՐՁԻՁՌՑԱԽՋՌՋՏՁՌՀԣՁՋՀԽՋԞԱԪԡԽՋՁԿՆԹՌՁՇՆԢ ԞՅԽՊՁԻԹՆՇԻՁԽՌՑ ԾՇՊԱԽՋՌՁՆԿԹՆԼԪԹՌԽՊՁԹՄՋԟԹՊՊԥԹՊԺՇՊԡՊՁՎԽԭԬԟՇՐԠԴԽՋՌԠՇՆՋՀՇՀՇԻՃԽՆԭԽՆՆՋՑՄՎԹՆՁԹ ԦՎԽՋԪՁԻՀԹԽՄԠԢԱԦԠՇՅՈՊԽՀԽՆՋՁՎԽԢՆՎՁՊՇՆՅԽՆՌԹՄԱՇՐՁԻՁՌՑԦՆԾՇՊՅԹՌՁՇՆՑՋՌԽՅգԲՋԽՊ ՋԤՍՁԼԽ ԱՁԼԽՈՇՇՄԻՁԽՆՌՁԾՁԻՇԾՌՏԹՊԽԪԻԨՁՆՄԽՑՎՁՄՄԽԠԹՄՁԾՇՊՆՁԹԪՇՍՆՌԡԦԹՆԼԴԞԟՊՍՆԿՋԞՋՁՅՈՄՁԾՁԽԼ ԼՇՋՁՆԿԹՈՈԹՊԹՌՍՋԾՇՊԾՁՋՀՌՇՐՁԻՇՄՇԿՑՋՌՍԼՁԽՋԴԹՌԽՊԯԽՋԽԹՊԻՀ ՈՊԹԿՍԽԧԟԪԽԹՋՍՊԽՅԽՆՌՇԾՈՇՄՄՍՌԹՆՌՌՇՐՁԻՁՌՑՌՇԾՁՋՀԟՁՇԹՋՋԹՑՅԽՌՀՇԼՋԾՇՊԹԻՍՌԽՌՇՐՁԻՁՌՑ ԴԹՌԽՊԯԽՋ ԬԢԠԡ ԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑԬԢԠԡԤՍՁԼԽՄՁՆԽԾՇՊՌՀԽՌԽՋՌՁՆԿԠՀԽՅՁԻԹՄՋ ԱԽՋՌ ԫՇԭԹՊՁՋԣՊԹՆԻԽՈՈ ԭԹԿԽՇԾ Page 162 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ Բ ԢԭԞ Ԡԣԯ ԭԹՊՌ ԣԽԼԽՊԹՄ ԦՆՋԽԻՌՁԻՁԼԽ ԣՍՆԿՁԻՁԼԽ ԹՆԼ ԯՇԼԽՆՌՁԻՁԼԽ ԞԻՌ ԤՇՇԼ ԩԹԺՇՊԹՌՇՊՑ ԭՊԹԻՌՁԻԽՋ ՌԹՆԼԹՊԼՋ ԣՁՆԹՄ ԯՍՄԽ ԬԾԾՁԻԽ ՇԾ ՌՀԽ ԣԽԼԽՊԹՄ ԯԽԿՁՋՌԽՊ ԫԹՌՁՇՆԹՄ ԞՊԻՀՁՎԽՋ ԹՆԼ ԯԽԻՇՊԼՋԞԼՅՁՆՁՋՌՊԹՌՁՇՆԲԤՇՎԽՊՆՅԽՆՌԭՊՁՆՌՁՆԿԬԾԾՁԻԽԴԹՋՀՁՆԿՌՇՆԡԠ ԲԢԭԞՀՇՊՌՌԽՊՅՅԽՌՀՇԼՋԾՇՊԽՋՌՁՅԹՌՁՆԿՌՀԽԻՀՊՇՆՁԻՌՇՐՁԻՁՌՑՇԾԽԾԾՄՍԽՆՌՋԹՆԼՊԽԻԽՁՎՁՆԿՏԹՌԽՊՋ ՌՇ ՅԹՊՁՆԽ ԹՆԼ ԽՋՌՍԹՊՁՆԽ ՇՊԿԹՆՁՋՅՋ ԱՀՁՊԼ ԽԼՁՌՁՇՆ ԬԾԾՁԻԽ ՇԾ ԴԹՌԽՊ Բ ԢՆՎՁՊՇՆՅԽՆՌԹՄ ԭՊՇՌԽԻՌՁՇՆ ԞԿԽՆԻՑԴԹՋՀՁՆԿՌՇՆԡԠԢԭԞԯ ԲԢԭԞԬԾԾՁԻԽՇԾԭՊԽՎԽՆՌՁՇՆԭԽՋՌՁԻՁԼԽՋԹՆԼԱՇՐՁԻՍԺՋՌԹՆԻԽՋԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌԽՊԻՊԽԽՆՁՆԿ ԭՊՇԿՊԹՅԱԽՋՌԤՍՁԼԽՄՁՆԽԬԠԭԭԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑԢԭԞԠ ԬԻՌՇԺԽՊԲԢՆՎՁՊՇՆՅԽՆՌԹՄԭՊՇՌԽԻՌՁՇՆԞԿԽՆԻՑԴԹՋՀՁՆԿՌՇՆԡԠ ԭԹԿԽՇԾ Page 163 of 260 ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ ԞԭԭԢԫԡԦԵԦԬԲԱԭԲԱԬԣԯԢԳԦԢԴԢԯ0ԱԞԱԦԱԦԠԞԩԳԢԯԦԣԦԠԞԱԦԬԫ Fisher's Exact Test ======NUMBER OF ------ IDENTIFICATION ALIVE DEAD TOTAL ANIMALS ------ CONTROL 24 0 24 0.001 20 4 24 ------ TOTAL 44 4 48 ====== Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 20. Since b is greater than 19.0 there is no significant difference between CONTROL and TREATMENT at the 0.05 level. Fisher's Exact Test ======NUMBER OF ------ IDENTIFICATION ALIVE DEAD TOTAL ANIMALS ------ CONTROL 24 0 24 0.0093 18 6 24 ------ TOTAL 42 6 48 ====== Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 18. Since b is less than or equal to 19.0 there is a significant difference between CONTROL and TREATMENT at the 0.05 level. Summary of Fisher's Exact Tests ------NUMBER NUMBER SIG GROUP IDENTIFICATION EXPOSED DEAD 0.05 ------CONTROL 24 0 1 0.001 24 4 2 0.0093 24 6 * ------ Test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Page 164 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.934 0.421 0.388 0.689 USE PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.51 0.11 0.06 7.43 1.33, 1.69 Dose1 4 1.55 0.16 0.08 10.44 1.29, 1.81 Dose2 4 1.33 0.19 0.10 14.35 1.03, 1.63 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.52 1.40 1.61 . . Dose1 1.54 1.39 1.75 102.58 -2.58 Dose2 1.29 1.15 1.59 87.85 12.15 ******************************************************************************** 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 2 9 2.26 0.160 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 1.51 . 1.53 ...... Dose1 1.55 0.916 1.53 0.654 . . . . . Dose2 1.33 0.226 1.33 0.084 0.170 . . . . ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 4.19 0.123 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 1.52 . . Dose1 1.54 0.889 0.614 Dose2 1.29 0.156 0.054 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences) Page 165 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.897 0.146 1.040 0.392 USE PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 3.18 0.59 0.30 18.62 2.23, 4.12 Dose1 4 3.11 0.71 0.36 22.94 1.97, 4.24 Dose2 4 2.69 0.27 0.13 10.03 2.26, 3.12 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 2.94 2.77 4.05 . . Dose1 2.96 2.41 4.10 97.80 2.20 Dose2 2.77 2.31 2.90 84.61 15.39 ******************************************************************************** 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 2 9 0.90 0.440 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 3.18 . 3.18 ...... Dose1 3.11 0.977 3.11 0.508 . . . . . Dose2 2.69 0.393 2.69 0.156 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 2 2.92 0.232 MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend Level Median MannWhit p-value Jonckheere p-value Ctrl 2.94 . . Dose1 2.96 0.889 0.614 Dose2 2.77 0.235 0.094 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences) Page 166 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.958 0.749 0.033 0.968 USE PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 51.25 1.26 0.63 2.46 49.25, 53.25 Dose1 4 52.00 1.41 0.71 2.72 49.75, 54.25 Dose2 4 51.00 1.41 0.71 2.77 48.75, 53.25 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 51.00 50.00 53.00 . . Dose1 51.50 51.00 54.00 101.46 -1.46 Dose2 51.50 49.00 52.00 99.51 0.49 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 0.58 0.578 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 51.25 . 51.63 ...... Dose1 52.00 0.666 51.63 0.734 . . . . . Dose2 51.00 0.952 51.00 0.504 0.574 . . . . ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 0.80 0.671 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 51.00 . . Dose1 51.50 0.467 0.822 Dose2 51.50 1.000 0.561 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences) Page 167 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.905 0.184 1.641 0.247 USE PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 62.25 2.22 1.11 3.56 58.72, 65.78 Dose1 4 62.75 5.12 2.56 8.16 54.60, 70.90 Dose2 4 62.25 1.89 0.95 3.04 59.24, 65.26 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 62.00 60.00 65.00 . . Dose1 61.50 58.00 70.00 100.80 -0.80 Dose2 61.50 61.00 65.00 100.00 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 2 9 0.03 0.972 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 62.25 . 62.50 ...... Dose1 62.75 0.969 62.50 0.626 . . . . . Dose2 62.25 1.000 62.25 0.618 0.977 . . . . ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 0.04 0.980 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 62.00 . . Dose1 61.50 1.000 0.442 Dose2 61.50 1.000 0.530 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences) Page 168 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.869 0.063 2.862 0.109 USE PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1440000 518716.4 259358.2 36.02 614606.5, 2265393 Dose1 4 950000.0 574224.1 287112.1 60.44 36281.29, 1863719 Dose2 4 1430000 1600458 800229.1 111.92 -1116686, 3976686 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1350000 960000.0 2100000 . . Dose1 1070000 160000.0 1500000 65.97 34.03 Dose2 740000.0 440000.0 3800000 99.31 0.69 ******************************************************************************** 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 2 9 0.30 0.749 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 1440000 . 1440000 ...... Dose1950000.0 0.732 1190000 0.438 . . . . . Dose2 1430000 1.000 1190000 0.467 0.791 . . . . ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 1.42 0.491 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 1350000 . . Dose1 1070000 0.346 0.124 Dose2 740000.0 0.494 0.153 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences) Page 169 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.672 <.001 8.295 0.009 USE NON-PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 27478.00 44186.91 22093.45 160.81 -42833.2,97789.23 Dose1 4 8064.75 9220.67 4610.33 114.33 -6607.39,22736.89 Dose2 4 350353.0 699764.8 349882.4 199.73 -763129, 1463835 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 8450.00 12.00 93000.00 . . Dose1 8046.50 66.00 16100.00 29.35 70.65 Dose2 665.00 82.00 1400000 1275.03 -1175.03 ******************************************************************************** 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 2 9 0.90 0.439 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 27478.00 . 128631.9 ...... Dose1 8064.75 0.997 128631.9 0.722 . . . . . Dose2350353.0 0.453 128631.9 0.756 0.485 . . . . ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 0.04 0.981 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 8450.00 . . Dose1 8046.50 1.000 0.500 Dose2 665.00 0.889 0.500 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences) Page 170 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.943 0.538 0.989 0.409 USE PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 12.50 1.91 0.96 15.32 9.45, 15.55 Dose1 4 11.30 3.22 1.61 28.48 6.18, 16.42 Dose2 4 8.23 3.26 1.63 39.66 3.03, 13.42 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 12.00 11.00 15.00 . . Dose1 11.50 7.20 15.00 90.40 9.60 Dose2 8.60 4.70 11.00 65.80 34.20 ******************************************************************************** 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 2 9 2.36 0.149 Dunnett - testing each trt mean signif. different than control Williams - test assumes dose-response relationship, testing negative trend Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC Level Mean Dunnett Isotonic Williams Tukey p-values p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 Ctrl 12.50 . 12.50 ...... Dose1 11.30 0.784 11.30 0.339 . . . . . Dose2 8.23 0.112 8.23 0.039 0.329 . . . . ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 3.93 0.140 MannWhit - testing each trt median signif. different from control Jonckheere - test assumes dose-response relationship, testing negative trend Level Median MannWhit p-value Jonckheere p-value Ctrl 12.00 . . Dose1 11.50 0.775 0.328 Dose2 8.60 0.135 0.028 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2 Page 171 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.874 0.074 0.161 0.854 USE PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 1.33 0.32 0.16 24.16 0.82, 1.83 Dose1 4 1.45 0.38 0.19 26.11 0.85, 2.05 Dose2 4 1.29 0.26 0.13 20.27 0.87, 1.71 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 1.20 1.10 1.80 . . Dose1 1.30 1.20 2.00 109.43 -9.43 Dose2 1.35 0.96 1.50 97.36 2.64 ******************************************************************************** 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 2 9 0.27 0.769 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 1.33 . 1.39 ...... Dose1 1.45 0.811 1.39 0.692 . . . . . Dose2 1.29 0.983 1.29 0.551 0.770 . . . . ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 0.54 0.764 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 -1.20 . . Dose1 -1.30 0.467 0.178 Dose2 -1.35 1.000 0.380 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL Williams >highest dose (no sign. differences) Jonckheere >highest dose (no sign. differences) Page 172 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet 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) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value . . . . NO DATA FOR TEST ******************************************************************************** Page 173 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.953 0.686 1.098 0.374 USE PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 26.00 2.94 1.47 11.32 21.32, 30.68 Dose1 4 29.50 5.00 2.50 16.95 21.54, 37.46 Dose2 4 12.50 6.45 3.23 51.64 2.23, 22.77 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 25.50 23.00 30.00 . . Dose1 27.00 27.00 37.00 113.46 -13.46 Dose2 15.00 3.00 17.00 48.08 51.92 ******************************************************************************** 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 2 9 12.84 0.002 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 26.00 . 27.75 ...... Dose1 29.50 0.534 27.75 0.769 . . . . . Dose2 12.50 0.008 12.50 0.002 0.003 . . . . ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 8.46 0.015 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 27.00 0.225 0.930 Dose2 15.00 0.067 0.052 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere >highest dose (no sign. differences) Page 174 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.904 0.180 2.959 0.103 USE PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 15.85 6.95 3.48 43.87 4.78, 26.92 Dose1 4 19.00 12.91 6.45 67.95 -1.54, 39.54 Dose2 4 3.01 2.35 1.17 78.05 -0.73, 6.74 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 17.50 6.40 22.00 . . Dose1 14.00 10.00 38.00 119.87 -19.87 Dose2 2.55 0.83 6.10 18.97 81.03 ******************************************************************************** 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 2 9 3.91 0.060 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 15.85 . 17.43 ...... Dose1 19.00 0.827 17.43 0.687 . . . . . Dose2 3.01 0.110 3.01 0.039 0.064 . . . . ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 7.38 0.025 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 17.50 . . Dose1 14.00 1.000 0.500 Dose2 2.55 0.067 0.010 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2 Page 175 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ test for fish screen study - Phosmet ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility ) TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 Use parametric analyses if neither test rejected, otherwise non-parametric analyses. Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion Test Stat P-value Test Stat P-value 0.987 0.998 3.109 0.094 USE PARAMETRIC TESTS ******************************************************************************** BASIC SUMMARY STATISTICS Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval Ctrl 4 97.00 2.16 1.08 2.23 93.56, 100.44 Dose1 4 95.75 1.89 0.95 1.98 92.74, 98.76 Dose2 4 90.25 4.79 2.39 5.30 82.63, 97.87 Level Median Min Max %of Control(means) %Reduction(means) Ctrl 96.50 95.00 100.00 . . Dose1 96.50 93.00 97.00 98.71 1.29 Dose2 90.00 85.00 96.00 93.04 6.96 ******************************************************************************** PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Analysis of Variance (ANOVA) - overall F-test Numerator df Denominator df F-stat P-value 2 9 4.97 0.035 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 97.00 . 97.00 ...... Dose1 95.75 0.810 95.75 0.356 . . . . . Dose2 90.25 0.029 90.25 0.009 0.090 . . . . ******************************************************************************** NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests Kruskal-Wallis test - equality among treatment groups Degrees of Freedom TestStat P-value 2 5.10 0.078 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 96.50 . . Dose1 96.50 0.775 0.328 Dose2 90.00 0.125 0.016 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL Williams Dose2 Jonckheere Dose2 Male tubercle score (exact Mann-Whitney) Obs conc endpoint 1 0 23 2 0 30 3 0 26 Page 176 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ Obs conc endpoint 4 0 25 5 1 37 6 1 27 7 1 27 8 1 27 9 2 17 10 2 16 11 2 14 12 2 3 13 3 . 14 3 . 15 3 . 16 3 . Page 177 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ The NPAR1WAY Procedure Wilcoxon Scores (Rank Sums) for Variable end point Classified by Variable conc Sum of Scor Expected Std Dev Mean conc N es Under H0 Under H0 Score 0 4 29.0 26.0 5.846522 7.250 1 4 39.0 26.0 5.846522 9.750 2 4 10.0 26.0 5.846522 2.500 Average scores were used for ties. Kruskal-Wallis Test Chi-Square 8.4645 DF 2 Asymptotic Pr > Chi-Square 0.0145 Exact Pr >= Chi-Square 0.0017 Page 178 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ The NPAR1WAY Procedure Page 179 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ The NPAR1WAY Procedure Wilcoxon Scores (Rank Sums) for Variable endpoint Classified by Variable conc Sum of Expected Std Dev Mean conc N Scores Under H0 Under H0 Score 0 4 13.0 18.0 3.380617 3.250 1 4 23.0 18.0 3.380617 5.750 Average scores were used for ties. Wilcoxon Two-Sample Test Statistic (S) 13.0000 Normal Approximation Z -1.3311 One-Sided Pr < Z 0.0916 Two-Sided Pr > |Z| 0.1832 t Approximation One-Sided Pr < Z 0.1124 Two-Sided Pr > |Z| 0.2249 Exact Test One-Sided Pr <= S 0.1000 Two-Sided Pr >= |S - Mean| 0.2000 Z includes a continuity correction of 0.5. Kruskal-Wallis Test Chi-Square 2.1875 DF 1 Pr > Chi-Square 0.1391 Page 180 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ The NPAR1WAY Procedure Page 181 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ The NPAR1WAY Procedure Wilcoxon Scores (Rank Sums) for Variable endpoin t Classified by Variable conc Sum of Expected Std Dev Mean conc N Scores Under H0 Under H0 Score 0 4 26.0 18.0 3.464102 6.50 2 4 10.0 18.0 3.464102 2.50 Wilcoxon Two-Sample Test Statistic (S) 26.0000 Normal Approximation Z 2.1651 One-Sided Pr > Z 0.0152 Two-Sided Pr > |Z| 0.0304 t Approximation One-Sided Pr > Z 0.0336 Two-Sided Pr > |Z| 0.0671 Exact Test One-Sided Pr >= S 0.0143 Two-Sided Pr >= |S - Mean| 0.0286 Z includes a continuity correction of 0.5. Kruskal-Wallis Test Chi-Square 5.3333 DF 1 Pr > Chi-Square 0.0209 Page 182 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ The NPAR1WAY Procedure Page 183 of 260 ԭԹԿԽՇԾ ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԭՀՇՋՅԽՌ ԢԭԞԪԯԦԡԫՍՅԺԽՊ The NPAR1WAY Procedure ԭԹԿԽՇԾ Page 184 of 260 DATA EVALUATION RECORD PHOSMET Study Type: OCSPP 890.1400, In vivo Hershberger Assay EPA Contract No. EP10H001452 Task Assignment No. 2-4-2012 (MRID 48618706) Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive Arlington, VA 22202 Prepared by Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B Durham, NC 27713 Primary Reviewer Signature: Rebecca Bryan, B.S. Date: 12/20/2011 Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 1/03/2012 Program Manager: Signature: Jack D. Early, M.S. Date: 1/04/2012 Quality Assurance: Signature: Jack D. Early, M.S. Date: 1/04/2012 This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel. Page 185 of 260 The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document). Page 186 of 260 /11 vivo Hershberger Assay (2011)/ Page I of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 Primary Reviewer: Greg Akerman, Ph.D. Signature: _e,;...... ,..1£.9,,...... :::.L...-- Health Effects Division Da te: ,,.--....;~""-~~!...,.._ Secondary Reviewer: John Liccione. Ph.D. Signature: ::::zS~~==~z:= Health Effects Division Date: =-~'64~..+--!'-.;2--- DATA EVALUATION RECORD STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 PC CODE: 059201 DP BARCODE: D397165 TXR#: 0056199 CAS#: 732-11-6 TEST MATERIAL (PURITY): Phosmet (96.8% a.i.) SYNONYMS: 0 , 0-dimethyl S-phthalimidomethylphosphorodithioate CITATION: Zorrilla, L. (2011 ). The Hershberger assay with phosmet in castrated CRL:CD(SD) IGS Rats. Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: C 194-200, October 26, 2011. MRID 48618706. Unpublished. SPONSOR: Gowan Company, P.O. Box 5569, Yuma, AZ TEST ORDER#: EDSP-059201 -54 EXECUTIVE SUMMARY: The Hershberger Assay consists of androgenic and anti androgenic components. To screen for potential androgenic activity, phosmet (96.8% a.i., Batch # 6QH9118000) in 0.5% aqueous carboxymethyl cellulose was administered daily via oral gavage to 59- to 60-day old, castrated male Sprague Dawley rats (8/dose group) at dose levels of 0 (vehicle), 5 or 25 mg/kg/day. An androgenic positive control group consisted of 10 castrated rats dosed by subcutaneous injection with 0.4 mg/kg/day testosterone propionate (TP) prepared in com oil. To screen for potential anti-androgenic activity phosmet (96.8% a.i., Batch# 6QH91l8000) in 0.5% aqueous carboxymethyl cellulose was administered daily via oral gavage to 59 to 60-day old, castrated male Sprague Dawley rats (8/dose group) at dose levels ofO (vehicle), 5, 15, or 25 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by subcutaneous injection. An anti-androgenic positive control group consisted of 10 castrated rats exposed to 0.4 mg/kg/day TP and 3.0 mg/kg/day flutamide (FT). Flutamide and TP were prepared in com oil. For both components of the assay, the animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent tissues. Page 187 of 260 In vivo Hershberger Assay (2011)/ Page 2 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 All animals survived until scheduled termination. Cholinergic signs (tremors) and a 12% decrease in terminal bodyweight was seen at 25 mg/kg/day suggesting that the high dose may have been excessive. In the androgen agonist assay, clinical signs in the 25 mg/kg/day group included tremors, uncoordinated movements and porphyrin staining around the eyes. No clinical signs were observed in the 5 mg/kg/day group. Body weights in the 5 and 25 mg/kg/day groups were comparable to controls throughout the duration of the assay. At 25 mg/kg/day, mean overall (Days 1-11) body weights gains were decreased (p≤0.05) by 69%. Animals dosed with 0.4 mg/kg/day TP had increased (p≤0.05) mean body weights of 12% on Day 11, and increased mean overall body weight gains (p≤0.05) of 105%. There were no significant increases in the accessory sex organ weights in animals dosed with the test compound. Animals in the 0.4 mg/kg/day TP group had accessory sex organ weight increases (p≤0.05) as follows: 1926% in seminal vesicles; 1264% in ventral prostate; 251% in levator ani-bulbocavernosus (LABC); 794% in Cowper’s gland; and 72% in glans penis. All tissue weight CVs were below the maximum values recommended by the test guideline. The performance criteria indicated that this assay was performing as expected. In the anti-androgen assay, clinical signs in the 25 mg/kg/day group included tremors, and uncoordinated movements. No clinical signs were observed in the 5 or 15 mg/kg/day groups. At 25 mg/kg/day, mean body weights were decreased (p≤0.05) by 12% on Day 11, and overall body weight gains were decreased (p≤0.05) by 60%. There were no effects on body weights or body weight gains in the 5 and 15 mg/kg/day group, or the positive control group. At 15 mg/kg/day, the weights of seminal vesicles were decreased 25% (p≤0.05). At 25 mg/kg/day, accessory sex organ weight decreases (p≤0.05) were noted as follows: 50% in seminal vesicles; 40% in ventral prostate; 27% in LABC; 34% in Cowper’s gland; and 10% in glans penis. Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) accessory sex organ weights as follows: 85% in seminal vesicles; 76% in ventral prostate; 50% in LABC; 66% in Cowper’s gland; and 21% in glans penis. All tissue weight CVs were below the maximum values recommended by the test guideline. The performance criteria indicated that this assay was performing as expected. Phosmet was negative for androgenic activity as no statistically significant increases in organ weights were seen in two or more of the five androgen sensitive tissues in the androgen agonist assay. However, phosmet was positive for anti-androgenicity in the Hershberger assay as statistically significant decreases in organ weights were seen in all five androgen sensitive tissues in the androgen antagonist assay. However, the decreases in androgen-sensitive tissue weights were seen at a dose (25 mg/kg/day) that resulted in overt toxicity (12% decrease in body weight and clinical signs), which confounds the interpretation of the results. . 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 188 of 260 In vivo Hershberger Assay (2011)/ Page 3 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 I. MATERIALS AND METHODS A. MATERIALS 1. Test Facility: Integrated Laboratory Systems, Inc. Location: Durham, NC Study Director: L. Zorrilla Other Personnel: Not provided Study Period: April 28, 2011-May 9, 2011 2. Test Substance: Phosmet Description: Off white solid Source: Gowan Company (commercial technical product) Batch #: 6QH9118000, expires January 31, 2012 Purity: 96.8 % Stability: Stable in the vehicle for up to 11 days at 2-8°C. CAS #: 732-11-6 Structure: 3. Reference Androgen: Testosterone propionate (TP) Supplier: Sigma-Aldrich Company (St. Louis, MO) Lot #: 048K1328 Purity: 100.2%, expires March 17, 2012 CAS # : 57-85-2 4. Reference Anti-androgen: Flutamide (FT) Supplier: Sigma-Aldrich Company (St. Louis, MO) Lot #: 107K1293 Purity: >99% CAS # : 13311-84-7 5. Solvent/Vehicle Control (Phosmet): Carboxymethyl cellulose (in sodium phosphate buffer) Supplier: Sigma-Aldrich Company (St. Louis, MO) Lot #: 100M0113 Rationale (if other than water): CMC (0.5%) was the vehicle used in several sponsor studies where th test substance was administered by gavage. Final concentration: 0.5% 6. Solvent/Vehicle Control (Testosterone propionate and Flutamide): Corn Oil Supplier: MP Biomedicals, LLC (Solon, OH) Lot/Batch #: 7862K Rationale (if other than water): Not provided. Final concentration: Not applicable Page 189 of 260 In vivo Hershberger Assay (2011)/ Page 4 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 7. Test Animals: Species: Rat (castrated males) Strain: Sprague Dawley (Crl:CD[SD]) Age/weight at dose initiation: Post-natal day (PND) 59-60; 240.0-313.5 g Source: Charles River Laboratories International, Inc., Raleigh, NC Housing: 2/cage in polycarbonate cages with absorbent heat-treated hardwood bedding (Northeastern Products Corp., Warrensburg, NY) Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad libitum; low phytoestrogen with total geinstein equivalents = 6.2 µg/g diet. Water: Reverse osmosis treated tap water, ad libitum Environmental conditions: Temperature: 22-26ºC Humidity: 33-48% Air changes: Not provided Photoperiod: 12 hrs light/12 hrs dark Acclimation period: 10 days at test facility B. STUDY DESIGN 1. In life dates: Start: 4/28/11 End: 5/9/11 2. Study Design: The present Hershberger Assay was conducted to screen for both potential anti-androgenic and androgenic activity. The test substance was administered daily via oral gavage to groups of castrated male rats both without TP (androgenic activity) and with a daily dose of TP (0.4 mg/kg/day) by subcutaneous injection (anti-androgenic activity). 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. Positive androgenic activity is defined as a significant increase in two or more organ weights compared to the vehicle control. 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: Following a 10-day acclimation period at the test facility, castrated male rats (surgical manipulation performed by Supplier at PND 42) were administered the test substance from PND 59-60 to PND 68-69. Rats were euthanized on PND 69-70, approximately 24 hours after the last dose and necropsied for organ weight measurements. 4. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1. Statistical analysis indicated that there were no significant differences in group means at study initiation. Page 190 of 260 In vivo Hershberger Assay (2011)/ Page 5 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 Table 1. Study design a Test group Dose (mg/kg/day) # of Males Androgen Agonist Assay Vehicle control (0.5% CMC) 0 8 Low 5 8 High 25 8 Positive control (0.5% CMC) +TP b 0.4 8 Anti-Androgen Assay Vehicle control (0.5% CMC) +TP b 0 8 Low (+TP) 5 8 Mid (+TP) 15 8 High (+TP) 25 8 Flutamide (oral gavage) + TP (s.c.), positive control 3 8 a Data were obtained from Tables 1 and 2 on page 17 of the study report. b Same animals dosed for both androgen agonist (serves as positive control) and anti-androgen (served as vehicle control) assays. This did not affect the outcome or conclusions of this study. 5. Dose Selection Rationale: The phosmet dose levels were selected based on the results from an acute oral gavage study (MRID 46593101), repeated-dose studies (MRIDs 44811801, 41520001, and 41916401), and a non-GLP six week range-finding study (no MRID or study number). Mortality was observed in ≥75 mg/kg treatment groups for the acute study. No mortality was observed in the repeated-dose dietary studies conducted at doses of up to 98.3 mg/kg/day. Cholinergic clinical signs were observed in 36 mg/kg males in the acute oral gavage study; however, no effects were observed at 22.5 mg/kg. In the range finding study, no cholinergic effects were observed for dietary doses up to 49.2 mg/kg/day and body weights were reduced 11.2% at 22.5 mg/kg/day. Based on these results, doses of 0, 5 and 25 mg/kg/day were used for both the androgen agonist and anti- androgen assays. 6. (a) Dose Preparation: Dose formulations were prepared once prior to initiation of treatment and twice during treatment by initially suspending the appropriate amounts of test substance in 0.5% aqueous carboxymethyl cellulose. Flutamide and TP were prepared daily as solutions in corn oil. Homogeneity and concentration analyses were performed on all dose formulations. Stability analyses were performed on 1.00, 3.00, and 5.00 mg a.i./mL formulations prior to administration following storage at 2-8°C for up to 8 days. In a previous storage stability study1, phosmet (lot number not reported) was determined to be stable in the 0.5% aqueous carboxymethyl cellulose for up to 11 days (temperature not reported). 1 Reibach, R. 2011. Storage Stability of Phosmet in 0.5% Aqueous Carboxymethylcellulose. Unpublished study report prepared by Smithers Viscient Inc., Study No. 12791-6139. Page 191 of 260 In vivo Hershberger Assay (2011)/ Page 6 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 (b) Dose Analysis Results Homogeneity (%CV): 2.45-5.45% Stability (% of Day 0): 98-104% following storage for up 8 days at 2-8°C. Concentration (% of nominal): 87-105% 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: Test formulations were administered to the animals daily via oral gavage (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw, and 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: Body weights, body weight gains and accessory sex organ weights were analyzed using studentized residual plots for outliers and Levene’s test for homogeneity of variance. Heterogeneous data (glans penis weights) were transformed and re-analyzed using Kruskal-Wallis and Dunn’s test. Body weights, body weight gains, and accessory sex organ weights were analyzed using one-way ANOVA followed by Dunnett’s t-tests. The positive controls were compared to the vehicle controls using the t-test. Significance was denoted at p≤0.05. The statistical analyses were considered adequate. C. METHODS 1. Clinical Examinations: Animals were checked for mortality or moribundity twice daily on weekdays and once daily on weekends. Rats were also checked for clinical signs of toxicity prior to dose administration and at termination. If adverse clinical signs were seen, additional observations were noted. 2. Body Weight: Animals were weighed at randomization, daily prior to dosing throughout the dosing period, and at termination. 3. Food Consumption (Optional): Food consumption was not determined. 4. Serum Hormone Measurements (Optional): Serum hormone measurements were not conducted. 5. Dissection and Measurement of Tissue and Organ Weights: On PND 69-70 (approximately 24 hours after the final administration of the test substance), all rats were weighed, anesthetized by CO2 inhalation followed by cervical dislocation and subjected to a gross necropsy. The five mandatory androgen-dependent organs (ventral prostate, seminal vesicles with coagulating glands and fluid, LABC, Cowper’s gland, and glans penis) were excised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400). Page 192 of 260 In vivo Hershberger Assay (2011)/ Page 7 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 II. RESULTS A. OBSERVATIONS 1. Mortality: All animals survived until scheduled termination. 2. Clinical signs of toxicity: In the androgen agonist assay, clinical signs at 1-4 hours after dosing included one 25 mg/kg/day rat with tremors on Days 3-9 and the same animal showed uncoordinated movement on Days 4 and 5. Six other 25 mg/kg/day rats also exhibited tremors 1-4 hours post-dose on Days 3-9 (7 total rats). At 24 hours after dosing, one 25 mg/kg/day rat exhibited porphyrin staining around the eyes on Day 9. No clinical signs were observed in the 5 mg/kg/day group. Table 2. Incidence of Clinical Observations in the Androgen Agonist Assay a,b Dose (mg/kg/day) Positive Control Observation Vehicle Control Vehicle + TP 5 25 (0.4) b # # # # # # # # Obs. Exam Obs. Exam Obs. Exam Obs. Exam Porphyrin staining 0 8 0 8 0 8 1 8 around eyes a Data were obtained from page 83 of the study report. b The observations of tremors were reported on page 21 of the study report but were not included in the clinical observation tables in the appendices. # Obs. = number of observed incidences. # Exam = number of animals examined. In the anti-androgen assay, clinical signs at 1-4 hours after dosing included one 25 mg/kg/day rat with tremors on Days 3 and 7 and uncoordinated movement on Day 3. Another 25 mg/kg rat exhibited tremors on days 3-9 and uncoordinated movement on day 5. Six other 25 mg/kg/day rats also exhibited tremors between Days 4 and 8. At 24 hours after dosing, no clinical signs were observed in any dose group. No clinical signs were observed in the 5 or 15 mg/kg/day groups. Table 3. Incidence of Clinical Observations in the Anti-Androgen Assay a Dose (mg/kg/day) Vehicle TP + FT Observation Control Positive 5 15 25 + TP (0.4) b Control # # # # # # # # # # Exam Obs. Exam Obs. Exam Obs. Exam Obs. Exam Obs. None a Data were obtained from page 84 of the study report. b The observations of tremors were reported on page 22 of the study report but were not included in the clinical observation tables in the appendices. # Obs. = number of observed incidences. # Exam = number of animals examined. Page 193 of 260 In vivo Hershberger Assay (2011)/ Page 8 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data are presented in Table 4 for the agonist assay. Body weights in the 5 and 25 mg/kg/day groups were comparable to controls throughout the duration of the assay. At 25 mg/kg/day, mean overall (Days 1-11) body weights gains were decreased (p≤0.05) by 69%. Animals dosed with 0.4 mg/kg/day TP had a mean body weight increase (p≤0.05) of 12% on Day 11, and increased (p≤0.05) mean overall body weight gains of 105%. Table 4. Selected Group Mean (±SE) Body Weights and Cumulative Body Weight Gains (g) in the Androgen Agonist Assay a Dose (mg/kg/day) Positive Control Study Day # Vehicle Control Phosmet 5 Phosmet 25 Vehicle + TP (0.4) b N Mean SD N Mean SD N Mean SD N Mean SD 1 8 274.0 19.2 8 276.6 17.2 8 276.7 19.0 8 275.4 19.5 4 8 282.2 21.5 8 298.8 18.3 8 284.9 22.0 8 278.8 21.6 7 8 290.3 23.0 8 314.9 23.5 8 295.9 23.5 8 280.3 19.8 11 8 305.1 28.0 8 340.5* 28.5 8 311.5 27.6 8 285.1 19.7 (↑12) BWG 8 31.1 12.3 8 63.9* 11.9 8 34.8 11.3 8 9.7* 10.7 (days 1-11) (↑105) (↓69) a Data were obtained from Tables 6 on page 23 and Appendix V on pages 92-93 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses. b The positive control group for the androgen agonist assay is same as the vehicle control group for the anti-androgen assay. This did not affect the outcome or conclusions of this study. N = Number of animals in the group SE= Standard Deviation * Significantly different from vehicle controls at p<0.05. Body weight and body weight gain data for the anti-androgen assay are presented in Table 5. At 25 mg/kg/day, mean body weights were decreased (p≤0.05) by 12% on Day 11, and mean overall body weight gains were decreased (p≤0.05) by 60%. There were no effects from phosmet on body weights or body weight gains in the 5 and 15 mg/kg/day group, or in the positive control group. Page 194 of 260 In vivo Hershberger Assay (2011)/ Page 9 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 Table 5. Selected Group Mean (±SD) Body Weights and Cumulative Body Weight Gains (g) in the Anti- Androgen Assay a Dose (mg/kg/day) Study Vehicle Control TP + FT Phosmet 5 Phosmet 15 Phosmet 25 Day # + TP (0.4) b Positive Control N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 1 8 276.6 17.2 8 275.5 15.6 8 277.4 18.5 8 279.8 12.8 8 275.4 14.2 4 8 298.8 18.3 8 292.7 18.7 8 295.8 19.1 8 296.8 15.7 8 282.4 14.7 7 8 314.9 23.5 8 306.2 23.2 8 313.8 20.1 8 313.8 15.8 8 290.3 12.9 11 8 340.5 28.5 8 329.8 25.2 8 337.4 21.4 8 339.3 17.4 8 301.0* 17.1 (↓12) BWG 8 63.9 11.9 8 54.3 26.2 8 60.0 8.8 8 59.5 10.8 8 25.6* 15.4 (days 1-11) (↓60) a Data were obtained from Table 7 on pages 24 and Appendix V on pages 93-94 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses. b The vehicle control group for the anti-androgen assay is same as the positive control group for the androgen agonist assay. This did not affect the outcome or conclusions of this study. N = Number of animals in the group SD= Standard Deviation * Significantly different from vehicle controls at p<0.05. C. FOOD CONSUMPTION (OPTIONAL): Food consumption was not determined (optional). D. SERUM HORMONE CONCENTRATIONS (OPTIONAL): Serum hormone concentrations were not determined. E. ORGAN WEIGHTS: Accessory sex organ weights for the androgen agonist assay are presented in Table 6 for the agonist assay. There were no treatment-related effects in any of the accessory sex organs of animals dosed with the phosmet. Animals in the 0.4 mg/kg/day TP group had accessory sex organ weight increases (p≤0.05) as follows: 1926% in seminal vesicles; 1264% in ventral prostate; 251% in LABC; 794% in Cowper’s gland; and 72% in glans penis. The CVs for all treatment groups were compared to the performance criteria values as stated in the Guidelines. All %CV values were less than the maximum recommended values. Page 195 of 260 In vivo Hershberger Assay (2011)/ Page 10 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 Table 6. Accessory Sex Organ Weights (mg) from Androgen Agonist Assay in Sprague-Dawley Rats a Dose (mg/kg/day) Positive Control Organ Vehicle Control Phosmet 5 Phosmet 25 Vehicle + TP (0.4) b N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV Seminal 8 33.7 3.6 11 8 39.1 9.2 24 8 30.4 3.8 12 8 682.9* 135.7 20 vesicles (↑1926) Ventral 8 15.5 3.1 20 8 16.1 2.7 17 8 13.2 2.0 15 8 211.4* 37.6 18 prostate (↑1264) 8 143.8 14.1 10 8 158.3 21.1 13 8 120.5 19.1 16 8 505.4* 41.9 8 LABC (↑251) Cowper’s 8 5.1 1.1 21 8 5.6 1.5 27 8 4.8 1.0 22 8 45.6* 9.6 21 glands (↑794) 8 47.3 3.6 8 8 49.2 3.6 7 8 44.9 4.4 10 8 81.2* 8.5 11 Glans penis (↑72) a Data were obtained from Table 8 on page 26 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses. b The positive control group for the androgen agonist assay is same as the vehicle control group for the anti-androgen assay. This did not affect the outcome or conclusions of this study. N = Number of animals in the group SD= Standard Deviation CV= Coefficient of Variation * Significantly different from vehicle controls at p<0.05. Accessory sex organ weights for the anti-androgen assay are presented in Table 7. At 15 mg/kg/day, the weights of seminal vesicles were decreased by 25% (p≤0.05). At 25 mg/kg/day, accessory sex organ weight decreases (p≤0.05) were noted as follows: 50% in seminal vesicles; 40% in ventral prostate; 27% in LABC; 34% in Cowper’s gland; and 10% in glans penis. Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) accessory sex organ weights as follows: 85% in seminal vesicles; 76% in ventral prostate; 50% in LABC; 66% in Cowper’s gland; and 21% in glans penis. The CVs for all treatment groups were compared to the performance criteria values as stated in the Guidelines. All %CV values were less than the maximum recommended values. Page 196 of 260 In vivo Hershberger Assay (2011)/ Page 11 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 Table 7. Accessory Sex Organ Weights (mg) from Anti-Androgen Agonist Assay in Sprague-Dawley Rats a Dose (mg/kg/day) Organ Vehicle Control TP + FT Phosmet 5 Phosmet 15 Phosmet 25 +TP (0.4) b Positive Control N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV N Mean SD CV Seminal 8 682.9 135.7 20 8 612.7 123.6 20 8 510.5* 55.1 11 8 339.4* 60.8 18 8 103.3* 26.8 26 vesicles (↓25) (↓50) (↓85) Ventral 8 211.4 37.6 18 8 184.8 28.8 16 8 181.9 23.4 13 8 127.6* 16.0 13 8 50.2* 10.9 22 prostate (↓40) (↓76) 8 505.4 41.9 8 8 515.3 65.0 13 8 465.5 60.9 13 8 367.6* 39.5 11 8 250.8* 45.0 18 LABC (↓27) (↓50) Cowper’s 8 45.6 9.6 21 8 41.5 9.2 22 8 40.0 5.8 15 8 30.2* 7.1 24 8 15.6* 1.5 10 glands (↓34) (↓66) Glans 8 81.2 8.5 11 8 79.9 3.3 4 8 78.0 5.5 7 8 73.0* 3.8 5 8 64.5* 7.4 12 penis (↓10) (↓21) a Data were obtained from Tables 9 on page 27 of the study report. Percent differences from controls were calculated by the reviewers and included in parentheses. b The vehicle control group for the anti-androgen assay is same as the positive control group for the androgen agonist assay. This did not affect the outcome or conclusions of this study. N = Number of animals in the group SD= Standard Deviation CV= Coefficient of Variation * Significantly different from vehicle controls at p<0.05. Page 197 of 260 In vivo Hershberger Assay (2011)/ Page 12 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 F. MACROSCOPIC PATHOLOGY: No gross observations were made at necropsy. III. DISCUSSION AND CONCLUSIONS A. INVESTIGATOR’S CONCLUSIONS: In the agonist assay, final body weights of animals administered phosmet were not statistically different compared to the vehicle control (0.5% carboxymethylcellulose) animals. Mean body weight gain was significantly decreased in animals administered 25 mg/kg/day phosmet compared to vehicle control rats. In addition, cholinergic clinical signs (e.g. tremors) were observed following dose administration of 25 mg/kg/day phosmet on 7 of 10 days of the study. Phosmet administered at dose levels of 5 and 25 mg/kg/day to castrated SD male rats did not increase androgen dependent tissue weights. In the androgen antagonist assay, final animal body weights and mean body weight gain were significantly decreased in rats co-administered 25 mg/kg/day phosmet and TP compared to control rats. Cholinergic clinical signs were observed following co- administration of 25 mg/kg/day phosmet and TP on 7 of 10 days of the study. Phosmet (25 mg/kg) co-administered with TP was associated with significant decreases in LABC, glans penis, ventral prostate, Cowper's glands, and seminal vesicle weights, and a decrease in seminal vesicle weight with 15 mg/kg/day phosmet and TP compared to the control group (0.5% carboxymethylcellulose and TP). Using the castrated rat model Hershberger Assay, oral administration of phosmet up to 25 mg/kg/day resulted in cholinergic clinical signs and body weight effects, but did not show any androgen agonist activity. Co-administration of 25 mg/kg/day phosmet and TP was also associated with cholinergic clinical signs, bodyweight effects, and a reduction in androgen-dependent tissue weights. B. AGENCY COMMENTS: All animals survived until scheduled termination. In the androgen agonist assay, clinical signs in the 25 mg/kg/day group included tremors, uncoordinated movements and porphyrin staining around the eyes. No clinical signs were observed in the 5 mg/kg/day group. Body weights in the 5 and 25 mg/kg/day groups were comparable to controls throughout the duration of the assay. At 25 mg/kg/day, mean overall (Days 1-11) body weights gains were decreased (p≤0.05) by 69%. Animals dosed with 0.4 mg/kg/day TP had increased (p≤0.05) mean body weights of 12% on Day 11, and increased (p≤0.05) mean overall (Days 1-11) body weight gains of 105%. There were no significant increases in the weights of the five target accessory sex organs of animals dosed with the test compound. Animals in the 0.4 mg/kg/day TP group had accessory sex organ weight increases (p≤0.05) as follows: 1926% in seminal vesicles; 1264% in ventral prostate; 251% in LABC; 794% in Cowper’s gland; and 72% in glans penis. The performance criteria indicated that this assay was performing as expected. In the anti-androgen assay, clinical signs in the 25 mg/kg/day group included tremors, and uncoordinated movements. No clinical signs were observed in the 5 or 15 mg/kg/day groups. At 25 mg/kg/day, mean body weights were decreased (p≤0.05) by 12% on Day 11, and decreased (p≤0.05) overall body weight gains of 60%. There were no effects on body weights or body weight gains in the 5 and 15 mg/kg/day group, or the positive control group. At 15 mg/kg/day, the weights of seminal vesicles were decreased 25% (p≤0.05). At 25 mg/kg/day, accessory sex organ weight decreases (p≤0.05) were noted as follows: 50% Page 198 of 260 In vivo Hershberger Assay (2011)/ Page 13 of 13 PHOSMET/059201 OCSPP 890.1400/ OECD 441 in seminal vesicles; 40% in ventral prostate; 27% in LABC; 34% in Cowper’s gland; and 10% in glans penis. Animals dosed with 0.4 mg/kg/day TP + 3 mg/kg/day FT had decreased (p≤0.05) accessory sex organ weights as follows: 85% in seminal vesicles; 76% in ventral prostate; 50% in LABC; 66% in Cowper’s gland; and 21% in glans penis. The performance criteria indicated that this assay was performing as expected. All tissue weight CVs were below the maximum recommended values indicated in the test guideline. Administration of phosmet up to 25 mg/kg/day did not result in an increase in any of the five target organ weights indicating that the test substance is negative for androgenic activity. At 25 mg/kg/day, significant decreases were seen in all five of the target organ weights indicating that phosmet is positive for potential anti-androgenic activity in this bioassay. Cholinergic signs (tremors) and a 12% decrease in terminal bodyweight was seen at 25 mg/kg/day suggesting that the high dose may have been excessive (overt toxicity). No statistically significant increases in organ weights were seen in two or more of the five androgen responsive tissues in the androgen agonist assay. Phosmet was negative for androgenic activity. Statistically significant decreases in organ weights were seen in all five androgen responsive tissues only at the high dose in the androgen antagonist assay. Phosmet was positive for potential anti-androgenic activity in this Hershberger assay. However, the decreases in androgen-sensitive tissue weights were seen at a dose (25 mg/kg/day) that resulted in overt toxicity (12% decrease in body weight and clinical signs), which confounds the interpretation of the results. C. STUDY DEFICIENCIES: None Page 199 of 260 DATA EVALUATION RECORD PHOSMET Study Type: OCSPP 890.1450, Female Pubertal Assay EPA Contract No. EP10H001452 Task Assignment No. 2-61-2012 (MRID 48673003) Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive Arlington, VA 22202 Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B Durham, NC 27713 Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 5/7/2012 Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 5/18/2012 Program Manager: Signature: Jack D. Early, M.S. Date: 5/21/2012 Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/21/2012 This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel. Page 200 of 260 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 260 PHOSMET I 059201 Primary Reviewer: Linda Taylor. Ph.D. Health Effects Division Secondary Reviewer: Elizabeth Mendez, Ph.D. Health Effects Division DATA EVALUATION RECORD STUDY TYPE: Female Pubertal Assay; OCSPP 890.1450; OECD None. PC CODE: 059201 DP BARCODE: D400672 TXR#: 0056199 CAS No.: 732-11-6 TEST MATERIAL (PURITY): Phosmet (96.8%) SYNONYMS: S-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl] 0, 0-dimethyl phosphorodi thioate CITATION: Zorilla, L. (2012). Pubertal developmental and thyroid function with phosmet in juvenile/peripubertal CRL: CD (SD) IGS female and male rats. Integrated Laboratory Systems, Inc., Durham, NC, Laboratory Study No.: Cl 94-300, February 24, 2012. MRID 48673003. Unpublished. SPONSOR: Gowan Company, P.O. Box 5569, Yuma, AZ TEST ORDER#: EDSP-059201-54 EXECUTIVE SUMMARY: In a female pubertal assay (MRID 48673003), 16 Sprague Dawley (Crl:CD®(SD) IGS) rats/dose group were treated daily by oral gavage with phosmet (96.8% a.i., Batch #: GQH9118000) in aqueous 0.5% carboxymethylcelJulose (CMC) at doses of 0, 10, or 20 mg/kg/day (5 mL/kg dosing volume) from post-natal day (PND) 22 to 42 or 43. The animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and body weight at day of attainment was recorded. After sacrifice on PND 42/43, serum thyroxine (T4) and thyroid stimulating hormone (TSH) concentrations were analyzed using radioimmunoassays. Various clinical chemistry parameters were measured in the serum. Liver, kidney, pituitary, adrenals, thyroid, ovaries, and uterus were weighed and examined macroscopically; microscopic examination was also conducted on the thyroid, ovaries, uterus, and kidneys. No treatment-related effects were observed on mortality, organ weights, serum TSH concentrations, clinical chemistry, or gross pathology. Changes (NS) in ovary, uterus, thyroid, and pituitary weights were noted in the low- and high-dose animals. There were no treatment related histopathological findings noted in the ovaries, uterus, thyroid, or kidneys. In addition, no treatment-related effects were noted on cycle status, mean age at first estrus, mean cycle length, percent cycling, or percent regularly cycling. Page 202 of 260 Female Pubertal Assay (2012) / Page 2 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None One 10-mg/kg/day rat exhibited localized head tremors on a single day, and tremors were observed at various times post-dose in up to 10/16 10-mg/kg/day rats on 10 days and up to sixteen 20-mg/kg/day rats at 18 days. No other treatment-related clinical signs were observed in animals from either dose group. Decreased body weights (p<0.05; ↓9% to 10%) were observed at 20-mg/kg/day during PND 26 to 36. No significant change in mean final body weight or body weight gain was observed in either treatment group compared to the vehicle control group. The day of VO was comparable among the groups. Unadjusted and adjusted body weights at VO were decreased (p<0.05) by 9% each in the 20 mg/kg/day group compared to control. No treatment-related effect was noted on cycle status, mean age at first estrus, mean cycle length, percent cycling, or percent regularly cycling. At 20 mg/kg/day, the number of ovarian antral follicles was increased (↑93%), and the number of atretic follicles was decreased (↓29%). All other values in the treated groups were similar to control. Serum T4 concentrations were increased (↑24 to 38%) in the treated groups. A marginal decrease in the thyroid colloid area and increase in the follicular cell height was observed in animals administered phosmet (10 and 20 mg/kg) compared to control. There were no effects on liver, kidney, pituitary, adrenal, uterus, or thyroid organ weights at either dose level compared to the control. Thyroid effects (increased serum T4 levels and thyroid histopathology) were seen at both doses. However, these doses (10 and 20 mg/kg/day) were determined to be excessive based on persistent tremors seen at both doses and severe body weight loss at the high dose. Additionally, data from the existing toxicity database show that lower doses should have been tested in this assay. Following the agency’s determination, the registrant submitted additional information (MRID 49610501) in response to the agency’s review of this assay. The submitted data show that tremors were seen in 10/16 rats at the 10 mg/kg/day dose level (during 10 days) and all animals at the 20 mg/kg/day dose (during 18 days). The severity of the tremors ranged from “not characterized to moderate.” Upon review of these data, the agency reaffirms its previous conclusion that both doses tested in this assay were excessive. The assay does NOT satisfy the EDSP Tier 1 Test Order requirements for a Female Pubertal Assay (OCSPP 890.1450) since overt toxicity was seen at both doses tested. However, a new assay is not required at this time since the results from a repeat assay would have no impact on the risk assessment. The point of departure (PoD) for the phosmet risk assessment is based on cholinesterase inhibition at substantially lower doses than the ones tested in this assay. For additional information see the EDSP Tier 1 Weight of Evidence Analysis for Phosmet (TXR 0057145). COMPLIANCE: Signed and dated GLP Compliance, Data Confidentiality, and Quality Assurance statements were provided. Page 203 of 260 Female Pubertal Assay (2012) / Page 3 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None I. MATERIALS AND METHODS A. MATERIALS 1. Test Facility: Integrated Laboratories Systems, Inc. Location: Durham, NC Study Directors: L. Zorilla. Other Personnel: S. Borghoff, study toxicologist M. Boyle, study pathologist Study Period: May 25, 2011 through February 24, 2012 2. Test Substance: Phosmet Description: Off white solid Source: Gowan Company Batch #: 6QH9118000 (expires January 31, 2013) Purity: 96.8% a.i. Stability: Report stated that the compound is stable in the vehicle at 1–10°C for up to 15 days CAS #: 732-11-6 O Structure: S P N S OCH3 OCH3 O 3. Vehicle: 0.5% Carboxymethylcellulose in sodium phosphate buffer 4. Test Animals: Species: Rat (females) ® Strain: Sprague-Dawley (Crl:CD (SD) IGS) Age/Mean Weight PND 22 / 45.9–67.8 g at Study Initiation: Source: Charles River Laboratories International, Inc. (Raleigh, NC) Housing: Parental dams were individually housed in polycarbonate cages with micro-isolator tops and absorbent, heat-treated, hardwood bedding; each dam was housed with her litter until weaning on PND 21. After weaning, up to 2 females were housed per cage. Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad libitum. It was stated that the total genistein equivalents of genistein plus daidzein was determined to be 8.0 μg/g of feed. Water: Reverse osmosis-purified tap water, ad libitum Environmental Temperature: 19–24°C Conditions: Humidity: 43–78% Air changes: Not reported Photoperiod: 14 h light / 10 h dark B. STUDY DESIGN 1. In-Life Dates: Start: July 1, 2011 End: December 12, 2011 2. Mating: Time-mated pregnant nulliparous dams (age not reported) were received from the supplier on gestation day (GD) 8 and were allowed to deliver naturally and rear their pups to PND 21. Between postnatal days (PND) 3 and 5, litters with the same date of birth were standardized to 8 pups with equal numbers of males and females. Page 204 of 260 Female Pubertal Assay (2012) / Page 4 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None 3. Animal Assignment: Following weaning on PND 21, animals were assigned to study using a procedure that stratifies animals across groups by body weight such that mean body weight of each group was not statistically different from any other group using analysis of variance. Care was taken to avoid placing littermates in the same dose group. The test groups are provided in Table 1. TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Females Control 0 16 Low 10 16 High 20 16 a Data were obtained from Table 1 on page 16 of the study report. 4. Dose Selection Rationale: The doses were selected based on the results of several previous studies. A 7-day relative sensitivity study (MRID 44839301) compared the toxic responses of PND 11 rat pups versus adult rats following repeated gavage administration of phosmet. No treatment-related effects on body weight or cholinergic clinical signs were seen in either pups or adults at the highest tested dose (HTD) of 5 mg/kg/day, however significant cholinesterase inhibition was evident (e.g. responses were 57-62% of controls in adult RBC and 81-86% of controls in adult brain at the HTD). The U.S. EPA (ToxSAC memorandum, May 6, 2009) calculated a BMD10L of 0.60 mg/kg/day for adult rat RBC cholinesterase inhibition and a BMD10L of 2.84 mg/kg/day for brain cholinesterase inhibition from the study data. Body weight effects were observed in a rat teratology study (MRID 41962902) that treated pregnant dams with 10 consecutive gavage doses of phosmet from GD 7–16. At the end of dosing, a statistically significant reduction in body weight gain was observed at 15 mg/kg/day (the HTD). This was associated with a 7% reduction in body weight, compared to controls. Probable treatment-related cholinergic clinical effects (shaking and/or stains around mouth) were seen in a few animals (4/24) after 6 days of dosing through the end of dosing in the 15 mg/kg/day group. There was no unambiguous evidence of treatment- related cholinergic clinical signs or body weight effects at 5 or 10 mg/kg/day. In a recent Uterotrophic assay (MRID 48618707), there were no body weight effects after 3 consecutive days of gavage dosing at 5 or 20 mg/kg/day. However, reduced activity was noted in a few of the 20 mg/kg/day animals on the third dosing day, within several hours of dosing. In a recent Hershberger assay (MRID 48618706), there were treatment-related cholinergic clinical signs in the agonist and antagonist cohorts at 25 mg/kg/day after ≥3 consecutive dosing days. These effects were typically noted within a few hours of dose administration but did not persist to the pre-dosing examination on the following treatment day. Treatment-related cholinergic clinical signs were not seen at ≤15 mg/kg/day. Statistically significant reductions in body weight gain were found for both agonist and antagonist Page 205 of 260 Female Pubertal Assay (2012) / Page 5 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None cohorts at the 25 mg/kg/day dose. Additionally, mean final body weight was significantly reduced at the 25 mg/kg/day treatment level as compared to controls in the antagonist portion of the study. No body weight effects were noted at ≤25 mg/kg/day in the agonist cohort. The highest doses tested in the relative sensitivity and Hershberger studies appear to bracket the appropriate range for the highest dose for the phosmet pubertal assays (>5 and <25 mg/kg/day), based on the target of achieving significant body weight effects while avoiding severe clinical signs. Considering the longer exposure intervals employed in the pubertal assays, compared to those used in the other gavage studies, the available data suggest that 20 mg/kg/day will elicit measurable body weight effects in the pubertal assays. It should be noted that it may not be possible to avoid the occurrence of some cholinergic clinical signs with an organophosphate such as phosmet, given the target to elicit body weight effects. However, such effects, if present, should be transient and would not be anticipated to be severe and persist, based on the 25 mg/kg/day data from the Hershberger assay. A robust existing database shows that substantial cholinesterase inhibition will occur at 10 and 20 mg/kg/day. Both selected doses significantly exceed the regulatory benchmark of 0.6 mg/kg/day, which is the EPA-calculated BMD10L, for RBC cholinesterase inhibition in the repeated-dose relative sensitivity study. Both doses also exceed the EPA-calculated BMD10 for brain cholinesterase inhibition (3.86 mg/kg/day) following repeated exposures. Accordingly, the selected doses ensure that an appropriately conservative experimental challenge is employed in the phosmet pubertal screening assays. 5. Dose Preparation and Analysis: Phosmet dose formulations were prepared at ILS in 0.5% aqueous carboxymethylcellulose five times during the study at concentrations of 2 and 4 mg/mL. Formulations were dispensed into vials to be used daily during the study. Dose concentrations were adjusted to correct for purity of phosmet. Samples of dose formulations prepared on June 28, 2011 and July 6, 13, 19, and 26, 2011 were collected from the top, middle, and bottom of the formulation and sent to Smithers Viscient (Wareham, MA) for analysis. Smithers Viscient analyzed, in duplicate, samples received for concentration and homogeneity. The report stated that dose formulations of phosmet in 0.5% aqueous carboxymethylcellulose held from 1 to 10°C for up to 15 days were considered stable (Dix, 20121); however, no data were submitted to allow independent verification. Results Stability (% of time zero): Reported in Study No. 12791.6159. Concentration (% of nominal): 96–106% Homogeneity (%CV): 2.3–11.8% The analytical data indicated that the mixing procedure was adequate and the variation between nominal and actual dosage to the animals was acceptable. Homogeneity as %CV only exceeded 10% for the 2 mg/mL formulation on July 13, 2011. 