Document oez5Z7LZQ1bw82YyLB3Rxv22r

TNO-report "i'Vi -ri-:."; CONFIDENTIAL P-1 4 / Z 2 b 3 7 S & T he effect o f 3 Perfluo rinated A lkyl SULPHONATED ON CHOLESTEROL/BiLE ACID METABOLISM IN 15 % -FAT FED E3-LEIDEN T ransg enic m ice In Vivo and on fatty acid CONVERSION INTO CHOLESTEROL IN RAT hepatocytes In Vitro. Project / study num ber 031.10074 / 3M-02 TNO Quality o f Life TNO Biosciences Gaubius laboratory Zernikedreef 9, P.O. Box 2215 2301 CE Leiden The Netherlands Phone+31 7 1 5 1 8 1 2 0 9 Fax +31 71 518 1901 Authors: L.H. Cohen E.J. Pielerman E. Hoegee-de Nobel In assignment of: 3M, Medical Department Study monitor John L. Butenboff, Ph D.. CIH, DABT Corporate Scientist Medical Department 3M Center, Building 0220-02-E -02 S tP au l, Minnesota 55144-1000 USA Phone: +1.651.733.1962 Fax: +1.651.733.1773 E-mail: itbutenhoff@mmm.ccm Study director: Dr. Louis H. Cohen Status and Date: Final version: 04 Decem ber 2006 Previous versions: Draftl: 18 October 2006 Draft2: 30 October 2006 Draft3: 28 November 2006 Number of pages: 35 Authorization by Dr. P.H.A. Quax, 1 of 35 TNO-report of study 3M-Q2 Contents PFAS in ApoE3Leiden mice Final report Testing facility Contributors 1. Introduction 2. Study design 3. Results of in vivo experiment (ApoE3Leiden mice) 3.1. Body weight and food intake measurements 3.2. Plasma measurements 3.3. Measurements in metabolic cages 3.3.1 Food and drink intake 3.3.2 Moving (X) activity 3.3.3 0 2-consumption, C 0 2.production and respiratory exchange ratio (RER) 3.3.4 Energy expenditure 3.4 Fecal bile acid, sterol, and fatty acid measurements 3.5 Intestinal cholesterol absorption 3.6 Perigonadal fat tissue weight 3.7 Liver weight and hepatic lipid contents 3.8 Liver enzyme activities: HMG-CoA reductase, Cholesterol 7a-hydroxylase and sterol 27-hydroxylase 4. Results of in vitro experiment (rat hepatocytes) 5. Summary and conclusions 5.1. Effects of PFBS in ApoE3Leiden mice 5.2. Effects of PFHS in ApoE3Leiden mice 5.3. Effects of PFOS in ApoE3Leiden mice 04-12-2006 page 3 page 3 page 3 page 4 page 6 page 6 page 7 page 13 page 13 page 15 page 15 page 17 page 18 page 22 page 23 page 23 page 25 page 30 page 32 page 32 page 32 page 33 P-2 2 of 35 TNO-report of siudv 3M-02 Testing facility Business Unit Biosciences TNO Quality of Life Gaubius Laboratory Zernikedreef 9 P.O. Box 2215 2301 CE Leiden Phone: +31 71 5181209 PFAS in ApoESLeiden mice Final report fax: +31 71 5181901 Contributors Study director: Dr. Louis H. Cohen Phone: +31 71 5181469 E-mail: louis.cohen@tno.nl Experimental work: Ing. Christa van Thiel Ing. Bep Hoegee Ing. Anita van Nieuwkoop Ing. Angela Koudijs Ing. Wim van Duyvenvoorde Ing. Koen van der Mark Ing. Ronald van der Sluis Dr. Peter Voshol Ing. Elsbet Pieterman Advisor : Dr. Hans Princen Prof. Dr. Louis M. Havekes 0^-12-2006 P-3 1. INTRODUCTION 3M has observed that the compounds of interest have strong cholesterol lowering effects in animal studies. In order to obtain more insight into the possible mechanism of action the effects of the compounds on cholesterol and bile acid synthesis in primary culture of rat hepatocytes and on bile acid uptake in hamster ileum pieces in vitro have been investigated in a previously performed study. From this study it was concluded that the compounds possibly increased hepatic bile acid synthesis, however indirect effect on sterol synthesis might be involved as well. In the present study the in vivo effect of the test compounds on cholesterol and bile acid metabolism have been investigated in ApoE3Leiden transgenic mice. This animal model shows a humanlike lipoprotein pattern and, unlike wild type mice, is sensitive for lipid lowering drugs like statins. Furthermore, the plasma cholesterol levels can be elevated by adjustment of the diet. Additionally, in the present project information on the effects of the test compounds on hepatic cholesterol synthesis have been obtained via an in vitro study, in which the conversion of long and short chain fatty acids into cholesterol in the presence of the compounds have been investigated. 3 of 35 1 TNO-reoort of study 3M-02 PFAS in ApcEslLeiden mice Final report 04-12-2006 2. STUDY DESIGN The design for the performance of this study has been described in the final study protocol of 08-05-2006. Deviations from this protocol have been mentioned below. Approval for this study by the TNO Animal Experiment Committee has been obtained on 06-062006 under number DEC#2125. The final study design and the concomitant measurement points have been depicted in scheme 1. week of treatment -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 group 1 (n=10); 15 % -fa t (H F ) diet; control group 2 (n=10); HF + 0.01% PFBS group 3 (n=10); HF + 0.03% PFBS group 4 (n=10); HF + 0.006% PFHS group 5 (n=10); HF + 0.02% PFHS group 6 (n=10); HF + 0.003% P FO S group 7 (n=10); HF + 0.01% PFO S *- Body weight and food intake Plasma ketone bodies Plfi Ptdsjnasar Intestinal cholesterol uptake Fecal kpid excretion (sterols, BA, FA) Liver microsomes; Perigonadal fa t weight ( 4 = 4 mice/group 1, 3 , 4 , 6) (p = pooled per group) x X Scheme 1. Experimental design of the study After a 3 weeks run-in period on a semi synthetic high-fat (HF; 14% lard, 1% com oil and 0.25% cholesterol) diet, 75 male ApoE3Leiden mice (11-12 weeks of age at start of run-in) were randomized on basis of body weight, plasma cholesterol and triglycerides (measured after 4 h fasting). Then, the groups received respectively HF-diet (control group 1) and 0.01%, 0.03% of PFBS (groups 2 and 3), 0.006% PFHS (group 4) and 0.003% PFOS (group 6) in the HF- diet for the following 10 weeks. Groups 5 and 7 received respectively 0.02% PFHS and 0.01% PFOS in the diet, but after 2 weeks of treatment the mice lost on average 3 grams of bodyweight (see Fig. 3.1.1) and food intake has steadily decreased (see Fig. 3.1.2). For this reason it was decided to stop with these treatments and to return to the high fat control diet until the end of the study (this deviation of the protocol has been discussed with the study monitor by E-mail correspondence of 5 ,6 and 18 July 2006). A further deviation concerns the plasma ketone bodies determination. These compounds can only be detected in plasma in a proper way after an overnight fasting period (16 h). For this reason it could not be measured in the same plasma samples of weeks 0 and 8 (taken after 4h fasting) and therefore, it was decided to perform this determination in week -1 and week 6 of treatment (see E-mail correspondence 13 and 30 June 2006). At weeks 0, 1, 4 and 8 plasma cholesterol and triglyceride levels after 4h-fasting have been measured. In weeks 1 (7 days of treatment), 4 and 8, 15pl-plasma samples have been sent to the 3M company for the determination of the test compound concentrations. P-5 TNO-rsport of studv 3M-02_____________ PFAS in ApoE3Leiden mice_______________G4--12-2006 Final report As originally planned, at weeks 0, 4 and 8, lipoprotein profiles were analyzed (at week 0, the average values of three subpools were determined, at week 4 and 8, one pooled sample per group was analyzed). Since the week 4-lipoprotein patterns, especially of groups 5 and 7, were different from expected, extra lipoprotein profiles have been analyzed in treatment-week 6 (see E-mail correspondence with study monitor of 24 July 2006). The latter plasma samples were obtained after overnight fasting (necessary for ketone body determination), which in our experience does not give different results compared to lipoprotein profiles of 4h-fasting samples. In weeks 0, 4, 6 (extra measure points; see E-mail correspondence of 24 July 2006) and 8, plasma ALAT values were determined in pooled samples per group. In week 5-6 feces has been collected from 3 subgroups per treatment-group during 3 periods of 2-3 days, and fecal bile acids, fatty acids and sterol quantities and composition were determined. Initiated by the E-mail correspondence of 25 July 2006, feces has been collected in week 4 as well. However, up to now, we see no reason yet to investigate it further. In week 7, the metabolic activity of 4 animals per group from groups 1 , 3 , 4 and 6 have been analyzed in metabolic cages. The following parameters have been measured: food intake, drink intake, feeding bouts, total physical activity, 0 2-consumption and C 0 2-production. From these parameters the respiratory exchange ratio (RER) and 0 2-consumption-related body energy expenditure have been calculated. Because the food can only be supplied in powdered form in the metabolic cages, during this period (week 7 - 7.5), the other animals in this study also received powdered food. Hereafter all animals received food in chunks again. In week 8-9 intestinal cholesterol uptake has been determined. In week 10 the mice were sacrificed, plasma was collected by means of heart punction, the liver and perigonadal fat were taken out and weighed, two small pieces of the liver were quickly frozen in liquid nitrogen for determination of lipids and eventual RNA analysis, and microsomes and mitochondria have been isolated from the remaining part of the liver. HMG-CoA reductase and cholesterol-7a-hydroxylase activity have been determined in the microsomal preparations, sterol-27-hydroxylase activity has been measured in the mitochondrial fractions. 5 of 35 P-6 TNO-repoil o ` study 3M-02 PFAS in ApoE3Leideo mice Fina! report 04-12-2006 3. RESULTS OF IN VIVO EXPERIM ENT (APOE3LEIDEN MICE) The resulting individual primary data and subsequent calculated data are provided in a separate appendix to this report. The mean values per group are presented in the figures and tables below. Significance of differences was calculated using the non-parametric Mann-Whitney U test and the resulting p-values are given in the appendix. In the figures, a colored asterisk at a separate measure point indicate a statistically significant difference (p<0.05) between the value of the indicated group (of the same color) and that of the control group. In the tables, this difference has been indicated by a yellow background. 3.1. Body w eight and food intake measurements As shown in Figs. 3.1.1 and 3.1.2, the treatment with the high concentrations of PFHS and PFOS (groups 5 and 7) caused already from the start a remarkable decrease in body weight and food intake. After the change back to the control diet in week 2, these animals return to normal food intake, comparable to all other groups. Subsequently, group 5 animals gained weight again up to the control values, however, the body weight of the group 7 mice, although increasing again, stayed behind during the whole treatment period. The PFBS treated mice showed the same body weight increase as the controls. The Groups 4 and 6 (low concentrations of PFHS and PFOS) lost weight significantly in the last weeks of the study when compared to control animals. This is in line with the loss of fat tissue as shown below. Figure 3.1.1 Body weight Values are means S.D. --G roup 1:HF control --G roup 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS --G roup 4: HF + 0.006 % PFHS Group 5: HF + 0.02 % PFHS --G roup 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS Except for the deviations in food intake behavior of groups 5 and 7 (see above), all other animals ate about the same throughout the treatment period (Fig 3.1.2). 6 of 35 TNO-reoort of study 3M-02 Figure 3.1.2 Food intake Values are means S.D. PFAS in ApoE3Leiden mice Final report 04-12-2006 G rou p 1:H F co n trol - - G ro u p 4 : H F + 0.00 6% P F H S G ro u p 7: H F + 0.01 % P F O S ~ r G rou p 2: H F + 0.01 % P F B S - * G ro u p 5: H F + 0.0 2 % P F H S G ro u p 3: H F + 0.03 % P F B S G ro u p 6: H F + 0.003% P F O S p.7 3.2. Plasma measurements The following parameters have been determined in plasma in the course of the study: cholesterol (Fig. 3.2.1), triglycerides (Fig. 3.2.2), lipoprotein profiles (Fig 3.2.3 - 3.2.5), ALAT (Fig. 3.2.6) and ketone bodies (Fig. 3.2.7). Figure 3.2.1 Plasma cholesterol Values are means S.D. --G ro u p 1: HF control Group 2: HF + 0.01 % PFBS -sfe-Group 3: HF + 0.03 % PFBS --G ro u p 4: HF + 0.006 % PFHS ~ Group 5: HF + 0.02 % PFHS --G ro u p 6: HF + 0.003 % PFOS < Group 7: HF + 0.01 % PFOS As seen in other animal studies, PFHS and PFOS strongly decreased plasma cholesterol. The reversal to control diet in the groups 5 and 7 resulted in a return to control values for 7 of 35 // p.8 TNO-report of siuav 3M-02______________Pi~AS in ApoE3Leiden m;ce______________ 04-12-2006 Final report the PFOS group (7), however this return was delayed until week 8 for the PFHS group (5). The PFBS treatment did not decrease significantly the cholesterol levels. More or less the same results have been obtained for the plasma triglyceride (TG) levels. Figure 3.2.2 Plasma triglyceride Values are means S.D. --G ro u p 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03% PFBS --G ro u p 4: HF + 0.006 % PFHS -S -G ro up 5: HF + 0.02 % PFHS --G ro u p 6: HF + 0.003 % PFOS --G ro u p 7: HF + 0.01 % PFOS PFHS and PFOS decreased TG levels, but also the highest concentration of PFBS (group 3) after 8 weeks of treatment; the lowest concentration of PFBS, group 2, was not different from control. TG levels rose again in the group 7 after return to the control diet, however it remained lower than the control TG level in week 8. Group 5 TG-levels turn back to control levels after the shift to control diet. The differentiation into lipoproteins showed more differences than observed with the lipid levels alone. The patterns have been determined by measuring the cholesterol contents of the different column fractions, which indicate all lipoprotein fractions (Figs 3.2.3a, 3.2.4a, 3.2.5a), and by measuring the phospholipid content, which is major a constituent of the HDL fractions (Figs 3.2.3b, 3.2.4b, 3.2.5b). The PFBS treatment resulted in only minor changes in the lipoprotein fractions in time. VLDL seems to be increased a little, but IDL/LDL and HDL are somewhat lower than these fractions in the control patterns. The low concentration of PFHS (group 4) showed the appearance of a lipoprotein fraction which is located before the HDL faction and half overlapping with the IDL/LDL fraction. Since phospholipids are a major constituent, this fraction has been designated as large, less-dense HDL. It might arise from an impaired clearance of smaller HDL by the liver. This fraction appeared to increase upon longer treatment with PFHS. Concomitantly, VLDL and IDL are strongly decreased, which might indicate a decrease in production/secretion of apoB-containing lipoproteins by the liver. The 4-week pattern of the group 5 animals (high cone. PFHS; after 2 weeks on control diet again) resembled very much the group 4 pattern with a major increase in the large HDL. However, after 6 and 8 weeks, the lipoprotein profiles went back to the control patters with again more VLDL, IDL and small HDL. 8 of 35 P-9 TNO-report of study 3M-02_____________ PFAS in ApoE3Leiden mice______________ 04-12-20C6 Final report Figure 3.2.3a Pooled plasma lipoprotein (cholesterol) profiles ; PFBS treatment Week 0 Week 4 {- -C ro u p 1: HF co n tro l | I'-* --G roup 1: HF control - -G ro u p 2; HF 0.P1 % PFBS - -G ro u p 3 : HF 0.03 % PFBS | fraction Akta Week 6 [" -G ro u p 1: HF co n tro l --P--Group 2: H F*C ,01 % PF8S - -G ro u p 3: HF- 0.63% PFBS | fraction Akta Week 8 [- -G ro u p 1 H F control -*-G ro u p 2 : HF 0,01 % PFBS - -G ro u p 3: H f + O .M SPFBS | fraction Akta fraction Akta Figure 3.2.3b Pooled plasma lipoprotein (phospholipid) profiles ; PFBS treatment WeekO ['- -G ro u p 1: HF control"] Week 4 [- -G ro u p 1: HF control - -G ro u p 2: HF 0.01 % PFBS - -G ro u p : HF *C.Q3 % PFBS [ fraction Akta Week 6 p -G ro u p t : HF co n tro l - -C ro u p 2: HF 0J1 K PFBS - -G ro u p 3: HF OJO % PFBS | fraction Akta Week 8 (- " Croup 1: HF eontrot - -G ro u p fc H f 0.01 % PFBS - -G ro u p > W * 0,03% PFBS | TNO-reoorl of study 3M-02 P r AS in ApoE3Le:den mice Final report 04-12-2006 Figure 3.2.4a Pooled plasma lipoprotein (cholesterol) profiles ; PFHS treatment WeekO Week 4 {- -G ro u p 1: HF cantre! j - -G ro u p 1: HF co n tro l - -G ro u p P: HF 0.006 %PFHS Group S: HF . OSS % PPHS | Week 6 fraction Akta p o -G ro u p 1; HF com m i HP- Group <: HF > 0 . % P T H -*-6 r u p & H f 0 . % PFHS | Week 8 fraction Akta {- -G ro u p CMF co n tro l Group 4 : PFHS - 6foup 5; 0,02 % PFHS | fraction Akta fraction Akta Figure 3.2.4b Pooled plasma lipoprotein (phospholipid) profiles ; PFHS treatment Week 0 {- -G ro u p 1: HF co n tro l | Week 4 (- -G ro u p 1:H F co n tro l - -G ro u p 4: HF *0 8 0 6 % PFHS - Group S; HF 0.02 % PFHS { fraction Akta Week 6 p - G ro u p 1: HF control - -G ro u p 4: HF 8.006 % PFHS Group S: HF * M 2 % PFHS [ fraction Akta Week 8 j- -G ro u p 1: HP control - -G ro u p 4 HF *0.001% PFHS Group 8: HF 0,02 % PFHS [ fraction Akta /V TNO-report of study 3M-Q2 PFAS in ApoE3Leiden mice Final report 04-12-2006 Figure 3.2.5a WeekO Pooled plasma lipoprotein (cholesterol) profiles ; PFOS treatment [- -C ro u p 1: HF co n tro l | Week 4 Group V. BF control - -G fo u p t : HT 0.803 UPFO Group 7: HF O.Ot % PFOS [ fraction Akta Week 6 f^ G ro u p 1 :HF control - -G ro u p + .H P * 0,0ft3% RFOS G roup7: HF 0.01 % PfO S | fraction Akta Week 8 -p o o p 1: HF control -- -G roup I : HP 0.003 % PPOS Group 7: HF * 0.01 % PF08 I fraction Akta fraction Akta Figure 3.2.5b Pooled plasma lipoprotein (phospholipid) profiles ; PFOS treatment WeekO EEGroup 1: HF control j Week 4 [- -G ro u p 1: HF control - -G ro u p 6: HP 04)63 % PFOS > Group 7: HF 0.01 % PFQ8 | fraction Akta Week6 p > -G ro u p 1: HF control "G ro u p 8: HF * 0.003 % PFOS Gron 7; HF 0.81 % PFOS | fraction Akta Week 8 [- -G ro u p 1: HF control - -G ro u p *: HF 0 .0 % PFOS Group 7: HF 0.01 % PFO$ [ 11 of 35 /5 TNQ-reoort of study 3M-02 PFAS in ApoE3Leiden mice Final report 04-12-2006 The lipoprotein profiles of the PFOS treated animals are remarkable. The low concentration (group 6) showed major the large-HDL fraction which is steadily increasing during the treatment. ApoB-eontaining lipoproteins have been decreased. Similar as with PFHS, VLDL and IDL fractions have been decreased. Group 7, which showed a recovery of plasma TC/TG levels after 2 weeks control diet, has VLDL levels similar to the control group again, but showed low IDL and a very large "large"-HDL peak, which remained the same over the rest of the treatment period. A possible explanation could be that liver VLDL production has recovered (or is enhanced), but lipoprotein clearance is still impaired. It all might be the result of the long half-life of PFOS in the body. This explains also the gradually increase of the "!arge"-HDL peak in time in the low concentration PFOS group 6, which in week 8 has become as large as this peak in the group 7 plasma. Since the liver seems to be strongly involved in the mechanism of action of PFHS and PFOS, the liver enzyme alanine aminotransferase (ALAT) has been determined a few times more than originally planned (Fig. 3.2.6). Figure 3.2.6 Pooled plasma ALAT activity Group 1: HF control G roup 2: HF + 0.01 % PFBS G roup 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS G roup S: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS 500 week 0 week 4 week 6 (o/n fast) week 8 At all measure points ALAT has been increased in the PFOS-treated groups (groups 6 and 7), although from week 2 group 7 received the control diet again. PFHS increased ALAT as well (group 4), but here the return to control diet (group 5) caused a (partial) recovery of this activity. Although plasma ALAT activity is often used as a marker for liver damage, the increase in the plasma can be the result as well of an induction of the expression of the liver enzyme by the treatment, but can also result from the enlargement of the liver as seen in this study (see Fig. 3.7.1). PFBS had no or only a minor effect on plasma ALAT levels. 12 of 35 p. 13 TNQ-report of study SM-02______________PFAS in ApoE3Le;tien mice_ PiO.il report Q4-12-200G Since it was expected that the test compounds might have an effect on fatty acid metabolism, plasma ketone bodies have been determined before and after 6 weeks of treatment. As shown in Fig. 3.2.7, PFHS and, even more so, PFOS increased significantly the plasma ketone body levels. The two weeks of treatment with the highest dose, followed by 4 weeks of control diet (groups 5 and 7) still resulted in increased levels in week 6. PFBS did not significantly increase the ketone bodies. Figure 3.2.7 Plasma ketone bodies Values are means S.D. Group 1: HF control B G roup 2: HF + 0.01 % PFBS G ro u p 3: HF + 0.03 % PFBS G roup 4: HF + 0.006 % PFHS S Group 5: HF + 0.02 % PFHS G ro u p 6: HF + 0,003 % PFOS G ro u p 7: HF + 0.01 % PFOS So, PFHS and PFOS induced fatty acid oxidation, which is supported by the results of the analysis in metabolic cages performed in week 7 of treatment. 3.3. Measurements in metabolic cages After 7 weeks of treatment, 4 mice of the different treatment groups, the HF control group (group 1), the 0.03% PFBS group (group 3), the 0.006% PFHS group (group 4) and the 0.003% PFOS group (group 6) have been analyzed individually for 3 days in metabolic cages (after 1 day of acclimatization in the same cage). The following parameters have been measured: food and drink intake (Figs. 3.3.1.1 and 3.3.1.3), feeding size bouts (Fig. 3.3.1.2), moving activity (X activity; Fig. 3.3.2), 02-consumption and C 0 2-production (Figs. 3.3.3.1 and 3.3.3.2). From the latter parameters the respiratory exchange ratio (RER; Fig. 3.3.3.3), which gives information on the substrate that is used for energy generation, and additionally, the 0 2-consumption-related energy expenditure (Fig. 3.3.4) have been calculated. 