Document 2NaJqwn5aKjMnnY9rJjVpEVjr
*
"NorthwestBioanalytical
R`eSptourdty NNoo.. NNWWBBRS9999--111147
Quantitative Determination of PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570 in Human
Serum by LC/MS/MS
Sample Analysis Report for Protocol EPI-PEuro
Northwest Bioanalytical (NWB)
A Division of NWT Inc. 1121 East 3900 South
Salt Lake City, UT 84124
PREPARED FOR:
3M Environmental Technology and Services (3M)
935 Bush Avenue
St. PaulMN, 55133
= on
= 35 pr (IE
w OF
AUTHOR;
/
David Vollmer, Ph.D., NWB Project Manager
Kae APPROVED FOR REL}
Y:
RodgerL. Foltz, Ph.D, Laboratory Director
Page
DATE: 1L// G9
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DATE: _ 1/1/74
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Northwest Bioanalytical
`Study No. NWBS99-117
QUALITY ASSURANCE STATEMENT
LABORATORY:
SPONSOR:
Northwest Bioanalytical (NWB) A Division NWT, Inc. 1121 East 3900 South Salt Lake City,UT84124
3M Environmental Technology Services (3M)
935 Bush Avenue St. Paul, MN 55133
. COMPOUNDS):
PFOS, PFOSA, PFOSAA, PFHS, POAA, M556 and M570
NWB STUDY NUMBER:
'NWBS99-117
SPONSOR STUDY NUMBER: EPI-PEuro
NWB STUDY TITLE:
`Extraction and Analysisof PFOS and other Fluorochemicals in `Human Serum by HPLC/Electrospray Mass Spectrometry
`The clinical sample analysis study described in this report is not included within the definition ofa GLP
regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelines ofthe U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD Principles ofGood Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the SafetyofDrugs (Ordinance No.
21, PAB Notification No. 424.) The following inspections were performed by the NWB QAU per SOP.
Inspection and Reporting Statement.
Inspection Date
27-30 Aug 1999 03 Sep 1999 08 Sep 1999
Hoa 0117-D2e2cS1e9p991999
PhaseofStudy
Sample Receipt Analytical Plan Draft Analytical Plan
Eton RSeapmoprlteDArnaaflytsRiasw Data
Date Inspection Report Issued To
NWBProjectManager ~~*NWBManagement
30 Aug 1999
31 Aug 1999
03 Sep 1999
30 Sep 1999
08 Sep 1999
30 Sep 1999
nhl 0222 DSeepc 11999999
Bhim 3300 SDeepc 11999999
*Reports to NWB Management are issued monthly.
SE Ay cst
AORPASAAR A eR TO
this final report accurately reflect the raw data.
lN.p.A A0)A c,
"Shaundel
Percey,B.S., M.T., A8
=
Quality Assurance Compliand
list
02
Date
re
ore Bi
ys sn
Report No. NWBR99-114
Compliance Statement
`The clinical sample analysis study described within this report is not included within the
definitionof a GLP regulated nonclinical study set forth in Title 21of the U.S. Code of
Federal Regulations Part 58. However, to the bestofour knowledge, this project was
conducted in accordance with the guidelinesof the U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 58) and according to the `methods and procedures described within this report. In addition, the study followed the guidelines of the OECD Principles of Good Laboratory Practice and the Japanese MHW `Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the
`Safetyof Drugs (Ordinance No. 21, PAB Notification No. 424.) There were no known incidents that affected the quality or integrity of the project or data reported. This report
represents an accurate record of the raw data.
VR
David Vollmer, Ph.D.
NWB Project Manager
Rodgef L. Foltz, Ph.D.
NWB Laboratory Director
_ofefse
Date
12/1/15
Date
:
! Northwest Bioanalytical
TABLE OF CONTENTS
ReSptourdty NNoo.. NNWWBBRS99S9.-111147
QUALITY ASSURANCE STATEMENT wc. srssssnnion
' LI OFTS ABLT ES ......corvrorennrissssissssssssssssssssins
-
ars ------------
4. RESULTS AND DISCUSSION .......ovvsseurrrsrsssssssmsssssssssssssssssssssssssssssssssssssssssssssssssssnsss 10
LIST OF TABLES
Table 1. Calibration Curve Summaryfor PFOS in Human Serum.............coeecosersssssssssssssnne1s2s.