1 Dix, M. (2012). Storage Stability of Phosmet in Aqueous, Buffered Carboxymethylcellulose. Unpublished study report prepared by Smithers Viscient Inc. Study No. 12791.6159. Page 206 of 260 Female Pubertal Assay (2012) / Page 6 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None 6. Dosage Administration: Dose formulations were administered by oral gavage at a dose volume of 5 mL/kg body weight. The dose formulations were administered for 21 or 22 consecutive days on PND 22–42/43. Dosing occurred 24-hours (±2 hours) from the previous dose. Dose volume was adjusted daily based on individual animal body weight. The dosing sequence was stratified across all dose groups; one animal from each group and then repeated until all animals were dosed. 7. Statistics: Descriptive statistics (mean, standard deviation, coefficient of variance, and sample size) were calculated using Microsoft Excel 2003/2007 (Redmond, WA). Data sets listed below were analyzed using Statistical Analysis System version 9.2 (SAS Institute, Cary, NC). Studentized residual plots were used to detect possible outliers in the data and Levene’s test was used to assess homogeneity of variance. Heterogeneous data were transformed (logarithm, square root, and multiplicative inverse), and if still heterogeneous, analyzed using the non-parametric Kruskal-Wallis and Dunn’s test. If data sets were not homogenous, ANCOVA analyses were not performed. Trend tests were performed on body weight and tissue weight data sets and reported when significant (p<0.05) for endpoints that did not show any significant pairwise comparisons. Classification Procedure Initial body weights Homogeneous data sets: Final body weight (use last day all body weights collected) One-way analysis of variance (ANOVA) followed by pair- Final body weight gain (use last day all body weights wise comparisons performed using Dunnett’s two-tailed collected) t-tests Age and body weight at VO Age at first estrus Cycle length Ovarian counts (follicles, cysts, corpora lutea) Tissue weights Two-way ANOVA with treatment and necropsy day (if >1 Relative tissue weights (liver, kidneys, pituitary, and day) as main effects. adrenals) Pair-wise comparisons were performed using Dunnett’s Hormone levels two-tailed t-tests. Clinical chemistry levels Initial body weights Two-way analysis of covariance (ANCOVA) with PND 21 Final body weight (use last day all body weights collected) body weight (weight on day of allocation) as the covariable. Final body weight gain (use last day all body weights Pair-wise comparisons were performed using Dunnett’s collected) two-tailed t-tests. Age and body weight at VO Tissue weights In the instances where VO had not occurred prior to necropsy, the last day of examination plus 1 was used as the age at VO and terminal body weight was used for body weight at VO. In instances when at least 1 animal in any group exhibited incomplete VO, including partial threads for > 3 days, the day partial separation was first recorded was used in the analyses. Cycling status Chi-square analysis Percent of animals cycling regularly Thyroid scoring (colloid area and follicular cell height) Fisher’s Exact test Page 207 of 260 Female Pubertal Assay (2012) / Page 7 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None Significance was reported as exact p-values. The statistical analyses were considered appropriate. C. METHODS 1. Mortality and Clinical Examinations: Animals were observed twice daily for mortality and moribundity on weekdays and once daily on weekends and holidays. Rats were observed prior to allocation of animals to study groups, daily prior to dose administration, and prior to euthanasia. 2. Body Weight: Body weights were collected weekly following birth, prior to allocation of animals to study groups, daily prior to dose administration, and prior to euthanasia. 3. Food Consumption: Food consumption was not measured. 4. Vaginal Opening: Beginning on PND 22, all animals were examined daily for onset of VO. VO was recorded as: VO not initiated, appearance of a pin hole, vaginal thread, or complete VO. Age and weight on the day of completion of VO were recorded. 5. Estrous Cyclicity: Beginning on the day of complete VO through euthanasia, vaginal smears were prepared daily, evaluated and classified as diestrus, proestrus, or estrus; metestrus was classified as diestrus. The overall cycling pattern for each female was characterized as regular, irregular, or not cycling. Regular cycling was defined as having recurring 4 to 5 day cycles, while irregular cycling was defined as having cycles with diestrus for a period >3 days or estrus ≥3 days. Animals were not considered to be cycling if there was ≥3 consecutive days of estrus or ≥5 consecutive days of diestrus. Mean cycle length was defined as the number of days from one diestrus to the next diestrus. Incomplete cycles were not used in calculating mean cycle length. In instances where the time between VO and humane euthanasia did not allow observation of more than 1 complete cycle, classification was based on data available and the assumption that animals were cycling regularly if the partial data fit the definition, and cycling irregularly if the study ends without being able to distinguish between irregular and not cycling. 6. Sacrifice and Pathology: At least 2 hours after the final dose administration, animals were humanely euthanized by decapitation, in the same order as they were dosed. After euthanasia and prior to tissue collection, trunk blood was collected, and serum was obtained. All serum samples were stored at or below −70°C. Serum samples were shipped on dry ice to AniLytics Inc. (Gaithersburg, MD) for analysis. a. Hormone Analysis: T4 and TSH were measured in serum using radioimmunoassays (RIAs). Samples were assayed in duplicate and in conjunction with multiple quality control samples. Page 208 of 260 Female Pubertal Assay (2012) / Page 8 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None b. Clinical Chemistry: The following CHECKED (X) parameters were examined. ELECTROLYTES OTHER X Calcium X Albumin X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus Total cholesterol X Potassium Globulins X Sodium Glucose ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Albumin/globulin ratio (calculated) Lactic acid dehydrogenase (LDH) Bile acids X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransferase (AST/also SGOT) X Sorbitol dehydrogenase 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 and grossly observed from all study animals. UROGENITAL OTHER X Ovaries (paired, without oviducts)*+ X Thyroid a*+ X Uterus*+ X Liver* X Kidneys (paired)*+ X Adrenals (paired)* X Pituitary* a Thyroid was weighed post-fixation. * Weights required based on guideline 890.1450. + Histopathological examination required based on guideline 890.1450. Uterus (without fluid), left ovary, left kidney, and thyroid were fixed in 10% neutral buffered formalin for at least 24-hours. After fixation, tissues were transferred to 70% histology grade alcohol, histologically processed, embedded, sectioned, and stained with hematoxylin and eosin. Histologically-processed tissues (thyroid, kidneys, uterus, and ovary) from all animals were microscopically evaluated. Five random sections of the ovary were examined for follicular development (including presence/absence of tertiary/antral follicles, presence/absence of corpora lutea, changes in corpus luteum development, and changes in number of both primary and atretic follicles) in addition to any abnormalities/lesions, such as ovarian atrophy. Two sections of each of the 2 lobes of the thyroid were examined and evaluated for follicular cell height and colloid area. A 5-point Page 209 of 260 Female Pubertal Assay (2012) / Page 9 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None grading scale (1 = shortest/smallest; 5 = tallest/largest) was used, and the thyroids were evaluated relative to photomicrographs supplied by the U.S. EPA. II. RESULTS A. Mortality: All animals survived. B. Clinical Signs of Toxicity: No treatment-related clinical signs were observed in animals from any dose group 24 hours post-dose; i.e., at the subsequent time of dosing. One 10 mg/kg/day rat exhibited localized head tremors on one day, and tremors were observed at various times post-dose in up to ten 10 mg/kg/day rats on 10 days and up to sixteen 20 mg/kg/day rats on 18 days. C. General Growth and Vaginal Opening: Body weights, body weight gains, age of attainment of VO, weight at VO, and proportion/incidence of unopened vagina are presented in Tables 2 and 3 below. Unadjusted and adjusted body weight at VO were decreased (p<0.05) by 9% each in the 20 mg/kg/day group compared to controls. No significant change in mean final body weight or body weight gain was observed in females administered 10 or 20 mg/kg/day phosmet compared to the vehicle control group. The treated groups displayed body weight gain deficits that diminished with time on test; e.g., at 20 mg/kg/day, body weight gain was ↓ 30% during Days 22-23; ↓20% during Days 26-29; and ↓6% during Days 29-36 (Table 2). At 20 mg/kg/day, body weights were decreased (p≤0.05) by 8–10% from PND 26–36. A water bottle malfunction is thought to have led to a body weight loss (compared to the previous day) in half the control group on PND 36–37. The report stated that it was likely that the terminal body weights of the 20 mg/kg/day phosmet group would have been ~90% of vehicle control body weights had the water bottle malfunction in the control not occurred. It is to be noted that all of the control females had attained VO by PND 36 so the malfunction had no impact on this parameter for this group. All but one of the 10 mg/kg/day females and all but two of the 20 mg/kg/day females had attained VO by PND 36 also. TABLE 2. Body Weight and Body Weight Gain (grams) a Days Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet PND 22 57.0±4.9 56.8±4.2 56.6±5.0 PND 27 82.8±7.0 80.9±6.7 75.9±6.5 ↓8%* PND 29 95.8±8.2 92.5±7.3 86.3±7.7 ↓10%* PND 36 140.9±11.9 134.3±9.9 128.5±13.4 ↓9%* PND 37 139.6±12.1 139.7±9.4 131.6±14.9 ↓6% PND 42 169.1±15.1 165.1±11.2 163.7±17.5 ↓4% PND 22-23 4.0 3.4 ↓15% 2.8 ↓30% PND 23-26 17.0 15.6 ↓8% 12.5 ↓26% PND 26-29 17.8 16.7 ↓6% 14.4 ↓20% PND 29-36 45.1 41.8 ↓8% 42.2 ↓6% Page 210 of 260 Female Pubertal Assay (2012) / Page 10 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None TABLE 2. Body Weight and Body Weight Gain (grams) a PND 26-36 62.9 58.5 ↓7% 56.6 ↓10% PND 36-37 -1.3 5.4 3.1 PND 38-42 21.6 19.8 25.5 ↑18% PND 22-36 83.9 77.5 ↓8% 71.9 ↓14% PND 22-42 112.1 108.3 ↓4% 107.1 ↓4% a Data were obtained from Appendix IV, pages 101-103 of the study report. * p<0.05; body weight gains calculated by reviewer. All performance criteria were met; however, weaning weight (PND 21) was not reported. TABLE 3. General Growth and Vaginal Opening (VO) a Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet Parameter Evaluated CV CV CV N Mean SD (%) N Mean SD (%) N Mean SD (%) Initial body weight U 16 57.0 4.9 8.5 16 56.8 4.2 7.4 16 56.6 5.0 8.8 (PND 22; g) A 16 57.0 ------16 56.9 ------16 56.5 ------111.5* U 16 122.1 11.3 9.2 16 115.6 13.7 11.9 15 9.8 8.8 Body weight at (↓9) vaginal opening (g) 111.6* A 16 122.1 ------16 115.6 ------15 ------(↓9) Final body weight U 16 169.1 15.1 8.9 16 165.1 11.2 6.8 16 163.7 17.5 10.7 (g) A 16 169.1 ------16 165.3 ------16 163.4 ------Final body weight U 16 ------16 97.6 ------16 96.8 ------(% of control) A 16 ------16 97.8 ------16 96.6 ------ Body weight gain U 16 112.1 12.1 10.8 16 108.3 8.7 8.0 16 107.1 14.8 13.8 (final – initial; g) A 16 112.1 ------16 108.4 ------16 106.9 ------ Age at vaginal U 16 33.2 1.2 3.7 16 32.9 2.0 6.0 15 33.4 1.5 4.5 opening (PND) A 16 33.2 ------16 32.9 ------15 33.4 ------Proportion unopened 0/16 0/16 1/16 (#/N) a Data were obtained from Table 7 on page 27 of the study report. Percent difference from controls (calculated by reviewers) is presented parenthetically. 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 * Significantly different from controls at p<0.05. D. Organ Weights: Organ weights at necropsy are presented in Table 4 below. No significant differences were observed in liver, kidney, adrenal, pituitary, thyroid, uterus (wet or blotted), or ovary weights of the treated groups compared to controls, but there was a dose- related significant trend (p≤0.05) in relative liver weights (↑3-5%). Although adrenal Page 211 of 260 Female Pubertal Assay (2012) / Page 11 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None weights in both groups were comparable to/ slightly lower than (↓2-3%) the control, the adrenal weight of the control was below the acceptable range of the performance criteria. Compared to the low range value, the adrenal weights were ↓16-18%. Thyroid weight was slightly increased (↑6%) at 10 mg/kg/day and slightly decreased (↓8%) at 20 mg/kg/day. Pituitary weights were slightly lower than control (↓6% and ↓8%) at 10 mg/kg/day and 20 mg/kg/day, respectively. The following performance criteria were not met: mean adrenal weight of 32.9 mg (acceptable range: 38.34–48.84); and %CV=11.4 for kidneys (top acceptable range: 10.76). TABLE 4. Organ Weights at Necropsya Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet Organ CV CV CV N Mean SD (%) N Mean SD (%) N Mean SD (%) U 16 7.52 0.67 9.0 16 7.60 0.66 8.7 16 7.74 1.15 14.8 Liver A 16 ------16 ------16 ------(g) 4.57 4.66 R* 16 4.45 0.2 4.6 16 0.25 5.5 16 0.36 7.6 (↑3) (↑5) U 16 1.33 0.15 11.4 16 1.33 0.07 5.1 16 1.32 0.14 10.8 Kidneys A 16 ------16 ------16 ------(g) R 16 0.78 0.06 7.3 16 0.8 0.05 6.4 16 0.8 0.04 5.6 U 16 9.9 1.3 13.3 16 9.4 1.2 12.6 16 9.2 1.6 17.0 Pituitary 9.4 9.2 A 16 9.9 ------16 ------16 ------(mg) (↓6) (↓8) R 16 5.8 0.7 11.8 16 5.7 0.7 13.1 16 5.6 1 16.9 U 16 32.9 4.9 14.9 16 32.1 6.5 20.4 16 31.8 5.5 17.2 Adrenals 31.8 A 16 32.9 ------16 32.1 ------16 ------(mg) (↓4) R 16 19.7 3.7 18.9 16 19.4 4.3 22.3 16 19.5 4.3 22.2 55.0 56.7 Ovaries U 16 59.8 11.9 19.9 16 6.6 12 16 10 17.7 (↓8) (↓6) (mg) A 16 59.8 ------16 55.1 ------16 56.6 ------383.4 373.5 U 16 418.6 206 49.2 16 194.8 50.8 16 153.3 41.1 Uterus, wet (↓8) (↓11) (mg) 372.4 A 16 418.6 ------16 384.4 ------16 ------(↓12) 301.1 321.3 Uterus, blotted U 16 330.3 74.9 22.7 16 90.8 30.1 16 65.8 20.5 (↓9) (↓3) (mg) A 16 330.3 ------16 301.8 ------16 320.5 ------22.0 19.1 Thyroid, fixed U 16 20.6 3.1 15.1 16 4.4 19.9 16 3.7 19.5 (↑6) (↓8) (mg) A 16 20.6 ------16 22 ------16 19.1 ------a Data were obtained from Table 8 on page 29 of the study report. No statistical differences were noted in pair-wise comparisons of treated groups with control. 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 R Organ-to-body weight ratio (relative to body weight) * Significant trend (p<0.05) Page 212 of 260 Female Pubertal Assay (2012) / Page 12 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None E. Estrus Cyclicity: Estrus cycle data are provided in Table 5. No treatment-related effect was noted on cycle status at necropsy, mean age at first estrus, mean cycle length, percent cycling, or percent regularly cycling. Page 213 of 260 Female Pubertal Assay (2012) / Page 13 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None TABLE 5. Estrous Cyclicitya Mean Age at Mean Cycle Regularly Cycle Status at Necropsy (# Females) Treatment First Estrus Length Cycling Cycling Not Groups N (PND) (days) (%) (%) Diestrus Proestrus Estrus Cycling Vehicle 16 35.1±2.2 4.1±1.4 93.75 87.5 5 2 9 1 10 mg/kg/day 16 35.6±3.2 3.5±0.9 100 87.5 10 3 3 0 Phosmet 20 mg/kg/day 16 35.4±2.7 3.9±0.9 93.3 86.7 6 1 8 1 Phosmet a Data were obtained from Tables 8 and 9 on pages 29-30 of the study report. No statistical differences were noted in pair- wise comparisons of treated groups with control. N Number of animals * Significantly different from controls at p<0.05. F. Clinical Chemistry and Hormone Levels: Clinical chemistry and hormone levels are presented in Table 6 below. Serum T4 levels were increased (p<0.05) by 24–38% in the treated groups. The CV (28.7) was slightly greater than the performance criteria mean (21.38) but was less than the top of the acceptable range (29.39). Serum TSH levels in the treated groups were slightly greater than (↑2%-6%) the control. The following clinical chemistry parameters differed from control in one or both treatment groups, although statistical significance was not always attained (NS): (i) minor increases in sodium of 4–6% at 10 mg/kg/day and 20 mg/kg/day (p<0.05); (ii) increases in alanine aminotransferase of 11–17% (NS at 20 mg/kg/day); (iii) increases of alkaline phosphatase (↑14-24%, NS at 10 mg/kg/day); (iv ) increases in bilirubin at 10 mg/kg/day and 20 mg/kg/day (↑23-38%; NS); (v) increases in blood urea nitrogen (↑8-23%, NS at 10 mg/kg/day); and (vi) increases in sorbitol dehydrogenase (↑13-33%, NS at 10 mg/kg/day). It is to be noted that several of the control values are outside the normal range. For example, the control ALP value is 246 U/L and the range provided in Table 12 of the study report is 17-183 U/L; control ALT value is 94 U/L, whereas the range is 20-56 U/L. It is to be noted that Table 12 of the study report is labeled as female data, but the same hormone and clinical chemistry ranges are listed in Table 25 of the study report as male ranges. TABLE 6. Hormone Levels and Clinical Chemistry a Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet Parameter CV CV CV Evaluated N Mean SD (%) N Mean SD (%) N Mean SD (%) Hormones Serum T4, Total 3.84* 4.27* 16 3.09 0.89 28.7 16 1.12 29.1 16 1.06 24.9 (µg/dL) (↑24) (↑38) Serum TSH 1.50 1.57 16 1.47 0.72 50.0 16 0.70 46.4 16 1.02 65.0 (ng/mL) (↑2) (↑6) Page 214 of 260 Female Pubertal Assay (2012) / Page 14 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None TABLE 6. Hormone Levels and Clinical Chemistry a Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet Parameter CV CV CV Evaluated N Mean SD (%) N Mean SD (%) N Mean SD (%) Clinical Chemistry 147* 150* Sodium (mEq/L) 16 141 4 3 16 2 2 16 2 2 (↑4) (↑6) Potassium (mEq/L) 16 8.2 0.7 8.4 16 8.4 1 11.9 16 8.6 0.7 7.8 110 114 Chloride (mEq/L) 16 104 2 2 16 2 2 16 2 2 (↑6) (↑14) 10.8* Calcium (mg/dL) 16 10.3 0.4 4.3 16 0.4 3.5 16 10.5 0.7 6.4 (↑5) Phosphorus 16 9.7 0.5 5.4 16 10.0 1.0 9.7 16 10.0 1.0 9.9 (mg/dL) AST (U/L) 16 281 48 17 16 284 87 31 16 294 74 25 104* 110 ALT (U/L) 16 94 10 11 16 21 20 16 21 19 (↑11) (↑17) GGT (U/L) 16 0.1 0.5 400 16 0.2 0.5 290 16 0 0 280 305* ALP (U/L) 16 246 48 19 16 49 18 16 63 21 (↑14) (↑24) 14 16* BUN (mg/dL) 16 13 2 16 16 2 14 16 2 15 (↑8) (↑23) Creatinine (mg/dL) 16 0.40 0.05 12.9 16 0.41 0.06 14.1 16 0.38 0.05 14.3 Total Bilirubin 0.16 0.18 16 0.13 0.11 86.7 16 0.11 66.9 16 0.18 97.1 (mg/dL) (↑23) (↑38) 17 20* SDH (U/L) 16 15 5 35 16 5 29 16 5 27 (↑13) (↑33) Total Protein 16 5.5 0.3 5.4 16 5.7 0.4 6.6 16 5.5 0.4 7.4 (g/dL) Albumin (g/dL) 16 4.3 0.3 6.3 16 4.5 0.3 6.9 16 4.3 0.4 8.7 a Data were obtained from Table 10 on page 30 and Table 11 on page 32 of the study report. Percent difference from controls (calculated by reviewers) is presented parenthetically. N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p<0.05. F. Gross pathology: There was a focus on the median liver lobe proximal to the hilus observed in one animal administered 20 mg/kg/day phosmet. No other gross observations were noted in female rats surviving to the scheduled termination. G. Histopathology: The incidences of histopathological findings of the thyroid gland are presented below in Table 7; follicular cell shape was not reported. A marginal decrease in the colloid area and increase in the follicular cell height was observed in animals administered phosmet (10 and 20 mg/kg) compared to controls. The number of rats with the largest follicular colloid area (Grades 4 or 5) included 14/16 in the control, 10/16 at 10 mg/kg/day and 8/16 at 20 mg/kg/day. Likewise, the number of rats with the shortest follicular cell height (Grades 1 or 2) included 14/16 in the control, 10/16 at 10 mg/kg/day and 8/16 at 20 mg/kg/day. Page 215 of 260 Female Pubertal Assay (2012) / Page 