3.3.1 Food and drink intake No significant differences have been observed between the groups in food intake, feeding bout size and drink intake, although the PFHS treated mice might have eaten somewhat more. 13 of 35 /7 TNO-renort of study 3M-02 PFAS in ApoE3Le;den mice Final report 04-12-2006 Figure 3.3.1.1 Food intake --Group 1: HF control Group 3: HF + 0.03 % PFBS --Group 4: HF + 0.006 % PFHS --Group 6: HF + 0.003 % PFOS Figure 3.3.1.2 Feeding bout size Time (h) Group 1: HF control --Group 3: HF + 0.03 % PFBS --Group 4: HF + 0.006 % PFHS --Group 6: HF + 0.003 % PFOS 0.08 Figure 3.3.1.3 0 12 Drink intake Time -Group 1: HF control --Group 3: HF + 0.03 % PFBS - Group 4: HF + 0.006 % PFHS --Group 6: HF + 0.003 % PFOS TNO-report o*' study 3M-02 P- AS in ApcF3Leids" mice Final report 04-12-2006 3.3.2 Moving (X) activity No significant differences have been observed between the groups in moving activity. Figure 3.3.2 X Activity --Group 1: HF control Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS G ro u p 6: HF + 0.003 % PFOS 3.3.3 (^-consumption, C 0 2-production and respiratory exchange ratio (RER) it is remarkable to notice that differences in these parameters have been observed between the groups, while using rather small groups. 0 2-consumption of the PFHS and PFOS treated animals was significantly increased, while no difference between control and PFBStreated mice have been found. Figure 3.3.3.1 0 2-consumption Group 1: HF control Group 3: HF + 0.03 % PFBS G r o u p 4: HF + 0.006 % PFHS G r o u p 6: HF + 0.003 % PFOS 5000 atOf>i 4000 E 3000 o> 2000 O > 1000 0 0 12 24 36 48 60 72 Time (h) 15 of 35 TNO-repori of study 3M-02 Pr AS in ApoESLeiden mice Final report 04-12-2006 C 0 2-production of the PFHS and PFOS groups was found to be increased as well, although in much less time points compared to the increase in 0 2-consumption. For this reason the RER for these groups are lower (significantly for PFOS, but not yet statistically significantly for PFHS) than the RER of the control and PFBS-treated group. This is an indication that fat is preferentially used for energy generation over the use of carbohydrates. This is in agreement with an increase in fatty acid oxidation as reflected by an increased plasma ketone body level in PFHS- and PFOS-treated mice and also for the decrease in fat tissue as found later in this study. Figure 3,,3.3.2 C 0 2-production Group 1: HF control Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS --Group 6 : HF + 0.003 % PFOS Time (h) Figure 3.3.3.3 Respiratory exchange ratio (RER) Group 1: HF control Group 3: HF + 0.03 % PFBS G r o u p 4: HF + 0.006 % PFHS G r o u p 6: HF + 0.003 % PFOS 16 of 35 TNQ-repoft of siudv 3M-02 PFAS in AooE3Leider. mies Final report 04-12-2006 3.3.4 Energy expenditure Congruent with these observations it has been calculated that the 02-consumptiondependent energy expenditure has been increased as well by the PFHS and PFOS treatm ent. Figure 3.3.4 Energy expenditure --G roup 1: HF control Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS G r o u p 6: HF + 0.003 % PFOS 0 12 24 36 48 60 72 Time (h) The differences observed in the metabolic cage analysis might be more pronounced by using larger groups in eventual future studies (preferable n = 8). TNO-report o;' study 3M-C2 PFAS in ApoE3Leiden mice Final report 04-12-2006 3.4. Fecaf bile acid, sterol and fatty acid measurements In week 6 of treatment, feces have been collected and its lipid and bile acid contents determ ined. Fecal bile acids have been decreased by PFHS (0.006%; group 4) and even more by PFOS (0.003%). Although groups 5 and 7 were back on control diet for 4 weeks, both these compounds have still kept the bile acid excretion below the control values. PFBS-treatment seems to have increased the fecal bile acid excretion, which was even significantly different from the control group for group 2. Figure 3.4.1 Bile acid excretion Values are means S.D. Group 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS Group 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS S: OO H a. 1 Table 3.4.1 Fecal bile acid composition group 1 % of bile adds | fa-murichoate|[deoxycholatelI cholate |I lithocholae IG-muricholatelIw-muricholatelI hyodeo/urso I 6.1 2.8 14.7 5.7 7.9 2.7 9.6 4.1 20.4 5.0 39.8 8.8 1.5 0.8 oup 2. HF + 0.0 : % PFBS 4.8 1.2 12.4 2.1 8.4 1.2 10.7 2.4 19.6 7.2 42.2 t 4.4 1.9 1.0 4.2 1.3 12.8 1.2 7.6 2.3 13.1 4.5 19.9 4.1 40.9 3.4 1.4 0.9 3ip HF+tjO'6 PFHS 7.1 2.3 11.3 1.8 10.2 4.2 10.9 3.3 17.2 4.5 40.9 3.8 2.3 2.2 T:T'.' - " . - 5.3 0.8 13.9 + 2.4 8.5 3.7 16.0 5.2 16.8 5.7 36.6 7.0 v a # ;u f C i~ir ' ' 0C 3 ? F 0 3 6.8 2.0 15.9 5.5 14.3 4;0 11.3 3.2 12.5 3.5 37.0 t 5.7 2.3 1.4 -- r is ia :b271 22.1 5 5 16.4 4.9 6.1 2.4 16.8 4.0 24.2 4.01 3.5 3.5 The latter increase with PFBS did not change the fecal bile acid composition. Concomitantly with the decrease by PFHS and PFOS, a change in composition of excreted bile acids has been observed as well, which can result from a change in bile acid synthesis pathways in the liver, but also from a change in intestinal bile acid conversion. The latter might also happen in the liver after reabsorption and return to the liver. 0- 18 of 35 TNO-repon of study 3M-02 PFAS in ApoE3Le:den mice Final report 04-12-2006 Only in group 4 (PFHS) was fecal neutral sterol excretion significantly increased. Fecal neutral sterols, which are primarily composed of cholesterol, are determined by food intake, intestinal absorption and liver production. Since the diet used contained 0.25% cholesterol, food intake plays a major role. This is more or less the same in all groups. Since intestinal cholesterol absorption is also the same or increased (see Fig.3.5.1), most probably the significantly increased fecal sterol excretion in group 4 (PFHS) is the result of an increased hepatic cholesterol synthesis. This is in agreement with the increased HMG-CoA reductase activity observed (see Fig. 3.8.2). Although no differences have been seen in the quantity of excreted plant sterols, which originate from the diet, the composition was slightly changed in the PFHS and PFOS groups. It might be the result of a change in intestinal absorption/resecretion. Figure 3.4.2 Sterol excretion Values are means S.D. Group 1: HF control G roup 2: HF + 0.01 % PFBS Group 3: H F + 0.03 %PFBS Group 4: HF + 0.006 % PFHS G roup 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS 100 (0 eO 3(p to 8> o X 3o E 0) E ta +4>) "n) (0 O (U Tf3-- o f-- O E 3. 80 60 40 20 0 neutral sterols phytosterols Table 3.4.2a Fecal neutral sterol composition % of neutral sterols coprostanol coprostanon cholesterol cholestanol lathosterol 0.0 0.0 0.0 0.0 94.4 0.4 2.7 0.2 2.9 0.3 0.0 0.0 0.0 0.0 95,0 , 0.6 2.3 0.8 2.7 0.3 0.0 0.0 0.0 0.0 94.6 0.7 2.7 0.4 2.7 0.6 0.0 0.0 0.0 0.0 95.5 0.3 2.4 0.3 2.1 0,1 0.0 0.0 0.0 0.0 94.4 1.4 3.1 1.5 Z s * m m 0.0 0.0 0.0 0.0 94.3 0.7 0 .6 - m m >fsQ|2 0.0 0.0 0.0 0.0 93.7 0.7 4.6 ' f m 19 of 35 TNO-repprt of study 3M-02 FFAS in AooE3Leiden mice Final report 04-12-2006 Table 3.4.2b Fecal phytosterol composition group ca m p este ro l 26.2 0.8 G roup 2: HF + 0.01 ;'o p FBS 26.2 1.0 26.5 0.7 Group 4; HF + 0.006 % PFHS 1.1 26.6 1.1 Group 6. HF + 0 0Q3 .J ! 1tV/}W*> f W M l - S 8 w % of phytosterols stigm asterol 3-sitosterol 8.2 1.1 65.6 1.1 8.1 1.7 65.7 1.5 7.7 1.9 65.9 1.7 4.3 2.0 6 7 3 * :;4 .3 -' 6.5 2.7 66.9 2.1 3.6 0.5 #68S8lllfc3'O iL 5.0 3.5 '672lr H 2.9 6-sitostanol 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Figure 3.4.3 Fatty acid excretion Values are means S.D. Group 1: HF control iG ro u p 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS G roup 5: HF + 0.02 % PFHS G roup 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS Table 3.4.3 Fecal fatty acid composition C14:0 2.0 0.4 1.9 0.5 1.8 0.3 1.7 0.3 2.0 0.5 1.8 0.4 2.2 0.4 C16:0 46.0 3.9 44.1 t 2.7 45.1 2.3 43.6 1.9 45.5 3.2 45.6 2.9 48.2 2.4 % of fatty adds C16:1 C18 0.38 0.04 46.0 3.7 0.35 0.13 47.5 2.9 0.39 0.04 47.0 2.4 0.35 0.02 48.6 1.9 0.39 0.04 46.2 3.2 0.34 0.03 46.6 3.0 0.35 0.03 43.5 2.7 C18:1 4.8 0.5 5.2 0.6 4.8 0.3 4.8 0.4 5.1 0.7 4.8 0.3 4.9 0.5 C18:2 0.30 0.04 0.30 t 0.05 0.31 0.02 0.31 0.03 0.29 0.03 029 0.03 038 t o;oo C18:3 0.55 0.21 0.62 0.10 0.58 0.08 0.60 0.07 0.57 0.10 0.56 0.09 0.46 i : 006 Fecal fatty acids are derived from the fat from food intake (TG) and lipids produced by the liver (PL). Most of the groups (some have large SD) showed no significant difference from control. Group 4 (PFHS) had significantly increased fecal fatty acids, whereas group 7 (2 weeks PFOS and 4 weeks control) showed a strong decrease, with a concomitant small change in fatty acid composition. 20 of 35 TNO-report of study 3M-02 PFAS in ApoE3Leiden mice Final report 04-12-2C06 From the difference between the total fatty acid (TG) intake per subgroup and total fatty acid excreted in the feces, the fatty acid balance has been calculated. The values are expressed as the difference between total fatty acid input and output in pmol fatty acid per 100 gram mouse per day. As shown in Fig. 3.4.5, all groups have more or less the same fat intake, except that in group 7 this is significantly higher. This is of interest in the light of the loss of fat tissue in the same group (see Fig. 3.6.1). Figure 3.4.5 Fatty acid balance Values are means S.D. G ro u p 1: H F control B G ro u p 2: H F + 0.01 % P F B S G ro u p 4: H F + 0.006 % P F H S B G ro u p 5: H F + 0.02 % P F H S B G ro u p 7: H F + 0.01 % P F O S G rou p 3: H F + 0.03 % P F B S G ro u p 6: H F + 0.003 % P F O S 8000 g g 6000 C10 3O s5> E~ 5(o0 U1) 4000 i2 2000 E 0 21 of 35 TNIO-reuort of study 3M-02 F~AS in ApoE3Leiden mice Final report 04-12-2006 3.5. Intestinal cholesterol absorption The intestinal cholesterol absorption was assessed by the fecal dual-isotope method. In week 8 all mice were individually housed and received an oral dose of 200 pi o f olive oil, containing 1 pCi [14C]-cholesterol and 1 pCi [3H]-sitostanol, per mouse. Bodyweight and food intake were monitored, and feces were collected for the following 4 days. Feces were homogenized, pooled per mouse over the 4-day period, and dissolved in ethanolic potassium (3mol/l, 60% ethanol). Radioactivity was determined in the fecal samples to assess the ratio of [14C]-labeled cholesterol and [3H]-labeled sitostanol. Sitostanol was used as the reference compound since it is known to be poorly absorbed (< 3 %) in mice. The formula used to calculate cholesterol absorption was: % cholesterol absorption = ([14CJ/[3H]-dosing mixture - [14C]/[3H]-feces) / [14C]/[3H]-dosing mixture * 100. Figure 3.5.1 Intestinal cholesterol absorption Values are means S.D. Group 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS Group 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS 60 week 8-9 Both PFOS-treated groups 6 and 7 have increased intestinal cholesterol absorption. Since all groups have about the same cholesterol intake via the food and the same fecal cholesterol output (except group 4), this increase must be caused by an increased hepatic cholesterol synthesis. Indeed, the increased HMG-CoA reductase activity in these groups (see Fig. 3.8.2b) suggests an increased hepatic cholesterol synthesis. Although not significantly, the intestinal cholesterol absorption of group 4 (PFHS) seems to be slightly increased as well, which, beside additionally an increased fecal excretion, might be explained as well by an increased hepatic cholesterol synthesis. 22 of 35 TNO-renort of study 3M-02 PFAS in AooE3Leiden mice Final report 3.6. Perigonadal fat tissue weight Figure 3.6.1 Perigonadal fat weight Values are means S.D. 04-12-2006 G roup 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS G roup 4: HF + 0.006 % PFHS Group 5: HF + 0.02 % PFHS G ro u p 6: HF + 0.003 % PFOS G roup 7: HF + 0.01 % PFOS 1.2 PFHS (group 4) and PFOS (groups 6 and 7) strong reduced perigonadal fat tissue. Although not measured, this might reflect a decrease in total body fat tissue. This notion is supported by the decrease in body weight. Plasma leptin levels might be measured, which should be found to be decreased as a reflection of decreased fat tissue. The reduction in perigonadal fat tissue was not seen in the PFBS-treated groups and the PFHS group 5. 3.7. Liver weight and hepatic lipid contents Livers are strongly increased by PFOS (groups 6 and 7) and PFHS (group 4). The return to control diet o f the group 5 PFHS-treated animals caused a return to only slightly but still significantly enlarged livers. When expressed as % of total body weight (Fig. 3.7.1b), the PFBS-treated group 3 also had slightly but significantly enlarged livers when compared to controls. As shown in Fig. 3.7.2, the lipids, triglycerides and cholesterylesters, have been increased in the livers of PFOS-treated groups 6 and 7. In the other livers these contents were comparable with that of the control mice. Free cholesterol contents have not been changed by any of the compounds. TNO-repcrt of study 3M-02 PFAS in ApoE3Leiden mice Final report 04-12-2006 Figure 3.7.1 a Liver weight Values are means + S.D. Group 1: HF control G roup 2: HF + 0.01 % PFBS BGroup 3: HF + 0.03 % PFBS G roup 4: HF + 0.006 % PFHS Group 5: HF + 0.02 % PFHS G roup 6: HF + 0.003 % PFOS G roup 7: HF + 0.01 % PFOS week 10 Figure 3.7.1 b Liver weight as % of body weight Values are means S.D. G roup 1: HF control Group 2: HF + 0.01 % PFBS G roup 3: HF + 0.03 % PFBS G roup 4: HF + 0.006 % PFHS G roup 5: HF + 0.02 % PFHS G roup 6: HF + 0.003 % PFOS G roup 7: HF + 0.01 % PFOS 24 of 35 TNO-report of study 3M-02 PFAS in ApoE3leiden ^lice Final report Figure 3.7.2 Lipid contents liver Values are means S.D. 04-12-2006 Group 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4; HF + 0.006 % PFHS G roup 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS | 300 3v Q. 250 O> 301 2 0 0 - 0> 1 5 0 - iC 1 0 0 - c oo 5 0 -a a 0- Free cholesterol Triglycerides Cholesterolester 3.8. Liver enzyme activities: HMG-CoA reductase, cholesterol 7a-hydroxylase and sterol 27-hydroxylase. Immediately after sacrifice, livers have been removed and microsomes and mitochondria have been isolated from the liver tissue. Thereafter, HMG-CoA reductase and cholesterol-7ahydroxylase activity have been determined in the microsomal preparations and sterol-27hydroxylase activity in the mitochondria. The yield of microsomes per g liver is about the same in all groups (Fig. 3.8.1) Fig. 3.8.1 Microsomal preparations Group 1: HF control iG ro u p 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS Group 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS m Group 7: HF + 0.01 % PFOS TNO-report of siudv 3M-C2 PFAS in ApoE3Leiden mice Final report 04-12-2006 Although HMG-CoA reductase activity was rather low in all preparations and therefore SD's are rather large, PFHS and PFOS seem to have increased this activity. This is significantly different from control for PFHS group 4. Since the yield of the microsomes per g liver is more or less the same and large differences between the liver weights have been observed, the activity has been recalculated and expressed as total activity per liver (Fig. 3.8.2b). This might indicate that large differences in total hepatic cholesterol synthesis between the groups exist. Total HMGCoA reductase activity per liver has been increased significantly in groups 4 (PFHS), 6 (PFOS) and 7 (PFOS). Figure 3.8.2a HMG-CoA reductase activity in liver microsomes Values are means S.D. Group 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF 0.006 % PFHS Group 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS 20 Figure 3.8.2b Total HMG-CoA reductase activity per liver Values are means S.D. Group 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF * 0.006 % PFHS G roup 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS 3 Group 7: HF + 0.01 % PFOS > 1000 > O ^ Ec= 800 I o E 600 oe o. o | 400 0~ 1 x rMa O 200 * 26 of 35 TNO-report of study 3 M - Q 2 _________PFAS in ApoE3Leiden mice_______________ 04-12-2006 Final report Figure 3.8.3a Cholesterol 7a-hydroxylase activity in liver microsomes Values are means S.D. Group 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS Group 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS 8 > p. 27 Figure 3.8.3b Total cholesterol 7a-hydroxylase activity per liver Values are means S.D. Group 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS G roup 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS * 1 20-r O(0 100 - <0 (0 <0 S2 >O *E & >p m>J= > aCL - CO o I- One of the rate limiting enzymes of bile acid synthesis, the microsomal enzyme cholesterol 7ahydroxylase, has found to be decreased by PFHS (group 4) and by PFOS (groups 6 and 7). The activity in the other groups did not differ significantly from the control value (although that of group 2 seems to be somewhat enhanced). If the activity is expressed as total activity per liver a significant decrease by PFHS (group 4) and PFOS (group 6) remained, suggesting that total hepatic bile acid production might be reduced as well. 3/ 27 of 35 TNO-report of study 3M-02 F -^ S in ApoE3Leiden mice Final report 04-12-2006 The other regulatory enzyme of bile acid synthesis, sterol 27- hydroxylase was measured in the mitochondrial preparations. It was remarkable that the yield of mitochondria (mg/g liver; Fig. 3.8.4) seemed to be increased in the PFHS-treated and PFOS-treated groups, suggesting that these compounds increased the number of mitochondria. This might be related with the observed increase of fatty acid oxidation. Figure 3.8.4 Liver mitochondrial preparations Values are means S.D. Group 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS Group 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS As expresses per mg of mitochondrial protein sterol 27-hydroxylase was found to be decreased by PFHS (group 4) and PFOS (groups 6 and 7) as well. Figure 3.8.5a sterol 27-hydroxylase activity in liver mitochondria Values are means S.D. Group 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS Group 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS 4 Group 7: HF + 0.01 % PFOS 20 > = 15 W 28 of 35 TNO-report of study 3M-02 PFAS in ApoE3Leiden mice Final report 04-12-2006 However, taking into account of the differences in mitochondrial yields and in liver weights and expressing the activity per liver (Fig. 3.8.5b), the total sterol 27-hydroxylase activity was found to be increased in the same groups. Figure 3.8.5b Total sterol 27-hydroxylase activity p er liver Values are means S.D. G roup 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS G rou p 4: HF + 0.006 % PFHS B Group 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS 800 33 29 of 35 TNO-report of study 3M-02 PFAS in ApoE3Leiden mice Final report 04-12-2006 4. RESULTS IN VITRO EXPERIMENT {RAT HEPATOCYTES) From the first in vitro experiments performed in project 3M-1, it was suggested that the PFAS compounds might influence the internal acetate pool, which is the substrate for hepatic cholesterol synthesis. Since fatty acid catabolism is a source for acetate, the effect of the test compounds on the incorporation of different sized radiolabelled fatty acids into cholesterol has been investigated in the present project. As a positive control, a strong and specific inhibitor of cholesterol synthesis, rosuvastatin has been taken along in each separate experiment. Identical experiments have been performed 3 - 4 times (as mentioned at the figures) Figure 4.1 Incorporation of [14C]-acetate into cholesterol in rat hepatocytes in vitro Values are means S.D. (n = 4). [14C]-Acetate incorporation Figure 4.2 Incorporation of [14Cl-butyrate into cholestrol in rat hepatocytes in vitro Values are means S.D. (n = 4). [14C]-Butyrate incorporation 30 of 35 p. 31 TNO-reoort of studv 3M-02______________PFAS in ApoE3Leiden mice______________ 04-12-2006 Final re n d Rosuvastatin strongly inhibited the incorporation o f the short chain fatty acids acetate and butyrate into cholesterol (Figs. 4.1 and 4.2). Although there is a large variation in the data obtained for the test compounds in the individual experiments, on average there seems to be hardly an effect o f these compounds on cholesterol synthesis from acetate and butyrate. The incorporation of the longer fatty acid [14C]-laurate into cholesterol is also comparable to control values in the presence of the PFAS's (Fig. 4.3); however, there might be a slight concentration dependent decreasing effect of PFHS and PFOS. Rosuvastatin is again a much stronger inhibitor if the cholesterol synthesis from [14C]-laurate and also from [14C]palmitate (Fig. 4.4). Figure 4.3 Incorporation of [14C]-laurate into cholesterol in rat hepatocytes in vitro Values are means S.D. (n = 3). [14C]-Laurate incorporation Figure 4.4 Incorporation of [14C]-palmitate into cholestrol in rat hepatocytes in vitro Values are means + S.D. (n = 3). [14C]-Palmitate incorporation compound concentration (pmol/l) 3S TNO-rsport of study 3M-02 PFAS in AooE3Lfeiden mice Final report 04-12-2C06 The incorporation of palmitate, the longest fatty acid used in this study, into cholesterol was influenced in a somewhat remarkable way. In the presence of the different concentrations of the test compounds on average all values are lower than control values. But there is no obvious concentration dependency in this decrease. So, it is difficult to understand that all three compounds are influencing palmitate uptake/transport/degradation in the same concentration independent way. Since the solubility of this longer fatty acid in the medium is depending on several factors and the radiolabelled compound was present in tracer amounts, it might be that the test compounds have influenced its solubility and hence its availability for uptake in the cells. So, all together the test compounds have either no or only slight effects on the incorporation of short and longer radiolabelled fatty acids into cholesterol in rat hepatocytes under the conditions used. 5. SUMMARY AND CONCLUSIONS Although the test compounds have sim ilar chemical structures, they show differences in their biological behavior. For that reason they are discussed here separately. 