Document 4JZBKVO8KEj462MdMDY4VL5L1
CENTRE FOR XENOBIOTIC RESEARCH
UniversityofDundee, BiomedicalResearch Centre,Level5,NinewellsHospitaland Medical School,Dundee, DD1 9SY
Study Report Determinationof cyanideinsensitivPeaimitoyi-CoA oxidationinsamples from 3M
Environmental Laboratory - Covance Studies6329-183 and 6329-212 Study Number XR0108
Author: Approved:
t.M.Elcom,be C.R.EL
.e-
XR0108
PaLyeI of 3
Date:
I
Introduction
The purposeofthisstudywas toinvestigattehelevelsof PCOA oxidationinthefrozen liversamplesprovidedby 3M EnvironmentalLaboratoryfrom Covance studie6s329-183 and 6329-212.
2 TestSubstance
6329-183:PFOS, 20ppm, administeredby Covance untilweek 4. 6329-212:ETFOSE-OK 100 and 300ppm, administerebdy Covance untilweek 4.
3 TestingFacility
CentreforXenobioticResearch,UniversityofDundee, BiomedicalResearchCentre, Level5,NinewelisHospitaland MedicalSchool,Dundee,DDI 9SY, UK.
4 Homogenste Preparation
4.1 Reagents SET buffer0:.25M Sucrose,5mM EDTA and 20mM Tris-HCI,pH 7.4
4.2 Instruments Potter-Elvehjehmomogeniser
4.3 Method Frozen liversampleswere thawed on ice,blottedand weighed.A 25% homogenate was preparedinicecoldSET usinga Potter-Elvehjehmomogeniser(6passes).
5 Determinationof Cyanide-in.sensitPiavlemitoyi-CoOxidation The 25% homogenate liversampleswere dilutedI partin 10 partswithicecoldSET. These dilutedsampleswere usedtodeterminethePCOA activitfyo,llowingCXR SOP011.
6 StatisticAanlalysis A Student'Ts-Test(2-sidedw)as performedon theresults; statisticadlilfyferenftrom controlp<0.05; p<0.01; p<0.001.
XR0108
PaL7e2 of 3
7
Results
PCOA
Study /Group 6329-183/1 6329-183/5 6329-212/1 6329-212/4 6329-212/5
Oxidation(mnol NAD+
Dose (ppm) 0 20 0 100 300
reduced/min./mg. Week 4
(Mean S.D.)
3.533 0.453 6.634 1.093***
5.153 0.855 9.901 0.800***
6.317 1.290
t in)
% of Control 100.0 12.8 187.8 30.9*** 100.0 16.6 -192.1 15.5*** 122.6 25.0
PCOA Oxidation 6329-183(XR0108)
8-
7.
6. 5
4
T
3
2
0
0
20
Dose PFOS (ppm)
PCOA
12 io CL IE
6 +4
2 c
XR01OR
Oxidation
6329-212
(XR0108)
0
100
300
Dose ETFOSE-OH (ppm)
PaLye3 nf 3
PCOA Oxkiation
Study No.: XROIOO
Date:
10102199
6329-183
TreabrieFit sm* Number
Proteinsmwb Protein/" ASS (nigbo Vol4Ld) Incuvett* CHANGE(MIN
0
C92506H 6.092
so
0.152
Control C92524i4 7.ST7
so
0.197
C92526w 7.242
so
0.181
C92529M 7.635
50
0.191
C92S46H 6.BM
so
0.171
0.003021
0.003939 0.004776 0.003903 0.003966
PFOS
C92752W 6.00t
50
o.i5o
20ppa
C92759H 6.243
so
0.156
C92779W B.T73
50
0.189
C92793H 6.652
50
0.166
C92790H 6.425
50
0.161
0.00622
0.004SS2 0.007521 0.007921 0.006907
nffwff4AD#R.ed. nmol NAD+
hran
redtnkvnq
0.486
0.633 0.760 0.627 O.&IB
3.189
3.216 4.241 3.285 3.732
T.ODO
0.748 1.209 1.273 1.110