Table 2. Calibration Curve Summary for PFOSA in HUMID SERUM...
12
Table 3. Calibration Curve Summary for PFOSAA in Human Serum..............cccoommucnecrmnnn1n2:.
Table 4. Calibration Curve Summary for PFHS in Human Serum............cccceocovermmesensssrsneneec 13
Table 5. Calibration Curve Summary for POAA in Human Serum ...........cceewmureessussersssnnns13
Table 6. Calibration Curve Summary for M556 in Human Serum...............cceuwesssssssisissssnnnc 13
Table 7. Calibration Curve Summary for M570 in Human Serum...
eevssesesesesenrererens
14
Table 8. Back-Calculated ConcentrationsofCalibration Standards for PFOS in HUMAN SEMUM.......oorrerrenerrsssssssnssssssssssssssssssssssssssrsssssss
wornsneaen 15
Table 9. Back-Calculated ConcentrationsofCalibration Standards for PFOSA in HUMAN SEUM......ovrrreserrssrssssssrsssssssssssssssssssssssssss esses sssssssssssssssssss |.
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.
TT
Northwest Bioanalytical
RSetpuodrytNNoo.. NNWWBBRS9999--111147
Table 10. Back-Calculated Concentrations of Calibration Standards for TT
----
Table 11. Back-Calculated Concentrations of Calibration Standards for
J
--"
nel
Table 12. Back-Calculated Concentrations of Calibration Standards for
TT
OS
`Table 13. Back-Calculated Concentrations of Calibration Standards for
Table 14. Back-Calculated Concentrations of Calibration Standards for
MST0 i HUMAN SEIU...
18
`Table 15. Analytical QC Summary for PFOS in Human Serum
19
Table 16. Analytical QC Summary for PFOSA in HUMN SEU...
19
Table 17. Analytical QC Summary for PFOSAA in HUMAN SNM...
20
Table 18. Analytical QC Summary for PFHSin HUMGN SEUM .....vrnre 20
Table 19. Analytical QC Summary for POAA in HUMAN SERUM...
21
`Table 20. Analytical QC Summary for MSS6 in HUMAN SEU ....vrssnsnn21
`Table 21. Analytical QC Summary for M570 in HUAN SER ven 22
`Table 22. Study SAMPIE CONCENTALONS crv
23
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Northwest Bioanalytical
ReSptourdtyNNoo.. NNWWBBRS9999--111147
Quantitative Determination of PFOS, PFOSA, PFOSAA, PFHS, POAA, M556
and M570 in Human Serum by LC/MS/MS
Sample Analysis for Protocol EPI-PEuro
1. INTRODUCTION
`This report summarizes the analytical results from the quantitationof PFOS, PROSA, PFOSAA, PFHS, POAA, M556 and M570 in human serum samples for 3M Environmental Technology and Services in support of Protocol EPI-PEuro [5.1]. The validated LC/MS/MS method provided a lower limitof quantitation (LLOQ) of 31.4 (PFOS), 1.00 (PFOSA), 1.45 (PFOSAA), 2.61 (PFHS) and 5.27 (POAA) ppb. All analytes exhibited a linear response to approximately 500 ppb with the exception of PFOSAA, which had an upper limit of quantitation (ULOQ)of 270 ppb [5.2]. The nonvalidated method for the quantitation for M556 and M570 in human serum provided a lower limitof quantitation (LLOQ)of 1.00 and 5.00 ppb, respectively, and exhibited a linear response to 500 ppb for both analytes.
`The testing facility was 3M Corporate Occupational Medicine (Bldg. 220-3W-05, St. Paul, MN 55144-1000), and Jean Burris at the facility served as the Principal Investigator. The following is a list of NW supervisory personnel involved in the completionof this `work: David Vollmer, Ph.D. (NWB Project Manager); Rodger L. Foltz, Ph.D. (NWB Laboratory Director).