5.1. Effects of PFBS in ApoE3Leiden mice Both PFBS-treated groups 2 (0.01%) and 3 (0.03%) showed no or small differences with the control group 1. No differences were found in body weight, food intake, plasma ALAT and ketone bodies, metabolic parameters as measured in metabolic cages, fecal sterol and fatty acid excretion, intestinal cholesterol absorption, fat tissue weight and liver weight, lipid contents and enzyme activities. Small differences have been seen in plasma lipid levels: a slight decrease in cholesterol and triglycerides, which was reflected in minor changes of the lipoprotein patterns; VLDL seemed to be increased a little, but IDL/LDL and HDL were somewhat lower than these fractions in the control patterns. Interesting is the small but significant increase in fecal bile acid excretion in the group 2 mice. These mice showed also an increased hepatic cholesterol 7a-hydroxylase activity which might support the preceding notion, but because of the large standard deviation in this value it was not statistically significant. The suggested small increase in bile acid synthesis might be related to the slightly decreased plasma lipid levels. 5.2. Effects of PFHS in ApoE3Leiden mice The group 4 animals have been treated with 0.006% of PFHS throughout the 10 weeks of the study period. The higher dosed group 5 (0.02% of PFHS) showed already from the start deviations in food intake, which led to a large loss of body weight in the 2ndweek of treatment. Therefore, from week 2, these mice were put back on control diet and remained on that throughout the rest of the treatment period. This resulted in a recovery of these animals, which, after a shorter or longer period, showed similar values as measured in the control group; however, some differences remained. The PFHS-treatment in group 4 had the following effects: Although food intake did not change, the body weight steadily decreased in the last 3 weeks. This can be explained by the prominent loss in fat tissue (measured as perigonadal fat mass; it might be better to measure plasma leptin as a marker for whole body fat). The latter effect was probably been caused by increased fatty acid oxidation, what can be concluded from increased plasma ketone bodies and a preference for fat oxidation over carbohydrate oxidation, as measured in the metabolic cage analysis (tendency towards decreased RER; group size was rather small). Additionally, 02consumption and concomitant energy expenditure was enhanced in these animals. Another indication for increased oxidation is the increased quantity o f mitochondria, 32 of 35 p. 33 TNO-reoort of study 3M-02 PFAS in AooE3Leidan mice Final report 04-12-2006 isolated from the significantly enlarged livers. For further support of increased fatty acid oxidation, hepatic activity and/or mRNA's of enzymes involved in fatty acid oxidation, e.g. Acyl-CoA oxidase, should be measured. Plasma cholesterol and triglycerides were strongly decreased by PFHS throughout the treatment period, which was paralleled by a change in lipoprotein profile: ApoB-containing fractions (VLDL, IDL and LDL) were decreased; whereas, a shift in HDL towards larger, less dense particles was observed. These phenomena can be explained by a decrease in hepatic VLDL production and decreased uptake/conversion of HDL by the liver. In summary, liver functions have been changed, which is further supported by an increase in liver size/weight (liver lipid contents did not change), increased plasma ALAT values, increased HMG-CoA reductase activity and decreased cholesterol 7a-hydroxylase, which are rate lim iting enzymes in cholesterol and bile acid syntheses, respectively. Mitochondrial sterol 27-hydroxylase activity was changed as well. Changes in hepatic cholesterol/bile acid synthesis might have influenced the fecal lipid excretion and intestinal cholesterol absorption. PFHS increased fecal cholesterol and fatty acid excretion and decreased fecal bile acid excretion. On the other hand, changes in intestinal uptake/conversion and small changes in food intake cannot be ruled out in this respect. There is a trend observed towards increased intestinal cholesterol absorption by PFHS. Several of the liver effects o f PFHS are sim ilar to those of a PPARa agonist, e.g., like fibrates. For this reason it might be of use to further investigate the expression of other genes which are regulated by PPARa. As known, such compounds lower plasma lipids and decrease fat tissue as well, but their effect on intestinal functions is not known. As mentioned above, in the group 5 animals with a 2 weeks-treatment at the highest dose of PFHS, followed by 8 weeks on control diet again, the effects by PFHS as seen in group 4 disappeared, and the parameters measured returned more or less rapidly to control values. That was not the case in all instances the case: The livers remained a little enlarged and plasma ALAT stayed somewhat higher than control. Ketone bodies remained significantly increased up to week 6, and fecal bile acid excretion was still reduced. So it might be that during this period PFHS was not yet totally eliminated from the mice. The values of plasma PFHS concentrations, which will be determined by 3Mcompany, w ill give inside in this matter. 5.3. Effects of PFOS in ApoE3Leiden mice The effects o f PFOS (group 6; 0.003%) on the parameters measured in ApoE3Leiden mice in this study were very sim ilar to those of PFHS, however, the effects were in several instances more pronounced. That means that group 6 mice showed: Loss of (perigonadal) fat tissue, strongly increased plasma ketone bodies and a decreased RER, which showed a preference for fat oxidation over carbohydrate oxidation, and a concomitantly increased energy expenditure; all indications for an increased fatty acid oxidation. Additionally, increased quantities of mitochondria, have been isolated from the livers of these animals. Plasma cholesterol and triglycerides were strongly decreased, with a similar change in lipoprotein profile as observed with PFHS. ApoB-containing fractions were decreased; whereas, a shift in HDL towards larger, less dense particles was observed. Interestingly, the "large"-HDL peak increased in time, suggesting that PFOS is accumulating during the treatment period in this group. 33 of 35 TNQ-report of siudv 3M-02 PFAS in AooE3Leiden mice Final report 04-12-2006 Furthermore, the liver weight increased almost 3 times with concomitantly increased plasma ALAT levels. Liver lipids, triglycerides and cholesteryl esters were significantly increased. Hepatic microsomal HMG-CoA reductase activity was increased and cholesterol 7a-hydroxylase and mitochondrial sterol 27-hydroxylase activities were decreased. PFOS did not change fecal cholesterol and fatty acid excretion, and decreased fecal bile acid excretion and composition. On the other hand, the ratio of different neutral sterols in the feces has been changed, and PFOS increased significantly intestinal cholesterol absorption. The latter might be a reflection of increased hepatic cholesterol synthesis, but other changes in intestinal cholesterol metabolism cannot be ruled out. As with PFHS, the PFOS mechanism of action might be (partly) understood as working like a PPARa agonist. This notion needs further investigation. The observation in group 7 (2 weeks of a higher dose of PFOS followed by 10 weeks on control diet) are almost identical to those in group 6. And although cholesterol levels seem to recover after 4 weeks, the lipoprotein pattern continued to contain the same large "large"-HDL fraction. These all are indications that PFOS concentrations in the mice remained at a sim ilar level throughout the whole period and that PFOS is not eliminated readily from the body. PFOS is known to have a long elimination half-life in rats, monkeys, and humans, and determination of the plasma PFOS-concentrations at the end of the treatment period, should confirm this presumption. The only remarkable difference between group 6 and 7 is found in the fecal excretion data. Much less fatty acids have been found in the feces o f group 7, which also possess a slightly different composition of fatty acids. So, intestinal fatty acid absorption (and/or conversion) seemed to be increased when PFOS is withdrawn from the food. Table 5.1 gives an overview of the effects of PFBS, PFHS and PFOS treatment on the different measured parameters. 34 of 35 TNO-renort of study 3M-02 PFAS in ApoE3L&id&n mice Final 'eport 04-12-2006 Appendix to TNO-report of 3M-Q2 Final version CONFIDENTIAL 04-12-2006 APPENDIX TO TNO-REPORT OF STUDY 3M-02 Project / study number: 031.10074 / 3M -02 TNO Quality of Life TNO Biosciences Gaubius laboratory Zemikedreef 9, P.O. 80x2215 2301 CE Leiden The Netherlands Phone +31 71 5181209 Fax +31 71 518 1901 Authors: L.H. Cohen E J Pieterman E Hoegee-de Nobel In assignment of. 3M, Medical Department Study monitor John L. Butenhoff, Ph.D.. CIH, DABT Corporate Scientist Medical Department 3M Center, Building 0220-02-E -02 St.Paul, Minnesota 55144-1000 USA Phone: +1.651.733.1962 Fax: +1.651.733.1773 E-mail: ilbutenhoff@mmm.com Study director: Dr. Louis H. Cohen Status and Date: Final version: 04 December 2006 Previous versions: Draftl: 18 October 2006 Draft2:30 October 2006 Number of pages: 172 Authorization by Dr. P.H.A. Quax, Technology manager o f Vascular and Metabolic Diseases: Signature and date: 1 \ Page A1 of A172 p. 37 Appendix to TNO-report of 3M-02____________ Final version_________________________ 04-12-2006 Contents Contents page A02 A. Code E3L mice before and after matching A03 B. Bodyweight and food intake measurements B1. Individual bodyweights B2. Food intake measurements A04 A04 A07 C. Plasma measurements C1. Plasma cholesterol measurements C2. Plasma triglyceride measurements C3. Pooled lipoprotein profile measurements C4. Pooled plasma ALAT measurements C5. Plasma ketone bodies measurements A13 A13 A15 A17 A24 A25 D. Measurements in metabolic cages D1. Code mice and metabolic cages D2. Average bodyweight mice in metabolic cages D3. Food and drink intake measurements D4. X activity measurements D5. 0 2 consumption,C02 production and respiratory exchange ratio measurements D6. Energy expenditure A28 A28 A28 A29 A38 A41 A50 E. Fecal bile acid, sterol and fatty acid measurements A53 E1. Fecal weight measurements, bodyweight and food intake Measurements A53 E2. Fecal bile acid measurements A60 E3. Fecal sterol measurements A83 E4. Fecal fatty acid measurements A107 F. Intestinal cholesterol absorption A122 G. Liver weight, perigonadal fat weight and lipid contents liver G1. Liver weight and perigonadal fat weight G2. Lipid contents liver A127 A127 A129 H. HMG-CoA reductase and cholesterol-7a-hydroxylase activity in liver microsomes and sterol-27-hydroxylase activity in liver mitochondria A136 H1. HMG-CoA reductase and cholesterol-7a-hydroxylase activity in liver microsomes A136 H2. Sterol-27-hydroxylase activity in liver mitochondria A148 I. Measurements in vitro experiment (rat hepatocytes) A156 Page A2 of A172 +! Appendix to TNO-report of 3M-02 Final version Appendix A. Code E3L mice before and after matching mouse# | cane# ; before matching 658 86 663 86 712 93 716 93 752 98 758 98 763 99 784 102 790 102 791 102 641 84 645 84 646 84 674 88 737 96 738 96 740 96 760 99 765 99 785 102 650 85 678 88 690 90/91 699 90/91 729 95 754 98 756 98 759 98 766 99 793 103 681 89 686 89 704 92 705 92 706 92 710 92 768 100 771 100 792 103 795 103 665 87 666 87 667 87 671 87 679 88 700 90/91 730 95 731 95 744 97 799 103 714 93 721 94 727 94 769 100 774 100 776 101 778 101 782 101 783 101 797 103 652 85 657 86 670 87 680 89 687 89 696 90/91 720 94 724 94 761 99 762 99 excluded (655) 85 excluded (656) 86 excluded (741) excluded 742) 96 96 excluded (748) 97 | eartan I group# | mouse# | cage# I |L R 01 after matching | 12 00 20 00 02 11 00 02 12 10 3 21 02 01 7 4 10 /. f. 01 ; 20 00 21 1 10 01 02 01 11 10 01 20 12 02 10 10 4 "3 3! 02 4 ' 3 32 00 4 33 0 4 19 34 01 4 '9 35 02 4 19 36 00 4 20 37 11 4 20 38 00 4 21 39 11 21 4 0 1 0. !i '" ' 22/' * 's 4 1 - 0 1 12 'S hfip v ? ,V 4 2 * 2 2 -;v 43 r r 'K l- .y . 22 44* 12 i n * V SZ3' ' 45. 2 0 V 5H -: 46 0 11 (<y5 &- ' - * 2 5 ' - 47 - tZS * 48 00 J5 j. a 1 2 ` J "'-'5 jfffll 01 W 4% . .1 5" 10 12 .3 29 93 10 02 43 57 00 3 ' 29 01 -57 02 2 " 53 22 70 21 32 n2 o0 02 - MW* Pfl-SSSMIV.--' - 00 to4<38fta* f t ' .B 4 - .' 1 2 * * * * * Irffe a e a y 00 00 iM a M JK r'.S B .--: MBBKUE " P . - . 20 1 0 33W?.!. -.69.-'E 01 ;39 > *- ^ 7 0 1 12 00 21 0 20 04-12-2006 Page A3 of A172 Appendix to TNO-report of 3M-02 Final version Appendix B. Bodyweight and food intake measurements B1. Individual bodyweights Group Mouse# cage# run in run in run in run in run in run In run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in run in ran in run in run in run in run in AVERAGE SD run in run in run in run in run in 641 84 645 84 646 84 650 85 B52 8b 657 86 658 86 663 66 665 87 666 87 667 87 670 87 671 87 674 88 678 88 679 88 680 89 681 69 686 89 687 89 690 90/91 696 90/91 699 90/91 700 90/91 704 92 705 92 706 92 710 92 712 93 714 93 716 93 720 94 721 94 724 94 727 94 729 95 730 95 731 95 737 96 738 96 740 96 744 97 752 98 754 98 756 98 758 96 759 98 760 99 761 99 762 99 763 99 765 99 766 99 768 100 769 100 771 100 774 100 776 101 778 101 782 101 783 101 784 102 785 102 790 102 791 102 792 103 793 103 795 103 797 103 799 103 excluded (655) excluded (6561 excluded (741) excluded (742) excluded (748) 85 86 96 96 97 eartaq LR 10 21 02 01 u0 10 01 12 10 01 11 02 12 01 02 12 00 10 02 12 01 00 11 20 00 10 01 02 00 01 20 00 10 20 12 10 01 11 10 01 20 00 00 01 20 02 12 00 10 01 11 21 02 00 10 11 02 00 01 02 22 00 10 02 12 00 10 11 21 12 12 00 21 02 20 bodvweioht (a) week-2 week-1 week 0 30.0 30.1 29.2 27.5 28.1 28.7 31.2 30.9 30.6 27.5 27.6 28.5 iras 30.0 31.1 31.4 30.9 32.2 32.6 31.8 31.9 30.1 30.9 31.1 30.5 30.7 31.3 25.0 25.6 25.4 28.0 27.7 27.9 30.7 31.0 31.3 28.6 27.9 29.9 31.5 31.4 31.9 29.9 30.0 30.3 29.3 29.0 30.9 31.4 31.5 31.5 29.4 29.2 30.9 29.1 28.9 29.1 29.9 29.6 31.5 31.4 31.0 31.8 26.2 26.3 26.7 27.6 27.9 27.8 29.7 29.4 29.5 29.0 29.7 29.2 31.4 30.9 30.4 32.0 31.9 31.8 24.6 24.8 26.4 29.4 29.1 29.8 29.0 29.6 30.2 28.8 28.4 28.9 28.2 28.8 29.1 32.5 32.6 31.8 27.5 27.1 29.0 26.5 26.9 28.0 25.0 25.3 27.0 29.4 29.4 30.7 28.8 28.5 29.3 28.9 29.0 30.0 28.1 28.7 28.3 30.6 30.9 30.9 27.0 27.4 27.7 29.0 29.4 30.5 28.6 29.0 31.1 28.2 28.0 29.0 27.8 27.3 29.3 24.5 24.7 26.1 25.2 25.7 26.4 23.1 23.8 24.0 25.5 25.9 25.5 25.4 25.0 25.9 212 26.8 26.5 28.0 28.1 27.9 27.0 27.6 28.9 28.1 28.2 30.4 29.0 29.3 29.8 27.2 27.1 28.0 25.5 25.7 25.3 28.2 28.3 28.1 27.5 27.6 29.0 27.1 27.0 29.5 28.6 28.9 28.9 29.3 29.7 28.8 28.8 28.7 29.0 26.1 26.8 26.6 27.4 27.5 26.0 27.7 27.8 27.9 26.8 26.7 26.9 29.0 29.5 28.0 29.1 29.4 29.5 28.43 28.51 29.03 2.03 1.93 1.93 27.3 27.0 28.5 22.2 22.9 24.6 29.0 29.2 30.9 31.0 31.6 32.2 30.6 30.9 31.0 04-12-2006 Page A4 of A172 Appendix to TNO-report of 3M-Q2____________ Final version p. 40 04-12-2006 Group Mous# cage 11 21 32 42 53 6 i_ a _ 74 35 9& 10 5 " > > ' ~U' 11 6 12 6 13 6 14 7 15 8 a16 8 17 16 9 19 9 20 10 21 11 22 12 23 13 24 13 25 14 26 18 27 15 2B 1 5 29 16 30 17 -1 > 4 ' . .4 31 32 33 34 35 36 37 36 39 40 SO _____5 _____ s 5 *I 5 51 5& 55 5 5*. * AVERAG Jl i. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 18 18 19 19 19 19 20 20 21 21 22 22 22 22 23 24 25 25 26 27 28 29 29 30 30 31 31 31 31 32 *7` 1 "7 1 1 61 62 33 34 L - i l i _____1 ---- 1I--__ i 63 64 65 35 36 36 -- 1-- 66 -- I-- 1 67 37 38 ' r U : J 68 38 * 7 M 69 39 L - :.- 7 J 70 39 A B R A S E ________ r so i i eartag LR 01 12 00 20 00 02 11 week 0 31.9 31.1 29.8 28.9 30.5 29.3 25.9 00 28.9 02 12 29.0 26.6 29.19 1.85 10 21 29.2 28.7 02 01 30.6 31.9 10 01 30.0 28.3 20 00 21 10 30.9 26.4 26.5 26.8 29.13 1.80 0 02 285 30.3 0 1 10 0 31.8 27.8 27.0 31.1 20 29.0 12 02 0 26.1 27.9 27.9 28.74 1.82 0 20 00 10 0 02 00 1 223990..219 30.4 31 .a 26.4 28.9 29.8 00 1 26.0 26.9 0 0 221 20 01 26.94 31.95 31 25.4 27.9 29.9 30.9 29.5 30.7 1 0 02 29.3 27.7 29.5 2921 1.78 0 30.2 1 02 31.8 28.0 0 30.4 02 00 0 02 28.0 25.3 28.1 29.0 22 21 29.5 28.0 _ 2 10 02 00 12 00 00 20 10 01 28.83 1.79 3312.21 31.3 31.5 31.5 26.7 29.1 29.0 24.0 25.5 2 9 .1 9 : 2.88 week 1 31.4 29.8 30.2 29.4 30.0 28.4 25.8 29.7 28.9 26.2 28.96 1.76 29.4 28.5 30.7 31.7 30.2 28.2 31.6 2S.9 26.3 28.1 29.06 2.03 ..... W 29.2 32.4 27.8 26.3 30.8 30.1 26.4 27.5 27.8 28.57 2.00 30.5 9.3 28.6 31.0 31.6 25.6 26.3 30.3 27,5 261 28.88 2.04 29.5 24.7 26.9 29.4 29.4 28.4 27.9 27,9 26.3 28.0 27.84 1.53 229.2 31 27.1 30.8 28.0 24.8 27.8 27.7 29.8 27.5 28.39 1.61 30.1 302 28.8 30.6 30.4 26.2 28.2 28.4 234 5.2 I, 2 8.15 2.46 week 2 32.6 29.8 298 28.6 week 2.3 30.9 27.8 24.8 29.0 29.8 26.3 28.94 2.24 29.8 28.9 30.3 32.3 29.7 29.4 31.7 25.9 26.5 28.8 29.33 2.00 w s .... 29.7 32.0 28.6 26.1 30.0 29.9 25.3 27.8 27.4 2841 2.03 3209.22 29.0 31.6 314 25.6 29.1 30.7 28.0 26.3 29.11 202 25.6 23.8 26.8 25.4 26.9 2296..37 25.0 23.8 26.6 25.0 27.0 26.5 28.4 25.8 24.0 26.1 25.6 24.6 25.3 25.94 25.78 1.36 1.35 2B.8 31.7 27.6 30.9 284 24.9 28.1 26.4 320742 26.64 1.88 27.0 26.6 26.6 28.9 284 28.9 22 6 4 25 27.7 27.3 25.0 26.6 26.0 22.4 . 24.5 25.8 25.4 21.7 23.6 26.11 ZOO 25.43 1.82 week 3 32.5 29.6 29.6 27.9 30.5 26.1 25.0 26.6 2 9 ,7 26 28.79 2.15 302 29.0 29.3 31.7 30.2 29.6 31.7 25.6 2266.28 29.23 196 28.9 32.6 28.8 25.6 29.1 29.9 24.9 27.8 27.3 2 8 .2 0 220 29.7 9.2 28.9 30.8 31.1 25.7 28.3 302 274 27.0 28,83 1.74 25.6 24.8 27.0 25.7 27.9 27.9 284 28 0 254 27.0 26.77 1.30 28.7 314 27.1 30.5 28.1 25.1 27.7 282 30.0 27.1 28.39 1.85 262 25.0 24.7 27.8 27.1 24.6 25.5 25.3 2Z3 23.5 25.20 1.61 w eek 3.4 week 4 3Z6 29.9 29.8 27.9 304 27.6 25.7 28.1 29.2 26.1 28.73 2.06 31.0 28.9 294 31.9 30.2 29.4 31.8 26.4 27.0 28.3 29.43 W T ~1.87 ..... 29.0 3Z5 29.2 252 29.2 30.0 25.0 28.9 26.7 28.35 2.27 29.7 28.9 28.9 30.9 31.1 25.3 28.2 30.6 27.5 27.3 28 84 1 83 28.0 24.8 27.3 27.5 29.1 28.6 29.5 29.7 25.2 27.6 27.5 242 26.7 28.1 28.9 284 29.2 29.3 25.7 27.8 27.73 1.67 27.58 1.63 28.3 31.3 27.1 30.3 28.8 25.1 27.9 28.9 3207.26 28.55 1.87 26.0 26.3 24.1 24.7 25.0 25.3 27.7 27.9 26.8 27.1 I2 5 .2 25.3 I2 6.2 25.8 ii 28.1 23.1 2Z8 24.3 23.8 I I2 5 .4 4 , 25.51 1.37 1.50 B ocfyw eiohtl 9)_________ w eek 5 w eek 5.3 week 5.6 w eek 6 329 33.9 33.9 32.9 30.5 307 31.2 30.4 30.5 31.0 30.1 30.0 28.7 31.1 28.1 31.1 28.2 30.6 28.3 292 29.6 26.5 29.5 26.7 29.4 27.2 304 26.3 30.0 3 0 S3 0 . 5 27.027.1 30.4 30.6 27.2 30.6 31.0 27.3 30.0 29.76 29.91 29.97 29.47 1.90 2.14 Z02 1.89 314 29.4 299 32,9 33.0 29.8 31.2 32.8 33.1 30.0 32.0 33.1 322 30.0 312 33.6 31.0 30.2 30.8 30.1 31.0 29.8 30.1 30.0 32.9 26Z 32.8 26.5 32.8 26.6 32.5 26.3 26.6 28.6 26.9 29.0 26.6 28.8 26.9 29.0 29.91 2.31 3Q29 2.33 30.38 2.48 30.18 Z34 ~ si 288 . 28.8 9 8 28 28.4 292 329 322 32.3 32.3 28.9 25.8 28.8 25.8 28.9 26.3 292 26.1 29.0 28.7 28.5 28.8 30.2 30.1 30.1 29.6 25,1 29.2 27.1 25.0 29.0 27.0 24.7 29.7 27.0 25.1 294 27.6 28.52 221 28.32 2.07 28.47 Z12 2.59 ZOO 302 29.3 30.3 29.3 30.2 29.2 296 28.7 28.8 304 29.2 30.9 28.9 30.7 29.6 30.5 30.7 25.3 27.3 31.0 25.6 27.0 30.7 25.8 26.7 30.7 262 26.9 302 30.2 30.2 30.1 27.1 27.5 27.7 27.7 27.2 27.7 272 27.7 28.68 1.80 28 89 1.82 28.73 1.78 28.72 1.62 28.6 25.0 274 26.9 25.2 27.6 30.0 25.3 27.8 29.1 25.5 2 8 .6 282 292 28.5 28.6 28.1 29.7 28.8 30.1 28.2 30.3 28.5 30.3 28.6 30.1 28.6 30.1 29.3 26.5 293 27.3 29.9 27.2 29.7 27.0 28.0 28.07 1.51 27.7 28.19 1.37 2 8 .t 2189524 28.4 " 28.9 1.42 28.5 31.5 274 28.4 31.3 27.3 28.5 31.5 27.2 28.7 314 27.0 30.3 28.7 25.5 27.6 28.9 30.2 28.8 25.6 27.6 29.1 30.0 284 254 27.8 29.1 29.7 28.0 25.3 26.9 28.8 304 26.8 30.3 26.7 30.7 26.7 30.9 26.3 28.56 1.62 1.9928.53 28.53 28.30 1.77 i 1.87 , 2 8 .0 27.2 26.1 28.3 274 28.2 I 27.7 27.3 i 282226 28.7 266 285 27.4 28.2 29.1 27.8 26.3 28.9 27.6 26.1 262 26.5 2Z8 24.6 26.1 26.1 !2 6 .0 262 26.7 : 26.9 23.8 23.0 25.1 24.6 262 26.7 272 23.6 25.7 26.32 1.64 I !2 6.48 , 2 6 .4 5 , 2 6.96 1.42 1.65 1.60 w eek 6.3 week 7 332 33.5 302 30.2 30.6 30.5 28.4 31.1 26.5 30.8 29.6 29.8 26.5 2 .0 5T o -3 0 .8 30.7 30.9 27.1 27.6 29.78 1.99 30.02 1.90 32.8 33.1 30.0 31.8 29.5 3Z0 33.1 33.0 30.3 30.9 32.7 25.9 26.5 29.1 304 29.5 314 262 27.7 29.5 30.31 2.53 3023 223 29.1 292 32.5 29.3 264 29.1 29.9 30.1 332 29.3 272 29.4 302 254 29.6 27.8 302 26.3 30.4 28.9 28.66 1.98 9.50 1.87 2922 9 J 29.4 29.4 29.8 30.5 30.0 30.5 31.1 26.5 28.0 30.7 264 27.1 30.9 27.5 27.5 30.7 27.9 28.3 29.09 1.81 29.04 1.54 29.8 25.6 292 30.5 25.9 29.6 29.1 298 29.1 30.6 28.6 30.6 302 30.0 302 27.3 30.5 272 28.6 29.0 28.86 29.26 1.48 1.57 28.7 28.9 3Z1 274 32.0 27 6 302 26.5 302 28.1 25.6 27.2 28 6 252 26.7 28.5 30.6 26.6 304 26.3 28.55 1.98 28.40 2.01 28.5 27.8 28.6 284 27.0 28.7 26.8 28.6 282 27.1 26.7 27.3 2253..48 27.05 1.50 28.3 272 262 27.1 24.0 . 