15 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None TABLE 7. Incidence of Histopathological Lesions of the Thyroid Gland a Parameter Evaluated Colloid Area Follicular Cell Height Treatment Groups Incidence Incidence Grade b Grade O E O E 1 0 16 1 1 16 2 0 16 2 13 16 Vehicle Control 3 2 16 3 2 16 4 13 16 4 0 16 5 1 16 5 0 16 1 0 16 1 0 16 2 0 16 2 10 16 10 mg/kg/day 3 6 16 3 6 16 Phosmet 4 10 16 4 0 16 5 0 16 5 0 16 1 0 16 1 1 16 2 1 16 2 7 16 20 mg/kg/day 3 7 16 3 7 16 Phosmet 4 7 16 4 1 16 5 1 16 5 0 16 a Data were obtained from Table 14 on page 35 of the study report. b A 5-point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used. O No. Observed E No. Examined The incidence of histopathological findings of the ovary, uterus, thyroid, and kidneys are presented in Table 8. In the ovary, the number of antral follicles was increased by 57% at 10 mg/kg/day (NS) and by 93% (<0.05) at 20 mg/kg/day. The number of atretic follicles was decreased by 24% (NS) at 10 mg/kg/day and 29% (<0.05) at 20 mg/kg/day. All other ovarian values in the treated groups were similar to the control. There were no treatment- related histopathological findings noted in the uterus, thyroid (except as noted in Table 6), or kidneys at any dose. Page 216 of 260 Female Pubertal Assay (2012) / Page 16 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None TABLE 8. Incidence of Histopathological Lesions of the Ovary, Thyroid, Uterus, and Kidneya Dose Level (mg/kg bw/day) Findings Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet # # # # # # Observed Examined Observed Examined Observed Examined Ovary [Mean±SD (CV)] Small follicles 27.7±9.6 (34.7)b 16 30.6±7.1 (23.2) 16 26.1±10.1 (38.7) 16 Medium follicles 13.7±4.0 (30.8) 16 14.4±3.1 (21.5) 16 14.6±5.0 (34.2) 16 Antral follicles 2.2±0.7 (31.8) 2.7±2.2* (81.5) 1.4±1.0 (69.9)b 16 16 16 [↑93] [↑57] Atretic follicles 6.0±2.7 (45.0) 5.6±1.5* (26.8) 7.9±2.5 (31.6)b 16 16 16 [↓24%] [↓29] Follicular cysts 0.025±0.1 0.013±0.05 16 16 (400.0)b 16 0.0±0.0 (384.6)b [↑92] Corpora lutea 6.8±1.9 (27.9)b 16 7.5±1.7 (22.7) 16 6.6±2.2 (33.3) 16 Thyroid Minimal C-cell 0 16 0 16 1 16 hyperplasia Uterus Minimal dilatation 7 16 5 16 2 16 Minimal eosinophilic 1 16 0 16 1 16 material Minimal inflammatory 3 16 0 16 6 16 cells/debris Mild inflammatory 0 16 0 16 1 16 cells/debris Minimal endometrial 6 16 7 16 7 16 folding Minimal inflammatory 1 16 0 16 3 16 cells in the endometrium Kidneys Minimal nephropathy 8 16 8 16 6 16 Minimal tubular dilatation 1 16 5 16 2 16 Minimal cysts 1 16 0 16 0 16 Minimal mineralization 0 16 1 16 0 16 a Data were obtained from Appendix X on pages 148, 150 and 152 of the study report. b (CV) * Significantly different from controls at p<0.05. III. DISCUSSION AND CONCLUSIONS A. INVESTIGATOR’S CONCLUSIONS: Administration of phosmet to intact juvenile/peripubertal female rats at a dose that met, but did not exceed the MTD, did not cause any changes in the evaluated endpoints to suggest a disruption in pubertal development or thyroid function. B. AGENCY COMMENTS: No treatment-related effects were observed on mortality, organ weights, serum TSH concentrations, clinical chemistry, or gross pathology. Changes (NS) in ovary, uterus, thyroid, and pituitary weights were noted in the low- and high-dose animals. There were no treatment-related histopathological findings noted in the ovaries, uterus, thyroid, or kidneys. In addition, no treatment-related effects were noted on cycle status, mean age at first estrus, mean cycle length, percent cycling, or percent regularly cycling Page 217 of 260 Female Pubertal Assay (2012) / Page 17 of 17 PHOSMET / 059201 OCSPP 890.1450/ OECD None One 10-mg/kg/day rat exhibited localized head tremors on a single day, and tremors were observed at various times post-dose in up to 10/16 10-mg/kg/day rats on 10 days and up to sixteen 20-mg/kg/day rats at 18 days. No other treatment-related clinical signs were observed in animals from either dose group. Decreased body weights (p<0.05; ↓9% to 10%) were observed at 20-mg/kg/day during PND 26 to 36. No significant change in mean final body weight or body weight gain was observed in either treatment group compared to the vehicle control group. The day of VO was comparable among the groups. Unadjusted and adjusted body weights at VO were decreased by 9% each in the 20 mg/kg/day group compared to controls. No treatment-related effect was noted on cycle status, mean age at first estrus, mean cycle length, percent cycling, or percent regularly cycling. At 20 mg/kg/day, the number of ovarian antral follicles was increased (↑93%), and the number of atretic follicles was decreased (↓29%). All other values in the treated groups were similar to control. Serum T4 concentrations were increased by 24–38% in the treated groups compared to the control, and TSH was slightly increased (6%) at 20 mg/kg/day. A marginal decrease in the thyroid colloid area and increase in the follicular cell height was observed in animals administered phosmet (10 and 20 mg/kg) compared to control. There were no effects on liver, kidney, pituitary, adrenal, uterus, or thyroid organ weights at either dose compared to control The high dose tested (20 mg/kg/day) was determined to be an excessive dose due to the occurrence of tremors in up to 16 of 20 rats at 20 mg/kg/day rats on 18 days. In addition, tremors were observed at various times post-dose in up to 10/16 10 mg/kg/day rats on 10 days. Also, decreased body weights (↓9%–10%) were observed at 20 mg/kg/day during PND 26–36. Following the agency’s determination, the registrant submitted additional information (MRID 49610501) in response to the agency’s review of this assay. The submitted data show that tremors were seen in 10/16 rats at the 10 mg/kg/day dose level (during 10 days) and all animals at the 20 mg/kg/day dose (during 18 days). The severity of the tremors ranged from “not characterized to moderate.” Upon review of these data, the agency reaffirms its previous conclusion that both doses tested in this assay were excessive. C. STUDY DEFICIENCIES: Dose levels tested were excessive. Page 218 of 260 DATA EVALUATION RECORD PHOSMET Study Type: OCSPP 890.1500, Male Pubertal Assay EPA Contract No. EP10H001452 Task Assignment No. 2-61-2012 (MRID 48673003) Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive Arlington, VA 22202 Prepared by CSS-Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B Durham, NC 27713 Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 5/7/2012 Secondary Reviewer: Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 5/17/2012 Program Manager: Signature: Jack D. Early, M.S. Date: 5/21/2012 Quality Assurance: Signature: Jack D. Early, M.S. Date: 5/21/2012 This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel. Page 219 of 260 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 220 of 260 Male Pubertal Assay (2012) I Page I of 15 PHOSMET I 059201 OCSPP 890.1500/ OECD None Primary Reviewer: Linda Tavlor. Ph.D. Health Effects Division Secondary Reviewer: John Liccione, Ph.D. Health Effects Division DATA EVALUATION RECORD STUDY TYPE: Male Pubertal Assay; OCSPP 890.1500; OECD None. PC CODE: 059201 DP BARCODE: D400672 TXR#: 0056199 CAS No.: 732-11-6 TEST MATERIAL (PURITY): Phosmet (96.8% a.i.) SYNONYMS: S-[(1,3-dihydro-l ,3-dioxo-2H-isoindol-2-yl)methyl] 0, 0-dimethyl phosphorodithioate CITATION: Zorilla, L. (2012). Pubertal developmental and thyroid function with phosmet injuvenile/peripubertal CRL:CD(SD) IGS female and male rats. Integrated Laboratory Systems, Inc., Durham, NC, Laboratory Study No.: Cl94-300, February 24, 2012. MRID 48673003. Unpublished. SPONSOR: Gowan Company, P.O. Box 5569, Yuma, AZ TEST ORDER#: EDSP-059201-54 EXECUTIVE SUMMARY: Jn a male pubertal assay (MRID 48673003), 16 Sprague-Dawley (Crl:CD®(SD) IGS) rats/dose group were treated daily via oral gavage with phosmet (96.8% a.i., Batch #: 6QH9118000) in aqueous 0.5% carboxymethylcellulose (5 mL/kg) at doses ofO, 10, or 20 mg/kg/day from post-natal day (PND) 23 to 53 or 54. The animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and body weight at day of attainment was recorded. Following sacrifice on PND 53154, total serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were analyzed by radioimmunoassay. Various clinical chemistry parameters were measured in the serum. Liver, kidney, pituitary, adrenals, seminal vesicles with coagulating glands, and ventral and dorsolateral prostate were weighed and examined macroscopically; in addition, microscopic examinations were conducted on the epididyrnides, testis, thyroid, and kidneys. All rats survived until study termination. Tremors were observed periodically in the majority of the rats at 20 mg/kg/day. No other clinical signs of toxicity were observed in the study at either dose. The decreases in body weight gains and final body weights were minimal (<10%) and not biologically significant. The small variations in clinical chemistry parameters observed at 20 mg/kg/day were not considered to be indicative of toxic effects on the kidney or liver. Page 221 of 260 Male Pubertal Assay (2012) / Page 2 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None There was no treatment-related effect on preputial separation at either dose level. The adjusted ventral prostate weights showed a linear trend of decreased weight with increasing dose; however, no statistically significant effect was detected between the treated groups and control. Both means were lower than historical control mean ventral weight for Sprague-Dawley male rats but were within the acceptable range. There were no effects on liver, kidney, pituitary, adrenal, testis, or other sex organ weights at either dose level compared to the control. Testosterone levels were lower in both dose groups compared to the control, but statistical significance was not attained and the means were within the expected range for Sprague-Dawley rats. It is to be noted that the standard deviation for testosterone in the 20 mg/kg/day dose group (1.62) was similar to the mean value (1.80). Serum T4 levels were increased in both treatment groups compared to the concurrent control and the mean value for Sprague-Dawley male rats, but they were within the acceptable range in the performance criteria. Additionally, these increases were within the range of control values observed at the analytical laboratory (3-7 µg/dL). TSH levels were slightly higher at both dose levels compared to the control, but the control value was below the acceptable range and the Guideline criteria for this parameter were not met. During the agency’s initial review, the doses (10 and 20 mg/kg/day) used in this study were considered excessive, based on the dose-selection rationale and the periodic occurrence of tremors in the majority of the males at 20 mg/kg/day. Subsequently, the registrant submitted additional data (MRID 4961502) in response to the agency’s review of this assay. The submitted data show at the 10 mg/kg/day dose level, mild tremors were seen during one day only in 6/16 animals. In contrast, the severity of tremors at the 20 mg/kg/day dose level ranged from mild to moderate and were seen on multiple days in 11/16 animals. Based on this additional information, the low dose was determined to be adequate with only the high dose showing evidence of overt toxicity. 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, and Quality Assurance statements were provided. Page 222 of 260 Male Pubertal Assay (2012) / Page 3 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None I. MATERIALS AND METHODS A. MATERIALS 1. Test Facility: Integrated Laboratories Systems, Inc. Location: Durham, NC Study Directors: L. Zorilla. Other Personnel: S. Borghoff, study toxicologist M. Boyle, study pathologist Study Period: May 25, 2011 through February 24, 2012 2. Test Substance: Phosmet Description: Off white solid Source: Gowan Company Batch #: 6QH9118000 (expires January 31, 2013) Purity: 96.8% a.i. Stability: Report stated that the compound is stable in the vehicle at 1–10°C for up to 15 days CAS #: 732-11-6 Structure: O S P N S OCH3 OCH3 O 3. Vehicle: 0.5% Carboxymethylcellulose in sodium phosphate buffer 4. Test Animals: Species: Rat (males) ® Strain: Sprague-Dawley (Crl:CD (SD) IGS) Age/Mean Weight PND 23 / 51.5–72.1 g at Study Initiation: Source: Charles River Laboratories International, Inc. (Raleigh, NC) Housing: Parental dams were individually housed in polycarbonate cages with micro-isolator tops and absorbent, heat-treated, hardwood bedding; each dam was housed with her litter until weaning on PND 21. After weaning, up to 2 males were housed per cage. Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad libitum. It was stated that the total genistein equivalents of genistein plus daidzein was determined to be 8.0 μg/g of feed. Water: Reverse osmosis-purified tap water, ad libitum Environmental Temperature: 19–24°C Conditions: Humidity: 43–78% Air changes: Not reported Photoperiod: 14 h light / 10 h dark B. STUDY DESIGN 1. In-Life Dates: Start: July 1, 2011 End: Approximately August 2, 2011 2. Mating: Time-mated pregnant nulliparous dams (age not reported) were received from the supplier on gestation day (GD) 8 and were allowed to deliver naturally and rear their pups to PND 21. Between postnatal days (PND) 3 and 5, litters with the same date of birth were standardized to 8 pups with equal numbers of males and females. Page 223 of 260 Male Pubertal Assay (2012) / Page 4 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None 3. Animal Assignment: Following weaning on PND 21, animals were assigned to study using a procedure that stratifies animals across groups by body weight such that mean body weight of each group was not statistically different from any other group using analysis of variance. Care was taken to avoid placing littermates in the same dose group. The test groups are provided in Table 1. TABLE 1. Study Design a Test group Dose (mg/kg/day) # of Males Control 0 16 Low 10 16 High 20 16 a Data were obtained from Table 2 on page 16 of the study report. 4. Dose Selection Rationale: The doses were selected based on the results of several previous studies. A 7-day relative sensitivity study (MRID 44839301) compared the toxic responses of PND 11 rat pups versus adult rats following repeated gavage administration of phosmet. No treatment-related effects on body weight or cholinergic clinical signs were seen in either pups or adults at the highest tested dose (HTD) of 5 mg/kg/day, however significant cholinesterase inhibition was evident (e.g. responses were 57-62% of controls in adult RBC and 81-86% of controls in adult brain at the HTD). The U.S. EPA (ToxSAC memorandum, May 6, 2009) calculated a BMD10L of 0.60 mg/kg/day for adult rat RBC cholinesterase inhibition and a BMD10L of 2.84 mg/kg/day for brain cholinesterase inhibition from the study data. Body weight effects were observed in a rat teratology study (MRID 41962902) that treated pregnant dams with 10 consecutive gavage doses of phosmet from GD 7–16. At the end of dosing, a statistically significant reduction in body weight gain was observed at 15 mg/kg/day (the HTD). This was associated with a 7% reduction in body weight, compared to controls. Probable treatment-related cholinergic clinical effects (shaking and/or stains around mouth) were seen in a few animals (4/24) after 6 days of dosing through the end of dosing in the 15 mg/kg/day group. There was no unambiguous evidence of treatment- related cholinergic clinical signs or body weight effects at 5 or 10 mg/kg/day. In a recent Uterotrophic assay (MRID 48618707), there were no body weight effects after 3 consecutive days of gavage dosing at 5 or 20 mg/kg/day. However, reduced activity was noted in a few of the 20 mg/kg/day animals on the third dosing day, within several hours of dosing. In a recent Hershberger assay (MRID 48618706), there were treatment-related cholinergic clinical signs in the agonist and antagonist cohorts at 25 mg/kg/day after ≥3 consecutive dosing days. These effects were typically noted within a few hours of dose administration but did not persist to the pre-dosing examination on the following treatment day. Treatment-related cholinergic clinical signs were not seen at ≤15 mg/kg/day. Statistically significant reductions in body weight gain were found for both agonist and antagonist cohorts at the 25 mg/kg/day dose. Additionally, mean final body weight was significantly Page 224 of 260 Male Pubertal Assay (2012) / Page 5 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None reduced at the 25 mg/kg/day treatment level as compared to controls in the antagonist portion of the study. No body weight effects were noted at ≤25 mg/kg/day in the agonist cohort. The highest doses tested in the relative sensitivity and Hershberger studies appear to bracket the appropriate range for the highest dose for the phosmet pubertal assays (> 5 and < 25 mg/kg/day), based on the target of achieving significant body weight effects while avoiding severe clinical signs. Considering the longer exposure intervals employed in the pubertal assays, compared to those used in the other gavage studies, the available data clearly suggest that 20 mg/kg/day will elicit measurable body weight effects in the pubertal assays. It should be noted that it may not be possible to avoid the occurrence of some cholinergic clinical signs with an organophosphate such as phosmet, given the target to elicit body weight effects. However, such effects, if present, should be transient and would not be anticipated to be severe and persist, based on the 25 mg/kg/day data from the Hershberger assay. A robust existing database shows that substantial cholinesterase inhibition will occur at 10 and 20 mg/kg/day. Both selected doses significantly exceed the regulatory benchmark of 0.6 mg/kg/day, which is the EPA-calculated BMD10L, for RBC cholinesterase inhibition in the repeated-dose relative sensitivity study. Both doses also exceed the EPA-calculated BMD10 for brain cholinesterase inhibition (3.86 mg/kg/day) following repeated exposures. Accordingly, the selected doses ensure that an appropriately conservative experimental challenge is employed in the phosmet pubertal screening assays. 5. Dose Preparation and Analysis: Phosmet dose formulations were prepared at ILS in 0.5% aqueous carboxymethylcellulose five times during the study at concentrations of 2 and 4 mg/mL. Formulations were dispensed into vials to be used daily during the study. Dose concentrations were adjusted to correct for purity of phosmet. Samples of dose formulations prepared on June 28, 2011 and July 6, 13, 19, and 26, 2011 were collected from the top, middle, and bottom of the formulation and sent to Smithers Viscient (Wareham, MA) for analysis. Smithers Viscient analyzed, in duplicate, samples received for concentration and homogeneity. The report stated that dose formulations of phosmet in 0.5% aqueous carboxymethylcellulose held from 1 to 10°C for up to 15 days were considered stable (Dix, 20121); however, no data were submitted to allow independent verification. Results Stability (% of time zero): Not reported Concentration (% of nominal): 96–106% Homogeneity (%CV): 2.3–11.8% The analytical data indicated that the mixing procedure was adequate and the variation between nominal and actual dosage to the animals was acceptable. Homogeneity as %CV only exceeded 10% for the 2 mg/mL formulation on July 13, 2011. Stability data should be submitted for review. 1 Dix, M. (2012). Storage Stability of Phosmet in Aqueous, Buffered Carboxymethylcellulose. Unpublished study report prepared by Smithers Viscient Inc. Study No. 12791.6159. Page 225 of 260 Male Pubertal Assay (2012) / Page 6 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None 6. Dosage Administration: Dose formulations were administered by oral gavage at a dose volume of 5 mL/kg body weight. The dose formulations were administered for 31 or 32 consecutive days on PND 23–53/54. Dosing occurred 24-hours (±2 hours) from the previous dose. Dose volume was adjusted daily based on individual animal body weight. The dosing sequence was stratified across all dose groups; one animal from each group and then repeated until all animals were dosed. 