6.885
4.792 7.141 7.658 6.913
MEAN
SD
3.633 1: OA63
G.GU t 1.093
SE 0.203
OA$9
%c
SD
SE
180.00 :k 12.920 5.733
187.76 t 30.934 13239
'wry
25% homogenates 6329-183,diluted1/10
)RRELATION COEFF
RERCEPT
OPE
RANGE Y RANGE
0
0.031
0
0.033
0.025
0.081
0.025
0.065
0.05
0.113
0.05
0.119
0.075
0.166
0.075
0.161
0.1
0.205
0.1
0.200
0.15
0.283
0.15
0.283
0.99857408 0.0326 1.686
Y CALC 0.033 0.033 0.075 0.075 0.117 0.117 0.159 0.159 0.201 0.201 0.286 0.286
No C92506M C92524M C92526M C92529M C92546M C92752M C92759M C92779M C92793M C92798M
GIVEN YI 0.231 0.336 0.283 0.319 0.261 .0.231 0.245 0.243 0.256 0.244
Standard curve
1/51diin. XR0108
GIVEN Y2 0.237 0.250 0.261 0.251 0.256 0.231 0.233 0.270 0.249 0.246
protein mg/ml 6.09 7.88 7.24 7.63 6.83 6.00 6.24 6.77 6.65 6.42
0.30-
0.25-
0.20-
0.15 --
ci C5
0.10-
0.05I
0.00 0
0.025
0.05
0.075
0.1
Proteinmg/mi
0.125
0.15
PCOA OxiMion
Study No.:
XRDIOS
Date:
10102"
TF*Abnent
Smnple Nwim
0 Contml
C90719H C90725H C90732H C90753H C9077SM
ETFOSE-OH IOOPPE
C90939H C90942M C90961H C9096ram
ETFOSE-OH 30uppo
c9o964m cgioosw C91024m C91033M C91037H
6329-212
Protein SmWie ProteinhW ONOW) Vol.fLd) ineuvene
6.032
so
0.151
0.837
so
0.186
6.082
so
0.167
6.954
so
0.174
6.985
so
0.175
6.122
so
0.153
6.712
50
0.168
6.712
so
0.168
6.939
50
0.173
6.213
so
6.773
so
7.287
so
6.758
so
O.W2
so
0.155 0.169 0.182 0.169 0.167
ABS CHANGEJMIN
0.005402 0,005967 0.00463 0.006220 0.004354
0.00841S 0.01027 0.01063 0.01IS9
0.006927 0.007351 0.005309 0.007947 0.005391
nnwNAD+ Red. nmol NAD+
hvdn
redtnwdnig
0.860 0.9m 0.744 1.001 0.700
5.70D 5.782 4.456 5.759 4.009
1.353 1.651 1.709 l.a63
1.114 1.182 0.854 1.778 O.W7
8.839 9.BM 10.184 10.741
7.170 6.9w 4.685 7.563 5.188
MEAN 5.163
9.901 6.317
so 1 0.855
1 O.M t l@M
SE Om
OADO 0.577
%c
so
SE
100.00 1 16.507 TAn
192.14 t 15.615 7.758 122.68 2 26.030 il.i"
.0wry
25% homogenates 6329-212, diluted1/10
.'ORRELATION NTERCEPT )LOPE
X RANGE 0 0
0.025 0.025 0.05 0.05 0.075 0.075.
0.1 0.1 0.15 0.15
COEFF
Y RANGE 0.031 0.033 0.081 0.065 0.113 0.119 0.166 0.161 0.205 0.200 0.283 0.283
0.99857408 0.0326 1.686
Y -CALC 0.033 0.033 0.075 0.075 0.117 0.117 0.159 0.159 0.201 0.201 0.286 0.286
No C90719M C90725M C90732M C90753M C90775M C90939M C90942M C90961M C90966M C90984M C91005M C91024M C91033M C91037M
GIVEN Yl 0.229 0.255 0.252 0.263 0.266 0.235 0.253 0.256 0.261 0.244 0.253 0.291 0.261 0.251
1/51diin.
GIVEN Y2 0.235 0.249 0.255 0.262 0.261 0.235 0.256 0.253 0.263 0.232 0.260 0.256 0.251 0.256
XROIOB
protein mg/ml 6.03 6.64 6.68 6.95 6.98 6.12 6.71 6.71 6.94 6.21 6.77 7.29 6.76 6.68
Standard curve
0.30-
0.25-
0.200c
0.15-
ci 0.10-
0.05-
0.00 0
0.025
0.05
0.075
0.1
Proteinmg/mi
0.125
0.15