252 27.11 I 123 week 8 33.8 312 302 28.6 31.0 29.7 27.5 31.0 30.7 27.3 30.10 1.94 34.1 292 322 33.7 29.9 30.5 312 26.4 28.7 29.7 30.59 Z32 302 30.0 33.1 292 27.0 30.1 30.1 236049 26.6 29.5a 1.91 29.7 29.0 302 30.3 30.8 262 27.6 30.9 26.1 28.5 2916 147 30.4 25.6 29.1 29.1 30.4 29.9 30.4 306 27.7 29.1 2923 1.56 28.8 32.1 26.8 30.3 282 2S.6 26.7 28.3 30.1 262 28.37 203 29.0 28.6 27.1 29.1 28.6 27.3 272 27.6 24.3 25.7 2 7 .4 5 1.53 week 8 4 week 9 342 31.0 302 31.1 30.0 26.5 30.8 29.9 27.3 284 312 30.0 27.7 31.9 33.1 30.6 27.1 304 27.0 30.11 2.03 30.34 2.32 33.7 292 33.1 332 34.6 30.1 336 33.6 30.8 31.0 31.7 262 28.9 295 312 312 324 27.8 29.8 30.1 30.82 229 31.46 2.14 302 30.1 30.6 30.0 332 29.3 272 31.0 302 334 294 272 314 30.7 26.1 31.1 282 26.3 31.6 28.1 29.73 2.05 2927 2.17 2289..70 29.8 304 30.7 264 284 20.1 29.6 302 30.9 264 272 30.6 27.0 30.4 27.6 284 272 28.6 28.88 1.50 28.68 1.55 304 254 29.1 302 25.0 28.9 292 30.1 29.3 29.6 292 29.7 30.7 31.0 30.5 28.1 28.9 30.4 28.5 29.0 29.22 29.16 127 1.64 28.5 32.1 26.7 28.1 264 322 26.6 27.8 274 252 26.8 282 302 26.3 27.1 25.7 26.7 27.9 30.0 262 27.96 27.87 1.96 : iU 294 28.7 27.8 292 28.6 274 29.7 292 28 2 29.8 28.9 27.8 272 27.8 24.7 26.3 27.6 28.0 25.0 26.6 , 27.75 , 28.10 I 1.45 I w e e k 10 34.3 314 30.5 26.9 32.0 29.3 26.0 332 31.7 27.4 30.67 226 35.4 30.6 332 33.6 30.6 30.7 32.9 28.3 30.8 30.1 31.65 2.12 31.0 30.9 33.6 284 272 30.9 31.3 25.9 3Z7 27.7 29.99 ! 2.54 i 282 28.1 292 29.6 30.3 26.2 26.7 29.7 26.8 28.1 2829 141 312 254 29.6 294 29.5 30.0 312 30.7 284 29.0 2945 1.70 27.8 31.3 262 27.7 2265.28 26.7 27.6 29.9 26.0 27.52 1.85 29.8 29.5 28.0 29.8 29.5 27.9 27.5 28.1 25.1 27.6 2828 145 Page A5 of A172 Appendix to TNO-report of 3M-02____________ Final version p. 41 04-12-2006 Page A6 of A172 Appendix to TNO-report of 3M-02____________ Final version B2. Food intake measurements p. 42 04-12-2006 cage# 84 85 86 87 88 89 90/91 92 93 94 95 96 97 98 99 100 101 102 103 number of Food on cage Food off cage mice week -2 (g) week -1 (g) 3 86.1 37.1 3 88.8 41.6 4 117.2 49.1 5 131.5 51.8 3 84.2 22.9 4 105.6 29.3 4 108.5 40.9 4 107.7 49.6 3 79.3 21 4 104.1 40 3 82.6 25.7 5 132.8 40 2 54.5 20.8 5 139.5 57.2 6 156.4 78.2 4 105.8 30.5 4 102.2 40.3 4 102.3 44.7 5 132.2 53.6 days 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Food intake (g/day/mouse) 2.72 2.62 2.84 2.66 3.41 3.18 2.82 2.42 3.24 2.67 3.16 3.09 2.81 2.74 2.17 3.14 2.58 2.40 2.62 2.80 0.32 Food on cage Food off cage week -1 (g) week 0 (g) 82 25 81.8 19.7 108.9 30.3 132.7 39.4 77.6 10.4 103.9 20.9 105.2 27.7 108.2 40.3 80.3 16.7 108.8 31.1 81.6 17.1 130.8 28.5 52.2 9.5 129.7 33.2 152.1 51.8 104.2 20.5 106.9 31 106.8 33.2 130.6 41.7 days 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Food intake (g/day/mouse) 2.71 2.96 2.81 2.67 3.20 2.96 2.77 2.43 3.03 2.78 3.07 2.92 3.05 2.76 2.39 2.99 2.71 2.63 2.54 2.81 0.22 Page A7 of A172 Appendix to TNO-report of 3M-02 Final version r " 1n 1 1 CMs (0Vg 8 C(SMI 8 09 08 00 0 s s o s 8 8 S3 s rsi o 9 rt 0 CO11 9- 0 0 CM 0h. 0 o O O N o 0 0 0 0 0 0 CM ri ri 0 0 ri d 0 ci ri r i ri 6 ri r i r i r i ri r i d ri r i r i ri d ri r i ri r i cmri ri d ri 0 r i r i ri 0 d 0 ri 0 0 ri ri i J | ID at to c 00 O 0 to 0 to to 0 0 o 0 0 D0 0 1 0 to 0 to 0 0 0 0 0 Is 52 81 0 CM9 co O 8c 8 w> 0) 0 m to 01 ' h to ri s at 0 (0 CM 0 s. 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Is Cs IC d ra.i in 2 If 9 N - - - K - - h> h~ - 3h. to CO0> 0 cm 9^ d 0 c~ r i CDc 0 <S o S 1r * * -- - o5 s ri 2 M3 SI CM r - K - 9- 9 M CMCMCMCMCM tr "1 at ft 0 9 OCM0 ri ri n d ri 8 ri 0 CMz 0 9* 0 9 0 00 CM 3 CCOM J 0 CMa n q 3 K n 5 gM ! to 0 O ri ! r0i N K 8 3 s S* 11 II 0 0 5 S 3 cn w CS CM r i si ri ri o 0 9 0 CM 9 O S 0 0 8 CM ri ri r i r i CM ri d ! i 0a o0 CM r i N CS CO0 0 0 0 ri ri ri ri is CS9- n 0 to 0 d 0 CM(S ^R 3 c Jf 0 CM5 i* ! l5a2 s N ID 00 K 8 CD 9N 11 U. s 9CM- cr S co m IC 0 0 0CM) s tr o tc T 05 9 S. 9 r- 0 o0n s d9 0 00 S (V o 0 o 0 0 to to ri 0 ri 0 s ri 0 00i SS ri ri1 1 CESO t'. I S8 s ni r CO 3?s M aas33s s d CO r ri d 0 n (Si ri ri ri 6 a s g CM0 c R 9*. a s d ri 0 o to jr tr s 0 0 CnO0 9. (M, 0 S 8 0 3 0 0 si r i d ri ri ri d ri CM ri r i r i ri ri 0 y f. <0 (OID ID T> - <p to - a 0 00 00 000 0 0 0 0 O0 0; 0 0 -1 o o 0 0 to 0 0 0' S3 S* CM e m d |l i c 0 csi 0 0 to CM0 ID d cCMri flO0 ri ri2 N. 0 0 CO J 0 d to 0 CS : 2 "1 ri0 to !0 0 0i J 11 a0 0 ri d S Is c ** . CD OJC A S Jj S p K. co CD gs 580 M; 0 n C0Ms g ri 0 N r* S3 00 8i 9 8 ririq 6 CM K Oi 00 MCM 0 CM0 8 00 o 0 0 0 to CM1 |8, R 8 s 3 "! s 1 a1 g 3 3 q g 8 8 (0Si nCO ri ri ri ri a S &0 ei csi esj 3 0COO n CMri O CMrri rri CM 8 53 q o ri ri ri ri ri s. d e 5 ri O 5 ri ri CM0 n ri d oO 0 O t0100 ri 0 0 S 0 0 0 CM 0 csi d ri K q csi d 10 9- 0 9N 0 0 0 ri: 00 ri d i to at IS to to o 8s e T" Oj cO0) sn 3 CPDi tNo CM8 Is ri Sie o * to U> to 0 li u. 13 " " (St i6 c ?p - CM0 to (0 to 0 0 0 0 0 0 to 0 0 ri CM s in n 0 N s0 0 0 w0 n rrii 0 506 0 0 00 s 3 0 0 :0 0 CSC n3 CM -m - CM- to 9~ 0 "- - " - - ! (M s 0 0 9- 0 0 8 ri 0ri i ri dat 0 3 00 0 0 - .0 0 d ri 2' 9- 0 0 C at CO i ;io*pd0 0 Si 4n 00000 " to 0 0| 0 0 0 tO "1 0 csi 9wat iR CSI a) l~ 0 0 3F r 8 Iri CM 0 0 - ri 80 to 0 |0 9 3 0d 0 r <M CMCM i0 o>O CM - - CM- - CM CM CM C0M f ! rr (M S a0<o <M 8 - - - CM- CMet 0 00 0 00 00 0 0 p. 43 04-12-2006 'age A 8 o f A172 Appendix to TNO-report of 3M-02 Final versipn 04-12-2006 Group cage# t 2 3 4 S number of mice 2 2 2 1 3 63 71 83 92 10 1 11 1 12 1 13 2 14 1 15 3 16 1 17 1 /v/ERAGE :>n ...... . 18 19 20 21 22 23 24 25 26 27 2 4 2 2 4 1 1 2 1 1 28 29 = 30 31 32 1 2 2 4 1 J 33 7 34 7 35 77 36 37 38 39 AVERAGE so _______ i 1 1 1 2 1 2 2 Food on cage Food off cage days week 4 fo) week 5(g) 19.0 5.5 55.8 22.0 5.5 51.7 18.3 5S 26.2 10.0 5.5 83.0 34 5.5 74.4 26.0 5J> 29.7 10.8 5.5 75.5 24.6 5.5 50.2 23.6 5.5 25.7 9.5 5.5 26.5 9.8 5.5 29.5 14.7 5.5 55.8 18.2 5.5 30.2 16.1 5.5 84.8 42.1 55 25.7 10.0 5.5 35.6 20.9 5.5 53.0 107.9 58.2 58.0 19.4 5.S 52.0 5.5 26.7 5.5 29.S 5.5 105.0 30.5 30.2 53.0 28.5 27.6 60.2 4.5 16.8 4 15.7 4.5 222 4.5 15.4 4.5 14.3 4.5 30.2 50.5 54.1 102.0 30.0 16.3 5.5 182 5.5 22.6 5.5 45.9 5.5 17.1 5.5 33.2 17.4 4.5 31.4 16.7 4.5 41.8 29.1 4.5 45.3 16.2 4.5 26.2 13.8 4.5 565.5 35.9 AJS 55.0 33.8 4j Food intake Food on cage Food off cage p/day/mouse week 5 (g) week 6 (g) 285 60.2 22.1 3.05 60.7 19.5 3.04 572 16.8 295 31.6 10.7 2.93 77.1 19.2 296 0.08 293 85.4 19.8 3.44 37.4 12.4 3.08 852 142 240 51.9 13.6 295 33.8 13.4 296 027 3.04 2.69 36 37.4 1165..72 3.42 55.9 7.1 2.56 35.4 15.6 2.59 89.8 32.7 2.85 27.4 2.67 312 8 9.4 283 0.31 3.05 57.7 16.5 254 106.9 332 286 53.4 11.1 2.59 57.7 17.7 276 024 249 109.2 32.3 3.04 41.4 20.1 322 32.5 10.6 3.42 61.1 15.5 291 32.8 12.5 2.96 31.6 102 3.01 0.32 2.53 36.3 16.6 294 60.8 20.3 2.86 52.6 12.3 2.55 105.5 36.7 234 34.8 16 2.64 025 3.51 332 12.7 3.27 35.4 16.3 2.82 39.9 20 323 78.1 38.8 2.76 31.1 1 12.1 329 612 24.9 2.36 52.9 15 3.03 0.40 i days 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Food intake g/day/roouse 2.72 2.94 2.69 2.99 2.76 2.86 0.12 3.12 3.57 3.38 2.74 2.89 3.14 0.34 2.90 3.03 3.49 2.83 2.72 2.77 3.11 2.98 0.26 2.94 2.63 3.02 2.86 2.86 0.17 2.75 3.04 3.13 3.28 2JK) 3.06 3.02 0.18 2.81 2.89 2.88 2.46 2.69 2.75 0.18 2.93 2.73 2.84 2.81 2.71 2.59 2.71 2.76 0.11 Food on cage Food off cage days week 6 fg) week 7(g) 78.3 61.7 3 73.4 55.5 3 67 48.9 3 38.3 29.1 3 94.8 71 3 97.1 69,2 3 42 32.8 3 85.6 54.1 3 60.9 46.3 3 46.3 37.3 3 39.9 40.9 56.5 46.1 114.5 34.9 37.9 303 3 31.4 3 39.2 3 37.8 3 91.4 3 26.2 3 29.5 3 61.5 130.6 70 67.9 42.6 3 98.9 3 50.3 3 50.3 3 138.4 52.3 37.3 74.9 39.9 35.4 107.2 44.3 27.6 53.6 31.1 26.8 3 3 3 3 3 3 44.3 34.9 3 66.8 48.7 3 60 42.9 3 142 112.9 3 46.7 39 3 35.7 27.4 3 42.4 33.8 3 46.1 36.9 3 90.6 73.3 3 39.7 30.7 3 76.5 60.4 3 65.9 50.8 3 Food intake g/day/mouse 2.77 2.93 3.02 3.07 2.64 2.90 0.18 3.10 3.13 3.50 2.43 3.00 3.03 0.39 3.20 3.17 2.88 2.77 2.57 2.90 2.80 2.90 0.22 3.15 2.64 3.28 2.93 3.00 0,28 2.60 2.67 3.23 3.52 2.93 2.87 2.97 0.35 3.13 3.02 2.85 2.43 2.57 2.80 0.30 2.77 2.87 3.07 2.88 3.00 2.68 2.52 2.83 0.19 Page A9 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Group cage# mouse# Food on cage Food offcage days Food intake Food on cage Food off cage days Food intake week 7 (g) week 7.5 (g) g/day/mouse week 7.5(g) week 8 (g) g/day/mouse 11 438.1 428.4 3 3.22 29.8 15.7 4 3.52 MC101 2 metabolic cage 20.3 12.8 3 2.51 MC 105 3 metabolic cage 18.6 8.7 3 3.29 MC 201 4 metabolic cage 23.5 15.0 3 2.84 35 485.4 | 476.5 3 2.96 26.1 13.9 4 3.05 MC206 6 metabolic cape ! 19.2 8.3 3 3.63 47 496.1 3 2.86 29.2 18.2 4 2.75 5 8,9,10 425.4 397.3 3 3.12 54.7 25.8 4 2.41 3.04 3.00 0.16 0.45 2 6 11,12,13 435 407.6 3 3.04 55.9 12.5 4 3.62 7 14 429.7 421.0 3 2.91 21.9 11.9 4 2.51 2 8 15,16,17 475.5 442.1 3 3.71 44.3 11.7 4 2.72 2 9 18,19 450.2 431.3 3 3.15 33.6 8.4 4 3.15 2 10 20 425.7 9.5 3 2.94 32.1 17.3 4 3.70 AVERAGE 3.15 3.14 S D 0.33 0.53 MC 102 21 metabolic cage 35.6 26.2 3 3.12 MC 106 22 metabolic cage 29.4 21.4 3 2.67 13 23 455.8 445.8 3 3.35 33.1 20.3 4 3.20 MC 202 24 metabolic cape 26.8 17.7 3 3.02 14 25 430.2 418.8 3 3.79 36.9 24.7 4 3.06 15 26,28 465.3 449.8 3 2.58 45.1 20.5 4 3.08 MC206 27 metabolic cape 32.9 25.1 3 2.61 16 29 437.8 428.5 3 3.10 26.3 15.5 4 2.71 17 30 429.1 421.5 3 2.53 44.1 34.7 4 2.36 | 3.07 2.87 I 0.53 0.29 4 MC 103 31 metabolic cage 44.3 35.6 3 2.89 4 MC 107 32 metabolic cage 51.2 43.3 3 2.62 4 19 33,34,35,36 486.1 448.3 3 3.15 84.3 36.0 4 3.02 4 MC 203 37 metabolic cage 33.1 23.1 3 3.34 4 MC207 38 metabolic cage 32.6 23.2 3 3.14 4 21 39,40 436.2 419.7 3 2.75 44.2 27.0 4 2.15 AVERAGE 2.95 2.86 SD 0.28 0.42 5 22 41,42,43,44 505.9 470.3 3 2.97 109.2 63.4 4 2.86 5. 23 45 443.9 434.1 3 3.26 41.4 31.1 4 2.57 5 24 46 438.7 428.7 3 3.35 32.5 18.9 4 3.41 5 25 47,48 443.3 423.9 3 3.24 61.1 38.2 4 2.86 5 26 49 428.6 419.7 3 2.98 32.8 20.9 4 2.98 5 27 50 436.1 427.5 3 2.86 31.6 22.2 4 2.36 AVERAGE 3.11 2.84 SP 0.20 0.36 6 MC104 51 I metaboliccage 38.2 27.5 3 3.58 6 29 52 I 432.6 424.2 3 2.81 27.6 15.6 4 3.00 MC 108 53 metabolic cage 33.1 25.6 3 2.49 6 MC204 54 metabolic cage 28.4 20.7 3 2.57 MC208 55 metabolic cage 33.1 24.7 3 2.81 6 31 56,57,58,59 409.8 371.4 3 3.20 87.4 46.3 4 2.57 3 32 60 441.6 433.5 3 2.69 21.7 11.4 4 2.58 AVERAGE SD 2.90 2.80 0.27 0.39 7 33 61 439.2 429.8 3 3.12 33.1 22.4 4 2.68 7 34 62 431.4 422.0 3 3.15 32.5 22.5 4 2.51 7; 35 63 442.7 434.7 3 2.67 39.4 27.5 4 2.98 36 64,65 445.6 427.0 3 3.10 66.1 39.3 4 3.35 ' 7 - 37 66 426.1 417.7 3 2.79 30.2 18.8 4 2.84 7 38 67,68 465.5 446.0 3 3.25 65.8 40.7 4 3.14 39 69,70 445.2 423.9 3 3.55 52.1 32.5 4 2.45 AVERAGE SD ___ 3.09 0.29 2.85 0.33 M3, ween 7-7.5: all mice are put on powdered food because of metabolic cages of part of mice Page A10ofA172 Appendix to TNO-report of 3M-02____________ Final version Group Mouse# cage# 11 21 32 42 53 63 74 85 95 10 5 11 12 13 14 15 16 17 18 19 20 3 21 22 23 24 25 26 27 28 29 30 6 6 6 7 8 8 8 9 9 10 11 12 13 13 14 15 15 15 16 17 4 4 4 & AVERAGE so 5 15 5 5 . 5 5 5 5 AVERAGE SO 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 S3 7 . " T- A-s 'T fW 7' 7i 7- 7' 7 7 AVERAGE SD 61 62 63 64 65 66 67 68 69 70 18 18 19 19 19 19 20 20 21 21 22 22 22 22 23 24 25 25 26 27 28 29 29 30 30 31 31 31 31 32 33 34 35 36 36 37 38 38 39 39 Food on cage Food off cage days Food intake week 8 (g) week 9 (g ) g/day/m ouse 34.3 11.7 " T ~ 3.23 27.1 5.8 7 3.05 28.6 7.9 7 2.96 44.1 24.6 7 2.79 36.9 15.6 7 3.05 43.3 20.5 7 3.26 42.2 22.2 7 2.86 342 10.1 7 3.45 33.0 12.1 j 7 2.98 32.9 8.6 7 3.47 3.11 0.23 34.1 9.5 7 3.52 36.5 16.4 7 2.87 28.4 6.3 7 3.16 29.5 11.4 7 2.58 28.3 5.6 7 3.22 27.1 3.2 7 3.42 36.0 15.9 7 2.87 44.3 22.2 7 3.16 45.2 25.0 7 2.88 33.9 9.3 7 3.52 3.12 4.6 0.32 12.0 7 3.15 35.1 14.2 7 2.98 29.8 7.7 7 3.16 27.6 8.2 7 2.77 44.1 18.8 7 3.62 44.3 23.4 7 2.99 47.2 26.3 7 2.99 34.9 13.8 7 3.01 33.2 13.1 7 2.87 3 l6 12.6 7 2.86 ___ 3.04 0.24 33.1 11.5 3.08 34.2 15.5 7 2.67 45.7 24.8 7 2.98 36.0 14.0 7 3.15 45.7 23.0 7 3.24 44.1 25.6 7 2.65 36.9 17.2 7 2.81 32.1 10.7 7 3.06 22.5 1.2 7 3.04 33.4 1Z3 7 3.02 2.97 0.20 44.1 19.5 ~~T" 3.52 37.7 18.9 i 2.68 45.1 21.2 7 3.42 44.2 25.6 7 2.66 35.6 15.2 7 2.91 43.2 19.8 7 3.35 44.8 26.1 7 2.67 42.5 18.6 7 3.42 29.6 7.9 7 3.10 34.3 15.6 7 2.67 3.04 0.36 33.9 10.7 T " 3.32 32.4 14.6 7 2.55 35.6 18.6 7 2.43 41.8 19.9 7 3.13 42.1 22.0 7 2.87 44.0 23.1 7 2.98 43.3 23.1 7 2.88 32.5 9.8 7 3.24 28.2 7.1 7 3.01 29.4 12.0 7 2.49 __ 2.89 0.31 34.4 14.3 2.87 45.2 21.3 7 3.41 30.1 8.8 7 3.05 229.8 7,7 7 3.16 27 3.8 7 3.34 33.4 12.5 7 2.99 31.5 10.2 7 3.04 46.2 26.1 7 2.87 44.1 23.2 7 2.99 32.5 14.4 7 2.58 3.03 0.24 50 p. 46 04-12-2006 Page A11 ofA172 Appendix to TNO-report of 3M-02____________ Final version p. 47 04-12-2006 Page A12ofA172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Appendix C. Plasma measurements C1. Plasma cholesterol measurements group Mous* cage* MrttM I Measured cholesterol (mmolrt) | dilution I L R week 0 week 1 week 4 week8 week 0 week 11 week 4 week 8 weekO 1 1 0 1 3.219 2.814 172 211 2 2 3 3 6.436 2 1 1 2 3.446 3.088 599 394 2 2 3 3 6.892 3 2 0 0 3.633 3.17$ 2.120 1.725 2 2 3 3 7.266 4 2 2 0 3.260 2.556 1.911 1.877 2 2 3 3 6.520 5 3 0 0 1.847 2.612 042 1.733 2 2 3 3 3.694 6 3 0 2 3.819 3.458 2.033 500 2 2 3 3 7.638 7 4 1 1 4.055 3.958 1.815 166 2 2 3 3 8.110 8 5 0 0 3.949 3,668 2.347 295 2 2 3 3 7.898 9 5 0 2 4.590 3.257 1.972 1.816 2 2 3 3 9.180 10 5 1 2 3.706 3.015 1.911 1.854 2 2 3 3 7.412 7.10 1.45 11 6 1 0 3.98 3.901 625 2.181 2 2 3 3 7.996 12 6 2 1 1.791 2.145 1.110 0.867 2 2 3 3 3.582 13 6 n r 2 4.426 3.281 2.094 2.287 2 2 3 3 8.856 14 7 n r 1 4.274 2.878 1.859 1.353 2 2 3 3 8.548 15 8 1 0 4.453 3.080 443 2.272 2 2 3 3 8.906 16 8 0 1 1.774 2.306 1.057 0.837 2 2 3 3 3.548 17 8 2 0 1.750 2.322 1.563 1.209 2 2 3 3 3.500 18 9 0 0 4.079 3.450 2.233 2.151 2 2 3 3 8.158 19 9 2 1 4.055 2.959 2.233 2.439 2 2 3 3 8.110 20 10 1 0 3.633 3.233 2.085 2.029 2 2 3 3 7.266 Li X 6.85 2.33 21 11 0 1 4.201 3.217 1.850 2.439 2 2 3 3 8.402 22 12 0 2 3.446 3.007 1.87$ 2.021 2 2 3 3 6.892 23 13 0 1 4.047 3.257 077 1.771 2 2 3 3 8.094 124 13 1 4.071 2.999 1.920 2.318 2 2 3 3 8.142 125 14 T l 3.649 2.685 1.406 1.763 2 2 3 3 7.298 26 15 0 3.893 741 1.885 1.870 2 2 3 3 7.786 1 227 15 2 0 3.130 2.798 1.737 1.703 2 2 3 3 6.260 28 15 3.519 3.201 1.850 2.219 2 2 3 3 7.038 29 16 0 2 4.290 3.466 521 2,431 2 2 3 3 8.580 30 17 1 0 2.083 1.968 1.153 1.315 2 2 3 3 4.166 7.27 1.31 31 18 1 0 3.974 1,566 1.327 1.125 2 2 3 3 7.948 22 18 0 2 3.625 1.622 1.327 1.353 2 2 3 3 7.250 133 19 0 0 4.104 2.057 1.476 1.422 2 2 3 3 8.206 34 19 0 3.122 1.912 1.284 1.186 2 2 3 3 6.244 4 35 19 0 1 3.698 2.322 1.380 1.490 2 2 3 3 7.396 4 36 19 0 2 1.701 1.904 1.301 1.209 2 2 3 3 3.402 37 20 0 0 3.576 1.895 1.467 1.452 2 2 3 3 7.152 4 38 20 1 1 3.511 1.879 1.354 1.156 2 2 3 3 7.022 4 39 21 0 0 3.917 2.467 1.754 1.672 2 2 3 3 7.834 40 21 1 1 2.010 1.557 1.293 1.429 2 2 3 3 4.020 6.65 1.65 s* 41 22 1 0 1.661 1.871 1.179 1.072 2 2 3 3 3.322 s 42 22 0 1 3.617 1.525 1.197 1.885 2 2 3 3 7.234 5 43 22 1 1 3.682 1.839 1.162 1.983 2 2 3 3 7.364 5 44 22 1 2 3.795 1.670 1.168 1.490 2 2 3 3 7.590 5 45 23 1 2 4.323 3.056 1.258 2.591 2 2 3 3 8.646 5 46 24 2 0 3.901 2.226 1.388 2.454 2 2 3 3 7.802 5 * 47 25 0 1 3.641 1.465 0.813 0.746 2 2 3 3 7.282 48 25 1 1 3.065 1.638 1.449 1429 2 2 3 3 6.130 5 ,' 49 26 0 0 1.669 2.008 1.354 1247 2 2 3 3 3.338 50 27 1 2 2.708 2.274 1.467 2.097 2 2 3 3 5.416 m 6.41 '' f 1.85 51 28 0 1 2.862 1.992 1.101 1.794 2 2 3 3 5.724 52 29 1 0 3.138 2.604 1.101 1.247 2 2 3 3 6.276 53 29 1 2 4.086 2.556 1.223 1.953 2 2 3 3 8.172 54 30 1 0 3.316 2.258 1.110 1.611 2 2 3 3 6.632 55 30 0 2 3.S11 2.492 1.136 1.543 2 2 3 3 7.022 56 31 0 0 3.584 2.508 1.110 1.968 2 2 3 3 7.168 57 31 0 1 1.523 1.654 1.214 0.928 2 2 3 3 3.046 58 31 0 2 3.795 2.330 1.005 1.452 2 2 3 3 7.590 59 31 2 2 3.357 2.024 1.249 1.763 2 2 3 3 6.714 60 32 2 1 3.365 2.065 1.310 386 2 2 3 3 6.730 AVERAGE 651 AD 1.39 61 " 33" T T 3.462 1 646 2.338 2.113 2 " 1 3 3 6.924 62 34 1 0 3.763 1.775 3.174 2.021 2 2 3 3 7.526 * 1* 63 35 0 2 3.024 1.541 573 2.447 2 2 3 3 6.048 64 36 0 0 3.479 1.549 2.242 280 2 2 3 3 6.958 65 36 1 2 3.324 1.501 1.920 1.816 2 2 3 3 6.648 in I 7\ 1 66 37 0 0 3.357 1.561 2.077 1.832 2 2 3 3 6.714 67 38 0 0 4.241 1.879 1.998 1.733 2 2 3 3 8.482 68 38 2 0 4.274 1.928 1.963 158 2 2 3 3 8.548 69 39 1 0 3.917 1.639 1.824 1.695 2 2 3 3 7.834 17 70 39 0 1 3.795 1.783 1.894 1.505 2 2 3 3 7.590 .E-KM SO 7.33 0.81 [ wxiludeo 655 I T " T T 6.230 2 12.460 axckideo 656 86 0 0 1.336 2 672 excluded 741 96 2 1 1.360 2 2.720 excluded 742 96 0 2 0.792 2 1.564 excluded 748 97 2 0 0.776 2 1 552 Cholesterol mmoVU week 1 week 4 5.628 6.516 6.176 7.797 6.352 6.360 5.112 5.733 5.224 6.126 6.916 6.099 7.916 5.445 7.336 7.041 6.514 5.916 6.030 5.733 6.32 68 0.90 0.70 7.802 7.875 4.290 3.330 6.562 6.282 5.756 5.577 6.160 : 7.329 4.612 ! 3.171 4.644 4.689 6.900 6.699 5.918 6.699 6.466 6.255 5.81 5.79 1.12 1.60 6.434 5.550 6.014 5.628 6.514 6.231 5.998 5.760 5.370 4.218 5.482 5.655 5.596 5.211 6.402 5.550 6.932 7.563 3.936 3.459 5.67 5.46 0.84 1.10 3.130 3il 3.244 3.981 4.114 4.428 3.824 3.852 4.644 4.140 3.808 3.903 3.790 4.401 3.758 4.062 4.934 5.262 3.114 3.879 3.84 4.19 0.61 0.43 3.742 3.537 3.050 3.591 3.678 3.340 3.486 3.564 6.112 3.774 4.452 4.164 2.970 2.439 3.276 4.347 4.016 4.062 4.548 4.401 3.92 3.74 0.94 0.57 3.984 3305 5.208 3.303 5.112 3.669 4.516 3.330 4.984 3.406 5.016 3.330 3.308 3.642 4 660 3.015 4.048 3.747 4.130 3.930 4.50 3.47 0.62 0.27 3.292 7.014 3.550 9.522 3.082 7.719 3.098 6.726 3.002 5.760 3.162 6.231 3.758 5.994 3.856 5.889 3.678 5.472 3.566 5.682 3.40 6.60 0.31 1.24 week 8 6.633 7.182 5.175 5.631 5.199 7.500 6.498 6.885 5.448 5.S62 6.17 0.87 6.543 2.601 6.861 4.059 6.816 2.511 3.627 6.453 7.317 6.087 5.29 1.88 7.317 6.063 5.313 6.954 5.269 5.610 5.109 6.657 7.293 3.945 5.96 1.10 3.375 4.059 4.266 3.558 4.470 3.627 4.356 3.468 5.016 4.287 4.05 0.53 3.216 5.655 5.949 4.470 7.773 7.362 2.238 4.287 3.741 6.291 5.10 1.81 5.382 3.741 5.859 4.833 4.629 5.904 2.784 4.356 5.289 7.158 4.99 1.23 6.339 6.063 7.341 6.840 5.448 5.496 5.199 6.474 5.085 4.515 5.88 0.88 I Page A13ofA172 52- Appendix to TNO-reoort of 3M-02____________ Final version p. 49 04-12-2006 Page A14ofA172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 C2. Plasma triglyceride measurements group Mouse# cage# eartaq LR 1 1 TT 1 2 1 12 3 2 nr 0 4 2 rr 0 5 3 00 6 3 02 7 4 11 8 5 00 9 5 02 10 5 1 2 Measured TG (mmolA) dilution week 0 week 1 week 4 week 6 week 0 week 1 week 4 week6 0.445 0.387 0.257 0298 2 2 3 3 0.467 0.270 0263 0266 2 2 3 3 0.950 0.731 0290 0.233 2 2 3 3 0.706 0.609 0.329 0277 2 2 3 3 1.666 0.583 0214 0.277 2 2 3 3 0.645 0.472 0345 0244 2 2 3 3 0.595 0.625 0247 0.471 2 2 3 3 0.495 0.482 0208 0.293 2 2 3 3 0.562 0.477 0296 0233 2 2 3 3 0.756 0.456 0.186 0212 2 2 3 3 11 6 1 0 0.564 0.434 0274 0.255 2 2 3 3 12 6 2 1 0.695 0.667 0.356 0.336 2 2 3 3 13 6 0 2 0.639 0.445 0219 0255 2 2 3 3 14 7 0 1 0.961 0.535 0219 0222 2 2 3 3 15 8 1 0 1.366 0.842 0.340 0.461 2 2 3 3 16 8 0 1 0.833 0.688 0.422 0.655 2 2 3 3 17 8 2 0 0.417 1.080 0.515 0.585 2 2 3 3 18 9 0 0 0.589 0.376 0.257 0.201 2 2 3 3 19 9 2 1 0.467 0.477 0252 0.320 2 2 3 3 20 10 1 0 0.501 0.355 0.214 0 .1 2 2 3 3 21 11 1 0.573 0.276 0.121 0.190 2 2 3 3 22 12 0 2 0.606 0307 0.148 0201 2 2 3 3 23 13 0 " T l 0.978 0.762 0.307 0.233 2 2 3 3 24 13 1 T 0.661 0.932 0.372 0.168 2 2 3 3 25 14 1 0 0.795 0.456 0.143 0.190 2 2 3 3 26 15 0 0.656 0.482 0.148 0244 2 233 27 15 2 0 0,478 0.376 0.143 0.152 2 2 3 3 28 15 1 2 0.528 0.382 0.137 0.195 2 2 3 3 29 16 0 2 0.412 0.403 0208 0.336 2 2 3 3 30 17 T l 0 0.900 0.673 0.312 0369 2 2 3 3 31 18 i o 0.856 0244 0.137 0.082 2 2 3 3 A 32 18 0 2 0.634 0.281 0.143 0.082 2 2 3 3 4 33 19 0 0 0.445 0.276 0.121 0.087 2 2 3 3 4 34 19 1 0 0.506 0.233 0.099 0.103 2 2 3 3 4 35 19 0 1 0.589 0.207 0.132 0.093 2 2 3 3 4 '4 36 19 0 2 0.489 0.297 0.230 0.206 2 2 3 3 37 20 0 0 0.739 0.329 0.132 0.082 2 2 3 3 44 38 20 1 0.994 0.419 0.148 0.082 2 23 3 39 21 0 0 0.562 0.270 0.104 0.103 2 2 3 3 4 40 21 1 1 0.673 0.339 0.166 0.190 2 2 3 3 ;ARAG .,, * 41 22 20 0.229 0.376 0.121 0.120 233 e 42 22 0 0.456 0.212 0.132 0.228 2 233 5 43 22 1 0.512 0.165 0.099 0.206 2 2 3 3 5 44 22 2 1.194 0.202 0.132 0.222 2 2 3 3 . 45 23 1 2 0.928 0.218 0.099 0.358 2 2 3 3 s 46 24 2 0 0.634 0.181 0.110 0.320 2 2 3 3 5 47 25 0 1 0.722 0.260 0247 0.309 2 2 3 3 48 25 0.678 0207 0.208 0.250 2 2 3 3 25 49 26 0 0 0.589 0.323 0.870 0.103 2 2 3 3 5 50 27 1 0.551 0.186 0.115 0260 2 2 3 3 AVERAGE S l> . 51 0 0.728 0.424 0.110 0.131 2 2 3 3 52 29 1 0 0.545 0.773 0.170 0.125 2 2 3 3 53 29 1 2 0.850 0.620 0208 0.201 2 2 3 3 254 30 0 1.227 0.916 0.159 0.136 233 2-; 55 30 0 2 0.667 0.720 0.170 0212 233 256 31 6 0 0.606 0.487 0.159 0.226 233 257 31 0 1 0.168 0244 0.154 0.114 233 256 31 2 0.628 0.588 0.154 0.179 233 259 31 2 n r 1 0.578 0.419 0.154 0.168 233 260 32 T 1 0.467 0.366 0.126 0.325 2 33 2sv 7 . 61 33 2 0 1.349 0.196 0.263 0.168 2 33 27 62 34 1 0 0.717 0228 0.361 0.163 233 2r 63 35 0 2 0.650 0.249 0.356 0212 233 __ 64 36 0 0 0.856 0244 0.203 0.217 2 2 3 3 *7 65 36 1 2 0.784 0.265 0.236 0260 2 2 3 3 7 66 37 0 0 0.539 0.329 0318 0212 2 2 3 3 7 67 38 0 0 0.606 0202 0.312 0.201 2 2 3 3 7 * 68 38 2 0 0.426 0.265 0.225 0.379 2 2 3 3 7 69 39 1 0 0.573 0302 0230 0212 2 2 3 3 70 39 0 1 0.473 0.255 0.132 0.136 2 2 3 3 AVERAGE " .-XU- ,, excluded excluded excluded excluded excluded 655 656 741 742 748 85 T T 86 0 0 96 ! 