7. Statistics: Descriptive statistics (mean, standard deviation, coefficient of variance, and sample size) were calculated using Microsoft Excel 2003/2007 (Redmond, WA). Data sets listed below were analyzed using Statistical Analysis System version 9.2 (SAS Institute, Cary, NC). Studentized residual plots were used to detect possible outliers in the data and Levene’s test was used to assess homogeneity of variance. Heterogeneous data were transformed (logarithm, square root, and multiplicative inverse) and if still heterogeneous analyzed using the non-parametric Kruskal-Wallis and Dunn’s test. If data sets were not homogenous, ANCOVA analyses were not performed. Trend tests were performed on body weight and tissue weight data sets and reported when significant (p<0.05) for endpoints that did not show any significant pairwise comparisons. Classification Procedure Initial body weights Homogeneous data sets: Final body weight (use last day all body weights collected) One-way analysis of variance (ANOVA) followed by pair- Final body weight gain (use last day all body weights wise comparisons performed using Dunnett’s two-tailed collected) t-tests Age and body weight at PPS Tissue weights Two-way ANOVA with treatment and necropsy day (if >1 Relative tissue weights (liver, kidneys, pituitary, and day) as main effects. adrenals) Pair-wise comparisons were performed using Dunnett’s Hormone levels two-tailed t-tests. Clinical chemistry levels Initial body weights Two-way analysis of covariance (ANCOVA) with PND 21 Final body weight (use last day all body weights collected) body weight (weight on day of allocation) as the covariable. Final body weight gain (use last day all body weights Pair-wise comparisons were performed using Dunnett’s collected) two-tailed t-tests. Age and body weight at PPS Tissue weights In the instances where PPS had not occurred prior to necropsy, the last day of examination plus 1 was used as the age at PPS and terminal body weight was used for body weight at PPS. In instances when at least 1 animal in any group exhibited incomplete PPS, including partial threads for >3 days, the day partial separation was first recorded was used in the analyses. Thyroid scoring (colloid area and follicular cell height) Fisher’s Exact test Significance was reported as exact p-values, except no p-value was reported for Dunn's Test (p<0.05). The statistical analyses were considered appropriate. C. METHODS 1. Mortality and Clinical Examinations: Animals were observed twice daily for mortality Page 226 of 260 Male Pubertal Assay (2012) / Page 7 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None and moribundity on weekdays and once daily on weekends and holidays. Rats were observed prior to allocation of animals to study groups, daily prior to dose administration, and prior to euthanasia. 2. Body Weight: Body weights were collected weekly following birth, prior to allocation of animals to study groups, daily prior to dose administration, and prior to euthanasia. 3. Food Consumption: Food consumption was not measured. 4. Preputial Separation (PPS): Beginning on PND 30, males were examined daily following dosing, and PPS was noted as: progression not initiated, partial separation, a persistent thread of tissue between the glans and prepuce, or complete PPS. 5. Sacrifice and Pathology: At least 2 hours after the final dose administration, animals were humanely euthanized by decapitation, in the same order as they were dosed. After euthanasia and prior to tissue collection, trunk blood was collected, and serum was obtained. All serum samples were stored at or below −70°C. Serum samples were shipped on dry ice to AniLytics Inc. (Gaithersburg, MD) for analysis. a. Hormone Analysis: Testosterone, T4, and TSH were measured in serum using radioimmunoassays (RIAs). Samples were assayed in duplicate and in conjunction with multiple quality control samples. b. Clinical Chemistry: The following CHECKED (X) parameters were examined. ELECTROLYTES OTHER X Calcium X Albumin X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus Total cholesterol X Potassium Globulins X Sodium Glucose ENZYMES X Total bilirubin X Alkaline phosphatase (ALP) X Total protein Cholinesterase (ChE) Triglycerides Creatine phosphokinase Albumin/globulin ratio (calculated) Lactic acid dehydrogenase (LDH) Bile acids X Alanine aminotransferase (ALT/also SGPT) X Aspartate aminotransferase (AST/also SGOT) X Sorbitol dehydrogenase 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 and grossly observed from all study animals. Page 227 of 260 Male Pubertal Assay (2012) / Page 8 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None UROGENITAL OTHER X Testes (left and right separately)*+ X Thyroid a*+ X Epididymides (left and right separately)*+ X Liver* X Seminal vesicle plus coagulating glands (with and without fluid)* X Adrenals (paired)* X Ventral prostate* X Pituitary* X Dorsolateral prostate* X Levator ani/bulbocavernosus (LABC) muscle complex* X Kidneys (paired)*+ Gross lesions and masses a Thyroid was weighed post-fixation. * Weights required based on guideline 890.1500. + Histopathological examination required based on guideline 890.1500. Left kidney and thyroid were fixed in 10% neutral buffered formalin for at least 24-hours. Left testis and left epididymis were each placed in Bouin’s fixative for 18- to 24-hours and then washed in 70% histology grade alcohol. After fixation, tissues were transferred to 70% histology grade alcohol, histologically processed, embedded, sectioned, and stained with hematoxylin and eosin. Histologically-processed tissues (thyroid, kidneys, epididymis, and testis) from all animals were microscopically evaluated. Two sections of each of the 2 lobes of the thyroid were examined and evaluated for follicular cell height and colloid area. A 5-point grading scale (1 = shortest/smallest; 5 = tallest/largest) was used, and the thyroids were evaluated relative to photomicrographs supplied by the U.S. EPA. II. RESULTS A. Mortality: All animals survived. B. Clinical Signs of Toxicity: No treatment-related clinical signs were observed in animals from any dose group 24 hours post-dose. However, it was noted in the report that tremors were observed in 6/16 animals receiving 10 mg/kg/day (during one day only) and in eleven 20 mg/kg/day rats (on 16 days). C. General Growth and Preputial Separation: Body weights, body weight gains, age of attainment of preputial separation (PPS), weight at PPS, and proportion/incidence of unseparated preputia are presented in Table 2 below. Body weight gains (adjusted and unadjusted) were decreased (p≤0.05) by 6-7% in the treated groups. Final body weights (adjusted and unadjusted) were decreased by 6% (p≤0.05) at 10 mg/kg/day and by 4% (NS) at 20 mg/kg/day. There was no treatment-related effect on PPS at either dose level. Although adjusted age at PPS showed a statistically significant increase at 10 mg/kg/day (PND 45.4 treated vs. PND 43.7 controls; 1.7 days), this difference is considered to be within biological variation. Body weight at PPS, age at partial PPS, and initial body weight were similar in the treated groups compared to the control. Page 228 of 260 Male Pubertal Assay (2012) / Page 9 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None The following performance criteria were outside of the specified values: %CV=11.7% for body weight at PPS (top of acceptable range: 7.57%); and %CV=7.7% for final body weight (top of acceptable range: 7.47%). Additionally, weaning weight (PND 21) was not reported. TABLE 2. General Growth and Preputial Separation (PPS) a Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet Parameter Evaluated CV CV CV N Mean SD (%) N Mean SD (%) N Mean SD (%) Age at PPS U 15 43.6 2.1 4.8 16 45.3 2.4 5.3 15 44.7 1.5 3.3 (PND) A 15 43.7 ------16 45.4* ------15 44.6 ------Body weight at U 15 226.1 26.5 11.7 16 225.4 17.6 7.8 15 228.5 17.0 7.5 PPS (g) A 15 227.0 ------16 226.4 ------15 227.6 ------U 12 41.7 0.9 2.1 14 41.1 1.4 3.4 9 41.4 1.1 2.7 Age at partial PPS (PND) A 12 41.7 ------14 41.1 ------9 41.4 ------ Initial body U 16 62.2 4.1 6.6 16 61.9 5.0 8.1 16 63.8 4.1 6.4 weight (PND 23; g) A 16 62.2 ------16 62.1 ------16 63.5 ------294.5* 300.3 U 16 313.2 24.0 7.7 16 17.8 6.1 16 18.6 6.2 Final body (↓6) (↓4) weight (g) 295.0* 299.8 A 16 313.3 ------16 ------16 ------(↓6) (↓4) Final body U 16 ------16 94.0 ------16 95.9 ------weight (% of control) A 16 ------16 94.2 ------16 95.7 ------237.5* 239.9* Body weight U 16 256.3 21.6 8.4 16 18.1 7.6 16 19.0 7.9 (↓7) (↓6) gain 237.8* 239.6* (final – initial; g) A 16 256.3 ------16 ------16 ------(↓7) (↓7) Proportion 1/16 0/16 1/16 unseparated (#/N) a Data were obtained from Table 21 on page 45 of the study report. Percent difference from controls (calculated by reviewers) is presented parenthetically. U Unadjusted for body weight on PND 23 A Adjusted for body weight on PND 23 N Number of animals examined SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p<0.05. D. Organ Weights: Organ weights at necropsy are presented in Table 3 below. The adjusted ventral prostate weights showed a linear trend of decreased (p≤0.05) weight with increasing dose (↓9-15%); however, no statistically significant effect was detected between the treated groups and control using Dunnett’s test. It is noted that some of the criteria values CV(%) were greater than the specified range. No other trend or statistically significant effect was detected for the sex tissues or any other organ weighed. The following performance criteria were outside of the specified ranges: (i) mean kidney weight of 2.07 g (acceptable range: 2.242–3.050 g); (ii) %CV=29.9% for seminal vesicles with coagulating gland and fluid (top acceptable range: 21.06%); (iii) %CV=23.3% for Page 229 of 260 Male Pubertal Assay (2012) / Page 10 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None ventral prostate (top acceptable range: 22.32%); (iv) %CV=27.4% for left epididymis (top acceptable range: 16.39%; right epididymis was within acceptable range); and (v) mean thyroid weight of 27.0 mg (acceptable range: 14–26 mg). TABLE 3. Organ Weights at Necropsy a Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet Organ CV CV CV N Mean SD (%) N Mean SD (%) N Mean SD (%) U 16 14.39 1.59 11 16 13.84 1.43 10.4 16 13.79 1.36 9.9 Liver A 16 14.39 - - 16 13.87 - - 16 13.76 - - (g) R 16 4.51 0.25 5.5 16 4.62 0.31 6.8 16 4.54 0.26 5.7 U 16 2.07 0.17 8.1 16 2 0.18 9.1 16 2.06 0.21 10.4 Kidneys A 16 2.07 - - 16 2 - - 16 2.06 - - (g) R 16 0.65 0.03 4.2 16 0.67 0.04 5.7 16 0.68 0.05 7.4 U 16 10 1.5 14.9 16 9.2 1.7 18.5 16 9.8 1.2 12.5 Pituitary A 16 10 - - 16 9.2 - - 16 9.8 - - (mg) R 16 3.1 0.5 16 16 3.1 0.6 18 16 3.2 0.4 13.4 U 16 49.8 10.5 21 16 44.7 8.6 19.3 16 45.1 5.9 13 Adrenals A 16 49.9 - - 16 44.9 - - 16 45 - - (mg) R 16 15.6 2.7 17.1 16 14.9 2.6 17.4 16 14.9 1.9 12.9 Seminal vesicle U 16 558 167 29.9 16 497.9 112.7 22.6 16 476.5 96.8 20.3 + coagulating gland, with fluid (mg) A 16 558 - - 16 498.2 - - 16 476.2 - - Seminal vesicle U 16 354.7 83.8 23.6 16 316 54.1 17.1 16 320.5 57.3 17.9 + coagulating gland, without A 16 354.7 - - 16 316.1 - - 16 320.3 - - fluid (mg) U 16 238.8 55.7 23.3 16 216.6 43.9 20.3 16 205.3 36.8 17.9 Ventral 217.7 204.1 prostate (mg) A c 16 238.9 - - 16 - - 16 - - (↓9) (↓15) Dorsolateral U 16 148.8 27.6 18.5 16 140.3 31.5 22.5 16 146.8 33.9 23.1 prostate (mg) A 16 148.8 - - 16 140.1 - - 16 146.9 - - LABC U 16 529.8 62.9 11.9 16 513.4 66.8 13 16 549.6 61.4 11.2 (mg) A 16 529.8 - - 16 513.5 - - 16 549.6 - - Epididymis, left U 15 b 191.8 52.6 27.4 16 209.5 22 10.5 16 205.4 18.4 9 (mg) A 15 b 204.3 - - 16 209.9 - - 16 205.2 - - Epididymis, U 16 212.9 14.6 6.9 16 211.9 25.2 11.9 16 208 16.2 7.8 right (mg) A 16 - - - 16 - - - 16 - - - Testis, left (mg) U 15 b 1343.3 372.6 27.7 16 1406.1 111.1 7.9 16 1367.1 93.3 6.8 A 15 b 1432.6 - - 16 1406.8 - - 16 1366.6 - - Testis, right U 16 1481.1 154.9 10.5 16 1403.4 106 7.6 16 1396.2 107 7.7 (mg) A 16 1481.2 - - 16 1403.8 - - 16 1395.8 - - Page 230 of 260 Male Pubertal Assay (2012) / Page 11 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None TABLE 3. Organ Weights at Necropsy a Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet Organ CV CV CV N Mean SD (%) N Mean SD (%) N Mean SD (%) Thyroid, fixed U 16 27 8.9 33.1 16 23.8 3.7 15.4 16 25.3 4 15.8 (mg) A 16 25 - - 16 23.8 - - 16 25.3 - - a Data were obtained from Table 22 on pages 47–48 of the study report. No differences (p≤0.05) were noted between treated groups and controls. b One animal removed due to potential recording error. c Significant trend (p<0.05) U Unadjusted for body weight on PND 23 A Adjusted for body weight on PND 23 R Organ-to-body weight ratio (relative to body weight) SD Standard Deviation CV Coefficient of Variation E. Clinical Chemistry and Hormone Levels: Clinical chemistry and hormone levels are presented in Table 4 below. Serum T4 levels were increased (p<0.0001) at both dose levels (↑48% at 10 mg/kg/day and ↑43% at 20 mg/kg/day) compared to the control. Serum TSH was slightly increased (↑7%- 8%) to a similar extent at both dose levels compared to the control, although the control value was low (3.63 ng/mL) compared to the acceptable range (4.212-24.112 ng/mL) and the performance criteria were not met. Testosterone levels in the treated groups were lower than the control (↓18% at 10 mg/kg/day and ↓12% at 20 mg/kg/day), but statistical significance was not attained, and the means were within the background levels. The following performance criteria were outside of the specified ranges: mean serum TSH level of 3.63 ng/mL (acceptable range: 4.212–24.112 ng/mL); and %CV=61.2% for serum TSH (top acceptable range: 58.29%). The following clinical chemistry parameters differed (p<0.05) in the 20 mg/kg/day group compared to controls: (i) sodium (↓2%); (ii) potassium (↓8%); (iii) phosphorus (↓9%); (iv) aspartate transferase (↓14%); and (v) creatinine (↓12%). The values for sodium and aspartate transferase were within the range of control values; all values for potassium and phosphorus were slightly above the range of control values; and all values for creatinine were slightly below the range of control values. These small variations were not considered to be indicative of toxic effects on the kidney or liver. All measured clinical chemistry parameters in the 10 mg/kg/day group were similar to controls. TABLE 4. Hormone Levels and Clinical Chemistry a Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet Parameter CV CV CV Evaluated N b Mean SD (%) N Mean SD (%) N Mean SD (%) Hormones Serum T4, 6.10* 5.91* 15 4.13 0.72 17.3 16 0.81 13.3 16 0.99 16.7 Total (↑48) (↑43) Page 231 of 260 Male Pubertal Assay (2012) / Page 12 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None TABLE 4. Hormone Levels and Clinical Chemistry a Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet Parameter CV CV CV Evaluated N b Mean SD (%) N Mean SD (%) N Mean SD (%) (µg/dL) Serum TSH 15 3.63 2.22 61.2 16 3.92 2.24 57.0 16 3.89 2.53 65.0 (ng/mL) Serum testosterone 15 2.04 0.92 45.1 16 1.67 0.99 59.0 16 1.80 1.62 90.1 (ng/mL) Clinical Chemistry Sodium 150* 15 153 2 1 16 151 4 2 16 3 2 (mEq/L) (↓2) Potassium 8.4* 15 9.1 0.6 6.8 16 8.7 0.6 7 16 0.8 9.2 (mEq/L) (↓8) Chloride 15 114 2 2 16 112 2 2 16 113 2 2 (mEq/L) Calcium 15 10.9 0.3 2.6 16 10.8 0.5 4.8 16 10.8 0.3 3.1 (mg/dL) Phosphorus 9.7* 15 10.7 0.9 8.8 16 10.4 0.9 8.3 16 0.8 8 (mg/dL) (↓9) 186* AST (U/L) 15 217 42 19 16 213 43 20 16 26 14 (↓14) ALT (U/L) 15 93 15 16 16 97 14 14 16 90 11 12 GGT (U/L) 15 0 ------16 0.2 0.5 290 16 0.2 0.8 400 ALP (U/L) 15 318 72 23 16 325 62 19 16 320 58 18 BUN 15 14 2 13 16 15 3 19 16 15 2 15 (mg/dL) Creatinine 0.39 0.38* 15 0.43 0.06 14.2 16 0.07 17.3 16 0.06 15.4 (mg/dL) (↓10) (↓12) Total Bilirubin 15 0.1 0.15 146 16 0.08 0.1 133 16 0.18 0.16 92.1 (mg/dL) SDH (U/L) 15 20 3 17 16 22 3 16 16 20 4 18 Total Protein 15 5.8 0.3 5.3 16 5.7 0.3 5 16 5.6 0.3 5.1 (g/dL) Albumin 15 4.2 0.3 6.3 16 4.2 0.3 6 16 4.1 0.3 6.7 (g/dL) a Data were obtained from Table 23 on page 50 and Table 24 on page 51 of the study report. Percent difference from controls (calculated by reviewers) is presented parenthetically. SD Standard Deviation CV Coefficient of Variation * Significantly different from controls at p<0.05. * Significantly different from controls at p<0.01. * Significantly different from controls at p<0.001. F. Gross pathology: There were no effects of treatment observed at necropsy. In one control rat, the right testis was noted as enlarged, the left testis and epididymis were noted as markedly small, and the seminal vesicle was also noted as being small. No other gross observations were noted in male rats surviving to the scheduled termination. Page 232 of 260 Male Pubertal Assay (2012) / Page 13 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None G. Histopathology: The incidences of histopathological findings of the thyroid gland are presented below in Table 5; follicular cell shape was not reported. A marginal increase in the colloid area and decrease in the follicular cell height was observed in animals administered phosmet (10 and 20 mg/kg) compared to controls. The number of rats with Grade 3 follicular colloid area decreased in a dose-related manner from 10/16 in the control (62%), to 6/16 at 10 mg/kg/day (38%), and 3/16 at 20 mg/kg/day (18%). Similarly, the number of animals having Grade 4 increased in a dose-related manner from 6/16 in the control (38%), to 10/16 at 10 mg/kg/day (62%), and 12/16 at 20 mg/kg/day (75%). There was a dose-related increase in the number of rats with Grade 2 follicular cell height (6/16 in the control (38%), 10/16 at 10 mg/kg/day (62%), and 12/16 at 20 mg/kg/day (75%), and a dose-related decrease in the number of the animals with Grade 3 follicular cell height (10/16 in the control (38%), 6/16 at 10 mg/kg/day (62%), and 3/16 at 20 mg/kg/day (18%). TABLE 5. Incidence of Histopathological Lesions of the Thyroid Gland a Parameter Evaluated Colloid Area Follicular Cell Height Treatment Groups Incidence Incidence Grade b Grade b O E O E 1 0 16 1 0 16 2 0 16 2 6 16 Vehicle Control 3 10 16 3 10 16 4 6 16 4 0 16 5 0 16 5 0 16 1 0 16 1 0 16 2 0 16 2 10 16 10 mg/kg/day 3 6 16 3 6 16 Phosmet 4 10 16 4 0 16 5 0 16 5 0 16 1 0 16 1 0 16 2 1 16 2 12 16 20 mg/kg/day 3 3 16 3 3 16 Phosmet 4 12 16 4 1 16 5 0 16 5 0 16 a Data were obtained from Table 26 on page 53 of the study report. b A 5-point grading scale (1 = smallest/ shortest; 5 = largest/tallest) was used. O No. Observed E No. Examined The incidence of histopathological findings of the testes, epididymides, liver, thyroid, and kidneys are presented in Table 6. There were no treatment-related histopathological findings noted in the testes, epididymides, or kidneys at any dose. Page 233 of 260 Male Pubertal Assay (2012) / Page 14 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None TABLE 6. Incidence of Histopathological Lesions of the Epididymides, Testis, and Kidneya Dose Level Vehicle Control 10 mg/kg/day Phosmet 20 mg/kg/day Phosmet Findings # # # # # # Observed Examined Observed Examined Observed Examined Epididymides Minimal interstitium inflammation 5 16 6 16 4 16 Minimal aberrant cells in tubule 15 16 15 16 13 16 lumen Mild aberrant cells in tubule lumen 0 16 0 16 1 16 Marked atrophy 1 16 0 16 0 16 Testis Minimal cellular debris in the 0 16 4 16 2 16 seminiferous tubules Minimal atrophy/hypoplasia 2 16 1 16 0 16 Marked atrophy/hypoplasia 1 16 0 16 0 16 Kidneys Minimal nephropathy 13 16 8 16 8 16 Minimal tubular dilatation 4 16 4 16 2 16 Minimal cysts 0 16 0 16 1 16 a Data were obtained from Appendix X on pages 160, 162, and 164 of the study report. III. DISCUSSION AND CONCLUSIONS A. INVESTIGATOR’S CONCLUSIONS: Administration of phosmet to intact juvenile/peripubertal male rats at a dose that met, but did not exceed the MTD, did not cause changes in any endpoints evaluated to suggest a disruption in pubertal development or thyroid function. B. AGENCY COMMENTS: No treatment-related related effects were observed on survival, body weight at PPS, age at partial PPS, organ weights, serum TSH and testosterone levels, clinical chemistry, necropsy, or histology at dose up to 20 mg/kg/day. No clinical signs of toxicity were observed at either dose level at the time of dosing throughout the study. However, tremors were observed periodically in the majority of the rats at 20 mg/kg/day. The decreases in body weight gains and final body weights were minimal and not biologically significant. The small variations in clinical chemistry parameters observed at 20 mg/kg/day were not considered to be indicative of toxic effects on the kidney or liver. There was no treatment-related effect on PPS at either dose level. Page 234 of 260 Male Pubertal Assay (2012) / Page 15 of 15 PHOSMET / 059201 OCSPP 890.1500/ OECD None The adjusted ventral prostate weights showed a linear trend of decreased weight with increasing dose; however, no statistically significant effect was detected between the treated groups and control using Dunnett’s test. Both means were lower than historical control mean ventral weight for Sprague-Dawley male rats but were within the acceptable range. Serum T4 levels were increased in both treatment groups compared to the concurrent control and mean values for Sprague-Dawley male rats, but they were within the acceptable range in the performance criteria. Additionally, these increases were within the range of control values provided by the analytical laboratory (3-7 µg/dL). A marginal decrease in the follicular cell height and an increase in the colloid area were observed in animals administered phosmet at both dose levels compared to controls. There were no treatment-related histopathological findings in the testes, epididymides, or kidneys at either dose level. During the agency’s initial review, the doses (10 and 20 mg/kg/day) used in this study were considered excessive, based on the dose-selection rationale and the periodic occurrence of tremors in the majority of the males at 20 mg/kg/day. Subsequently, the registrant submitted additional data (MRID 4961502) in response to the agency’s review of this assay. The submitted data show at the 10 mg/kg/day dose level, mild tremors were seen during one day only in 6/16 animals. In contrast, the severity of tremors at the 20 mg/kg/day dose level ranged from mild to moderate and were seen on multiple days in 11/16 animals. Based on this additional information, the low dose was determined to be adequate with only the high dose showing evidence of overt toxicity. 