NWB SOPs were used in the conductofthis study and were available to study personnel in electronic and/or hard copy formats
Date Study Initiated: September 7, 1999 Date Analyses Completed: September 20, 1999 `The clinical study described inthis report is not included within thedefinition ofaGLP regulated nonclinical study. However, Northwest Bioanalytical conducts al studies within the guidelinesofthe U.S. FDA Good Laboratory Practice Regulations for
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Northwest Bioanalytical
ReSptourdtyNNoo.. NNWWBBRS9999--111147
Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD Principles of Good Laboratory Practice and the Japanese MEW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424). Any changes to or deviations from the original protocol (Analytical Plan) were documented through approved protocol amendments or deviation `memos and are retained within the raw data.
2. METHODOLOGY
`The validated assay used for this study is Meth98082.00 and was reported in Northwest Bioanalytical report NWBR99-005 [5.2],
Samples for this study were received at NWB on the following date:
Receipt Date August 27, 1999
Numberof Samples Received 18
Storage Condition (except during analysis)
-20C
Reference Material
Analyte LotNumber Purity Expiration Date Source Storage Conditions
PFOS (FC95) 193 100%
PFOSA PFOSAA (EC-129) PFHS
214 100% 617 538%
S398182 100%
12312010
12312000 12312000
12312000
3M Room Temperature
(@ry)
3M RoomTemperature 3M Room Temperature
3M
~20
*POAA 2s 100% 12312010 3M Room Temperature
(FC 143)
(dry)
MsS6
NB 989% 12312000 3M Room Temperature
113047-80
M570 11850626 99.75% 12312000 3M Room Temperature SDS (sodium 17HO459 99% 1162001 Sigmp Room Temperature dodecylsulfate) = sAnyanloyntyemnoaumsewsituhseBdRtOrSou(gFhCo-u9t5)is report do not imclud thei FC Wenner; for example, PFOS 7s
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Northwest Biosalytical
ReSptourdytNNoo.. NNWWBBRS9599--111147
Nine calibration standards were prepared fresh daily by adding the appropriate spiking standard solution into 0.200mL ofhuman serum. The final calibration standard concentrations in human serum for each analyte were as follows:
Analyte
PFOS PFOSA PFOSAA PFHS POAA Mss6 M70
Calibrdgion Standard Concentrations (ppb)
31.4,35.f, 40.4, 55.4, 80.4,130, 280, 430, 530 1.00, 5.40, 10.0,25.0, 50.0, 100, 250, 400, 500 1.45,3.60, 6.29, 14.4, 27.8, 54.7, 135, 216, 270 264BBN1.6,26.6, 51.6, 102,252, 402, 502 5277927,14.3,29.3, 54.3, 104, 254, 404, 504 1.00, 5.00, 10.0, 25.0, 50.0, 100, 250, 400, 500 1.00, 5.00, 10.0, 25.0, 50.0, 100, 250, 400, 500
Analytical QCs were prepared in human serum on September 13, 1999 and frozen in a "20C freezer. Analytical QC atthe following levels were assayed in duplicate in cach analytical run:
Analyte
PFOS PFOSA PFOSAA PFHS POAA M56 M570
OC Concentrations (ppb)
Low (50.4) and High (380) Low (20.0) and High (350) Low (11.7) and High (189) Low (21.6) and High (352) Low (24.3) and High (354) Low (20.0) and High (350) Low (20.0) and High (350)
After the addition ofHPLC-grade water and sodium dodecyl sulfate (SDS, internal
standard) to 0.200 mLof human serum, the serum mixture is made basic with the addition of 0.5 M tetrabutylammonium hydrogen sulfate (TBA, pH 10) and 0.25 M carbonate buffer. This mixture is then extracted with methyl-tert-butyl ether. Afler sufficient mixing, the sample is centrifuged. The organic layer is transferred via pipette into a clean test tube. The organic layer is then evaporated to dryness and the sample is
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"Northwest Bioanalytical
ReSptourdtyNNoo.. NNWWBBRS9999--111147
reconstituted into 2 mM ammonium acetate water:methanol (50:50 v/v). The extracts are then analyzed by liquid chromatography/tandem mass spectrometry using negative-ion electrospray ionization and multiple reaction monitoring.