2 1 96 0 2 97 2 0 1.954 0.978 0.917 0.251 0256 2 2 2 2 2I week 0 0.69 0.934 1.9 1.412 3.332 1.29 1.19 0.99 1.124 1.512 1.46 0.73 1.168 1.39 1.278 1.922 2732 1.666 0.834 1.178 0.934 1.002 1.41 0.57 1.146 1.212 1.956 1.322 1.59 1.312 0.956 1.056 0.824 1.8 1.32 0.36 1.712 1.268 0.89 1.012 1.178 0.978 1.478 1.988 1.124 1.346 1.30 0 0.456 0.912 1.024 2.388 1.856 1.268 1.444 1.356 1.178 1.102 1.30 0.53 1.4& 1.09 1.7 2.454 1.334 1.212 0.336 1.256 1.156 0.934 1.29 0.54 2.696 1.434 1.3 1.712 1.568 1.078 1-212 0.656 1.146 0.946 1.40 0.53 3.908 1.956 1.834 0.502 0.512 TG immol/n week 1 week 4 0.774 0.771 0.54 0.789 1.462 1.218 1.166 0.944 125 0.964 0.87 0.987 0.642 1.035 0.741 0.624 ! 0.954 0.888 0.912 0.558 1.02 0.79 026 0.16 0.888 1.334 0.69 0.822 1.068 0.657 1.07 0.657 1.684 1.376 1.02 1266 2.16 0.752 1.545 0.771 0.954 0.71 0.756 0.642 1.18 0.92 0.46 0.30 0.552 0.363 0.614 1.524 0.444 0.921 1.864 1.116 0.912 0.964 0.429 0.444 0.752 0.764 0.429 0.411 0.806 1.346 0.624 0.936 1.01 0.61 0.43 027 0.488 0.411 0.562 0.429 0.552 0.466 0.363 0297 0.414 0.594 0.658 0296 0.69 1 0.396 < 0.838 0.54 0.678 0.444 0.312 0.558 0.58 0.43 0.12 0.12 0.752 0.363 0.424 0.396 0.33 0297 0.404 0.436 0.362 0.396 0.297 023 0.52 0.414 0.741 0.624 0.646 0.372 0.345 0.47 0.42 0.14 0.16 0.848 1.546 1.24 023 021 0.624 1.832 1.44 0.974 0.477 021 0.477 0.488 1.176 0.462 0.462 0.838 0.732 0.462 0278 1.11 0.47 0.41 0.06 0.392 0.456 0.789 1.083 0.498 1.068 0.468 0.53 0.609 0.708 ! 0.658 0.404 0.53 0.604 0.51 0.51 0.08 0.954 0.936 0.675 0.69 0296 0.79 022 week 8 0.894 0.798 0.699 0831 0.831 0.732 1.413 0.879 0.699 0.636 0.84 022 0.765 1.008 0.765 0.666 1283 1.965 1.755 0.603 0.96 0.585 1 . 0.49 0.57 0.603 0.699 0.504 027 0.732 0.456 0.585 1.008 1.107 0.68 021 0246 0246 0281 0.309 0279 0.618 0246 0.246 0.309 0.57 0.33 0.14 0.36 0.684 0.618 0.666 1.074 0.96 0.927 0.75 0.309 0.78 0.76 0.21 0.393 0275 0.603 0.408 0.636 0.684 0.342 0.537 0.504 0875 0.55 0.19 0.504 0.489 0.636 0.651 0.78 0.636 0.603 1.137 0.636 0.408 0.65 020 I Page A15of A172 54 Appendix to TNO-report of 3M-02 Final version 04-12-2006 SS Page A16ofA172 Appendix to TNO-report of 3M-02 Final version C3. Pooled lipoprotein profile measurements 04-12-2006 WeekO fraction 1 2 3 4 5 6 7 S 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured cholesterol (mM) pooled sample 1 0.008 0.002 0.021 0.043 0.292 0.295 0.166 0.109 0.087 0.109 0.115 0.147 0.222 0.266 0.257 0.216 0.232 0.295 0.307 0.229 0.147 0.093 0.071 0.040 dilution cholesterol (mM) 2 0.016 2 0.004 2 0.042 2 0.086 2 0.584 2 0.590 2 0.332 2 0.218 2 0.174 2 0.218 2 0.230 2 0.294 2 0.444 2 0.532 2 0.514 2 0.432 2 0.464 2 0.590 2 0.614 2 0.458 2 0.294 2 0.186 2 0.142 2 0.080 measured cholesterol (mM) pooled sample 2 0.012 0.008 0.012 0.068 0.248 0.235 0.131 0.093 0.075 0.078 0.100 0.134 0.188 0.222 0.229 0.194 0.210 0.288 0.288 0203 0.122 0.090 0.049 0.040 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 cholesterol (mM) 0.024 0.016 0.024 0.136 0.496 0.470 0262 0.186 0.150 0.156 0.200 0268 0.376 0.444 0.458 0.388 0.420 0.576 0.576 0.406 0244 0.180 0.098 0.080 measured cholesterol (mM) pooled sample 3 0.012 0.015 0.005 0.062 0.270 0.285 0.163 0.144 0.103 0.103 0.134 0.181 0.282 0.314 0.279 0241 0.238 0.339 0.370 0270 0.166 0.093 0.059 0.040 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 cholesterol (mM) 0.024 0.030 0.010 0.124 0.540 0.570 0.326 0.288 0.206 0.206 0.266 0.362 0.564 0.628 0.558 0.482 0.476 0.678 0.740 0.540 0.332 0.186 0.118 0.080 Average plasma cholesterol (mm ol/l) 0.021 0.017 0.025 0.115 0.540 0.543 0.307 0.231 0.177 0.193 0.233 0.308 0.461 0.535 0.510 0.434 0.453 0.615 0.643 0.468 0.290 0.184 0.119 0.080 sd 0.005 0.013 0.016 0.026 0.044 0.064 0.039 0.052 0.028 0.033 0.034 0.049 0.095 0.092 0.050 0.047 0.029 0.055 0.086 0.068 0.044 0.003 0.022 0.000 PL fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured PL (mg/dl) pooled sample 1 0.000 0.000 0.000 0.000 3.776 4.429 2.305 1.706 1.597 1.652 1.978 3.503 5.083 8.623 7.752 7.261 9.059 12.762 15.104 12.436 10.094 5.954 3.939 1.760 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 PL (mM) 0.000 0.000 0.000 0.000 0.058 0.068 0.036 0.026 0.025 0.026 0.031 0.054 0.079 0.133 0.120 0.112 0.140 0.197 0.234 0.192 0.156 0.092 0.061 0.027 measured PL (mg/dl) pooled sample 2 0.000 0.000 0.003 0.984 3.513 4.235 2.584 2.016 1.706 1.861 2.016 3.409 5.732 7.022 8.416 7.435 8.622 15229 15.177 13.061 9.913 7.704 3.822 2.171 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 PL (mM) 0.000 0.000 0.000 0.015 0.054 0.065 0.040 0.031 0.026 0.029 0.031 0.053 0.089 0.109 0 .t3 0 0.115 0.133 0.235 0.235 0.202 0.153 0.119 0.059 0.034 measured PL (mg/dl) pooled sample 3 0.000 0.000 0.000 0.000 4.145 4.199 2.670 1.850 1.468 1.796 2.561 3.708 7.313 9.389 10.044 8.460 10.099 17.036 19.167 15.944 10.918 7.969 4.418 1.632 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 PL (mM) 0.000 0.000 0.000 0.000 0.064 0.065 0.041 0.029 0.023 0.028 0.040 0.057 0.113 0.145 0.155 0.131 0.156 0.263 0.296 0.246 0.169 0.123 0.068 0.025 Average plasma PL(mM) 0.000 0.000 0.000 0.005 0.059 0.066 0.039 0.029 0.025 0.027 0.034 0.055 0.093 0.129 0.135 0.119 0.143 0.232 0.255 0.214 0.159 0.111 0.063 0.029 sd 0.000 0.000 0.000 0.009 0.005 0.002 0.003 0.002 0.002 0.002 0.005 0.002 0.018 0.019 0.018 0.010 0.012 0.033 0.036 0.029 0.008 0.017 0.005 0.004 Page A17 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 week 4 Cholesterol fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured cholesterol (mM) pool group 1 0.006 0.006 0.006 0.028 0.136 0.139 0.096 0.074 0.071 0.083 0.126 0.197 0.272 0.284 0.241 0.204 0.278 0.442 0.429 0.265 0.151 0.099 0.065 0.040 Average dilution cholesterol (mM) group 1 2 0.012 2 0.012 2 0.012 2 0.056 2 0272 2 0.278 2 0.192 2 0.148 2 0.142 2 0.166 2 0.252 2 0.394 2 0.544 2 0.568 2 0.482 2 0.408 2 0.556 2 0.884 2 0.858 2 0.530 2 0.302 2 0.198 2 0.130 2 0.080 measured cholesterol (mM) pool group 2 0.012 0.009 0.009 0.062 0.185 0.157 0.099 0.074 0.071 0.077 0.120 0.173 0.225 0.231 0.219 0.207 0.253 0.358 0.327 0.204 0.123 0.080 0.049 0.034 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average cholesterol (mM) group 2 0.024 0.018 0.018 0.124 0.370 0.314 0.198 0.148 0.142 0.154 0.240 0.346 0.450 0.462 0.438 0.414 0.506 0.716 0.654 0.408 0.246 0.160 0.098 0.068 measured cholesterol (mM) pool group 3 0.009 0.012 0.009 0.031 0.108 0.142 0.089 0.077 0.068 0.083 0.108 0.157 0.204 0.207 0.182 0.179 0.222 0.337 0.352 0.238 0.136 0.086 0.049 0.031 Average diution cholesterol (mM) groups 2 0.018 2 0.024 2 0.018 2 0.062 2 0.216 2 0.284 2 0.178 2 0.154 2 0.136 2 0.166 2 0.216 2 0.314 2 0.408 2 0.414 2 0.364 2 0.358 2 0.444 2 0.674 2 0.704 2 0.476 2 0.272 2 0.172 2 0.098 2 0.062 fra c tio n 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 m easured cholesterol (m M ) pool group 4 0 .0 2 0 0 .0 1 4 0 .0 2 7 0 .0 3 0 0 .0 8 4 0 .0 5 8 0 .0 4 8 0 .0 3 0 0 .0 2 7 0 .0 3 0 0.054 0 .1 0 7 0 .2 1 5 0 .3 2 4 0 .3 2 4 0.271 0.231 Q.203 0 .1 6 0 0 .1 0 7 0 .0 6 4 0 .0 4 5 0 .0 3 6 0 .0 2 4 d ilu tio n 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 A ve ra g e cholesterol (m M ) group 4 0 .0 4 0 0 .0 2 8 0.054 0 .0 6 0 0 .1 2 8 0 .1 1 6 0 .0 9 6 0 .0 6 0 0 .0 5 4 0 .0 6 0 0 .1 0 8 0.214 0 .4 3 0 0 .6 4 8 0 .6 4 8 0 .5 4 2 0 .4 6 2 0 .4 0 6 0 .3 2 0 0 .2 1 4 0 .1 2 8 0 .0 9 0 0 .0 7 2 0 .0 4 8 m easured ch olesterol (m M ) d iu tio n pool group 5 0 .0 0 8 2 0 .0 0 8 0 .0 0 5 2 2 0 .0 2 4 0 .0 7 6 2 2 0 .0 7 9 0.051 2 2 0 .0 3 6 2 0 .0 3 3 2 0 .0 3 0 0 .0 3 0 2 2 0.061 0 .1 3 2 0 .2 0 9 0 .2 5 9 0.231 0 .2 0 9 0 .2 0 9 0 .1 7 5 0 .1 1 6 0 .0 7 3 2 2 2 2 2 2 2 2 2 2 0 .0 3 9 0 .0 3 6 2 2 0 .0 1 7 2 A ve ra g e cholesterol (m M ) group 5 0 .0 1 6 0 .0 1 6 0 .0 1 0 0 .0 4 8 0.152 0 .1 5 8 0 .1 0 2 0 .0 7 2 0 .0 6 6 0 .0 6 0 0 .0 6 0 0 .1 2 2 0 .2 6 4 0 .4 1 8 0 .5 1 8 0 .4 6 2 0 .4 1 8 0 .4 1 8 0 .3 5 0 0 .2 3 2 0 .1 4 6 0 .0 7 8 0 .0 7 2 0 .0 3 4 m easured cholesterol (m M ) pool group 6 0.011 0008 0011 0 .0 2 4 0.054 0 .0 7 3 0.039 0 .0 3 6 0 .0 2 7 0 .0 3 0 0 .0 3 6 0.064 0 .1 3 5 0 .2 1 5 0249 0 .2 1 8 0 .1 9 7 0 .1 8 4 0 .1 6 0 0110 0 .0 6 4 0 .0 3 9 0 .0 2 7 0 .0 1 4 d ilu tio n 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 A ve ra g e m easured cholesterol (m M ) cholesterol (m M ) group 6 pool group 7 0 .0 2 2 0 .0 1 6 0 .0 1 6 0 .0 2 2 0 .0 1 3 0 .0 1 0 0 .0 4 8 0 .1 0 8 0 .0 2 5 0 .1 2 0 0 .1 4 6 0 .0 7 8 0.126 0 .0 6 5 0 .0 7 2 0 .0 5 4 0 .0 4 9 0.031 0 .0 6 0 0 .0 7 2 0 .0 3 4 0 .0 4 3 0 .1 2 8 0 .2 7 0 0 .0 9 5 0 .2 4 6 0 .4 3 0 0 .4 9 8 0 .4 3 6 0 .3 9 4 0 .4 3 6 0 .5 2 5 0 .4 5 8 0 .3 5 9 0 .3 6 8 0 .3 2 0 0 .2 2 0 0.128 0.078 0 .2 7 7 0 .2 0 3 0 .1 2 9 0 .0 8 3 0 .0 4 9 0.054 0 .0 4 0 0 .0 2 8 0 .0 2 5 d ilu tio n 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 A verage cholesterol (m M ) group 7 0 .0 3 2 0 .0 2 8 0 .0 2 0 0 .0 5 0 0 .2 4 0 0 .2 5 2 0 .1 3 0 0 .0 9 8 0 .0 6 2 0 .0 6 8 0 .0 8 6 0 .1 9 0 0 .4 9 2 0 .8 7 2 1 .0 5 0 0 .9 1 6 0 .7 1 8 0 .5 5 4 0 .4 0 6 0 .2 5 8 0 .1 6 6 0 .0 9 8 0 .0 8 0 0 .0 5 0 57 Page A18ofA172 Appendix to TNO-report of 3M-02 Final version EL fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured PL(mg/dl) pool group 1 0.000 0.000 0.000 0.000 0.113 0.247 0.000 0.000 0.000 0.000 0.918 3.201 5.752 7.095 6.289 6.155 12.063 22.804 23.744 14.748 8.303 4.946 2.261 0.247 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL(mM) group 1 0.000 0.000 0.000 0.000 0.002 0.004 0.000 0.000 0.000 0.000 0.014 0.049 0.089 0.110 0.097 0.095 0.186 0.353 0.387 0.228 0.128 0.076 0.035 0.004 measured PL{mg/til) pool group 2 0.000 0.000 0.000 0.000 1.321 1.187 0.000 0.000 0.000 0.000 0.650 2.664 4.275 5.886 5.618 5.886 10.317 17.702 17.433 10.586 5.752 3.872 1.187 0.000 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group 2 0.000 0.000 0.000 0.000 0.020 0.018 0.000 0.000 0.000 0.000 0.010 0.041 0.066 0.091 0.087 0.091 0.160 0.274 0.270 0.164 0.089 0.060 0.018 0.000 04-12-2006 measured PL(mg/dl) pod group 3 0.000 0.000 0.000 0.000 0.000 0.516 0.000 0.000 0.000 0.000 0.381 1.992 3.604 4.141 4.409 4.678 8.840 17.031 19.448 12.331 7.363 4.812 1.321 0.000 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group 3 0.000 0.000 0.000 0.000 0.000 0.008 0.000 0.000 0.000 0.000 0.006 0.031 0.056 0.064 0.068 0.072 0.137 0.263 0.301 0.191 0.114 0.074 0.020 0.000 fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured PL(mgfdl) POOlQfDUp4 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.939 4.149 8.162 9.232 8162 6.557 6.82S 5.487 2.946 1.340 0 939 0.000 0000 caution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL(mM) group 4 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.015 0.064 0.126 0.143 0.126 0.101 0.106 0.085 0.046 0.021 0.015 0 000 0.000 measured PL(mgWI) poolgroup 5 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.742 4.551 6.825 6.825 6.022 7.226 6.022 3.481 1.875 0.672 0.000 0.000 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group 5 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.027 0.070 0.106 0.106 0.093 0.112 0.093 0.054 0.029 0.010 0.000 0.000 measured PL(mg/dO pool group 6 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.742 4.283 6.022 5.621 5.219 5.487 5.086 2.812 1.207 0.538 0.000 0.000 <audon 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL(mM) group 6 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.027 0.066 0.093 0.087 0.081 0.085 0.079 0.043 0.019 0.008 0.000 0.000 measured PUmgf<) pool croup 7 0.000 0.000 0.000 0.000 0.000 0.187 0.000 0.000 0.000 0.000 0.000 0.858 5.690 11.998 16.024 15.085 12.400 10.253 7.435 4.34B 2.603 1.529 0.321 0.000 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL{mM) group 7 0.000 0.000 0.000 0.000 0.000 0.003 0.000 0.000 0.000 0.000 0.000 0.013 0.088 0.185 0.248 0.233 0.192 0.159 0.115 0.067 0.040 0.024 0.005 0.000 Page A19ofA172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Week 6 Chotesterol fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured cholesterol (mM) pool group 1 0.010 0.001 0.004 0.025 0.135 0.135 0.083 0.065 0.056 0.074 0.129 0.223 0.293 0.293 0.238 0.183 0.241 0.368 0.347 0.220 0.132 0.092 0.062 0.044 Average dilution chotesterol (mM) group 1 2 0.020 2 0.002 2 0.008 2 0.050 2 0270 2 0.270 2 0.166 2 0.130 2 0.112 2 0.148 2 0.258 2 0.446 2 0.586 2 0.586 2 0.476 2 0.366 2 0.482 2 0.736 2 0.694 2 0.440 2 0.264 2 0.184 2 \2 0.124 0.088 measured chotesterol (mM) pool group 2 0.007 0.007 0.004 0.050 0.174 0.147 0.083 0.065 0.050 0.068 0.114 0.186 0.226 0211 0.180 0.162 0.220 0.274 0.241 0.153 0.086 0.059 0.035 0.022 dfiution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average cholesterol (mM) group2 0.014 0.014 0.008 0.100 0.348 0.294 0.166 0.130 0.100 0.136 0.228 0.372 0.452 0.422 0.360 0.324 0.440 0.548 0.482 0.306 0.172 0.118 0.070 0.044 measured cholesterol (mM) pool group 3 0.013 0.013 0.010 0.062 0.189 0.168 0.092 0.077 0.062 0.083 0.123 0.180 0.223 0.211 0.162 0.156 0.235 0.329 0.296 0.189 0.114 0.074 0.047 0.032 Average dilution chotesterol (mM) group 3 2 0.026 2 0.026 2 0.020 2 0.124 2 0.378 2 0.336 2 0.184 2 0.154 2 0.124 2 0.166 2 0.246 2 0.360 2 0.446 2 0.422 2 0.324 2 0.312 2 0.470 2 0.658 2 0.592 2 0.378 2 0.228 2 0.148 2 0.094 2 0.064 fraction 1 2 3 4 5 6 7 8 3 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured cholesterol (mM) poolgroup 4 0.016 0.004 0.010 0.022 0.068 0.056 0.034 0.031 0.025 0,028 0.059 0.138 0.251 0.315 0.300 0.217 0.168 0.129 0.0) 0.056 0.037 0.028 0.025 0.019 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average measured Average measured cholesterol (mM) chotesterol (mM) dilution chotesterol (mM) chotesterol (mM) group 4 pod group 5 group 5 pool group 6 0.032 0.010 2 0.020 0.016 0.008 0.004 2 0.008 0.025 0.020 0 044 0.010 2 0.020 0.077 2 0.154 0.016 0.037 0.138 0.112 0.068 0.062 0.050 0.056 0.118 0.276 0.248 2 0.496 20.193 0.386 20.117 0.234 20.080 0.160 20.071 0.142 20.077 0.154 20.129 0.258 20.220 0.440 0.071 0.071 0.037 0.034 0.031 0.037 0.053 0.120 0.502 0.281 2 0.562 0.214 0.630 0.251 2 0.502 0.266 0.600 0.202 2 0.404 0.266 0.434 0.156 2 0.312 0.211 0.336 0.205 2 0.410 0.156 0.258 0.281 2 0.562 0.126 0.160 0.254 2 0.508 0.080 0.112 0.150 2 0.300 0.049 0.074 0.092 2 0.184 0.037 0.056 0.056 2 0.112 0.025 0.050 0.043 2 0.086 0.016 0.038 0.037 2 0.074 0.013 dBution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average measured chotesterol (mM) cholesterol (mM) group 6 poolgroup 7 0.032 0.008 0 050 0.011 0.032 0.011 0.074 0029 0.142 0.093 0.142 0.081 0.074 0.051 0.068 0.045 0.062 0.036 0.074 0.045 0.106 0.075 0.240 0.142 0.428 0.306 0.532 0.425 0.532 0.407 0.422 0.306 0.312 0.206 0.252 0.139 0.160 0.081 0.098 0.054 0.074 0.039 0.050 0.033 0.032 0.026 0.026 0.017 diution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average cholesterol (mM) grotte 7 0.016 0.022 0.022 0.058 0.186 0.162 0.102 0.090 0.072 0.090 0.150 0.284 0.612 0.850 0.814 0.612 0.412 0.278 0.162 0.108 0.078 0.066 0.052 0.034 5? Page A20ofA172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 EL fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured PL(mgAU) pool group 1 0.000 0.000 0.000 0.000 1.035 1.166 0.246 0.115 0.115 1.035 2.349 4.977 6.948 7.605 6.685 6.685 11.415 19.168 18.248 11.678 7.342 4.977 2.086 0.772 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group 1 0.000 0.000 0.000 0.000 0.016 0.018 0.004 0.002 0.002 0.016 0.036 0.077 0.107 0.118 0.103 0.103 0.176 0.296 0.282 0.181 0.114 0.077 0.032 0.012 measured PL(mg/dl) pool group 2 0.000 0.000 0.000 0.000 1.823 1.297 0.903 0.640 0.246 0.509 1.823 4.057 5.502 6.159 5.897 6.028 9.313 13.649 12.729 8.525 4.714 2.874 1.035 0.115 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group 2 0.000 0.000 0.000 0.000 0.028 0.020 0.014 0.010 0.004 0.008 0.028 0.063 0.085 0.095 0.091 0.093 0.144 0.211 0.197 0.132 0.073 0.044 0.016 0.002 measured PL(mg/dl) pool group 3 0.000 0.000 0.000 0.000 1.429 1.429 0.115 0.115 0.000 0.640 2.743 3.400 4.451 4.845 4.188 5.108 9.050 14.963 14.306 9.182 5.239 3.268 1.035 0246 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group 3 0.000 0.000 0.000 0.000 0.022 0.022 0.002 0.002 0.000 0.010 0.042 0.053 0.069 0.075 0.065 0.079 0.140 0.231 0.221 0.142 0.081 0.051 0.016 0.004 fraction 1 2 3 4 5 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured PL(mg/dl) pool group 4 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0000 0.000 0.000 0.505 2 599 6.264 9.405 9.798 7.835 5.872 4.301 2.861 1.683 1.291 0.636 0.000 0.000 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group 4 0.000 0.000 0.000 0.000 0.000 0.000 0.00 0.000 0.000 0.000 0.008 0.040 0.097 0.145 0.151 0.121 0.091 0.066 0.044 0.026 0.020 0.010 0.000 0.000 measured PL(mg/dl) poolgroup 5 0.000 0.000 0.000 0.113 2.338 1.945 0.898 0.505 0.SO5 0.898 2.730 5.479 7.442 7.442 6.133 6.002 8.489 12.808 12.547 8.227 5.217 2.992 1.160 0.243 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group S 0.000 0.000 0.000 0.002 0.036 0.030 0.014 0.008 0.008 0.014 0.042 0.085 0.115 0.115 0.095 0.093 0.131 0.198 0.194 0.127 0.081 0.046 0.018 0.004 measured PL(mg/dl) pool group 6 0.000 0.000 0.000 0.000 0.000 0.505 0.000 0.000 0.000 0.000 0.374 2.338 5.348 7.966 8.882 7.049 5.610 4.039 2.599 1.552 0.898 0.505 0.000 0.000 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL(mM) group 6 0000 0.000 0.000 0.000 0.000 0.008 0.000 0.000 0.000 0.000 0.006 0.036 0.083 0.123 0.137 0.109 0.087 0.062 0.040 0.024 0.014 0.008 0.000 0.000 measured PUmg/dl) poolgroup 7 0.000 0.000 0.000 0.000 0.282 0.414 0.000 0.000 0.000 0.017 0.678 3.583 8.866 13.621 14.413 11.508 7.281 4.640 2.131 1.074 0.678 0.414 0.000 0.000 diution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) 9roup7 0.000 0.000 0.000 0.000 0.004 0.006 0.000 0.000 0.000 0.000 0.010 0.055 0.137 0.211 0.223 0.178 0.113 0.072 0.033 0.017 0.010 0.006 0.000 0.000 Page A21 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 W eek 8 Cholesterol fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured cholesterol (mM) poo) group 1 0.000 0.000 0.000 0.036 0.191 0.167 0.103 0.078 0.078 0.100 0.164 0.267 0.328 0.292 0.240 0.191 0.249 0.346 0.346 0.200 0.109 0.066 0.048 0.027 Average diution cholesterol (mM) group 1 2 0.000 2 0.000 2 0.000 2 0.072 2 0.382 2 0.334 2 0.206 2 0.156 2 0.156 2 0.200 2 0.328 2 0.534 2 0.656 2 0.584 2 0.480 2 0.382 2 0.498 2 0.692 2 0.692 2 0.400 2 0.218 2 0.132 2 0.096 2 0.054 measured cholesterol (mM) pool group 2 0.000 0.000 0.002 0.066 0.228 0.182 0.109 0.078 0.069 0.087 0.139 0.200 0.249 0.252 0.237 0.197 0.237 0.276 0.240 0.136 0.078 0.051 0.023 0.020 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average cholesterol (mM) group 2 0.000 0.000 0.004 0.132 0.456 0.364 0.218 0.156 0.138 0.174 0.278 0.400 0.498 0.504 0.474 0.394 0.474 0.552 0.480 0.272 0.156 0.102 0.046 0.040 measured cholesterol (mM) pool group 3 0.000 0.011 0.011 0.054 0.206 0.179 0.112 0.091 0.075 0.112 0.164 0.246 0.298 0.273 0.237 0.188 0.231 0.310 0.288 0.170 0.097 0.063 0.030 0.020 Average dilution cholesterol (mM) group 3 2 0.000 2 0.022 2 0.022 2 0.108 2 0.412 2 0.358 2 0-224 2 0.182 2 0.150 2 0.224 2 0.328 2 0.492 2 0.596 2 0.546 2 0.474 2 0.376 2 0.462 2 0.620 2 0.576 2 0.340 2 0-194 2 0.126 2 0.060 2 0.040 fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured cholesterol (mM) poolgroup 4 0.015 0.000 0.006 0.021 0.061 0.052 0.023 0.021 0.012 0.035 0.044 0.118 0571 0.357 0.363 0580 0.205 0.162 0.107 0.067 0.044 0.023 0.018 0.015 diution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average measured Average measured cholesterol (mM) cholesterol (mM) dilution cholesterol (mM) cholesterol (mM) group 4 poolgroup 5 group 5 pooioroupS 0.030 0.000 2 0.000 0.000 0.001 0.000 2 0.000 0.006 0.012 0.006 2 0.012 0.009 0.042 0.041 2 0.082 0.035 0.122 0.153 2 0.306 0.107 0.104 0.130 2 0.260 0.095 0.046 0.078 2 0.156 0.061 0.042 0.058 2 0.116 0.044 0.024 0,049 2 0.098 0 038 0.070 0.058 2 0.116 0.032 0.088 0.098 2 0.196 0.067 0.236 0.159 2 0.318 0150 0.542 0510 2 0.420 0565 0.714 0525 2 0.450 0.372 0.726 0508 2 0.416 0.377 0.560 0.179 2 0.358 0.300 0.410 0.324 0.214 0.199 2 0.398 0.257 2 0.514 0522 2 0444 0.239 0.196 0.150 0.134 0.144 2 0588 0.092 0.088 0.075 2 0.150 0.052 0.046 0.052 2 0.104 0.032 0.036 0.029 2 0.058 0.021 0.030 0.029 2 0.058 0.021 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average measured cholesterol (mM) cholesterol (mM) groupe poolgroup 7 0.001 0.012 0.000 0.000 0.018 0.070 0.003 0.041 0514 0.167 0.190 0.135 0.122 0.073 0.088 0.076 0.056 0.038 0.064 0.050 0.134 0.079 0.300 0.170 0.530 0.744 0.754 0.302 0.419 0.419 0.600 0.478 0.328 0.246 0.392 0.300 0.184 0.104 0.064 0.199 0.138 0.085 0.053 0.035 0.042 0.023 0.042 0.011 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average cholesterol (mM) group 7 0.000 0.000 0.006 0.082 0.334 0.270 0.146 0.112 0.076 0.100 0.158 0.340 0.604 0.638 0.838 0.656 0.492 0.398 0.276 0.170 0.106 0.070 0.046 0.022 Page A22ofA172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 EL fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured PL(mg/dl) pool group 1 0.000 0.000 0.000 0.000 2.067 1.800 0.601 0.201 0.334 1.400 3.266 6.599 8.998 8.732 7.132 7.265 11.931 20.595 18.596 11.531 7.265 4.733 2.467 0.867 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group 1 0.000 0.000 0.000 0.000 0.032 0.028 0.009 0.003 0.005 0.022 0.050 0.102 0.139 0.135 0.110 0.112 0.184 0.318 0.287 0.178 0.112 0.073 0.038 0.013 measured PL(mgMI) pool group 2 0.000 0.000 0.000 0.201 3.000 2.067 0.867 0.201 0.334 1.000 2.600 4.999 6.866 7.932 7.665 8.065 10.731 15.130 12.864 8.065 5.266 3.266 1.000 0.000 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2^ 2 2 2 2 2 2 2 2 Average PL (mM) group 2 0.000 0.000 0.000 0.003 0.046 0.032 0.013 0.003 0.005 0.015 0.040 0.077 0.106 0.123 0.119 0.125 0.166 0.234 0.199 0.125 0.081 0.050 0.015 0.000 measured PL(mg/dl) pool group 3 0.000 0.000 0.000 0.000 2.200 2.067 0.867 0.334 0.734 1.400 3.533 6.199 8.199 8.732 7.265 7.399 10.864 17.929 15.930 9.798 6.732 4.200 1.534 0.334 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group 3 0.000 0.000 0.000 0.000 0.034 0.032 0.013 0.005 0.011 0.022 0.055 0.096 0.127 0.135 0.112 0.114 0.168 0.277 0.246 0.151 0.104 0.065 0.024 0.005 fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 measured PL(mgftfl) poolgrow 4 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.048 253 8.185 12.587 12.987 10.453 8.052 6.184 4.183 2.583 1.916 1.115 0.000 0.000 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL(mM) group 4 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.040 0.127 0.195 0.201 0.162 0.124 0.096 0.065 0.040 0.030 0.017 0.000 0.000 measured PMmgfdl) pool group 5 0.000 0.000 0.000 0.000 1.515 1.249 0.181 0.000 0.000 0,448 1.515 3.916 5.517 6.8S1 6.851 7.118 9.786 14.454 13.521 8.318 4.450 3.116 0848 0.315 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL(mM) group 5 0.000 0.000 0.000 0000 0.023 0.019 0.003 0.000 0.000 0.007 0.023 0.061 0.085 0.106 0.106 0.110 0.151 0.223 0.209 0.129 0.069 0.048 0.013 0.005 measured PL(mg/dl) poolgroup 6 0.000 0.000 0.000 0.000 0.715 0.582 0.000 0 000 0000 0.000 0.715 3.783 8.452 12.720 13.120 11.386 9.519 8.318 5.917 3.650 2.983 2.182 0.315 0.000 (fihition 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) groups 0.000 0.000 0.000 0.000 0.011 0.009 0.000 0.000 0.000 0.000 0.011 0.058 0.131 0.197 0.203 0.178 0.147 0.129 0.091 0.056 0.046 0.034 0.005 0.000 measured PL(mg/dl) pool group 7 0.000 0.000 0.000 0.000 1.619 0.951 0.150 0.000 0.000 0.000 0.951 4.290 10.033 13.639 14.707 12.570 10.166 8.163 5.626 3.622 2.687 1.619 0.150 0.000 dilution 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Average PL (mM) group 7 0.000 0.000 0.000 0.000 0.025 0.015 0.002 0.000 0.000 0.000 0.015 0.066 0.155 0.211 0.227 0.194 0.157 0.126 0.087 0.056 0.042 0.025 0.002 0.000 Page A23 of A172 Appendix to TNO-report of 3M-02 Final version C4. Pooled plasma ALAT measurements 04-12-2006 group Group 1: HF control Group 2: HF + 0.01 % PFBS Group 3: HF + 0.03 % PFBS Group 4: HF + 0.006 % PFHS Group 5: HF + 0.02 % PFHS Group 6: HF + 0.003 % PFOS Group 7: HF + 0.01 % PFOS week 0 97.1 measured week 4 27.1 30.6 23.5 88.7 46 109 119 Pooled plasma ALAT (U/l) dilution week 4 week 6 (o/n fast) 3 81.3 3 91.8 3 70.5 3 266.1 3 138 3 327 3 357 84 113 108 213 130 246 307 week 8 57 134 87.2 370 180 394 450 Page A24of A172 Appendix to TNO-report of 3M-02____________ Final version p. 60 04-12-2006 C 5. P lasm a ketone bodies m easurem ents W eek-1 concentration 3dm (mmolA) 0.000 0.000 0.033 0.033 0.067 0.067 0.134 0.134 0.266 0.268 0.535 0.535 1.070 1.070 2.140 2.140 A **!