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: • CV=11.7% for body weight at PPS (top of acceptable range: 7.57%) • CV=7.7% for final body weight (top of acceptable range: 7.47%) • weaning weight (PND 21) was not reported • mean kidney weight of 2.07 g (acceptable range: 2.242–3.050 g) • CV=29.9% for seminal vesicles with coagulating gland and fluid (top acceptable range: 21.06%) • CV=23.3% for ventral prostate (top acceptable range: 22.32%) • CV=27.4% for left epididymis (top acceptable range: 16.39%; right epididymis was within acceptable range) • mean thyroid weight of 27.0 mg (acceptable range: 14–26 mg) • mean serum TSH level of 3.63 ng/mL (acceptable range: 4.212–24.112 ng/mL) • CV=61.2% for serum TSH (top acceptable range: 58.29%) Page 235 of 260 DATA EVALUATION RECORD PHOSMET Study Type: OCSPP 890.1550, Steroidogenesis Assay EPA Contract No. EP10H001452 Task Assignment No. 2-4-2012 (MRID 48618701) Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive Arlington, VA 22202 Prepared by Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B Durham, NC 27713 Primary Reviewer: Signature: Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 12/19/2011 Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 12/19/2011 Program Manager: Signature: Jack D. Early, M.S. Date: 12/27/2011 Quality Assurance: Signature: Jack D. Early, M.S. Date: 12/27/2011 This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel. Page 236 of 260 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 237 of 260 Steroidogenesis Assay (2011) I Page 1 of 14 PHOSMET I 059201 OCSPP 890.1550/ OECD None Primary Reviewer: Gregory Akerman Signature: ~,.If--:. Health Effects Division Secondary Reviewer: John Liccione, Ph.D. Signature:Date~ Health Effects Division Date. -- Template version 0812011 DATA EVALUATION RECORD STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550 PC CODE: 059201 DP BARCODE: 0397165 TXR#: 0056199 CAS No.: 732-11-6 TEST MATERIAL (PURITY): Phosmet (96.8% a.i.) SYNONYMS: S-[( l ,3-dihydro-l ,3-dioxo-2H-isoindol-2-yl)methyl] 0 ,0-dimethyl phosphorodithioate CITATION: Wagner, H. (2011). Phosmet: H295R Steroidogenesis Assay. CeeTox, Inc., Kalamazoo, Ml. Laboratory Study No. 9142V-100348STER, November 3, 201 I. MRID 48618701. Unpublished. SPONSOR: Gowan, 370 Main Street, Yuma, AZ 85364 TEST ORDER#: EDSP-059201 -54 EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48618701), H295R cells cultured in vitro in 24-well plates were incubated with phosmet (96.8% purity, Batch# GQH9118000) at concentrations of 100, IO, 1, 0.1, 0.01, 0.001, and 0.0001 µM for 48 hours in triplicate for four independent experiments. Run 1 was not analyzed because one of the forskolin concentrations on the quality control plate was different than the proposed concentration outlined in the test guideline (3.33 µM was used instead of l µM). Dimethyl sulfoxide (DMSO) was used as a vehicle, at a final concentration of 0.05% v/v. Testosterone and estradiol levels were measured using HPLC-MS/MS. A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels. Positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production. All 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. Neither testosterone nor estradiol concentrations were affected by phosmet. Decreases (p<0.05) of I 0-11 % in estradiol concentration were observed at 0.0001, 0.001, and 0.0 I µM of phosmet in Run 2, but not at higher concentrations nor at any concentration in the other runs. An increase Page 238 of 260 Steroidogenesis Assay (2011) / Page 2 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None (p≤0.05) of 10% in estradiol concentration was observed at 100 µM of phosmet in only one of the three runs. Based on hormone responses in each of three independent runs, phosmet 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 239 of 260 Steroidogenesis Assay (2011) / Page 3 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None I. MATERIALS AND METHODS A. MATERIALS 1. Test Facility: CeeTox Location: Kalamazoo, MI Study Director: Hilary Wagner, M.S. 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 Study Period: July 15, 2011 through November 2, 2011 2. Test Substance: Phosmet Description: Off-white crystals Batch # (expiration date): GQH9118000 (January 31, 2013) Purity: 96.8% a.i. Solubility (in solvent): Approximately 10−3.5 M Vapor pressure at 25°C: 0.065 mPa Stability: Not reported Storage conditions: Room temperature CAS #: 732-11-6 Molecular weight: 317.3 Structure: 3. Positive Control: Forskolin Description (molecular weight): White powder (410.50) Source: Sigma Aldrich (St. Louis, MO) Lot # (expiration date): 109K50571V (July 2016) Purity: 98% Solubility (in solvent): DMSO Storage conditions: Room temperature CAS #: 66575-29-9 4. Negative Control: Prochloraz Description (molecular weight): White powder (376.67) Source: Sigma Aldrich (St. Louis, MO) Lot # (expiration date): SZE6220X (August 8, 2012) Purity: 99.1% Solubility (in solvent): DMSO Storage conditions: Room temperature CAS #: 67747-09-5 Page 240 of 260 Steroidogenesis Assay (2011) / Page 4 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None 5. Solvent/Vehicle Control: Dimethyl Sulfoxide (DMSO) Description (molecular weight): Clear liquid (78.13) Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): RNBB7617 (Not provided) Purity: Not provided Storage conditions: Sigma-Aldrich (Cat. # D2650) CAS #: 67-68-5 Justification for choice of Not provided solvent: Final concentration in assay: 0.05% v/v 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/nutrient mixture F12 Ham Source: Sigma-Aldrich (St. Louis, MO) Lot # (expiration date): 021M8304 (not provided) HEPES: 15 mM; Sigma-Aldrich (St. Louis, MO); Lot # 021M8304 Sodium bicarbonate: Sigma-Aldrich (St. Louis, MO); Lot # 068K0105; purity not reported Nu-Serum: 2.5%; Becton-Dickinson (Franklin Lakes, NJ); Catalog # 355500; Lot # 81515); tested for background hormone concentrations by performing laboratory ITS+premix: Becton-Dickinson (Franklin Lakes, NJ); Catalog # 354352; Lot # 05245, 09233) 8. Test Cells: H295R human adrenocortical carcinoma cells (ATCC CLR-2128; lot # 7635054). Cells were used in the assay at passage 7.5 – 8.5 and were incubated in the stock medium supplemented with 10 µM 22R-hydroxycholesterol in ethanol 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 at least 4 additional passages x Total number of passages does not exceed 10 B. METHODS 1. Pre-Test Information: a. Hormone Assay Interference Test: A hormone assay interference test was not performed. b. Hormone Extraction: See Section on “Hormone Measurement Section.” c. Laboratory Proficiency Test: The laboratory proficiency test data were not provided. Page 241 of 260 Steroidogenesis Assay (2011) / Page 5 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None 2. Test Solutions: Phosmet was dissolved in DMSO to make a 200 mM stock solution and then serially diluted in DMSO. 22R-Hydroxycholesterol was dissolved in ethanol to make a 40 mM stock and then diluted in supplemented medium to a final concentration of 10 µM. Phosmet was then diluted 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 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 phosmet, with the final concentration of DMSO in the medium being held constant at 0.05% (v/v). The final ethanol concentration in the supplemented medium was 0.025% (v/v). No precipitation was visible. 3. Cell Plating and Preincubation: Cells were maintained in the Stock Medium described above. It was reported that hormone concentrations in undiluted Nu-Serum were 3754 pg/mL testosterone and 3846 pg/mL estradiol. H295R cells were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for seven or eight additional passages prior to use in the assay. The culture medium was supplemented with 10 uM 22R- hydroxycholesterol at the time of plating, dosing, and harvest. The concentration of 22R- hydroxycholesterol was chosen based on laboratory proficiency experiments previously conducted at the Performing Laboratory. The cells were plated into wells of a 24-well cell culture plate at a density of approximately 300,000 cells/mL. The cells were then placed into a 5% CO2 incubator at 37±2°C for approximately 24 hours prior to chemical exposure. Prior to dosing, the cells were checked microscopically for attachment and proper morphology. 4. Exposure: The medium was removed from the cells and replaced with 1 mL of medium containing 10 μM 22R-hydroxycholesterol and concentrations of test chemical in triplicate, according to the schematic presented in Table 1. TABLE 1. Dosing Schematic for the Exposure of H295R Cells to Phosmet (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 13 of the study report. A concurrent QC plate was included with each of the three independent runs of the test chemical plates to demonstrate the assay’s response to forskolin (an inducer of testosterone and estradiol production) and prochloraz (an inhibitor of testosterone and estradiol production). The QC plate was prepared and dosed in the same manner with either forskolin or prochloraz according to the schematic presented in Table 2. Page 242 of 260 Steroidogenesis Assay (2011) / Page 6 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None TABLE 2. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in µM).a 1 2 3 4 5 6 A Blankb Blank Blank Backgroundc Background Background B DMSO DMSO DMSO DMSO + MeOHd DMSO + MeOH DMSO + MeOH C Forskolin 1µM Forskolin 1µM Forskolin 1µM Prochloraz Prochloraz Prochloraz 0.1µM 0.1µM 0.1µM D Forskolin 10µM Forskolin 10µM Forskolin 10µM Prochloraz 1µM Prochloraz 1µM Prochloraz 1µM a Data were obtained from page 13 of the study report. b Blank wells received medium containing 22R-hydroxycholesterol. c Background wells received medium only. d MeOH = methanol was added to these wells for 30 minutes at room temperature following medium removal. Following dosing, the plates were incubated for 48±2 hours under the conditions previously described. After the 48±2 hour exposure, each well was examined under the microscope and images were taken of the vehicle control wells as well as the two greatest non-cytotoxic concentrations (based on observation under the microscope). The media was collected from all wells in two equal aliquots and stored at -80±10°C until shipment to the analytical laboratory for hormone measurement. 5. Cell Viability/Cytotoxicity Assay: After media removal, cell viability was measured by MTT assay. On the QC plates, wells designated to receive methanol (control wells for cell death measurements) were rinsed twice with phosphate buffered saline (PBS), then incubated in methanol for 30 minutes at room temperature. After the methanol incubation, the methanol-treated wells were rinsed again with PBS three times. Following media removal and/or methanol treatment, 0.5 mL of 0.5 mg/mL MTT (Sigma, Cat#M5655, Lot# MKBG1611V) 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 the 3 hour incubation, the MTT solution was removed and 0.5 mL of isopropanol was added to each well. Plates were incubated at room temperature for 20 minutes with shaking. Following this incubation, absorbance at 570 nm and 650 nm were measured on a Packard Fusion plate reader. The absorbance at 650 nm was subtracted from the absorbance at 570 nm to calculate the MTT value for each well. The change in cell viability was determined by comparing treated wells to the solvent control (SC) wells. A greater than 20% reduction in cell viability was considered evidence of cytotoxicity. 6. Hormone Measurement System: Testosterone and estradiol levels were measured at OpAns, LLC (Durham, NC). Testosterone and estradiol were extracted from H295R supplemented medium by liquid-liquid extraction using methyl tert-butyl ether (MTBE) 2 2 after the addition of [ H5]-testosterone and [ H5]-estradiol as internal standards. Extracts were analyzed by HPLC-MS/MS using positive ion multiple reaction monitoring. This method was validated over the range 100 to 100,000 pg/mL for testosterone and 10 to 10,000 pg/mL for estradiol (OpAns Document Number OPR-OPP-0006). The lower limit of quantification (LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol using a 300 µL aliquot of H295R supplemented medium. Freshly prepared calibration standards (n = 7) were prepared in supplemented medium for each run. QC samples spiked in supplemented medium at three concentrations (2 replicates per concentration), were included with each run. No study samples were re-analyzed during this study. Page 243 of 260 Steroidogenesis Assay (2011) / Page 7 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None 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) NR Solvent control within 75% range below maximum response on standard curve X 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. 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 p-values were less than or equal to 0.05 in either the normality or the homogeneity test, a log 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: Compared to the minimum detection level (MDL), basal hormone production in the SC wells was 18 to 30-fold MDL for testosterone and 17 to 23-fold MDL for estrogen. The between plate %CV for the SC based on absolute hormone concentrations was 26% for testosterone and 14% for estradiol. The %CVs for the absolute concentration within replicate wells of a plate were 4.87-6.68% for testosterone and 2.25-5.32% for estradiol. These values were below the maximum guideline recommended level of 30%. The Sponsor stated: “Four independent runs of the steroidogenesis assay were conducted. Run 1 was not analyzed because one of the forskolin concentrations on the quality control Page 244 of 260 Steroidogenesis Assay (2011) / Page 8 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None plate was different than the proposed concentration outlined in the test guideline (3.33 uM was used instead of 1 uM). Data from Run 1 will not be presented in this report as the media was not analyzed for testosterone and estradiol.” The concentrations of estradiol and testosterone observed in the other three assay runs are summarized in Table 3 and graphically depicted in Figures 1 and 2. Neither testosterone or estradiol concentration was affected by phosmet. Decreases (p<0.05) of 10-11% in estradiol concentration were observed at 0.0001, 0.001, and 0.01 µM phosmet, but not at higher concentrations nor at any concentration in the other runs. An increase (p≤0.05) of 10% in estradiol concentration was observed at 100 µM phosmet in only 1 of the three runs. TABLE 3. Mean (±SD) Hormone Concentrations Following Treatment with Phosmet for 48 Hours.a Nominal Statistical Concentration Run 2 Run 3 Run 4 Run 2 Run 3 Run 4 Meanb ± SDb Significance (µM) Testosterone (pg/mL) Fold Difference DMSO 3044±203 1801±93 2292±112 NA NA NA NA -- 0.0001 2778±108 1803±49 2351±19 0.91 1.00 1.03 0.98 0.06 — 0.001 2833±176 1858±75 2248±91 0.93 1.03 0.98 0.98 0.05 — 0.01 2781±40 1814±56 2303±134 0.91 1.01 1.00 0.97 0.06 — 0.1 2883±91 1904±56 2205±144 0.95 1.06 0.96 0.99 0.06 — 1 2981±113 1793±93 2422±14 0.98 1.00 1.06 1.01 0.04 — 10 2899±106 1892±108 2359±60 0.95 1.05 1.03 1.01 0.05 — 100 2813±84 1647±10 2223±41 0.92 0.91 0.97 0.93 0.03 — Estradiol (pg/mL) Fold Difference DMSO 227±12 171±4 206±8 NA NA NA NA -- 0.0001 202±4 166±4 217±2 0.89* 0.97 1.06 0.97 0.09 Run 2 0.001 203±4 167±3 208±3 0.90* 0.97 1.01 0.96 0.06 Run 2 0.01 202±3 164±5 214±8 0.89* 0.96 1.04 0.96 0.08 Run 2 0.1 212±9 175±3 203±11 0.93 1.03 0.99 0.98 0.05 — 1 222±9 161±4 218±1 0.98 0.94 1.06 0.99 0.06 — 10 222±6 175±7 226±3 0.98 1.03 1.10 1.04 0.06 — 100 239±4 170±2 226±5 1.06 0.99 1.10* 1.05 0.06 Run 4 a Data were obtained from pages 25-26 and 32-34 of the study report. The lower limit of quantification (LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol. b Calculated by the reviewers. Page 245 of 260 Steroidogenesis Assay (2011) / Page 9 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None FIGURE 1. Change in Testosterone Production Relative to Phosmet Concentration. Page 246 of 260 Steroidogenesis Assay (2011) / Page 10 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None FIGURE 2. Change in Estradiol Production Relative to Phosmet Concentration. B. CYTOTOXICITY: It was stated that cytotoxicity was not assessed in Run 3 because the plate was dropped by the plate reader prior to data acquisition. Less than 5% cytotoxicity Page 247 of 260 Steroidogenesis Assay (2011) / Page 11 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None was noted, except in the positive control (92-95% cytotoxicity). Data are summarized in Table 4 and depicted graphically in Figure 3. TABLE 4. Mean (±SD) MTT Cell Viability Results after Treatment with Forskolin, Prochloraz, or Phosmet for 48 Hours. a Concen. Cell Viability – Run 2 Cell Viability – Run 3 Cell Viability – Run 4 Compound (µM) Mean SD Mean SD Mean SD Blank NA 99.4 10.54 100.1 4.93 103.0 1.84 Background NA 102.5 5.95 98.7 0.69 104.0 1.76 SC + Methanol NA 4.9 0.99 5.7 0.64 8.3 1.79 Forskolin 1 103.9 3.88 97.2 2.2 107.3 0.91 Forskolin 10 105.1 4.9 97.5 2.67 106.7 4.11 Prochloraz 0.1 100.7 1.89 97.8 1.79 101.7 2.17 Prochloraz 1 101.1 2.01 96.1 2.79 105.0 4.29 Phosmet 0.0001 95.4 2.80 ND ND 95.6 2.97 Phosmet 0.001 98.3 1.60 ND ND 98.0 1.12 Phosmet 0.01 99.1 3.07 ND ND 97.7 0.92 Phosmet 0.1 97.8 2.59 ND ND 99.2 0.37 Phosmet 1 97.3 3.12 ND ND 101.6 3.54 Phosmet 10 97.6 3.51 ND ND 99.4 1.58 Phosmet 100 99.1 3.59 ND ND 99.4 4.51 a Data were obtained from pages 19-20 of the study report. NA Not applicable ND Not determined due to dropped plate Page 248 of 260 Steroidogenesis Assay (2011) / Page 12 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None FIGURE 3. MTT cell viability results for phosmet-treated cells. 