3. SAMPLE ANALYSIS
`The prepared samples, calibration standards and QCs were injected into the PE Sciex API 3000 LC/MS/MS system in a systematic order.
:
Chromatographic peakintegrationwas performed using MacQuan software (version 1.6).
Quadratic regression analysis (weighted 1/x) all analytes was performed using the peak
area ratio vs. concentration utilizing Watson DMLIMS software (version 5.4.10.2).
3.1. Acceptance Criteria
For an analytical run to be accepted, it must have met the acceptance criteria listed below which are consistent with regulatory and industry recommendations.
Calibration Curve
Each run will include duplicate calibration standards at 7 or more concentrations covering the lower to upper limitofquantitation. At least two-thirdsofthe
calibration standards back-calculated concentrations must be within + 15% (+ 20% for LLOQ)oftheir individual target concentrations. If acalibration standard is not within the acceptance criteria, it is deactivated. This process starts from the highest calibration standard down to the lowest until all the active standards are within the acceptance criteria.
LowerLimitof Quantitation
`The back-calculated concentrationsofat least one of the duplicate lowest points in the calibration curve must be within + 20%ofthe target concentrationto qualify as the LLOQ.Ifthis criterion is not met, the next level is subjected to the same test and the. LLOQ raised accordingly.
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Norwest Bioanalytical Quality Control Samples
ReSptourdtyNNoo.. NNWWBBRSS999-.111147
Each analytical run will include low and high QC samples in triplicate. The measured concentrationsofat least two-thirds of all analytical QCs must be within + 20% of their target concentrations. If study samples require dilution, a dilution QC is analyzed in duplicate for each dilution level. At least one dilution QC at cach level
`must be within + 20% in order to accept study sample results at that dilution level.
`The dilution QC acceptance was independentofthe undiluted analytical QC
:
acceptance.
4. RESULTS AND DISCUSSION
Each accepted run met the acceptance criteria set for the calibration curve points, for the lower limitofquantitation (LLOQ) and for the analytical quality control (QC) samples.
RIDunRPeRsOulSt RPeFsOuStA PFReOsSulAtA RPeRsuElSt RePsOuAlAt ReMsSuSl6t RMe5su1l0t Comments
_--_T -- scopiedaccepted accepted accepted _sceepted aceepied eaceceped
-
There were no known circumstances that may have affected the quality or integrityofthe data. In conclusion, based on the quality control and calibration curve data, it is our opinion that the data submitted to 3M Environmental Technology and Services for PFOS, PFOSA, PFOSAA, PHS, POAA, M556 and M570 are accurate for allofthe EPI-Puro study samples. Please note that the results for analytes M556 and M570 should be considered unvalidated data. 5. REFERENCES 5.1. 3M Protocol EPI-PEuro, "Analysisof Fluorochemicals in Pooled Sera from Europe,"
August 24, 1999.
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52. D. Vollmer, "Quantitative Determination of POS, PFOSA, PFOSAA, N-MeFOSEOH, N-EFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS," NWB Study Number NWBS98-082, NWB Report Number NWBR99-005, May 13, 1999.
`The raw data and final report for this study will be stored in the NWB Archives, 1121
East 3900 South, Salt Lake City, UT 84124 per regulations and contract agreement. After
submissionofthe final report to the Sponsor, remaining study samples will be stored
under required conditions until confirmationofSample Disposition/Refum Authorization
:
is received from the Sponsor. 3M Environmental Technoloagndy Services and 3M
Corporate Occupational Medicine will be notified concerning final dispositionofrecords
at completionofcontract obligations.