** 0.214 0114 0118 0115 0118 0122 0116 012 0118 0115 0118 0115 0117 0118 0117 0.22 A*# PC* 0.211 0114 0.222 012 0.223 0.239 0149 0157 0182 0164 0.348 0.358 0.462 0.484 0.689 0.718 netto -0.003 0 0.004 0.005 0.005 0.017 0.033 0.037 0.064 0.069 0.13 0.143 0145 0166 0.472 0.496 0.0 0 4 1.0 1.5 2.0 2.5 Kten beJK (wweW) yxs * b mouse# 641 645 646 650 652 6S5 656 657 658 663 685 666 667 670 671 674 678 679 680 681 686 687 690 696 699 700 704 706 706 710 712 714 716 720 721 724 727 729 730 731 73? 738 740 741 742 744 748 752 754 756 758 759 760 761 762 763 765 766 768 769 771 774 776 778 782 783 784 785 790 791 792 793 795 797 799 a: I 0.2283 1 b: I 0.0028 I AwPX 0.303* 0.321 0.3 0169 0193 0329 0.307 0.381 0.293 0.35 0.245 0176 0173 0.3S1 0.324 0.464 091 0447 0134 0.468 0352 0.467 0.356 0.274 0.3 0.305 0.311 0194 0192 0419 0.388 0169 0133 0.346 0102 0.347 0.29 0144 0117 017 0432 0.387 0.351 0149 0139 0142 0,26 0184 0166 0182 0133 0117 0.303 0193 0177 0197 0187 0109 0 383 0182 0.318 0157 0.31 0141 0198 0.282 0184 0.272 0.253 0.36 0192 0261 0271 0.339 0.301 A3 poet 0.766 0146 0.623 0.644 0.546 013 0.505 0.87 0.658 0.58 0489 015 0.668 0.621 0213 0.628 0.534 0.649 0579 0.736 0.608 0.638 0.644 0.546 0.563 0.S83 0.666 0.609 0.621 0.664 0.624 0.573 0.465 0.692 0.705 0.754 044 027 0.423 0.405 0.712 0.581 0.578 0.485 0418 0.516 0.429 0.445 0.631 0.558 0.664 0.613 0.605 0.639 0.623 0.69 0.62 0.627 0.632 0.693 0.533 0.554 0.521 0.466 0.596 0.556 0.653 0.645 0.4S2 0.589 0.58 0.403 0.561 0.712 0.572 Am netto 0.459 0.225 0.323 0.375 0153 0.201 0.198 0189 0.365 013 0.244 0.374 0.395 0.27 0189 0.164 0143 0102 0145 0.268 0156 0.171 0188 0.272 0.263 0.278 0.355 0.315 0.329 0145 0136 0184 0.132 0.346 0.403 0.407 0.15 0.126 0.106 0.135 018 0.194 0127 0136 0.179 0.274 0.169 0.161 0.265 0176 0.331 0196 0.302 0.346 0.346 0.393 0.333 0.318 0149 0.311 0115 0.197 0.211 0.225 0.296 0174 0.369 0.373 0.199 0.263 0.288 0.142 0.19 0.373 0.271 meas, concentration katonbocSs* (mmolfl) 2.00 0.97 1.40 1.63 1.10 0.87 0.86 115 1.59 1.00 1.06 1.63 1.72 1.17 1.25 0.71 1.05 0.87 1.06 1.16 1.11 0.74 115 1.18 1.14 1.21 1.54 1.37 1.43 1.06 1.02 113 0.57 1.50 1.75 1.77 0.64 0.54 0.45 0.58 1.21 0.64 0.98 1.02 0.77 1.19 0.73 0.69 1.15 110 1.44 1.26 1.31 1.50 1.50 1.71 1.45 1.38 1.08 1.35 0.93 0.85 0.91 0.97 1.29 1.19 1.60 1.62 0.88 1.14 115 0 61 0.82 1.82 1.17 dilution 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 Plasma keton- bodias (mmcM) 2.00 0.97 1.40 1.63 1.10 0.87 0.86 115 1.59 1.X 1.06 1.63 1.72 1.17 1.25 0.71 1.05 0.87 1.06 1.16 1.11 0.74 1.25 1.18 1.14 111 1.54 1.37 1.43 1.06 1.02 1.23 0.57 1.50 1.75 1.77 1.29 1.08 090 1.16 111 0.84 0.96 1.02 0.77 1.19 0.73 1.39 1.15 1.20 1.44 1.28 1.31 1.50 1.50 1.71 1.45 1.38 1.08 1.35 0.93 0.85 0.91 0.97 119 1.19 1.60 1.62 1.72 1.14 1.25 0.61 0.82 1.62 1.17 mousa# after matching 11 12 13 21 61 excluded excluded 62 1 2 41 42 43 S3 44 14 22 45 64 31 32 65 23 66 24 46 33 34 35 36 3 51 4 67 52 68 53 25 47 48 15 16 17 excluded excluded 49 excluded 5 26 27 6 28 18 69 70 7 19 29 37 54 38 55 56 57 58 59 8 20 9 10 39 30 40 60 50 Page A25 ofA172 Appendix to TNO-report of 3M-02____________ Final version p. 61 04-12-2006 W eek 6 concentration Kfttonbodies (mrnoW) A **p re A p p o s i A^o r>ao 0.000 0.000 0.256 0.254 0.242 0246 0.013 -0.008 0.033 0.255 0.251 0.004 0.033 0.258 0.254 -0.004 0.067 0.067 0.134 0.256 0.262 0.257 0.261 0.269 0.279 0.005 0.007 0.022 0.134 0.262 0.287 0.025 0.268 0.257 0.318 0.061 0288 0.256 0.319 0.063 0.535 0-258 0.388 a i3 0.535 0-257 0.3% 0.135 1.070 1.070 0.256 0.261 0.5 0.521 0.244 0.26 2.140 0.258 0.729 0.47 2.140 0.266 0.768 0302 y=ax * b Group * II 0.233 I Mouse# coge# 11 21 32 42 53 63 74 85 95 10 5 11 6 12 6 13 6 14 7 15 8 16 8 17 6 18 9 19 9 20 10 21 11 22 12 23 13 24 13 25 14 26 15 27 15 23 15 29 16 o 1> 31 16 32 18 33 19 34 19 35 19 36 19 37 20 4 38 20 39 21 40 21 - I s- \ s I I r I r s .4 5 -r 5. * -* 41 42 43 44 45 46 47 48 49 50 l ,_ i .iiirA H fc rT it- 51 52 53 54 55 56 57 58 60 22 22 22 22 23 24 25 25 26 27 28 29 29 30 30 31 31 31 31 32 -a -.. T________ !-------------- ? -- -- 61 62 63 t-. y . 64 tr 7 - 65 66 . . T * - - 67 .7 -- 68 -- 9 I 70 1 AfeC E ~ n : SO .... I 33 34 35 36 36 37 38 38 39 39 _ * a rt L 0 1 0 2 0 0 1 0 0 1 R 1 2 0 0 0 2 1 0 2 2 Amo pro 0.303 0.284 0.273 0.271 0.267 0.2? 0.263 02! 0.269 0.26 A u poet 0.443 0.402 0.383 0.359 0.382 0.363 0.424 0.38 0.387 0.379 1 5 0.289 0.413 2 1 0.288 0.343 Q 2 0273 0.382 0 1 0.288 0.468 1 0 0.282 0.395 0 1 0.299 0.347 2 0 0.306 0.364 0 0 0.271 0.413 2 1 0.267 0.366 1 0 0.27 0.383 0 0.279 0.443 0 2 0277 0.473 0 1 0.318 0.409 0.291 0.38 0 0262 0.421 0 1 0272 0.418 2 0 0261 0.419 2 0.269 0.374 0 2 028 0.445 0 0289 0.372 0 "6 .2 *4 0.409 0 2 0.266 0.381 0 0 0.275 0.424 1 0 0.275 0.439 0 1 0285 0.4S2 0 2 0.264 0.446 0 0 0263 0.417 1 1 0263 0.365 0 0 0272 0.49 1 1 o i h " 0.427 1 0 0259 ~ <U3 0 1 029 0.437 1 1 0299 0.541 1 2 0264 0.408 1 2 0.315 ossa 2 0 0.305 0.529 0 1 0.309 0299 1 1 0295 0.439 0 0 0253 0.44 1 2 0296 0.466 0 ' 1 0263 1 0 0261 1 2 0278 1 0 0262 0 2 0.262 0 0 0.276 0 1 0.264 0 2 0.274 2 2 0.283 2 1 .284 0.581 0.556 0.541 0.54 0.551 0.606 0.408 0.60$ 0.564 0.659 2 0 0.266 0251 1 0 0269 0.568 0 2 0266 0.566 0 0 0268 0.522 1 2 0.268 0.S41 0 0 027 0279 0 0 0.268 0215 2 0 0.269 0268 1 0 0.27? 0.618 0 1 0274 0.647 meas, concentracin dfcition Plasma keton- Am netto ketonbodies (mrnot/T) bodies {mmoM) 0.14 0.118 0.11 0.086 0.115 0.093 0.141 0.11 0.098 0.099 0.62 0.53 0.49 0.40 0.52 0.42 0.63 .49 0.44 0.45 3 1.87 3 1.59 3 1.4 3 120 3 1.55 3 127 3 1.88 3 1.48 3 1.33 3 1.34 0.124 0.055 0.19 0.16 0.113 0.048 0.056 0.142 0.099 0.113 025 0.26 0.49 0.80 0.51 0.23 026 0.63 0.45 0.51 i.5o 0.23 3 1.66 3 0.78 3 1.47 3 2.39 3 1.52 3 0.69 3 0.79 3 1.90 3 1.34 3 1.52 1.41 0.54 0.164 0.196 0.091 0.089 0.159 0.146 0.158 0.105 0.165 0.103 0.147 0.115 0.149 0.164 0.167 0.l 2 0.154 0.102 0218 0.154 0.178 0.147 0.242 0.124 0243 0224 0.09 Q.144 0.187 0.17 0.318 0295 0265 0.278 0 .2 0.327 0.144 0.331 0281 0.375 0.73 0.86 0.41 0.40 0.71 0.65 0.70 047 0.73 0.46 0.6S 0.52 0.66 0.73 0.74 0.72 0.68 0.46 0.96 066 0.78 0.65 1.06 0.55 1.07 0.98 0.41 0.64 023 0.75 1.39 1.29 1.16 1.22 1.26 1.43 0.64 1.44 1.23 1.63 3 3 3 3 3 3 3 3 3 3 I 3 3 3 3 3 3 3 3 3 31 3 3 3 31 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2.18 2.59 124 121 2.12 1.95 2.10 142 2.19 129 1.64 0.48 1.96 1.55 1.99 218 222 2.15 2.05 1.38 2.88 2.05 2.04 0.40 233 1.96 3.18 1.66 320 2S 123 1.92 248 226 232 065 4.16 3.87 3.48 3.65 3.79 428 1.92 4.33 3.68 4.90 3.61 0.78 0.285 125 3 3.74 0299 1.31 3 3.92 0298 1.30 3 3.91 0254 1.11 3 3.34 0273 1.19 3 3.58 0.309 1.35 3 4.05 0.347 121 3 4.54 0.299 1.31 3 392 0.341 1.49 3 4 46 0 3 3 1.62 3 4.87 4.03 047 Page A26ofA172 5 Appendix to TNO-report of 3M-02 Final version Group cage# _ eartag Plasma ketonbodies (mmol/l) w e ek-1 12 20 10 1.02 1.71 1.72 0.38 2.00 0.97 1.40 0.71 1.21 0.84 0.98 1.25 1.48 1.55 1,88 1.33 1.50 0.78 1.47 2.39 1.52 0.69 1.52 24 25 14 15 27 15 15 29 2.59 1.25 1.24 1.21 1.08 2.12 1.15 1.95 1.20 2.10 1.38 2.19 18 19 34 35 36 37 20 38 20 39 40 0.48 1.11 1.55 1.37 2.18 2.15 0.93 2.88 0.82 22 42 22 a 43 22 22 45 23 25 26 27 28 29 54 30 55 30 56 58 60 32 m ---- ' - 7 . - .} wRAe' V-SD excluded excluded (658) excluded (741) excluded (742) excluded (748) 65 67 68 70 33 34 35 36 36 37 38 38 39 1.06 1.72 1.25 1.21 1.19 1.17 1.22 1.75 0.85 0.91 1.29 1.62 1.25 2.33 3.18 2.95 1.23 0.65 3.65 4.28 1.92 4.33 4.90 0.78 0.74 4.0S 1.77 1.50 4.46 1.50 1.28 0.86 1.02 0.77 04-12-2006 Page A27ofA172 Appendix to TNO-report of 3M-02____________ Final version Appendix D. Measurements in metabolic cages D1, Code mice and metabolic cages p. 63 04-12-2006 D2. Average bodyweight mice in metabolic cages group# mouse# 2 3 4 6 21 22 24 27 4 31 4 32 4 37 4 38 6 51 6 53 6 54 6 55 metabolic cage# 101 105 201 205 102 106 202 206 103 107 203 207 104 108 204 208 Bodyweight (g) | Average bodyweight (g) begin metabolic cage end metabolic cage in metabolic cage 29.9 30.7 30.30 30.2 30 30.10 28 29.1 28.55 29.3 29.6 29.45 29.5 30.2 29.85 29.8 30.1 29.95 28.8 29.7 29.25 29.5 30.2 29.85 28.9 29.8 29.35 28.9 26.9 30.4 29 28.9 31.3 28.95 27.90 30.85 28.2 29.1 28.65 27.3 26.5 26.90 29.4 27.8 30.7 28.8 30.05 28.30 Page A28of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 D3. Food and drink intake measurements Food intake measurements Tim e (hr) 6.1 7 .4 8 .6 9 .9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 2 2 .4 23.6 2 4 .9 26.1 2 7 .4 2 8 .6 2 9 .9 31.1 3 2 .4 3 3 .6 3 4 .9 36.1 3 7 .4 3 8 .6 39.9 41.1 42.4 4 3 .6 4 4 .9 46.1 4 7 .4 4 8 .6 4 9 .9 51.1 5 2 .4 5 3 .6 5 4 .9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 6 4 .9 66.1 6 7 .4 68.6 6 9 .9 71.1 72.4 73.6 74.9 76.1 77.4 Accum ulated food intake (a) Average acc. SD ca g e 0101 ca g e 0105 ca ge 0201 ca g e 0205 fo o d in take (g) fo) 0.258 0.182 0 .1 1 4 0 .2 0 6 0.19 0.06 0.735 0.468 0 .2 7 3 0.555 0.51 0.19 0.888 0.696 0.733 0.777 0 .7 7 0 .0 8 1.017 0.851 0.926 0.997 0 .9 5 0 .0 8 1.205 0.946 1.021 1.184 1.09 0 .1 3 1.380 1.248 1.356 1.345 1.33 0.06 1.571 1.388 1.770 1.525 1.56 0.16 1.810 1.510 2.016 1.681 1.75 0.21 2.015 1.682 2.182 1.792 1.92 0 .2 2 2.184 1.747 2.294 1.898 2 .0 3 0 .2 5 2.402 1.837 2.324 1.992 2 .1 4 0 .2 7 2.458 1.940 2.623 2.060 2.27 0 .3 2 2.616 2.045 3.224 2 .1 5 2 2.51 0.54 2.724 2.188 3.390 2.308 2 .6 5 0.54 2.824 2.437 3.480 2.473 2.80 0.48 3.064 2.599 3.486 2.689 2 .9 6 0.40 3.300 2.792 3.510 2.748 3.09 0.38 3.564 3.013 3.525 2.995 3.27 0.31 3.879 3.109 3.676 3.304 3.49 0 .3 5 4.146 3.214 3.802 3.448 3 .6 5 0.41 4.399 3.284 3.980 3.750 3 .8 5 0 .4 6 4.531 3.372 4 .1 1 0 3.918 3.98 0 .4 8 4.743 3.412 4 .1 7 4 3.977 4 .0 8 0 .5 5 4.856 3.514 4 .2 8 0 4 .0 8 5 4 .1 8 0 .5 5 4.935 3.602 4.321 4.343 4 .3 0 0 .5 5 5.129 3.739 4.441 4 .4 8 8 4 .4 5 0.57 5.509 4,064 4 .4 6 9 4 .6 3 5 4 .6 7 0.61 5.770 4.341 4 .7 0 5 4 .8 1 5 4.91 0.61 5.968 4.531 4 .8 9 7 4 .9 4 7 5 .0 9 0 .6 2 6.096 4.684 5.032 5.059 5.22 0.61 6.260 4.869 5.144 5 .1 3 8 5 .3 5 0 .6 2 6.378 5.016 5.351 5.318 5.52 0.59 6.529 5.181 5.535 5.470 5.68 0.59 6.741 5.397 5.692 5.729 5.89 0.59 6.916 5.608 5.944 5.965 6.11 0.56 7.046 5.794 6 .0 6 0 6 .1 3 9 6.26 0.54 7.233 5.993 6 .3 2 8 6 .4 4 9 6.50 0.52 7,443 6.101 6 .6 1 5 6 .7 4 9 6 .7 3 0 .5 5 7.561 7.629 6.231 6.317 6 .7 7 6 7 .0 0 2 6 .8 7 9 7.007 6 .8 6 6.99 0 .5 5 0 .5 4 7.763 6.406 7 .1 7 5 7.153 7.12 0.56 8.003 6.555 7.392 7.280 7.31 0.59 8.140 6.690 7.504 7.345 7 .4 2 0.60 8 .2 9 7 6.796 7.656 7.432 7 .5 5 0 .6 2 8.485 6.840 7.831 7.553 7 .6 8 0.68 8.671 7.034 7 .9 2 2 7 .6 2 0 7.81 0 .6 8 8.766 7.102 7 .9 7 3 7.671 7 .8 8 0.69 8.926 7.156 8 .0 1 4 7.735 7 .9 6 0 .7 4 9.030 9.096 9.128 9.139 9.279 9.441 9.564 7.190 7.277 7.336 7.553 7.633 7.933 8.168 8.075 8.149 8.299 8.481 8.613 8.781 8.938 7.826 7.856 7.904 8.007 8.157 8 .2 7 7 8.488 8 .0 3 8 .0 9 8 .1 7 8 .3 0 8 .4 2 8.61 8 .7 9 0.76 0.76 0.75 0 .6 8 0 .7 0 0 .6 6 0.61 9.710 9.835 10.044 8.462 8.621 8.689 9 .2 0 7 9 .4 5 9 9.575 8.706 8 .9 7 0 9.184 9 .0 2 9 .2 2 9 .3 7 0 .5 5 0.53 0.58 Tim e w 61 7.4 8 .6 9 .9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 2 2 .4 2 3 .6 24.9 26.1 2 7 .4 2 8 .6 2 9 .9 31.1 3 2 .4 3 3 .6 3 4 .9 36.1 37.4 3 8 .6 3 9 .9 41.1 4 2 .4 4 3 .6 4 4 .9 46.1 4 7 .4 4 8 .6 4 9 .9 51.1 52.4 53.6 5 4 .9 56.1 57.4 58.6 5 9 .9 61.1 6 2 .4 6 3 .6 6 4 .9 66.1 6 7 .4 6 8 .6 6 9 .9 71.1 7 2 .4 73.6 7 4 .9 76.1 77.4 Accum ulated food intake .9) Average acc. SD ca ge 0102 ca g e 0106 ca g e 0202 ca g e 0206 fo o d in take (g) (fi) 0.215 0.190 0.188 0 .2 0 3 0.20 0.01 0 .5 4 8 0 .5 2 8 0 .6 4 3 0 .4 9 4 0 .5 5 0 .0 6 0 .8 3 4 0.665 0.961 0.615 0 .7 7 0 .1 6 0 .9 7 9 0 .8 2 2 1.200 0.745 0 .9 4 0 .2 0 1.156 1.081 1.312 0.890 1.11 0.18 1.416 1.340 1.505 1.058 1.33 0 .1 9 1.624 1.437 1.738 1.282 1.52 0 2 0 1.828 1.652 1.911 1.374 1.69 024 2 .0 0 5 1.696 2.105 1.503 1.83 0 .2 8 2 .1 6 4 1.772 2.282 1.601 1.95 0 .3 2 2 .2 0 0 1.844 2 .4 5 0 1.658 2 .0 4 0 .3 5 2 .2 9 0 1.877 2.731 1.761 2.16 0 .4 4 2.416 2.008 2.955 1.807 2 .3 0 0.51 2 .5 0 6 2.171 3.097 1.885 2.41 0.52 2 .6 3 8 2.304 3.284 2.120 259 0.51 2 .7 9 0 2.564 3.633 2.294 2 .8 2 0 .5 8 3.033 2.637 3.819 2.454 2 .9 9 0.61 3 .2 8 8 2.836 4.141 2.726 3 .2 5 0 .6 4 3.478 3.028 4.557 2.931 3.50 0 .7 4 3 .6 5 8 3.030 4.845 3 .0 8 7 3 .6 6 0 .8 4 3.815 3.033 4.958 3.251 3 .7 6 0.86 3.989 3.083 5.184 3 .4 6 6 3 .9 3 0.91 4 .1 6 0 3.140 5.331 3.592 4 .0 6 0 .9 5 4 .2 6 4 3 .2 0 0 5.478 3.741 4 .1 7 0.97 4 .3 8 5 4 .6 6 0 3.322 3.464 5.596 5.823 3.824 4.060 4 .2 8 4 .5 0 0.98 1.01 5.208 3.675 6.138 4.316 4 .8 3 1.07 5.472 3.794 6.381 4.484 5.03 1.13 5 .6 6 5 5.794 4.016 4.175 6.645 6.781 4.648 4.691 5 .2 4 5 .3 6 1.16 1.16 5.890 4 .3 7 5 6.887 4 .7 3 6 5.47 1.14 6 .0 5 9 6.241 6.399 4 .4 7 6 4.642 4 .8 3 9 7.033 7.204 7.392 4 .8 7 0 4 .9 6 5 5.180 5.61 5.76 5 .9 5 1.16 1.18 1.17 6.520 5.095 7.600 5.323 6.13 1.16 6.764 5.403 7.862 5.512 6 .3 9 1.16 7.101 7.424 7.608 5.597 5 .8 0 2 5.958 8.127 8.354 8.528 5.755 6.043 6.120 6 .6 5 6.91 7 .0 5 1.20 120 1.23 7.838 6.095 8.658 6.304 722 123 8.024 6 .2 4 0 8.807 6.420 7 .3 7 1.25 8.114 6 .3 0 0 8.955 6.534 7.48 1.27 8.228 6.388 9.050 6.656 7.58 1 .2 7 8.312 8.479 8.672 8.711 8.785 6 .4 6 0 6.546 6.724 6.908 7.060 9.159 9.299 9.494 9.698 9.857 6.748 6.817 6.931 6.987 7.086 7.67 7 .7 9 7 .9 6 8.08 820 1.28 1.32 1.35 1.36 1.37 8.830 7.178 9.942 7.105 8.26 1 .3 7 8 .8 4 4 7.263 10.006 7.200 8.33 1 .3 5 8 .9 0 3 8 .9 2 0 8 .9 9 6 9 .0 9 8 7.332 7.393 7.574 7.806 10.105 10.230 10.390 10.490 7.265 7.319 7.448 7.629 8.40 8.47 8.60 8.76 1.36 1.39 1.38 1.33 9.384 9.629 7.976 8.172 10.763 11.070 7.816 8.089 8.98 9.24 1.38 1.41 9.819 9.936 8.314 8.454 11.308 11.426 8.285 8.447 9 .4 3 9.57 1.44 1.42 Page A29ofA172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Accumulated food intake (q) cane 0103 cans 0107 cage 0203 cage 0207 0.211 0.360 0.336 0.424 0.535 0.688 0.784 0.907 0.656 0.881 1.069 1.157 0.844 1.075 1.187 1.292 0.952 1.146 1.318 1.510 1.045 1.181 1.282 1.410 1.490 1.611 1.612 1.682 1,355 1.470 1.719 1.825 1.475 1.518 1.796 1.867 1.612 1.603 1.896 2.072 1.724 1.856 2.060 2.173 1.840 2.142 2.138 2.328 2.066 2.412 2.257 2.463 2.272 2.408 2.630 2.775 2.440 2.575 2.629 2.771 2.588 2.702 2.899 2.822 2.952 3.350 2.849 3.090 3.486 2.957 3.204 3.580 3.247 3.880 3.988 4.074 3.592 4.090 4.394 4.524 3.788 4.209 4.849 4.851 3.849 3.935 4.315 4.435 5.167 5.255 5.095 5.180 4.033 4.112 4.297 4.530 4.596 4.917 5.345 5.513 5.702 5.222 5.308 5.423 4.563 4.733 4.925 5.184 5.370 5.644 5.847 5.977 6.195 5.572 5.836 5.974 5.058 5.163 5.862 6.025 6.305 6.418 6.104 6.208 5.285 5.412 6.249 6.429 6.561 6.782 6.264 6.450 5.592 5.945 6.233 6.682 6.866 7.064 7.023 7.303 7.717 6.601 6.770 6.965 6.686 7.269 8.032 7.365 7.036 7.546 8.318 7.866 7.312 7.920 8.558 8.118 7.437 7.544 8.042 8.134 8.678 8.803 8.400 8.589 7.578 7.667 8.166 8.205 8.931 9.030 8.735 8.832 7.792 7.871 8.260 8.338 9.197 9.339 8.900 9.022 7.998 8.416 9.506 9.156 8.093 8.503 9.620 9.257 8.272 8.345 8.426 8.474 8.596 8.664 8.858 9.226 9.590 9.961 10.204 8.585 8.669 8.750 8.848 8.900 9.017 9.148 9.256 9.385 9.515 9.712 9.680 9.753 9.904 10.001 10.123 10.340 10.445 10.689 10.994 11.208 11.433 9.388 9.484 9.569 9.658 9.772 9.883 10.118 10.283 10.529 10.865 11.104 Average acc. food intake (g) 0.33 0.73 0.94 1.10 1.23 1.36 1.47 1.59 1.66 1.80 1.95 2.11 2.30 249 2.63 2.80 2.99 3.33 3.80 4.15 4.42 4.61 4.70 4.78 4.88 5.08 599 5.48 5.68 5.83 5.95 6.09 6.27 6.47 6.72 6.99 7.34 7.69 7.98 8.14 8.27 8.35 8.43 8.54 8.64 8.77 8.87 8.98 9.06 9.16 9.25 9.35 9.48 9.64 9.86 10.12 10-39 10.61 SO (9) 0.09 0.16 0.22 0.19 0.24 0.25 0.23 0.22 0.20 0.23 0.20 0.20 0.18 0.17 0.18 0.16 0.22 0.30 0.38 0.41 0.52 0.64 0.63 0.62 0.65 0.62 0.56 0.56 0.55 0.55 0.55 0.56 0.59 0.62 0.57 0.61 0.55 0.54 0.52 0.54 0.56 0.61 0.62 0.63 0.66 0.69 0.70 0.66 0.66 0.69 0.71 0.72 0.77 0.76 0.74 0.76 0.78 0.79 eta colio c?i ".-s crone c HF 0.(T 3 V;. PFCS Time ! Accumulated food intake (9) Average acc. (hr) cage 0104 cage 0108 cane 0204 cage 0208 food intake (q) 6.1 0.204 0.119 0.211 0956 0.20 7.4 0.535 0.170 0.629 0.547 0.47 8.6 0.808 0.260 0.916 0.709 0.67 9.9 0.960 0.518 1.134 0.865 0.87 11.1 1.185 0.732 1.406 1.047 1.09 12.4 1.512 0.847 1.618 1.189 199 13.6 1.749 0.889 1.848 1.340 1.46 14.9 1.940 0.998 2.001 1.406 1.59 16.1 2.020 1.095 2.100 1.591 1.70 17.4 2.174 1.216 2.185 1.642 1.80 18.6 2.406 1.304 2.252 1.767 1.93 19.9 2.543 1.370 2.270 1.851 2.01 21.1 2.648 1.558 2.390 1.938 2.13 22.4 2.810 1.650 2.480 2.082 2.26 23.6 3.050 1.750 2.546 2.234 2.40 24.9 " l 3.301 1.898 2.689 2.577 2.62 26.1 3.423 2.071 2.851 2.815 27.4 3.778 2.310 3.211 3.149 2.79 3.11 28.6 4.101 2.641 3.553 3.436 3.43 29.9 4.314 2.965 3.990 3.637 3.73 31.1 4.529 3.048 4.316 3.864 3.94 32.4 4.663 3.078 4.572 3.978 4.07 33.6 4.720 3.203 4.695 4.070 4.17 34.9 4.865 3.307 5.002 4.232 4.35 36.1 4.989 3.385 5.161 4.322 446 37.4 5.264 3.615 5.358 4.501 4.68 38.6 5.554 3.670 5.466 4.702 4.85 39.9 5.835 3.735 5.640 4.919 5.03 41.1 6.067 3.862 5.747 5.126 42.4 6.303 ! 