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 Page 249 of 260 Steroidogenesis Assay (2011) / Page 13 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None ± SD) for the three assay runs are presented in Table 5. The minimum basal hormone production levels (500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both blank and SC wells. OCSPP 890.1550 guideline requirements for basal hormone production in SC wells, induction by 10 µM forskolin, and inhibition by 1 µM prochloraz were met for testosterone and estradiol. Basal hormone production in SC wells was ≥ 5-fold MDL for testosterone and ≥ 2.5-fold MDL for estrogen. Compared to SC, 10 µM forskolin on average induced testosterone 1.74-fold (instead of 2-fold as per the guideline) and estradiol by 9.55-fold. However, when the background is subtracted, 10 µM forskolin induced testosterone an average of 2.74-fold. Compared to SC, 1 µM prochloraz inhibited the synthesis of testosterone to 0.48-fold and estradiol to 0.35-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 19% for testosterone and 14% for estradiol, which meets the guideline criteria of ≤30% for the assay. The %CVs within each run for the QC plates were 2.19 to 2.64% for testosterone and 1.89 to 4.01% for estradiol. TABLE 5. Mean (±SD) Hormone Concentrations Following Treatment with Forskolin or Prochloraz for 48 Hours.a Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Meanb ± SDb Concentration (µM) Testosterone (pg/mL) Fold Difference (Relative to DMSO) Background 1493 1035 1359 0.57 0.58 0.59 0.58 0.01 Blank 2921 1799 2338 1.12 1.01 1.01 1.05 0.06 DMSO 2612 1782 2319 1.00 1.00 1.00 1.00 0.00 1 µM Forskolin 4325 2403 3108 1.66c 1.35c 1.34c 1.45c 0.18 10 µM Forskolin 5604 2561 3765 2.15d 1.44d 1.62d 1.74d 0.37 0.1 µM Prochloraz 2145 1505 1843 0.82 0.84 0.79 0.82 0.03 1 µM Prochloraz 1351 740 1168 0.52 0.42 0.50 0.48 0.05 Estradiol (pg/mL) Fold Difference (Relative to DMSO) Background 66 57 76 0.29 0.33 0.34 0.32 0.03 Blank 244 179 242 1.07 1.02 1.08 1.06 0.03 DMSO 229 175 223 1.00 1.00 1.00 1.00 0.00 1 µM Forskolin 1550 1014 1559 6.76 5.79 6.99 6.51 0.64 10 µM Forskolin 2396 1363 2321 10.46 7.78 10.41 9.55 1.53 0.1 µM Prochloraz 168 151 176 0.73 0.86 0.79 0.79 0.07 1 µM Prochloraz 77 62 78 0.34 0.36 0.35 0.35 0.01 a Data were obtained from pages 21-22 of the study report. The lower limit of quantification (LLQ) was 100 pg/mL for testosterone and 10 pg/mL for estradiol. b Calculated by the reviewers. c When the background is subtracted, the values are 2.53-, 1.83-, and 1.82-fold difference for Runs 2, 3, and 4, respectively, with a mean of 2.06-fold change. d When the background is subtracted, the values are 3.67-, 2.04-, and 2.51-fold difference for Runs 2, 3, and 4, respectively, with a mean of 2.74-fold change. III. DISCUSSION AND CONCLUSIONS A. INVESTIGATOR’S CONCLUSIONS: Phosmet did not induce or inhibit testosterone at any of the concentrations tested in the three independent runs of the assay. Based on the Page 250 of 260 Steroidogenesis Assay (2011) / Page 14 of 14 PHOSMET / 059201 OCSPP 890.1550/ OECD None results of the three independent assays using H295R cells, phosmet was not considered to be an inducer or inhibitor of estradiol or testosterone in this steroidogenesis assay. B. AGENCY COMMENTS: All performance criteria were met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate induction and inhibition with positive controls. Neither testosterone nor estradiol concentrations were affected by phosmet. Decreases (p<0.05) of 10-11% in estradiol concentration were observed at 0.0001, 0.001, and 0.01 µM phosmet, but not at higher concentrations nor at any concentration in the other runs. An increase (p≤0.05) of 10% in estradiol concentration was observed at 100 µM phosmet in only 1 of the three runs. Based on hormone responses in each of three independent runs, phosmet treatment did not result in a statistically significant reproducible alteration in testosterone or estradiol production. C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to have had an adverse impact on the results, interpretation or conclusions of this study: • Total number of cell passages was 12-13, exceeding 10. • The laboratory proficiency test data were not provided. • Mean±SD and %CV for the fold-differences were not reported. Page 251 of 260 DATA EVALUATION RECORD PHOSMET Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay EPA Contract No. EP10H001452 Task Assignment No. 2-4-2012 (MRID 48618707) Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive Arlington, VA 22202 Prepared by Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B Durham, NC 27713 Primary Reviewer Signature: Rebecca Bryan, B.S. Date: 12/05/11 Secondary Reviewer Signature: Michael E. Viana, Ph.D., D.A.B.T. Date: 12/07/11 Program Manager: Signature: Jack D. Early, M.S. Date: 12/15/2011 Quality Assurance: Signature: Jack D. Early, M.S. Date: 12/15/2011 This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by Dynamac Corporation personnel. Page 252 of 260 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 253 of 260 In vivo Uterotrophic Assay (2011) I Page I of 7 PHOSMET/ 059201 OCSPP 890.1600/ OECD 440 Primary Reviewer: Sheila Healy. Ph.D. Health Effects Division Date: --=-J.6..1....~::...L-_,.,__ Secondary Reviewer: _""'""Je=s=s""""R=o;...;.w'""l=an=d=------Signature: d;? ~ - ~ Health Effects Division Date: <9.LSi t r Template version 0912011 DATA EVALUATION RECORD STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440 PC CODE: 059201 DP BARCODE: D397165 TXR#: 0056199 CAS#: 732-11-6 TEST MATERIAL (PURITY): Phosmet (96.8% a.i.) SYNONYMS: S-[( 1,3 -dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl] 0 , 0-dimethyl phosphorodithioate; Imidan technical CITATION: Zorrilla, L., and Codrea, E. (20 11 ). The Uterotrophic Assay with Phosmet in Ovariectomized CRL:CD(SD) IGS Rats. Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study ID: C194-100, October 19, 2011. MRID 48618707. Unpublished. SPONSOR: Gowan Company, P.O. Box 5569, Yuma, AZ TEST ORDER#: EDSP-059201-54 EXECUTIVE SUMMARY: In an uterotrophic assay (MRID 48618707) conducted to screen for potential estrogenic activity, phosmet (96.8% a.i., Batch# 6QH9118000) in 0.5% carboxymethyl cellulose was administered daily via oral gavage to groups of eight ovariectomized female Sprague-Dawley rats at dose levels ofO (vehicle), 5, or 20 mg/kg/day on post-natal days (PND) 63-65. A positive control group was treated with 17a-ethynyl estradiol (EE) by daily oral gavage at a dose level ofO.l mg/kg/day. All animals were terminated and necropsied on PND 66 approximately 24 hours after the final dose to determine wet and blotted uterine weights. All animals survived until scheduled termination. Two 20 mg/kg/day rats had decreased movements at 1-4 h post-dosing on Days 1 and 2. Body weights in the dose groups were comparable to the controls throughout the duration of the assay. Uterine weights in the dose groups were comparable to the controls. Absolute wet and blotted uterus weights for the 0.1 mg/kg/day EE group were increased (p<0.05) by 407% and 23 1%, respectively. The dose levels selected for this study are adequate based on previously conducted studies which showed no mortality at doses up to 27 mg/kg/day and significant cholinesterase inhibition at 10 mg/kg/day. No statistically significant changes were seen in uterine weight in this assay. Phosmet was negative in the uterotrophic assay. Page 254 of 260 In vivo Uterotrophic Assay (2011) / Page 2 of 7 PHOSMET/ 059201 OCSPP 890.1600/ OECD 440 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 255 of 260 In vivo Uterotrophic Assay (2011) / Page 3 of 7 PHOSMET/ 059201 OCSPP 890.1600/ OECD 440 I. MATERIALS AND METHODS A. MATERIALS 1. Test Facility: Integrated Laboratory Systems, Inc. Location: Durham, NC Study Director: L. Zorrilla Other Personnel: Study Period: May 3-6, 2011 2. Test Substance: Phosmet Description: Off white solid Source: Gowan Company (commercial technical product) Batch #: 6QH9118000, expires January 13, 2012 Purity: 96.8 % a.i. Stability: Stable in the vehicle for up to 8 days at 1-10°C (Smithers Viscient Inc., Study No. 12791-6159) CAS #: 732-11-6 Structure: 3. Reference Estrogen: 17α-ethynyl estradiol (EE) Supplier Sigma Aldrich, St. Louis, MO Lot/Batch #: 090M1241V, expires February 2012 Purity: ≥98 % CAS # : 57-63-6 4. Solvent/Vehicle Control (Phosmet): Carboxymethyl cellulose Supplier Spectrum Chemical Mfg. Corp. Lot/Batch #: ZE0383, expires August 1, 2011 Rationale (if other than water) Not provided Final concentration 0.5% 5. Solvent/Vehicle Control (EE): Corn Oil Supplier MP Biomedicals, LLC, Solon, OH Lot #: 7862K, expires August 1, 2011 Rationale (if other than water) Not Provided Final concentration Not Applicable 6. Test Animals: Species: Rat (ovariectomized females) Strain: Sprague-Dawley (Crl:CD[SD] IGS) Age/weight at dose initiation: PND 63 days old / 236.6-298.7 g females Source: Charles River Laboratories International, Inc., Raleigh, NC Housing: 2/cage in polycarbonate cages with absorbent heat-treated hardwood bedding (Northeastern Products Corp., Warrensburg, NY) Diet: Teklad Global 16% Protein Rodent Diet (Teklad Diets, Madison, WI), ad libitum; low phytoestrogen with total genistein equivalents = 6.2 µg/g diet. Water: Reverse osmosis-treated tap water, ad libitum Environmental conditions: Temperature: 22-26ºC Humidity: 33-48% Air changes: Not provided Photoperiod: 12 hrs light/12 hrs dark Acclimation period: 8 days Page 256 of 260 In vivo Uterotrophic Assay (2011) / Page 4 of 7 PHOSMET/ 059201 OCSPP 890.1600/ OECD 440 B. METHODS 1. In Life Dates: Start: May 3, 2011 End: May 6, 2011 2. Study Design: Following an 8-day acclimation period, ovariectomized female rats (surgery performed by Supplier) were administered the test substance from PND 63-65. Rats were euthanized approximately 24 hours after the last dose and necropsied for uterine weight measurements. 3. Animal Assignment: Animals were randomly assigned, stratified by body weight, to the test groups noted in Table 1. Statistical analysis indicated that there were no significant differences between group mean weights at study initiation. Table 1. Study Design of Estrogen Agonist Assaya Test Group Dose (mg/kg/day) # of Females Vehicle Control 0 8 Low 5 8 High 20 8 17α-ethynyl estradiol (EE), Reference Estrogen 0.1 8 a Data were obtained from Table 1 on page 16 of the study report. 4. Dose Selection Rationale: The phosmet dose levels were selected based on the results from an acute oral gavage study (MRID 46593101) and repeated-dose studies (MRIDs 44811801, 41962902, 41520001, 41916401, and 44839301). Mortality was observed in ≥75 mg/kg treatment groups for the acute oral study. No mortality was observed in the repeated- dose dietary studies conducted at doses of up to 27 mg/kg/day, or in repeated-dose gavage studies with doses up to 15 mg/kg/day. An acute relative sensitivity study (MRID 47087401) compared the toxic responses of PND 11 rat pups versus adult rats following single-dose gavage administration of phosmet. No cholinergic clinical signs were found in either pups or adults at 10 mg/kg; however, significant cholinesterase inhibition was observed at this dose, especially in RBC. The adult rat RBC cholinesterase response was 62- 63% of controls at 10 mg/kg. EPA has calculated a BMD10L of 5.122 mg/kg for brain cholinesterase inhibition from the data. In the 7-day relative sensitivity study (MRID 44839301), no cholinergic clinical signs were observed for the 5 mg/kg/day dose group. Based on these results, doses selected for the main study were 0, 5 and 20 mg/kg/day. 5. Dose Preparation and Analysis: Dose formulations were prepared once prior to initiation of treatment by initially dissolving the appropriate amounts of test substance in 0.5% carboxymethyl cellulose. The reference estrogen, EE, was mixed with corn oil for dosing. Doses were adjusted daily based on individual body weight measurements. Homogeneity and concentration analyses were measured prior to administration in the 5 and 20 mg/kg/day formulations. In a previous storage stability study1, phosmet (lot number not reported) was 1 Reibach, R. (2011) Storage Stability of Phosmet in Aqueous, Buffered Carboxymethylcellulose. Unpublished study report prepared by Smithers Viscient Inc., Study No. 12791-6159. Page 257 of 260 In vivo Uterotrophic Assay (2011) / Page 5 of 7 PHOSMET/ 059201 OCSPP 890.1600/ OECD 440 determined to be stable in the 0.5% aqueous carboxymethyl cellulose for up to 8 days at 1- 10°C. Results of Dose Analysis Homogeneity (% CV): 2.45-14.1% Stability: Stable for up to 8 days following storage at 1-10°C (Smithers Viscient Inc., Study No. 12791-6159). Concentration (% of nominal): 81-109% (the 5 mg/kg dose measured at 4.06 mg/kg was only used on study day 1, and for days 2-3 the 5 mg/kg dose was 87% of nominal). The analytical data indicated that the mixing procedure was marginally adequate and that the variation between nominal and actual dosage to the animals was marginally acceptable. 6. Dosage Administration: Animals were administered the test formulations and/or EE or vehicle daily via oral gavage for three consecutive days in a dose volume of 5 mL/kg body weight. Dose volumes were adjusted daily based on the concurrent body weight measurement. 7. Statistics: Body weights, body weight gains, and uterine weights were analyzed using studentized residual plots for outliers and Levene’s test for homogeneity of variance. Body weights and body weight gains were analyzed using one-way ANOVA followed by Dunnett’s two tailed t-test. The wet and blotted uterine weights were analyzed by Kruskal- Wallis and Dunn’s test. Significance was denoted at p≤0.05. The statistical analyses were considered adequate. C. METHODS 1. Clinical Examinations: Animals were checked for mortality or moribundity twice daily on weekdays and once daily on weekends. Rats were also checked for clinical signs of toxicity prior to dose administration and three h (±30 min) post-dosing. 2. Body Weight: Animals were weighed at randomization, daily prior to dosing throughout the dosing period, and at termination. 3. Food Consumption (Optional): Food consumption was not measured. 4. Necropsy and Measurement of Uterine Weight: On PND 66 (approximately 24 hours after final administration of the test substance), all surviving animals were euthanized by CO2 inhalation followed by cervical dislocation and 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 moist filter paper to remove the luminal fluid while preventing desiccation and the blotted uterus was weighed. Page 258 of 260 In vivo Uterotrophic Assay (2011) / Page 6 of 7 PHOSMET/ 059201 OCSPP 890.1600/ OECD 440 5. Microscopic Examination (Optional): Microscopic examinations were not conducted. II. RESULTS A. OBSERVATIONS 1. Mortality: All animals survived until scheduled termination. 2. Clinical Signs of Toxicity: Two 20 mg/kg/day rats had decreased movements at 1-4 h post- dosing on Days 1 and 2. No clinical signs of toxicity were observed in the 5 mg/kg/day animals. B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain data are presented in Table 2. Body weights in the 5 and 20 mg/kg/day dose groups were comparable to controls throughout the duration of the assay. Table 2. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a Dose (mg/kg/day) Study Day Vehicle Control 5 20 EE (0.1) # N Mean SD N Mean SD N Mean SD N Mean SD 1 8 264.3 18.4 8 265.3 19.8 8 262.1 12.2 8 265.7 16.4 2 8 266.5 19.9 8 268.2 19.4 8 266.2 17.1 8 266.8 21.0 3 8 268.7 19.6 8 270.3 21.6 8 266.5 16.1 8 264.1 19.8 4 8 270.6 20.4 8 271.2 20.4 8 265.3 17.0 8 258.7 20.8 Body Weight 8 6.4 3.0 8 5.9 5.8 8 3.2 5.9 8 -7.0 5.8 Gain (1-4) a Data were obtained from Table 3 on page 20 and Appendix V on page 66 of the study report. N= No. of animals in the group SD = Standard Deviation * Significantly different from controls at p <0.05. C. FOOD CONSUMPTION (Optional): Food consumption was not measured. D. PATHOLOGY 1. Uterine Weights: Selected uterine weight data are presented in Table 3. Uterine weights in the dose groups were comparable to the controls. Due to an outlier (666 mg wet; 601 mg blotted), the uterine weight means for the 20 mg/kg/day dose group were based on 7 animals. The mean blotted vehicle control uterine weight was <0.04% of the mean vehicle control terminal body weight, which meets the guideline requirement. Absolute wet and blotted uterus weights for the 0.1 mg/kg/day EE group were increased (p<0.05) by 407% and 231%, respectively. These increased uterine weights were in the expected range. Page 259 of 260 In vivo Uterotrophic Assay (2011) / Page 7 of 7 PHOSMET/ 059201 OCSPP 890.1600/ OECD 440 Table 3. Uterine Weights from Estrogen Agonist Assay in SD Ratsa Dose (mg/kg/day) Reference Estrogen Parameter Vehicle Control 5 20 EE (0.1) N Mean SD N Mean SD N Mean SD N Mean SD Terminal BW (g) 8 270.6 20.4 8 271.2 20.4 8 265.3 17.0 8 258.7 20.8 Wet, absolute (mg) 8 70.9 12.0 8 74.1 6.5 7C 68.2 6.8 8 359.6* 94.8 (↑407) Wet, relative (%) b 8 0.026 0.004 8 0.027 0.001 8 0.026 0.004 8 0.140 0.039 Blotted, absolute (mg) 8 63.9 10.1 8 66.5 4.8 8 61.0 5.3 8 211.3* 29.7 (↑231) Blotted, relative (%) b 8 0.024 0.003 8 0.025 0.002 8 0.023 0.003 8 0.082 0.011 a Data were obtained from Table 4 on page 21 and Appendix VI on page 68 of the study report. b Relative wet and blotted uterine weights were calculated by the reviewer from the individual data. BW= body weight C Outlier removed N= No. of animals in the group SD = Standard Deviation * Significantly different from vehicle control at p < 0.05. 2. Microscopic Examination (Optional): Microscopic examinations were not conducted. III. DISCUSSION AND CONCLUSIONS A. INVESTIGATOR’S CONCLUSIONS: Phosmet administered at dose levels of 5 and 20 mg/kg/day to ovariectomized Sprague-Dawley rats did not induce a change in body weight, body weight gain or uterine weight, wet or blotted, as compared to the vehicle control. Using the ovariectomized rat model, Uterotrophic Assay (OPPTS 890.1600), oral administration of phosmet at dose levels up to 20 mg/kg/day is not estrogenic under these experimental conditions. B. AGENCY COMMENTS: All animals survived until scheduled termination. Two 20 mg/kg/day rats had decreased movements at 1-4 h post-dosing on Days 1 and 2. No clinical signs of toxicity were observed in the 5 mg/kg/day animals. Body weights in the dose groups were comparable to the controls throughout the duration of the assay. Uterine weights in the treatment groups were comparable to the controls. The mean blotted vehicle control uterine weight was <0.04% of the mean vehicle control terminal body weight, which meets the guideline requirement. Absolute wet and blotted uterus weights for the 0.1 mg/kg/day EE group were increased (p<0.05) by 407% and 231%, respectively. These increased uterine weights were in the expected range. No statistically significant changes were seen in uterine weight in this study. Phosmet was negative in the uterotrophic assay. C. STUDY DEFICIENCIES: None Page 260 of 260