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! Northwest Bioanalytical
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Table cwtntetton s-------- 1. Calibration Curve Summary for PFOS in Human Serum
[[r 7omom[ir Nummbger|[ow*oT mos[s*oT o[n[nas[|eoemeelr1u|soTe4l|m5on]) [ ererT rovo ar[Toom|w wer|n |
erm ------
Table 2. Calibration Curve Summary for PFOSA in Human Serum
[isalnl|=--[Iown I[onase [I averS]ooso| 30 | 0] [CvI eenT| T[ovowTor [iovaT 1 t[aaoti]oT woT|TT |]]
tse sreegrt
Table 3. Cv Calibration Curve Ae ser, Summary for PFOSAA in Human Serum
[smaol]I -- TIowEowosInovo [Iamassa ]sm| 5| |
[CvC eem|T TToT wT ows [owT eT romno es |av von
|TT] T]
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| Northwest Bioanalytical
Study No. NWBS99-117
Table cots 1. Atom set, 4. Calibration Curve Summary for PFHS in Human Serum [ il|=1 IEwowsIwI om [owes|as m |aor| | [ES vC E en|r TowowsooT O owesT |ammo|T ||
ret------ `Table coven remeron 5. Calibration Curve Summary for POAA in Human Serum
[= i7or | `Number ownsova [aos|ors|527 | | [er n T[vows[T ower[aT oe |omT ||T|
CeT taTt vetTe T pos teft]
Table 6. Calibration Curve Summary for M556 in Human Serum
[[ omef1 oie7 ]Jovo+mon 1 3sos|[a9 on[|uo9 asa| i1o01 n|lo0n|
[eT m | TTowowooT ma oT so |aoT r||T] Ce T rte T roem eoT tT]
Northwest Bioanalytical
ReSptourdty NNoo.. NNWWBBSR999S-111147
Table 7. Calibration Curve Summary for M570 in Human Serum Quadratic weighted . All concentrationsareexpres1spepdb.
RunDate | Run
a
Number
Cc [RSquared| LLOQ [ULOQ
17-Sep-1999| 1 | 0.000001| 0.002742 | 0.015241| 0.9984 [5[0500 0|
Le TT TTTT
* ALB ad C aecosficientswed todetfheiquandiaeic curve
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| Northwest Bioanalytical
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Table 8. Back-Calculated Concentrationsof Calibration Standards for PFOS in
re onion Human Serum
[|rRuweomoe |Re1 nNomerJ] 1o4[554[[r40e4|Ts5 [Tanaa 112a0 |a23e0|] so[500]] [ TTwT sloeole| a sea [rsear m onr[murs] e sol Tvn so]voa[aress[areern[[aava ae[]zsfwoesriotFe]] [mawel TToosl[e0s 3t5a[|at3a[[7aaa[2as2[fsaisJaosaJ[u1nao])
Tela alalatalsls]
Table 9. Back-Calculated Concentrationsof Calibration Standards for PFOSA in
Cites Atom Human Serum se str [I i759 a[roe 03[eala[E3d5[EoCz|Erd|EDE1D rr TuesT[reT n[iT[raat [T29oTv[]sa] w vseonl e Tleowoms[| TJsoe | m[]J[a[mraamoeas rf|s]a Jsuilaieo]]] [me] TToes a Tao faJetsalrafoloazlosue]]
* Sample deactivated as an outlier (>.+15% theoretical for non-LLOQ;> 20% theoretical for LLOQ)
:
! Northwest Bioanalytical
Study No. NWBS99-117
ilFt dl al Table 10. vn Ammn seeemena Back-Calculated Concentrations of Calibration Standards for PFOSAA in
[sI wimsENumCbeJr iEe E|veEo E|eaTr[FvaOr[E0E5S5|EI[Ea E| s {Trvio [erorowr suemw[r ovuenr[[ar ose sn [eor wno][ser vsaeiake] e ram 0w 0]owsa [[1o6s[[2wso[[0se 7o|1ss[[aassT]aroo2s2] TetatetaaTaTae]
Cte emma tg, Table 11. Back-Calculated ConcentrationsofCalibration Standards for PFHS in Human Serum
(sE wim[] 1T[eRmslnslCesasar|[EE RuEmaRfEsmRs|EwwooRr[EamI][EaawaneeEanra]] SseolT TToowmTo1saT[omaI msT[isaFi[oorrmr TrTm wTss ]ame|]
C e ew]TTTTaaell[aaeT ta[a1e0lJ[auasal]]oosorT[[ssotes[uauselTfsieos]]] tm 1 ot ene Oem)
i.