3.946 5.878 5.205 5.20 5.33 43.6 6,441 4.055 6.075 5.268 546 44.9 6.584 4.147 6.188 5.421 5.59 46.1 6.733 4.293 6.320 5.543 5.72 47.4 7.107 4.400 6.508 5.644 5J91 48.6 7.672 4.522 6.617 5.846 6.16 49.9 7.929 4.758 6.854 6.035 51.1 8.198 5.100 7.194 6.268 6.39 6.69 52.4 8.427 5.352 7.512 6.465 6.94 53.6 8.566 5.511 7.721 6.688 7.12 54.9 8.762 5.603 7.901 6.921 7.30 56.1 8.892 5.689 8.062 7.030 57.4 9.088 5.749 8.139 7.093 7.42 7.52 58.6 9.207 5.852 8.244 7.166 7.62 59.9 9.378 6.024 8.302 7.200 7.73 61.1 9.554 6.105 8.386 7.307 7.84 62.4 9.689 6.228 8.526 7.433 7.97 63.6 9.839 6.294 8.674 7.524 64.9 10.003 6.361 8.830 7.611 66.1 10.139 6.478 8.880 7.664 8.08 8.20 8.29 67.4 10.256 6.542 8.933 7.717 8.36 68.6 10.347 6.591 9.006 7.720 69.9 10.464 6.718 9.083 7.740 71.1 10.593 6.810 9.309 7.807 72.4 10.793 6.915 9.451 7.914 8.42 8.50 8.63 8.77 73.6 11.011 7.103 9.579 8.188 8.97 74.9 11.264 7.345 9.788 8.977 76.1 11.518 7.679 10.107 9.184 77.4 11.641 7.878 10.312 9.426 9.34 9.62 9.81 SO (g) 0.06 0.20 0.29 0.26 0.28 0.35 044 0.47 0.46 047 0.50 0.51 048 0.50 0.55 0,57 0.55 0.60 0.60 0.58 0.66 0.73 0.71 0.77 041 041 047 0.95 0.97 1.03 1.06 1.07 1.07 1.17 1.33 1.34 1.32 1.33 1.32 1.36 1.38 1.43 1.44 1.44 1.48 1.48 1.52 1.57 1.58 1.60 1.62 1.63 1.66 1.71 1.70 1.63 1.61 1.58 Page A30 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Accumulated Food intake; p-values Mann Whitney U-test group 1 vs 4 group 1 vs 6 3 vs 4 group 3 vs 6 I group 4 vs 6 1 nI nI p nln| P I nI n P 0.686 4 4 0.057 0.886 4 4 0.114 0.886 4 0.343 0.886 4 4 0.343 1.000 4 - 0.686 0.886 4 1.000 0.886 4 4 0.886 0.886 4 4 0.886 0.886 4 4 0.686 0.886 4 4 0.686 0.886 4 4 0.886 0.686 4 4 1.000 0.686 4 4 0.686 0.686 4 4 0.686 0.686 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.686 0.886 4 4 0.686 1.000 4 4 0.686 1.000 4 4 0.343 0.686 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.486 0.886 *4 ""4 0.486 1.000 4 4 0.486 1.000 4 4 0.486 1.000 4 4 0.686 1.000 4 4 0.686 1.000 4 4 0.886 1.000 4 4 0.686 1.000 4 4 0.486 1.000 4 4 0.686 0.886 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.486 0.886 4 4 0.686 1.000 4 4 0.686 1.000 4 4 0.686 1.000 4 4 0.686 1.000 4 4 0.686 0.886 4 4 0.686 0.686 4 0.886 4 4 0.686 0.686 4 0.886 4 4 0.686 0.686 4 0.886 4 4 0.686 0.686 4 0.886 4 4 0.686 0.486 ; 4 0.886 4 4 0.686 0.486 4 0.886 4 4 0.686 0.686 4 0.886 4 4 0.686 0.686 - 4 0.886 4 4 0.686 0.486 ' 4 0.886 4 4 0.686 0.343 4 0.886 4 4 0.686 p<0.05 Page A31 ofA172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Feeding bout size measurements Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46,1 47.4 48.6 49.9 51.1 52,4 53.6 54.9 56.1 57,4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Feeding bout size (q) Average feeding SD caqe 0101 C3Q60105 cage 0201 caae 0205 bout s in to) (fl) 0.045 0.036 0.018 0.044 0.036 0.013 0.043 0.019 0.032 0.025 0.030 0.010 0.002 0.029 0.034 0.018 0.021 0.014 0.018 0.002 0.012 0.025 0.014 0.010 0.017 0.014 0.010 0.008 0.012 0.004 0.022 0.032 0.053 0.022 0.032 0.015 0.024 0.013 0.038 0.017 0.023 0.011 0.023 0.017 0.018 0.013 0.018 0.004 0.013 0.010 0.005 0.013 0.010 0.004 0.014 0.005 0.012 0.012 0.011 0.004 0.023 0.013 0.002 0.003 0.010 0.010 0.006 0.005 0.078 0.012 0.025 0.035 0.019 0.014 0.015 0.008 0.014 0.005 0.008 0.028 0.021 0.014 0.018 0.009 0.017 0.020 0.000 0.025 0.018 0.011 0.016 0.005 0.003 0.011 0.009 0.006 0.038 0.030 0.000 0.016 0.021 0.017 0.012 0.012 0.005 0.026 0.014 0.009 0.040 0.011 0.021 0.028 0.025 0.012 0.029 0.011 0.021 0.009 0.018 0.009 0.015 0.010 0.000 0.042 0.017 0.018 0.025 0.002 0.016 0.004 0.012 0.011 0.011 0.008 0.006 0.003 0.007 0.003 0.007 0.009 0.008 0.015 0.010 0.004 0.016 0.016 0.008 0.033 0.018 0.011 0.031 0.004 0.009 0.010 0.014 0.012 0.026 0.046 0.011 0.015 0.025 0.016 0.026 0.025 0.025 0.015 0.023 0.005 0.018 0.012 0.013 0.010 0.013 0.003 0.012 0.017 0.010 0.011 0.013 0.003 0.021 0.014 0.013 0.017 0.016 0.004 0.008 0.018 0.024 0.017 0.017 0.007 0.018 0.023 0.021 0.021 0.021 0.002 0.023 0.020 0.018 0.020 0.020 0.002 0.009 0.023 0.021 0.017 0.018 0.006 0.021 0.017 0.012 0.028 0.020 0.007 0.025 0.014 0.038 0.030 0.027 0.010 0.010 0.014 0.025 0.029 0.020 0.009 0.008 0.009 0.008 0.011 0.009 0.001 0.011 0.009 0.023 0.015 0.015 0.006 0.013 0.013 0.021 0.016 0.016 0.004 0.027 0.019 0.012 0.012 0.015 0.016 0.000 0.007 0.014 0.014 0.011 0.005 0.007 0.029 0.009 0.004 0.015 0.013 0.018 0.009 0.012 0.014 0.005 0.011 0.011 0.002 0.024 0.020 0.005 0.007 0.008 0.004 0.008 0.000 0.008 0.012 0.010 0.005 0.013 0.008 0.003 0.008 0.002 0.002 0.001 0.001 0.002 0.001 0.008 0.001 0.002 0.017 0.018 0.009 0.010 0.015 0.017 0.002 0.047 0.012 0.018 0.009 0.017 0.014 0.019 0.017 0.003 0.014 0.001 0.021 0.015 0.027 0.010 0.010 0.009 0.014 0.025 0.016 0.006 0.006 0.009 0.008 0.015 0.008 0.019 0.021 0.038 0,002 0.035 0.013 0.020 0.027 0.828 0.016 0.010 0.011 0.010 0.010 0.015 0.014 0.012 0.003 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Feeding bout size to) Average feeding SD caqe 0102 cage 0106 caqe 0202 caqe 0206 bout size to) (fl) 0.045 0.043 0.032 0.041 0.040 0.006 0.018 0.018 0.038 0.010 0.021 0.012 0.024 0.011 0.033 0.014 0.021 0.010 0.017 0.026 0.025 0.015 0.021 0.006 0.036 0.024 0.008 0.015 0.021 0.012 0.002 0.019 0.026 0.020 0.017 0.010 0.036 0.017 0.018 0.020 0.023 0.009 0.012 0.008 0.024 0.008 0.013 0.008 0.015 0.011 0.019 0.011 0.014 0.004 0.013 0.001 0.013 0.007 0.009 0.006 0.010 0.008 0.015 0.012 0.011 0.003 0.001 0.004 0.028 0.005 0.010 0.012 0.018 0.018 0.024 0.003 0.016 0.009 0.012 0.019 0.009 0.015 0.014 0.004 0.009 0.007 0.025 0.020 0.015 0.009 0.015 0.028 0.036 0.027 0.027 0.009 0.028 0.009 0.021 0.012 0.018 0.009 0.028 0.031 0.046 0.029 0.034 0.008 0.020 0.001 0.027 0.021 0.017 0.011 0.011 0.000 0.024 0.009 0.011 0.010 0.024 0.003 0.016 0.029 0.018 0.011 0.022 0.008 0.012 0.009 0.013 0.006 0.000 0.000 0.023 0.008 0.008 0.011 0.014 0.011 0.010 0.018 0.013 0.004 0.013 0.012 0.015 0.012 0.013 0.001 0.055 0.010 0.031 0.032 0.032 0.018 0.044 0.025 0.028 0.016 0.028 0.012 0.009 0.012 0.032 0.020 0.018 0.010 0.027 0.031 0.012 0.009 0.020 0.011 0.006 0.015 0.011 0.003 0.009 0.005 0.014 0.011 0.020 0.012 0.014 0.004 0.010 0.009 0.010 0.010 0.010 0.001 0.023 0.017 0.018 0.016 0.019 0.003 0.017 0.032 0.017 0.022 0.022 0.007 0.005 0.033 0.024 0.011 0.018 0.013 0.038 0.022 0.025 0.024 0.027 0.007 0.032 0.019 0.032 0.024 0.027 0.006 0.030 0.018 0.020 0.023 0.023 0.005 0.013 0.008 0.014 0.000 0.009 0.006 0.025 0.015 0.011 0.026 0.019 0.007 0.015 0.010 0.023 0.008 0.014 0.007 0.014 0.007 0.006 0.013 0.004 0.015 0.017 0.010 0.010 0.011 0.006 0.004 0.013 0.011 0.007 0.002 0.008 0.005 0.020 0.001 0.022 0.012 0.014 0.010 0.008 0.030 0.022 0.011 0.018 0.010 0.004 0.009 0.007 0.002 0.006 0.003 0.011 0.019 0.020 0.010 0.015 0.005 0.000 0.007 0.002 0.011 0.007 0.005 0.005 0.006 0.013 0.001 0.014 0.006 0.004 0.009 0.007 0.005 0.004 0.005 0.000 0.013 0.014 0.011 0.023 0.023 0.009 0.018 0.010 0.002 0.022 0.014 0.006 0.019 0.015 0.005 0.005 0.006 0.033 0.008 0.041 0.019 0.025 0.015 0.019 0.017 0.023 0.008 0.022 0.021 0.032 0.017 0.024 0.016 0.006 0.006 0.006 0.015 0.010 0.013 0.011 0.004 Page A32 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Time (he) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26,1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Feeding boutsize (g) Average feeding caw 0103 caae 0107 cage 0203 cage 0207 bout size (g| 0.043 0.050 0.060 0.066 0.055 0.019 0.023 0.037 0.037 0.029 0.009 0.023 0.014 0.025 0.018 0.022 0.014 0.011 0.009 0.014 0.007 0.007 0.025 0.021 0.015 0.010 0.024 0.006 0.004 0.011 0.020 0.000 0.015 0.020 0.014 0.010 0.006 0.011 0.001 0.007 0.013 0.005 0.006 0.019 0.011 0.013 0.012 0.007 0.012 0.011 0.011 0.027 0.021 0.005 0.016 0.021 0.040 0.008 0.023 0.023 0.018 0.013 0.014 0.008 0.013 0.016 0.029 0.010 0.021 0.019 0.015 0.009 0.025 0.013 0.016 0.019 0.001 0.024 0.024 0.017 0.009 0.019 0.024 0.030 03)21 0.036 0.063 0.056 0.041 0.049 0.033 0.030 0.044 0.048 03)39 0.024 0.020 0.049 0.049 0.036 0.014 0.012 0.036 0.017 0.020 0.005 0.009 0.019 0.025 0.015 0.010 0.017 0.005 0.000 0.008 0.012 0.000 0.018 0.010 03)10 0.009 0.012 0.013 0.008 0.011 0.028 0.032 0.015 0.007 04)21 0.019 0.040 0.020 0.030 0.027 0.017 0.000 0.012 0.019 04)12 0.022 0.044 0.021 0.011 04)25 0.009 0.015 0.007 0.011 0.011 0.007 0.016 0.010 0.008 0.010 0.012 0.017 0.030 0.019 0.020 0.013 0.035 0.011 0.010 0.017 0.028 0.007 0.029 0.017 0.020 0.037 0.026 0.037 0.017 0.029 0.033 0.015 0.035 0.022 03)26 0.040 0.027 0.027 0.060 04)39 0.030 0.039 0.032 0.026 04)32 0.022 0.025 0.023 0.036 0.027 0.013 0.004 0.001 0.020 0.010 0.008 0.008 0.021 0.016 0.013 0.006 0.002 0.006 0.014 0.007 0.014 0.007 0.018 0.006 0.011 0.004 0.005 0.017 0.007 0.008 0.011 0-006 0.015 0.018 0.013 0.014 0.012 0.014 0.011 0.013 0.017 0.002 0.009 0.009 0.009 0.008 0.015 0.000 0.014 0.009 0.011 0.009 0.022 0.009 0.013 0.006 0.000 0.001 0.008 0.004 0.001 0.010 0.017 0.008 0.009 0.014 0.010 0.013 0.013 0.013 0.006 0.014 0.015 0.013 0.012 0.034 0.013 0.011 0.024 0.021 0.041 0.008 0.035 0.023 0.027 0.034 0.015 0.030 0.025 0.026 0.037 0.016 0.019 0.036 0.027 0.017 0.014 0.025 0.013 0.017 SO (B) 0.010 0.009 0.008 0.006 0.009 0.009 0.009 0.005 0.007 0.D03 0.010 0.013 0.004 0.008 0.007 0.011 0.009 0.013 0.009 0.016 0.011 0.009 0.007 0.007 0.002 0.012 0.010 0.009 0.014 0.003 0.004 0.008 0.012 0.010 0.010 0.009 0.016 0.005 0.006 0.009 0.006 0.005 0.006 0.006 0.004 0.002 0.006 0.007 0.006 0.004 0.007 0.002 0.004 0.011 0.015 0.008 0.011 0.005 Time (hr) 6.1 7.4 8.6 9.9 in 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 v otabciic ca es arjurj 6 ` 'r 0 003 RFC3 I Feeding bout size (g) I Average feeding cage 0104 caw 0108 cage 0204 caw 0208 bout size (g) 0.040 0.017 0.037 0.035 0.032 0.026 0.000 0.043 0.025 04)24 0.020 0.010 0.018 0.018 0.017 0.024 0.036 0.025 0.018 0.026 0.022 0.015 0.027 0.013 0.019 0.030 0.009 0.027 0.014 0.020 0.027 0.003 0.014 0.014 0.015 0.008 0.019 0.019 0.008 0.014 0.010 0.001 0.000 0.018 0.007 0.026 0.013 0.009 0.002 0.013 0.014 0.013 0.007 0.012 0.012 0.011 0.008 0.010 0.013 0.011 0.015 0.017 0.011 0.013 0.014 0.019 0.010 0.004 0.014 0.012 0.024 0.013 0.006 0.015 0.015 0.018 0.010 0.023 0.033 0.021 0.013 0.031 0.015 0.033 0.023 0.050 0.013 0.049 0.024 0.034 0.020 0.046 0.029 0.033 0.032 0.027 0.016 0.031 0.011 0.021 0.013 0.006 0.035 0.024 0.020 0.014 0.003 0.023 0.011 0.013 0.010 0.016 0.017 0.012 0.014 0.011 0.012 0.036 0.019 0.020 0.011 0.004 0.004 0.002 0.005 0.039 0.023 0.022 0.023 0.027 0.029 0.007 0.014 0.022 0.018 0.024 0.013 0.012 0.026 0.019 0.023 0.004 0.013 0.014 0.014 0.018 0.016 0.017 0.002 0.013 0.013 0.003 0.011 0.012 0.010 0.020 0.020 0.012 0.013 0.016 0.013 0.010 0.015 0.015 0.013 0.066 0.010 0.020 0.018 0.029 0.026 0.016 0.019 0.012 0.018 0.031 0.029 0.032 0.019 0.028 0.027 0.029 0.031 0.027 04)29 0.021 0.019 0.029 0.023 0.023 0.012 0.014 0.017 0.014 04)14 0.014 0.012 0.016 0.026 0.017 0.019 0.005 0.012 ! 0.005 0.010 0.015 0.006 0.005 0.011 0.009 0.015 0.014 0.014 0.001 0.011 0.014 0.015 0.001 0.006 0.009 0.023 i 0.010 0.014 0.013 0.015 0.012 0.013 0.014 0.010 0.012 0.016 0.001 0.021 0.008 0.012 0.015 0.017 0.005 0.009 04)12 0.010 0.002 0.005 0.008 0.006 0.012 0.008 0.007 0.001 0.007 0.011 0.003 0.009 0.000 0.006 0.012 0.014 0.006 0.007 0.010 0.009 0.014 0.027 0.003 0.013 0.022 0.008 0.011 0.020 0.015 0.028 0.028 0.021 0.087 0.041 0.027 0.021 0.019 0.015 0.021 0.020 0.031 0.036 0.030 0.029 0.007 0.016 0.013 0.016 0.013 SD (B) 0.010 0.018 0.004 0.008 0.006 0.010 0.010 0.006 0.008 0.010 0.003 0.002 0.003 0.006 0.007 0.010 0.010 0.018 0.011 0.009 0.013 0.008 0.003 0.012 0.004 0.008 0.010 0.007 0.008 0.008 0.005 0.004 0.002 04)25 0.006 0.006 0.002 0.004 0.002 0.006 0.007 0.005 0.007 0.007 0.006 0.002 0.009 0.006 0.004 0.005 0.005 0.004 0.010 0.007 0.031 0.005 0.007 0.004 Page A33of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Feeding bout size; p-values Mann Whitney U-test 1 vs 4 r group 1 vs 6 nlnT n group 3 vs 4 nlni n group 3 vs 6 I group 4 vs 6 1 n In I n InI nI P 4 4 0.029 4 4 1.000 4 4 0.886 4 4 0.057 4 4 0.486 4 4 0.200 4 4 0.886 4 4 0.343 4 4 0.486 4 4 0.886 4 4 0.886 4 4 0.200 4 4 0.886 4 4 0.200 4 4 0.886 4 4 1.000 4 4 0.686 4 4 0.343 4 4 0.343 4 4 0.343 4 4 0.886 4 4 0.886 4 4 0.343 4 4 0.200 4 4 0.114 4 4 0.686 4 4 0.343 4 4 0.343 4 4 0.486 4 4 0.343 4 4 0.886 4 4 0.886 4 4 0.886 4 4 0.886 4 4 0.114 4 4 0.886 4 4 0.686 4 4 0.057 4 4 0.029 4 4 0.343 4 4 0.486 4 4 0.686 4 4 1.000 4 4 1.000 4 4 0.886 4 4 0.686 4 4 1.000 4 4 0.486 4 4 0.114 4 4 0.343 4 4 0.686 4 4 0.343 4 4 0.886 4 4 0.343 4 4 0.886 4 4 0.343 4 4 0.886 4, 4 0.343 p<0.05 Page A34of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Drink intake measurements Tim e (hr) 6.1 7 .4 8 .6 9 .9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 4 4 .9 46.1 4 7 .4 4 8 .6 4 9 .9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 6 4 .9 66.1 67.4 68.6 6 9 .9 71.1 72.4 73.6 74.9 76.1 77.4 Accum ulated drink intake ml) ca g e 0101 ca ge 0105 cage0201 c a g e 0205 0 .2 0 5 0.065 0.129 0.230 0.720 0.337 0.591 0.580 0.946 0.658 0.782 0.846 1.053 0.791 0.784 1.083 1.209 0.845 0.805 1.297 1.284 1.166 0 .9 3 6 1.334 1.375 1.292 1.208 1.481 1.602 1.326 1.325 1.610 1.716 1.448 1.348 1.624 1.799 1.479 1.373 1.702 1.836 1.499 1.382 1.806 1.898 1.530 1.463 1.826 1.981 1.530 1.564 1.863 2.002 1.530 1.576 1.891 2.049 1.778 1.610 1.976 2.213 1.870 1.610 2 .0 2 4 2.280 2.047 1.796 2.024 2.465 2.264 1.927 2.098 2.714 2.349 2.268 2.291 3.045 2.480 2.638 2.436 3.250 2.581 2.921 2.691 3.375 2.669 3.022 2.949 3.523 2 .7 2 0 3.227 2.967 3.640 2.863 3.434 3.036 3.734 3.104 3.738 3.252 3.796 3.281 3.818 3.353 4 .0 5 3 3.602 4.089 3.434 4.358 3.893 4.292 3.680 4 .5 0 8 4.029 4.460 3.765 4 .5 2 4 4.072 4.5G8 3.832 4.654 4 .1 1 4 4 .5 1 7 3.885 4 .7 1 6 4.201 4.683 4.004 4.784 4.325 4.812 4.170 4.966 4.510 4.908 4.342 5.096 4.661 5.072 4.499 5.140 4 .9 2 5 5.175 4.538 5.200 5.018 5.251 4.664 5.343 5.110 5.469 4 .8 4 8 5.465 5.280 5.699 4 .9 2 2 5.530 5.423 5.895 5.037 5.694 5.540 6.008 5.175 5.892 5.799 6.279 5.348 6.146 5.984 6.385 5.428 6.297 6.086 6.578 5.481 6.399 6.137 6.760 5.557 6.604 6.372 6.937 5.612 6.604 6 .4 9 9 7.038 5.653 6.695 6.511 7.075 5.773 6.796 6.511 7.222 5.865 6.812 6.547 7.270 5.865 6.812 6.562 7.447 5.900 6.812 6.800 7.544 6 .0 5 6 6.942 6.848 7.694 6.148 7.192 7.031 7.822 6.297 7,311 7.293 7.852 6.387 7.535 7.560 8.022 6.608 7.639 7.701 8.317 6.845 7.875 7.752 8 .3 4 9 7.043 Average acc. drink in take (ml) 0 .1 6 0 .5 6 0.81 0.93 1.04 1.18 1 .3 4 1.47 1.53 1.59 1.63 1.68 1.73 1.75 1.85 1.93 2 .0 4 2.19 2.41 2.65 2 .8 6 3.00 3.11 3 .2 4 3 .4 6 3 .5 6 3.79 4 .0 6 4 .1 9 4 .2 3 4 .2 9 4 .4 0 4 .5 2 4 .6 8 4 .8 3 4 .9 4 5.03 5.19 5 .3 4 5.47 5.60 5 .8 3 5 .9 9 6.11 6.21 6.38 6 .4 5 6.51 6.60 6.62 6.68 6.80 6.91 7.09 7.21 7.43 7 .6 3 7 .7 5 SD (m l) 0.08 0.16 0.12 0 .1 6 0.25 0.18 0.12 0.16 0.17 0.20 0.22 0.21 0.22 0.23 0.20 0.25 0.20 0.23 0.21 0 .2 8 0.30 0.29 0 .3 4 0.36 0.33 0.28 0.33 0.32 0.36 0.34 0.36 0.35 0.32 0.30 0.30 0.29 0.27 0.27 0.33 0.35 0.35 0.38 0.41 0.47 0.51 0 .5 6 0.58 0.55 0.57 0.59 0.64 0.61 0.63 0.63 0.61 0.59 0.60 0.54 Tim e (hr) 6.1 7.4 8 .6 9 .9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 2 2 .4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 3 9 .9 41.1 4 2 .4 4 3 .6 4 4 .9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 6 3 .6 6 4 .9 66.1 6 7 .4 6 8 .6 6 9 .9 71.1 72.4 73.6 74.9 76.1 77.4 Accum ulated drink intake ml) cage 0102 cage 0106 cage 0202 cage 0206 0.125 0.077 0.074 0.092 0.387 0.392 0.569 0.388 0.699 0.496 0.967 0.560 0.883 0.510 1.102 0.676 0.912 0.691 1.253 0.864 0.994 0.800 1.449 1.024 1.176 0.824 1.634 1.276 1.318 0.834 1.800 1.400 1.462 0.848 1.850 1.400 1.565 0.861 2.153 1.450 1.592 0.872 2.254 1.500 1.600 0.880 2.311 1.500 1.659 0.880 2.578 1.548 1.730 0.923 2.743 1.616 1.768 1.003 2.804 1.780 1.837 1.013 3.065 1.842 1.898 1.024 3.283 1.988 2.045 1.078 3.512 2.146 2.210 1.179 3.904 2.280 2.357 1.194 4.171 2.418 2.539 1.205 4.361 2.606 2.710 1.216 4 .5 5 8 2.764 2.912 1.232 4 .6 6 9 2.906 2.968 1.245 4.869 2.988 3.101 1.302 4.941 3.026 3.251 1.360 5.004 3.248 3.547 1.386 5.263 3.424 3.787 1.453 5.465 3.520 3.955 1.557 5 .6 4 5 3.520 4.046 1.664 5.702 3.590 4.059 1.725 5.731 3.632 4.130 1.776 5.814 3.722 4.232 1.864 6 .0 4 8 3.788 4.293 1.925 6.185 3.874 4.387 2.098 6.361 4 .0 4 8 4.488 2.166 6.577 4.100 4.763 2.238 6.869 4.252 5.006 2 .3 8 4 6.988 4.420 5.194 2 .5 6 0 7.207 4 .6 0 8 5.344 2.587 7.337 4 .8 0 4 5.506 2.672 7.529 5.032 5.597 2.782 7.729 5.188 5.693 2 .8 3 8 7.884 5.352 5.742 2.869 8.100 5.420 5.842 2.960 8.217 5.510 6.122 3.056 8.334 5.646 6.157 3.131 8.496 5.700 6.176 3.146 8.674 5.836 6 .1 9 8 3.157 8.726 5.900 6 .2 1 3 6.235 3.168 3.224 8 .7 4 8 8.748 5.946 6.000 6 .2 5 0 6.301 3.272 3.341 8.748 8.975 6.000 6.072 6.358 3.434 9.104 6 .2 6 0 6.584 3.462 9.446 6.362 6.795 3.486 9.756 6.532 6.912 3.550 9.992 6 .7 3 0 6.998 3.640 10.177 6.890 Average acc. drin k in take (ml) 0.09 0.43 0.68 0.79 0.93 1.07 1.23 1.34 1.39 1.51 1.55 1.57 1.67 1.75 1.84 1.94 2.05 220 2 .3 9 2.53 2.68 2.81 2.93 3.02 3.09 322 3.41 3.56 3.67 3.75 3.79 3.86 3.98 4 .0 7 422 4 .3 3 4 .5 3 4 .7 0 4 .8 9 5.02 5.18 5 .3 2 5 .4 4 5.53 5.63 5.79 5.87 5 .9 6 6.00 6 .0 2 6 .0 5 6 .0 7 6 .1 7 6 .2 9 6.46 6 .6 4 6.80 6 .9 3 SD (m l) 0.02 0.09 0.21 0.26 0.24 027 0.33 0.40 0.41 0.53 0.57 0.59 0.70 0.75 0 .7 4 0.85 0.93 1.00 1.13 123 1.29 1.37 1.40 1.48 1.49 1.49 1.59 1.65 1.68 1.66 1.65 1.66 1.72 1.75 1.75 1.81 1.90 1.89 1.91 1.95 1.99 2.03 2.07 2.14 2.15 2.17 220 226 2.28 2.28 2.26 2 .2 4 2.30 2 .3 2 2 .4 4 2.56 2.63 2.67 Page A35 of A172 7/ Appendix to TNO-report of 3M-02 Final version 04-12-2006 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Accumulated drink intake ml) Average acc. cape 0103 cape 0107 cane 0203 cape 0207 drink Intake (ml) 0.131 0.362 0.214 0.306 025 0.361 0.782 0.880 0.849 0.72 0.479 0.996 1.327 1.135 0.98 0.574 1.207 1.512 1254 1.14 0.612 1.207 1.718 1.395 123 0.669 1.248 1.961 1.518 1.35 0.768 1.343 1.995 1.558 1.42 0.819 1.370 2.085 1.606 1.47 0.982 1.377 2.184 1.624 1.54 1.113 1.377 2.184 1.694 1.58 1.121 1.550 2.297 1.729 1.67 1.159 1.758 2.310 1.815 1.76 1.290 1.880 2.329 1.971 1.87 1.463 1.938 2.520 2.024 1.99 1.516 1.955 2.545 2.024 2.01 1.592 1.955 2.747 2.081 2.09 1.615 1.955 2.982 2.152 2.18 1.685 2.072 3.137 2.429 2.33 2.048 2.708 3.620 2.974 2.84 2.329 2.948 4.053 3.518 3.21 2.453 3.074 4.574 3.846 3.49 2.512 3.186 4.893 4.052 3.66 2.546 3.349 4.998 4.092 3.75 2.595 3.451 4.998 4.145 3.60 2.645 3.461 5.093 4.312 3.88 2.683 3.667 5.187 4.312 3.96 2.831 3.856 5.271 4.396 4.09 2.945 3.910 5.414 4.576 421 3.027 4.175 5.527 4.708 4.36 3.173 4.352 5.565 4.717 4.45 3.192 4.352 5.649 4.752 4.49 3.211 4.423 5.712 4.759 4.53 3.276 4.498 5.943 4.888 4.65 3.416 4.651 6203 4.950 4.81 3.585 4.763 6.422 4.950 4.93 3.753 4.859 6.766 5.025 5.10 4.051 5.005 7.046 5.350 5.36 4.366 5.263 7.388 5.870 5.72 4.579 5.632 7.642 6.395 6.06 4.579 5.794 7.854 6.743 624 4.579 5.868 7.892 7.018 6.34 4.579 5.930 8.100 7.227 6.46 4.699 6.011 8245 7.454 6.60 4.803 6.042 8.392 7.608 6.71 4.872 6.106 8.610 7.726 6.83 4.940 6.188 8.736 7.806 6.92 4.978 6.297 8.736 7.854 6.97 5.043 6.351 8.736 7.920 7.01 5.088 6.450 8.763 8.004 7.08 5.111 6.460 8.904 8.074 7.14 5.111 6.504 8.988 8.140 7.19 5.172 6.542 9.114 8.175 725 5.187 6.664 9284 8228 7.34 5.324 6.725 9.303 8.391 7.44 5.596 6.732 9.574 8.532 7.61 5.917 6.773 9.811 8.708 7.80 6.236 6.943 10.019 9.082 8.07 6.335 7.133 10.185 9297 824 so (ml) 0.10 024 0.36 040 046 0.54 0.51 0.53 0.50 0.46 0.49 047 0.43 043 0.42 048 0.58 0.62 0.65 0.74 0.92 1.04 1.05 1.02 1.06 1.06 1.02 1.04 1.05 0.99 1.02 1.03 1.10 1.14 1.16 1.25 125 127 129 1.39 1.44 1.54 1.57 1.60 1.67 1.69 1.67 1.64 1.64 1.69 1.73 1.75 1.79 1.77 1.78 1.78 1.78 1.80 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 474 48.8 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68 6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Metabcc c.::. os qrouy 6 HF* 0,003 ". PFOS I Accumulated drink intake Iml) Average acc. cape 0104 case 0108 caae0204 cage 0208 drink intake (ml) 0.134 0.711 0240 0288 0.151 0.724 0202 0.470 0.18 0.55 1.078 1282 1.576 1.877 2.179 2.394 2.567 2.669 0228 0.563 0213 0.883 0.896 0.941 0.992 1.085 1.159 1.269 1.505 1.667 1.818 1.957 2.016 2.018 0.616 0.716 0.840 0.976 1.062 1.100 1216 1.280 i 0.80 0.96 1.18 1.35 1.49 1.60 1.70 1.76 2.820 1.146 2.016 1.288 1.82 2.948 2.992 2.992 1253 1264 1278 2.016 2.016 2.016 1.360 1.422 1.446 1.89 1.92 1.93 3.012 1.312 2.016 1.560 1.98 3.135 1.312 2.088 1.576 2.03 3.196 1238 2.160 1.630 2.08 3.344 1.506 2.198 1.794 221 3.611 1.768 2.362 2.022 2.44 3.822 4.039 4.097 4.097 4.097 4.097 4.148 4.423 4.648 4.784 4.944 5.117 5.151 2.093 2.198 2217 2.394 2.557 2.642 2.720 2.774 2.832 2.864 2.902 3.024 3.024 2.907 3.308 3.438 3.620 3.978 4.104 4.111 4.176 4.363 4.437 4.464 4.529 4.698 2.198 2.390 2.534 2.664 2.808 2.940 3.000 3.134 3.342 3.468 3.480 3.494 3.506 2.75 2.98 3.10 3.19 3.36 3.45 3.49 3.63 3.80 3.89 3.95 4.04 4.09 5.156 3.046 4.698 3.550 4.11 5202 3.178 4.698 3.636 4.18 5263 5.535 3205 3234 4.698 4.806 3.732 3.858 422 4.38 5.879 5.964 6.137 6.392 6.681 6.871 6.989 7.342 7.431 7.497 3.624 3.898 4242 4.346 4.536 4.608 4.707 4.891 4.912 5.022 5.123 5.377 5.555 5.767 5.994 6.118 6.311 6.390 6.604 6.772 3.974 4.148 4.378 4.560 4.676 4.778 4.958 5.020 5.212 5.346 4.65 4.85 5.08 5.27 5.47 5.59 5.74 5.91 6.04 6.16 7.616 5.152 6.804 5.452 6.26 7.616 7.616 7.659 7.701 7.701 7.701 7.701 7.813 8.050 8.109 8.109 5.168 5216 5222 5232 5290 5.347 5.408 5.504 5.678 5.987 6242 6.804 6.804 6.804 6.804 6.804 6.804 6.804 6944 7.175 7.547 7.686 5.516 5.540 5.540 5.540 5.568 5.630 5.676 5.824 5.952 6.098 6.304 6.28 629 6.31 6.32 6.34 6.37 6.40 6.52 6.71 6.94 7.09 5D (ml) 0.05 021 0.39 0.37 0.41 0.50 0.61 0.69 0.73 0.73 0.77 0.78 0.78 0.77 0.75 0.81 0.82 0.81 0.82 0J0 0.85 0.83 0.80 0.79 0.77 0.74 0.80 0.85 0.88 0.93 0.95 1.00 0.98 0.93 0.93 0.98 1.04 0.99 0.92 0.98 1.04 1.09 1.09 1.17 1.18 1.17 1.16 1.14 1.12 1.13 1.15 1.12 1.09 1.06 1.06 1.10 1.06 0.95 75 Page A36of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 _ 0.3431 Page A 3 /o tA 1 (2 Appendix to TNO-report of 3M-02 Final version 04-12-2006 D4. X activity measurements Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 161 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32,4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43,6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 X activity per interval cage 0101 cage 0105 cage 0201 cage 0205 100.7 88.8 134.7 99.5 63.9 227.7 123.3 739 67.5 124.2 57.2 69.0 76.1 238.3 182.4 62.8 90.7 71.1 72.9 173.1 88.6 74.3 128.0 80.4 69.1 109.8 547 35.5 72.2 28.9 67.7 67.7 14.3 92.9 70.7 46.6 112.4 64.9 119.1 63.5 144.9 81.0 41.1 47.5 74,2 81.1 92.5 166.7 44.1 99.0 49.7 29.3 47.4 71.0 121.8 94.7 88.3 91.9 108.7 57.4 424.8 330.7 375.7 615.8 133.7 276.6 421.4 814.9 331.4 346.2 385.6 228.3 121.9 221.1 257.7 488.8 143.4 139.3 121.1 158.0 133.1 229.4 213.0 217.7 74.2 181.4 129.7 250.7 54.6 172.7 182.5 166.9 199.9 158.2 94.7 119.2 84.7 128.2 53.6 109.3 101.7 122.5 136.4 118.5 53.4 25.2 67.8 54.2 33.9 49.9 56.4 55.9 84.9 47.7 44.7 23.0 28.9 79.6 74.6 119.0 64.8 34.9 59.5 96.2 74.9 64.0 65.7 38.0 69.8 