Document 7RD5JQOjazRYYrR35x2rK09x6
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CENTRE FOR XENOBIOTIC RESEARCH
University of Dundee, Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, Dundee, DD1 9SY
Study Report Determination of cyanide insensitive Paimitoyl-CoA oxidation in samples from 3M
Environmental Laboratory - Covance Studies 6329-183 and 6329-212 Study Number XR0108
Author: .
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Approved: .
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XR0108
Pape 1 o f3
Date:.\ ^ J . r-.J .? .
002401
1 Introduction
The purpose o f this study was to investigate the levels o f PCoA oxidation in the frozen liver samples provided by 3M Environmental Laboratory from Covance studies 6329-183 and 6329-212.
2 Test Substance
6329-183: PFOS, 20ppm, administered by Covance until week 4. 6329-212: EtFOSE-OH, 100 and 300ppm, administered by Covance until week 4.
3 Testing Facility
Centre for Xenobiotic Research, University of Dundee, Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK.
4 Homogenate Preparation
4.1 Reagents
SET buffer: 0.25M Sucrose, 5mM EDTA and 20mM Tris-HCl, pH 7.4
4.2 Instruments
Potter-Elvehjem homogeniser
4.3 Method
Frozen liver samples were thawed on ice, blotted and weighed. A 25% homogenate was prepared in ice cold SET using a Potter-Elvehjem homogeniser (6 passes).
5 Determination of Cyanide-insensitive Palmitoyl-Co Oxidation
The 25% homogenate liver samples were diluted 1 part in 10 parts with ice cold SET. These diluted samples were used to determine the PCoA activity, following CXR SOP011.
6 Statistical Analysis
A Student's T-Test (2-sided) was performed on the results; * = statistically different from control p<0.05; ** = p<0.01; *** = p<0.001.
XR0108
Pa ee 2 of 3
002402
7 Results
PCoA Oxidation (nmol NAD+ reduced/min./mg. Protein)
Week 4
Study / Group
Dose (ppm)
(M e a n t S.D.)
% o f Control
6329-183 /1
0
3.533 0.453
100.0 12.8
6329-183/5
20
6.634 1.093***
187.8 30.9***
6329-212/1
0
5.153 0.855
100.0 16.6
6329-212 / 4
100
9.901 0.800***
192.1 15.5***
6329-212/5
300
6.317 1.290
122.6 25.0
PCoA Oxidation 6329-183 (XR0108)
D ose P FO S (ppm) P C o A Oxidation 6329-212 (XR0 10 8 )
XR0108
0 1 0 0 300 Dose Et F OS E - OH (ppm)
Pane 3 o f 3
002403
PCoA Oxidation
study No.: XR0108
Date:
10/02/99
6329-183
Treatm ent
Sam ple Protein Sam ple Protein/m l
ABS nmolNAD* Red. nm ol N AO .
Number (mg/ml) Vol.(ul) in cuvette CHANGE/MIN
Anin red/min/mg
0 C925Q6M 6.092 s o 0.152
C o n tro l
C 92524M 7.877
so
0.197
C 92526M 7.242 5 0 0.181
C 92529M 7.635 5 0 0.191
C 92546M 6.833 5 0 0.171
PFOS 20ppm
C92752M C92759M C92779M C92793M C92798M
6.001
6.243 6.773 6.652 6.425
SO 0.150
5 0 0.156 5 0 0.169 5 0 0.166 50 0.161
0 .0 0 3 0 2 1 Q .Q 03939 0.0 0 4 7 7 6 0.0 0 3 9 0 3 0 .003966
0 .0 0 6 2 2 0 .004652 0 .0 0 7 5 2 1 0 .007921 0 .006907
0.486 0.633 0.768 0.627 0.638
1.000 0.748 1.209 1.273 1.110
3.189 3.216 4.241 3288 3.732
6.665 4.792 7.141 7.658 6.913
I
002404
MEAN 3.533
SO SE
%C
SD SE
0.453
0.203
100.00 12.820 5.733
T -V A LU E (VS CONTROL)
6.634 t 1.063
0.486
187.76 * 30.636 13.835
*6.860
w ry 25% homogenates 6329-183, diluted 1/10
JRRELATION CO EFF
fE R C E P T
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 Y1 0.231 0.336 0.283 0.319 0.261 0.231 0.245 0.243 0.256 0.244
1/51diln.
GIVEN Y2 0.237 0.250 0.261 0.251 0.256 0.231 0.233 0.270 0.249 0.246
Standard curve
XR0108
protein mg/ml 6.09 7.88 7.24 7.63 6.83 6.00 6.24 6.77 6.65 6.42
002405
PCoA Oxidation
Study No.: XR010S
Date:
10/02/99
6329-212
T reatm ent
Sample Protein Sam ple Protein/M Number (mgftnl) Vol.(ul) in cuvette
0 C90719M 6.032 50 0.151
C ontrol
C90725M 6.637
50
0.166
C90732M 6.682 50 0.167
C90753M 8.954 50 0.174
C9775M 6.985 SO 0.175
EtFOSE-OH
C90939M 6.122
50
0.153
IQOppm
C90942M 6.712
50
0.168
C90961M 6.712 50 0.168
C9966M 6.939 50 0.173
EtFOSE-OH
C90984M 6.213
50
0.155
300ppat
C91005M 6.773
50
0.169
C91024M 7.287 50 0.182
C91033M 6.758 50 0.169
C91Q37M 6.682 50 0.167
A B S nm oiNAD. Red. nm ol N A D .
CHANG E/MIN
/min
red/min/mg
0.005402 0.005967 0.00463 0.00622B 0.004354
0.868
0.959 0.744 1.001 0.700
5.760
5.782 4.456 5.759 4.009
0.008415 0.01027 0.01063 0.01159
1.353 1.651 1.709 1.863
8.839 9.839 10.184 10.741
0.006927
0.007351 0.005309 0.007947 0.005391
1.114
1.182 0.854
1.278 0.867
7.170 6.980 4.685
7.563 5.188
002406
MEAN
SD SE
%C
SD SE
6.163 0.855
0.392
100.00 t 16.697
7.422
T -V A LU E (VS CONTROL)
9.901 0.900
0.400 192.14 16.616 7.769
0.317 1.290
0.677
122.69 t 26.030 11.194
-8.631 -1.662
.owry
25% homogenates 6329-212, diluted 1/10
1/51diln.
CORRELATION CO EFF
NTERCEPT
>LOPE
X 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 C90719M C90725M C90732M C90753M C90775M C90939M C90942M C90961M C90966M C90984M C91005M C91024M C91033M C91037M
GIVEN Y1 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
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
Standard curve
XR0108
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
002407
