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