|
Semen
sytem
Table 12. Back-Calculated Concentrationsof Calibration Standards for POAA in
Cet 1. ArmHuman Serum se
[[mSoerroinsalJr Name{]w8a2r7[a3an[11673T[s0[|sausaT[oinos]sata[asoe || E TesA [ows TYoE sa[Yeafon fos on[5 [smeo [oeowmToowwefoaasm[os0ms [onmaaa[sosr[20 2a7[orrs] [ow ma Toea sT[3o2[wua[a5n1u0fe a[o20fsstafao0[onsa]| Co Tel alal lalala a]e]
Table 13. Back-Calculated Concentrations of Calibration Standards for M556 in `Human Serum
Quadratic weighted 1/x. All concentrations are expressed as ppb.
[[seemtpJemNenter|]i50n0TJaown[0a0[3s50sso]i f[e0ofea[r0) T ir olasre r aT n[asa r [ror[an or[su]
[[show [iwT [wnme[Noer[[n 2e5m5[] ann e aveaanss] [C[ omes| w 150TTe eeal[NieaJsv 0]]5549Ta1oa[[2ae 0a[1aas[]i1o2s] Ce Tv falafel]
* Sample deactivated as an outlier (> 15% theoretical for non-LLOQ; > 20% theoretical for LLOQ)
Cr Northwest Bioanalytical
remy Study No. NWBS99-117
[om soe] 0 [so]ss] cnn nt Table 14. Back-Calculated Concentrations of Calibration Standards for M570 in. `Human Serum
[[ r rset]T Tor a]o wstnr [|3tmos[[2ar 7so 5a[]sotarr[|ovuesr[]l Tr [owss [o]v]
sveonl] [er]
vw[[aoom[1as]swaer[soas aua eze ae|] Toa so[aa[we[se[os es[x5]
C mel TIlaaoolloaalsaal|5aiTtoasl[asTeTesr]]
Smet5I eteret150 orere)
Table 15. nr Analytical QC Summary for PFOS in Human Serum
Run Number| Low QC High QC
I Tse]
Tee[ws 50.4 ppb
380 ppb
|
eSol Ta a| [wes lwe ea el o] se | C TT w 00 [so]
Table 16. or trTO Analytical QC `Summary for PFOSA in Human Serum
Run Number| Low QC
High QC
Tee]
T Tmw o||o eme]] 20.0 ppb.
350 ppb.
TTTs [ow
e se Tw w|| ew]| t welea oeiss|l wae || C ow t Fevear0] | we]
:
Northwest Bioanalytical
Study No. NWBS99-117
"Table 17. JO Analytical QC Summary for PFOSAA in Human Serum
Run Date
|Run Number|
Low QC
11.7 ppb.
High QC
189 ppb
Te ew]
Tew wm] [wee lT roie w s | ] mees]]
me] ws ae]
`Table 18. escape Analytical QC Summary for PFHS in Human Serum
T Tew] Tm a me[|n ooowwm || Run Date
|Run Number|
2L1o.w6pQpCb
|
High QC
352 ppb
ew|
[ee l r5 ie ms |a]]| C T5 e] s|
-
`Table 19. oem ss Analytical QC Summary for POAA in Human Serum
Run Date |Run Number
Low QC
24.3 ppb
High QC
354 ppb.
Cm me[oawi] l ei we [ |a| m e e T oess a [oowru]] C ml T T e] a]
Table 20. J Analytical QC Summary for M556 in Human Serum
Run Number|
Low QC
20.0 ppb
High QC
350 ppb
wese a em] TCvo T Te w wwmr [ Iem wTsI |a wo | TooeT | aT ss | |
mms
p-- en
Table 21. rence Analytical QC Summary for M570 in Human Serum
Run Number|
Low QC 20.0 ppb
High QC 350 ppb
Tw we swme] TTseo To me |m a ss]| CI oo T --oTI[ua Cow [50 as]
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