101.4 87.1 72.6 90.4 52.7 46.1 71.2 373.3 279.0 284.9 313.7 337.7 147.0 370.8 371.0 361.6 270.6 276.3 229.2 161.5 365.6 199.2 227.9 164.7 143.5 156.8 202.0 119.6 206.4 153.3 185.1 76.2 117.9 136.6 57.6 82.3 180.9 143.9 106.5 96.9 97.8 154.2 234.6 173.9 144.2 96.5 86.3 90.2 68.3 85.5 90.4 71.4 80.6 87.9 29.3 49.0 185.5 93.7 91.4 94.4 67.4 115.8 76.0 36.2 14.6 23.2 57.4 91.6 118.8 97.4 31.4 42.5 77.5 379 106.7 50.0 112.7 53.7 40.3 94.0 43.9 83.9 120.3 186.7 126.5 215.6 235.7 380.3 349.8 374.6 551.9 411.4 223.4 274.0 372.4 246.9 230.0 248.0 185.3 156.4 360.5 270.5 260.9 Average X a ctivity 105.9 122.2 79.5 139.9 102.0 92.8 67.3 59.1 56.1 90.0 78.6 103.6 55.5 83.7 86.6 436.8 411.7 322.9 272.4 140.5 198.3 159.0 132.9 154.3 94.0 119.8 50.2 49.0 50.1 75.5 63.9 60.7 82.7 65.1 312.7 306.6 284.4 238.6 166.8 166.1 97.1 128.4 145.9 125.2 83.6 67.3 104.9 88.4 32.9 84.8 66.0 64.2 85.5 191.1 414.2 320.3 227.6 262.1 SD 19.9 75.0 30.3 84.7 48.3 24.2 31.5 20.3 33.7 29.9 47.5 42.7 30.2 31.9 21.4 125.4 293.4 67.1 155.3 15.2 44.0 75.2 69.6 24.1 32.3 14.3 17.9 10.5 25.7 36.9 25.2 15.8 14.6 19.9 43.1 107.6 55.6 89.0 25.1 37.9 36.5 43.2 64.9 41.1 10.4 26.2 57.5 21.5 18.6 37.5 32.5 32.8 31.7 47.5 92.8 86.7 29.3 83.5 Time ... (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 X activity per interval cage 0102 cage 0106 cage 0202 cage 0206 92.2 254.5 88.7 185.0 92.7 238.4 92.6 375.7 79.1 99.1 110.4 111.4 131.6 82.0 45.9 221.7 134.8 124.3 259.8 60.1 54.7 138.5 174.2 39.8 134.4 53.3 68.6 182.7 59.0 82.5 103.6 47.8 122.4 105.6 146.3 18.4 84.7 29.6 245.5 93.8 158.6 172.4 76.4 41.6 21.0 39.6 50.1 14.5 93.0 113.8 169.7 118.6 111.5 160.0 89.9 126.0 158.6 55.4 169.7 72.4 454.8 579.9 431.8 455.2 432.8 621.4 618.1 933.4 277.4 351.9 422.6 616.8 325.6 48.8 191.6 130.5 262.1 373.9 151.6 282.2 95.4 52.6 238.0 216.4 127.8 215.5 260.5 148.1 99.6 135.3 127.1 112.9 199.2 141.3 217.7 267.4 135.8 276.8 172.6 73.8 109.4 98.3 200.9 103.7 58.6 61.8 132.5 28.6 61.9 88.5 155.1 26.9 60.3 164.0 35.5 11.7 47.7 32.8 87.4 223.3 69.2 57.8 71.1 68.3 131.2 97.6 173.2 60.2 88.0 78.5 143.7 50.2 120.8 63.9 104.5 30.4 361.3 203.4 323.6 485.2 527.6 382.4 442.4 1194.3 308.7 470.4 298.9 609.6 201.9 177.0 84.2 327.0 332.9 80.5 218.2 274.2 117.4 102.8 219.6 242.1 136.8 300.6 165.3 167.6 121.2 113.3 113.2 309.0 177.5 352.3 305.4 374.6 236.1 139.3 186.5 212.9 70.6 171.6 92.8 28.1 102.9 131.2 71.9 161.7 72.1 72.4 43.7 9.3 62.0 63.4 125.6 266.9 130.8 49.7 53.5 23.2 75.2 27.2 84.9 221.5 84.0 116.4 51.9 45.1 63.1 82.3 73.4 35.7 129.7 62.8 151.3 92.7 201.6 143.3 125.5 219.4 429.8 396.0 387.1 405.0 278.5 493.7 280.0 701.6 270.4 175.4 217.9 407.2 256.5 298.8 295.6 603.1 Average X a ctivity 155.1 199.9 100.0 120.3 144.8 101.8 109.8 73.2 98.2 113.4 112.3 31.3 123.8 121.9 114.0 480.4 651.4 417.2 174.1 267.5 150.6 188.0 118.7 206.4 164.8 128.1 70.4 83.1 67.9 97.8 66.6 115.6 90.1 79.9 343.4 636.7 421.9 19725 226.5 170.5 192.6 164.2 302.5 193.7 90.8 116.9 49.4 129.5 64.3 102.2 74.4 63.6 109.1 172.5 404.5 438.5 267.7 363.5 SD 79.9 135.9 15.0 76.2 83.5 64.9 60.0 24.9 55.7 92.5 63.4 16.4 32.6 29.4 58.5 67.2 207.6 145.7 116.7 91.3 90.6 61.2 15.8 52.1 85.1 48.8 44.0 54.2 67.1 86.8 6.0 48.1 39.2 40.8 116.1 376.5 147.8 100.1 108.0 70.6 73.4 96.6 88.2 41.5 60.2 38.4 29.9 96.3 46.3 83.4 32.8 2022 392! 45.1 18.4 202.5 100.8 160.9 77 Page A38 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 X activity per interval cane 0103 caqe 0107 cage 0203 cage 0207 75.4 118.1 92.7 258.6 91.1 61.6 100.7 151.8 32.3 128.9 85.4 124.6 88.7 58.6 66.1 53.7 56.0 47.9 89.9 144.3 79.3 139.0 66.9 185.7 103.8 31.0 -43.1 57.7 51.9 53.5 153.0 95.1 65.2 199.5 53.3 90.9 83.7 172.4 74.7 34.6 28.4 46.4 42.0 124.4 59.3 94.9 103.2 76.9 108.0 72.6 51.6 57.3 59.8 39.3 122.1 273.1 71.2 76.4 74.7 95.4 484.3 589.1 168.2 410.3 821.1 605.3 368.9 719.7 418.6 371.6 219.7 566.0 282.1 99.3 260.1 379.5 175.2 215.2 227.2 177.6 203.6 190.6 144.7 107.9 92.2 84.7 83.6 169.4 146.6 178.0 157.2 185.6 85.3 14.1 101.8 125.7 173.4 94.0 82.6 336.9 141.5 111.9 103.3 374.0 97.6 65.8 82.8 63.6 46.4 28.1 47.1 140.5 54.7 69.4 73.6 65.1 47.0 47.6 44.3 73.3 79.7 55.9 49.8 76.8 33.1 77.6 137.3 47.2 141.3 169.8 67.1 187.9 148.8 21.7 77.9 55.3 211.0 139.5 118.3 271.5 555.2 277.4 176.1 489.8 227.0 219.5 308.1 548.7 192.2 120.1 214.5 482.5 145.5 229.0 129.1 229.2 159.5 85.7 211.4 146.7 114.2 96.2 119.5 189.7 64.4 84.0 123.6 69.4 116.3 161.9 141.4 211.7 59.8 24.8 79.1 127.3 98.7 39.5 90.8 97.0 97.6 87.9 60.4 116.3 97.9 39.2 108.8 93.5 87.3 120.3 55.8 55.3 69.4 44.1 120.1 211.5 63.4 28.1 69.2 83.9 34.3 79.2 92.1 126.5 77.4 110.7 103.2 151.6 54.9 103.1 77.5 66.3 121.3 183.4 84.3 76.1 262.2 613.3 156.4 233.3 393.8 463.2 326.7 686.7 152.2 146.0 423.4 234.1 160.4 290.0 246.2 300.4 Average X activity 136.2 101.3 92.8 66.8 84.5 117.7 58.9 884 1022 914 60.3 83.6 724 123.6 79.4 413.0 628.8 394.0 255.3 198.8 161.7 107.5 166.9 81.7 171.7 182.7 77.5 65.5 65.7 53.1 65.6 73.8 141.5 75.9 185.1 374.6 325.8 252.3 1832 150.8 129.9 85.4 157.8 72.8 81JS 90.6 84.9 79.7 111.3 612 83.0 110.7 75.5 116.3 316.3 467.6 238.9 249.3 SD 83.5 37.6 44.8 15.5 43.8 552 31.9 47.5 66.7 58.1 43.4 19.6 25.4 105.7 10.9 178.9 194.4 142.7 1162 26.3 43.9 41.5 18.1 48.0 117.3 128.6 15.9 50.8 8.1 13.6 14.9 46.2 532 53.8 69.9 177.7 153.9 158.7 53.4 51.6 41.1 26.8 40.5 42.8 28.2 23.3 31.1 30.9 73.9 23.7 38.1 30.8 20.6 48.9 203.0 156.3 129.4 63.7 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 h- etafcCFC cages arouo 6 HF f 0 CC3 % PFOS X activity per interval Average cage 0104 cage 0108 cage 0204 cage 0208 X activity 152.1 223.2 114.3 130.4 155.0 136.3 217.5 107.7 211.5 168.3 37.5 132.4 78.6 35.1 70.9 120.1 164.6 61.9 77.3 106.0 165.9 124.0 96.1 64.6 112.7 64.9 56.9 76.3 42.0 60.0 99.3 51.5 54.9 136.8 85.6 66.3 168.5 118.2 51.8 101.2 35.2 74.0 67.3 72.4 62.2 29.9 94.3 55.5 23.5 50.8 75.5 152.4 123.9 48.4 100.1 94.7 30.6 41.5 67.2 58.5 61.8 90.8 77.4 96.6 81.7 41.2 176.7 52.9 68.4 84.8 154.0 98.7 119.9 115.8 122.1 248.5 353.9 134.8 128.7 216.5 432.4 353.1 703.1 283.1 442.9 225.6 313.2 437.8 367.4 336.0 250.2 403.1 203.5 248.2 276.3 130.8 117.3 152.3 164.3 141.2 194.5 243.2 125.1 138.7 175.4 136.1 54.8 78.9 156.4 106.6 138.1 137.0 183.6 133.4 148.0 250.1 184.4 140.2 133.4 177.0 164.8 39.0 140.4 63.1 101.8 111.8 88.9 133.9 108.8 110.9 81.0 99.0 41.5 98.2 79.9 64.7 12.4 52.8 123.7 63.4 29.8 48.0 55.2 50.8 46.0 36.2 141.9 24.8 28.7 57.9 73.6 35.9 147.9 112.9 92.6 75.9 45.5 63.5 32.1 54.3 42.3 63.0 57.3 78.0 60.2 38.4 84.7 46.8 106.0 69,0 140.0 152.0 217.4 78.4 147.0 405.1 292.8 232.2 226.6 289.2 131.5 332.3 228.9 252.9 236.4 212.6 410.7 208.7 ; 235.1 266.8 114.7 310.1 107.5 227.4 189.9 136.0 161.8 93.3 83.1 118.6 198.1 73.7 158.3 108.8 134.7 89.1 134.6 91.5 166.0 120.3 136.5 126.4 110.9 175.2 137.3 180.5 105.8 129.4 141.3 139.3 85.9 71.5 90.4 116.7 91.1 56.8 151.5 70.7 107.5 96.6 74.8 40.4 70.0 67.5 63.2 42.2 130.3 32.1 72.8 69.4 26.7 24.3 85.4 91.4 57.0 74.5 80.0 53.0 61.2 67.2 59.1 27.3 68.7 34.4 47.4 57.3 101.8 40.5 102.7 75.6 55.0 80.3 57.9 55.6 62.2 106.5 43.9 112.9 98.0 90.3 193.8 308.6 110.3 109.8 180.6 256.2 142.5 208.6 290.9 224.6 131.1 305.9 223.4 367.3 256.9 203.7 407.8 178.4 337.2 281.8 SD 48.0 54.7 45.6 46.2 43.0 14.4 40.5 53.2 18.2 32.1 46.8 28.6 15.5 62.3 23.2 106.9 183.9 89.5 87.3 21.1 54.3 47.6 23.8 53.7 60.3 18.4 26.9 46.0 11.2 56.2 48.5 19.3 14.8 31.9 57.0 82.9 82.7 96.7 97.1 36.8 54.7 36.9 27.4 31.2 18.9 42.4 15.5 44.2 36.4 12.3 19.7 31.6 12.1 31.5 94.0 64.3 102.5 109.1 Page A39ofA172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 X activity, p-values Mann Whitney U-test group 3 vs 6 I group 4 vs 6 1 InI nI P 4 4 0.486 4 4 0.114 4 4 0.886 4 4 0.200 4 4 0.343 4 4 0.057 4 4 0.486 4 4 0.886 4 4 0.686 4 4 0.343 4 4 0.200 4 4 0.200 4 4 0.486 4 4 0.886 4 4 mas 4 4 0.114 4 4 0.200 4 4 0.686 4 4 0.886 4 4 0.029 4 4 0.886 4 4 0.686 4 4 0.200 4 4 S.29 4 4 0.343 4 4 0.343 4 4 0.686 4 4 0.886 4 4 0.057 4 4 0.343 4 4 0.686 4 4 0.486 4 4 0.057 4 4 1.000 4 4 0.886 4 4 0.686 4 4 0.886 4 4 0.686 4 4 0.686 4 4 0.486 4 4 1.000 4 4 0.114 4 4 0.486 4 4 0.057 4 4 0.886 4 4 1.000 4 4 0.343 4 4 0.686 4 4 0.343 4 4 1.000 4 4 0.200 4 4 0.114 4 4 0.686 4 4 0.686 4 4 0.343 4 4 0.029 4 4 1.000 4. 4 0.686 p<0.05 Page A40of A172 Appendix to TNO-rePort of 3M-02 Final version 04-12-2006 D5. 0 2 consumption,C02 production and respiratory exchange ratio measurements 0 2 consumption Tim (hr) 6-1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39 9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 VOj (ml/Wkg mouse) Average VO? cage 0101 cao 0105 cao 0201 case 0205 (mt/h/kg mouse) 3055 3609 3572 3183 3355 2909 3769 3278 2987 3236 2786 3499 3070 3028 3096 3051 3540 3607 2913 3278 2741 3200 2943 3204 3022 2814 2966 3194 2903 2969 2636 3102 2744 2661 2786 2685 2756 2766 2705 2728 2399 2814 2770 2676 2665 2811 2699 2874 2756 2785 3115 2850 2610 2730 2826 2766 2917 2939 3098 2930 2602 2990 2628 2542 2691 2743 2708 2828 2892 2793 3001 2963 2979 2785 2937 3803 3693 3803 3939 3809 2959 3356 3539 3857 3428 3391 3518 3497 3027 3358 2921 3300 3385 3544 3287 2968 3266 3115 2992 3085 2981 3263 3316 3042 3150 2763 3180 3307 3199 3112 2742 3182 3401 2921 3062 3160 3090 3203 2842 3074 2844 3097 2702 2680 2831 2831 2938 3274 2716 2940 2671 2564 2826 2528 2647 2400 2696 2776 2658 2633 2690 2672 2630 2450 2611 2344 2796 2790 2828 2689 2542 2523 2642 2639 2587 2549 2679 2872 2558 2665 2757 2884 3000 2753 2849 2937 2860 2704 2758 2815 3585 3535 3750 3397 3566 3458 3277 3756 3348 3460 3320 3475 3528 3083 3352 2985 3469 3327 3112 3223 2933 3202 3213 3143 3123 3066 3289 3231 3090 3169 2657 3247 3191 2815 2977 2818 3230 3258 2922 3057 2822 2877 3274 3079 3013 2899 3156 3119 2652 2957 2683 2389 2820 2882 2794 2765 2648 2368 2736 2601 2451 2707 2939 2554 2663 2727 2568 2982 2495 2693 2399 2276 2392 2563 2407 2588 2697 2816 2448 2637 2392 2652 2564 2667 2569 2532 2656 2612 2506 2576 2612 2511 2816 2723 2666 3115 3156 3363 3306 3235 3315 3487 3910 3443 3539 3280 3320 3488 3288 3344 2917 2855 3251 3337 3373 3389 2915 3106 3114 3172 SD 278 389 296 347 224 162 216 39 186 75 216 136 203 83 102 101 375 228 264 136 164 239 290 162 192 240 134 163 110 231 63 151 118 104 146 211 199 216 130 108 287 220 206 233 84 262 212 216 118 157 126 70 132 119 258 98 234 244 Time M 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 VOj (ml/h/kg mouse) Average VOa caae0102 cage 0106 cage 0202 caqe 0206 (m i/h/kfl mouse) 3472 3395 3050 3369 3409 3203 3160 3368 3449 3514 3614 3293 3379 3446 3249 3172 3251 2926 3037 2788 2871 2799 3056 2601 2805 2750 3171 3833 3636 3312 3416 3230 2869 2856 2994 3079 2968 3173 2704 2718 2628 2607 2947 3125 3060 2695 2851 2875 2652 3007 2709 2795 2921 2587 3748 3978 3248 2692 3470 2582 2876 2690 2731 3042 2525 2513 2533 2616 2201 3117 3816 3395 3096 3097 3325 3518 3074 3117 3413 3103 3538 4154 4043 3549 3359 3388 3516 3552 3323 3477 3324 3366 3166 3179 3017 3282 3489 3179 2899 3070 2552 2576 2756 2662 2514 2879 2867 2788 3592 3820 3655 3071 3377 3131 3029 2895 3112 2751 2900 2538 2470 2540 3134 3181 3343 3070 2975 2822 2912 2931 2950 2735 2973 2910 3020 3832 3869 3441 3135 3366 3025 3078 2975 3100 3022 2991 2730 2725 2700 2806 2730 2797 2698 3046 3577 3645 3451 3181 3346 3086 3090 2863 3087 3056 2671 2444 2399 2342 2545 2404 2463 2455 2691 3248 3502 3217 3102 3005 2354 2485 2627 2525 3124 3499 3441 2990 2800 2879 3022 2881 3127 2850 3145 2789 2700 2391 2295 2324 2660 2428 2326 2864 3251 3329 2931 2951 3253 3350 3198 3342 3851 3852 3668 3312 3445 3554 3415 3403 3591 3467 3078 2892 2935 3212 2815 3006 2803 2845 3286 3489 4078 3610 3421 3571 2736 2734 2680 2752 3637 3908 3515 3294 3165 3289 3186 3262 3313 3039 2635 2715 2380 2899 2392 2846 2507 2482 2798 3160 3573 3467 3432 3348 2768 2842 2801 2916 3547 3776 3519 3194 3189 3202 3178 3102 3279 3103 2882 2710 2603 2711 2512 2645 - 2608 2553 2770 3190 3601 3406 3222 3219 SD 158 113 167 226 320 228 188 224 309 396 194 287 296 147 420 237 181 193 332 100 397 325 284 305 237 364 302 320 215 496 369 365 267 355 306 187 105 148 284 289 172 272 230 260 266 191 265 418 227 332 155 196 387 258 346 170 247 293 80 Page A41 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 $4.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 V02(mi/h/kg mouse) Average VO* cage 0103 cape 0107 cage 0203 cage 0207 (mVh/kg mouse) 3575 3707 3794 3663 3685 3438 3276 3450 3564 3743 3720 3572 3214 3551 3443 3298 3287 3705 3019 3327 3219 3263 3026 3409 3779 3672 3239 3345 3316 3422 3253 2782 3223 2912 3043 3148 2910 3420 2872 3088 3131 3100 3357 3284 3169 3191 2875 2771 3133 3086 3016 3076 2897 3165 2977 3223 2999 2870 2972 3084 3085 2998 2948 2834 2966 3053 3230 2966 3150 3152 3288 3341 3059 3128 3182 4037 4262 4423 4267 3777 4205 3640 4105 3969 4210 3794 3878 3828 3747 3812 3514 3369 3957 3750 3648 3148 3395 3529 3172 3640 3650 3236 3219 3388 3359 3149 2853 3984 3250 3309 3169 2970 3160 3016 2941 2850 2941 2907 2942 2796 3066 3211 2429 2889 3030 2788 2683 2931 2730 2681 2997 3271 3921 3604 3350 3543 3214 3085 3210 2981 2965 3440 3046 3297 3069 3309 3283 2655 2885 2530 2725 2754 2692 3180 3399 3018 3177 3218 2899 2876 2903 2836 2835 2981 3141 3205 2865 3259 2689 3005 3687 4214 3511 3964 3496 4094 3092 3677 3446 3987 3775 3674 3687 3386 4344 3911 3700 3750 3876 3680 3475 3342 3563 2927 3848 3707 3319 3329 3551 3326 3292 3037 3252 2949 2998 3028 2891 2898 2599 2763 2667 2847 2862 3237 3789 3950 3367 3409 3075 2999 3141 2581 2658 2847 2517 2895 2387 2486 2703 2910 2964 3292 4002 3705 2971 3211 3560 3737 3706 3450 3240 3009 3312 3049 3169 2988 3097 3004 3025 3296 3796 4281 4117 3938 3360 3049 3142 2942 2765 2797 2674 2635 3032 2671 2657 2767 2614 2919 3425 3810 3470 3360 3322 3206 3310 2980 2915 2920 2848 2869 2797 2727 2781 2882 2866 3186 3753 3937 3481 3480 SO 91 141 240 283 323 177 233 253 198 224 53 15$ 105 161 99 345 75 55 260 234 218 480 352 481 209 249 240 165 280 128 140 331 105 273 252 230 314 220 222 319 200 355 269 357 259 116 345 172 366 209 211 100 181 180 240 251 475 317 Time (hQ 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77A \ Hr *0 3 P FOS VO2(ml/h/kg mouse) Average VOj cage 0104 fcage 0108 cage 0204 cage 0208 ( m t/M m mouse) 3936 4321 3970 3628 3963 3779 3980 3795 3648 3800 3334 3807 3674 3379 3549 3695 3783 3579 3441 3624 3866 3646 3646 3141 3575 3633 3225 3349 3124 3333 3766 3107 3222 3167 3315 3592 3471 3249 3007 3330 3335 2903 3089 3039 3091 32 1 2 3171 2996 2777 3040 3726 3504 3191 2894 3329 3676 2963 2911 3007 3139 3305 3099 2949 3046 3100 3297 3319 2980 3014 3152 3731 3204 3078 3199 3303 4099 4024 3349 3392 3716 4351 3972 4050 3767 4035 3752 3870 4131 3917 3918 3941 4064 3600 3773 3645 3747 3497 3405 3446 3524 3948 3675 3488 3364 3619 3818 3178 3407 3492 3474 3918 3425 3579 3323 3561 3886 3428 3396 3234 3486 3850 3073 3304 3074 3325 3591 3040 3332 3159 3280 3316 3011 2944 3124 3099 3267 2725 2955 3142 3022 3116 2830 2972 3019 2984 3148 3212 2800 2929 3022 3309 2822 3150 3182 3116 3392 2842 2934 2916 3021 3310 2932 3103 3085 3108 3301 2919 2962 3075 3064 3857 3307 3541 3244 3487 4409 3774 3676 3620 3870 3834 3995 3818 3874 3880 4037 3881 3610 3592 3780 3747 3731 3514 3608 3650 3907 3451 3525 3338 3555 3965 3167 3620 3404 3539 3733 3412 3604 3462 3553 3792 3344 3376 3424 3484 3870 3327 3465 3392 3513 3598 3153 3213 3205 3292 3697 3167 3007 3175 3262 3151 2771 3077 2885 2971 3203 2934 2705 3044 2972 2953 2527 2901 2845 2806 3241 2925 2873 2718 2939 3204 2665 2843 2776 2872 3161 2950 2659 3014 2946 3089 2860 2748 2780 2869 3570 2917 2989 3115 3148 3761 3686 3194 3269 3477 3836 3329 3493 3677 3584 3648 3583 3465 3833 3632 3736 3613 3363 36 8 8 3600 so 283 136 229 148 307 220 304 258 180 198 364 360 150 180 291 400 242 158 202 153 254 265 260 280 366 239 163 236 119 191 208 250 155 171 278 365 80 217 110 247 339 145 208 245 205 301 174 209 192 220 233 211 154 293 287 220 154 166 Page A42 of A172 Appendix to TNO-rePort of 3M-02 Final versipn 04-12-2006 VO2, p-values Mann Whitney U-test group 3 vs 6 group 4 vs 6 I nI n| P n|n| p 4 0.029 m 0.200 H- w a rn E H 0.057 4 0.114 1 0.486 4 0.057 4 0.486 ^ B 4 0.200 ^ B 0.200 0.486 0.486 4 mom 0.686 -At 0.057 ^ B 0.200 4 0.200 ^ B 0.686 4 0.486 0.886 4 0.200 ^ B 0.343 4 0.200 ^ B 0.886 4 0.343 E H 0.200 4 0.114 4 0.343 r a 0.886 4 0.686 ^ B 0.486 4 0.486 ^ B 0.343 4 0.029 ^ B 0.343 0.029 ^ B 0.343 4 0.114 ^ B A 0.114 ^ B A 0.200 ^ B 0.686 0.200 0.486 4 .029* ^ B 0.343 4 0.200 0.200 4 0.200 ^ B 1.000 4 0.486 ^ B 0.486 4 0.200 ^ B 0.200 4 0.200 ^ B _/ 0.114 ^ B 4 0.686 E H 0.343 0.686 0.200 4 0.200 ^ B 0.114 4 0.200 ^ B 0.886 4 0.200 ^ B 1.000 4 0.686 E H 0.686 0.886 - 0.886 E K 9 0.486 . 0.029 ^ B 0.343 19.029 ^ B 0.886 A 0.029 E H 0.486 4 0.200 E H 0.343 4 0.200 ^ B 0.343 4 0.029 ^ B 0.343 4 0.200 ^ B 0.200 4 0.200 ^ B 0.114 4 0,029 E H 0.200 : 4 0.029 E H 0.114 _ 4 0.114 B H 0.686 J 4 0.486 0.486 4 0.114 ^ B 0.886 4 0.343 ^ B 0.343 4 0.114 ^ B 0.486 4 0.057 E H 0.486 : 4 0.686 4 4 E E E a 4 0.886 E r a 0.686 4 0.886 E H 0.114 4 0.200 E H 0.114 4 0.029 ^ B 0.486 ___4 0.057 B H 0.486 CC0OO0 1 00 p<0.05 8 Page A43 ofA172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 C 0 2 production Time 0) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26,1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 VC02(ml/h/kg mouse) Averagt VCO* cage 0101 cane 0105 case 0201 cage 0205 (ml/h/kg m o tn) 2929 3110 3620 2845 3126 2978 2781 2963 3408 3142 3025 3399 3042 3464 2787 2814 2655 3143 2945 3027 2773 2721 2478 2629 2718 2759 2619 2308 2664 3107 2736 2708 2841 2565 2379 2391 2749 2788 2553 2509 2418 2713 3008 2486 2362 2412 2563 3454 2775 3139 2804 2860 2861 2563 2599 2801 2500 2517 2627 2470 2748 2281 2448 2369 2594 2662 3258 3054 3010 2744 2610 2547 2403 2581 2571 2631 2574 2295 2211 2536 2132 2274 1943 1969 2228 2767 2844 2927 2559 2530 2501 2207 2361 2419 2465 2231 2617 3237 2971 3105 2718 2620 2568 2568 2436 2505 2421 2267 2362 2518 2440 2470 2244 2400 2551 2631 3226 2903 3083 3012 2778 2717 2640 2784 2393 2574 2152 2478 2122 2019 1693 2161 2025 2239 1950 2843 3169 3054 2840 2822 2567 2650 2357 2757 2357 2731 2602 3386 3388 3283 3414 3100 3243 3137 2976 2827 2436 2933 2813 2873 2656 2655 2505 2829 2874 2543 3635 3311 3407 3274 2975 3158 2997 3114 2915 2798 2597 2429 2445 2461 1936 2434 2320 2420 2546 3065 3474 3241 3150 2961 2267 2315 2233 2474 2041 2440 2402 3369 3044 2578 3120 2592 2750 2730 2197 2280 2334 2241 2191 2345 2095 2354 2168 2209 2334 2510 3092 2877 2777 2839 2727 2521 2341 2383 2423 2088 2128 1822 1985 1914 2008 1765 2073 1970 2267 2691 2866 2818 2531 2683 2438 2471 2490 2534 2306 2453 2546 3361 3044 3026 3014 2793 2856 2750 2552 2603 2423 2490 2498 2562 2485 2440 2341 2451 2588 2587 3303 3036 3069 2967 2773 2736 2595 2715 2576 2523 2363 2256 2191 2232 1943 2159 2090 2150 2248 2841 3088 3010 2770 2749 SD 348 311 176 333 76 229 157 190 130 248 351 152 184 207 99 91 256 309 318 238 285 270 327 260 68 321 276 227 290 163 161 265 222 71 233 199 261 233 152 295 297 312 239 305 258 299 193 312 185 28$ 162 221 244 162 297 182 289 185 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 VCO2(ml/h/kg mouse) Average VCO cage 0102 caga 0106 caga 0202 caga 0206 (ml/h/kg mouse) 3252 3003 2825 3076 3039 3223 3131 3321 3262 3234 2920 2824 3352 2920 3004 2958 2601 2985 2986 2883 3064 2849 3475 2676 3016 2699 2726 3161 2520 2776 2801 2151 2872 2743 2642 2572 2481 2697 2060 2452 2714 2400 3020 2190 2581 2585 2148 3083 2324 2535 2574 2524 2658 2156 2478 2184 2181 2852 2015 2303 2411 2426 3150 2209 2549 2300 2522 2630 2256 2427 2736 2188 3094 2372 2598 3201 3308 3794 3129 3358 3254 3232 3611 3130 3307 2985 3014 2775 2175 3335 3300 3196 2743 3073 2808 2775 2675 3305 2895 2913 2439 2504 1920 2269 3161 3092 2671 2606 2548 261 2610 2010 2869 2378 2472 2617 2140 2941 2590 2572 2477 2899 2465 1937 2788 2859 2206 2524 2485 255$ 2685 2169 3039 2355 2562 2683 2199 3043 2262 2547 2621 2408 2877 2293 2550 2474 2507 1986 2197 2836 2699 2555 2100 2463 2376 2548 2467 2105 2244 2784 2683 2263 2211 2425 2401 2764 2233 3009 2456 2615 3042 3429 2866 3157 3422 3477 3123 3172 3113 3309 3211 3047 3074 2715 3245 2949 2974 2936 3126 2912 3111 2508 3013 2564 2799 2896 2513 3032 2765 2802 2872 2595 2781 2578 2707 2476 2380 2820 2651 2582 2685 2602 2998 2616 2725 2488 2275 2807 2394 2491 2467 2477 2587 2067 2399 2183 2398 2543 2186 2320 1944 2326 2532 1776 2145 1948 2041 2791 2344 2281 2018 2013 2171 1721 1981 1832 1903 2347 2267 2007 1932 2169 2213 1916 2057 1855 1854 2316 1905 1982 2156 2564 1965 2510 2756 2797 2343 2630 2305 2625 3132 2821 3506 2895 3088 2833 2774 2869 2421 3164 3060 2960 2920 2956 2794 2464 2451 2963 2853 2683 SD 177 80 236 188 345 272 331 276 363 407 222 374 413 179 402 300 210 245 480 277 515 346 372 329 237 445 383 394 257 354 276 301 219 341 232 168 125 140 307 221 144 194 186 228 228 176 346 380 188 257 155 224 338 125 308 149 275 264 Page A44 of A172 Appendix to TNO-report of 3M-Q2 Final version 04-12-2006 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 VCOj (ml/h/kg mouse) Average VCO caqe 0103 case 0107 cape 0203 cane 0207 (ml/h/kg mouse) 3309 3390 3535 3348 3395 3243 3195 3526 3375 3335 2910 3285 3487 2961 3161 3013 2980 3360 2695 3012 2848 2670 3385 2981 2971 2803 3043 3222 3000 3017 2757 2834 2340 2384 2802 2957 2483 2469 2596 2661 2699 2685 2527 2316 2557 2641 2677 2628 2448 2598 2474 2658 2505 2519 2539 2501 3011 2615 2533 2665 2634 2835 2489 2379 2634 2636 2685 2634 2873 2708 2739 2772 2653 2586 2687 3504 3473 3331 3246 3389 3532 3535 36% 3585 3585 3236 3539 3498 3374 3412 3243 3256 3755 3475 3432 2940 3382 3493 3025 3210 3109 2912 3516 3001 3135 2575 2599 3708 2979 2965 2536 2804 3465 2755 2890 2419 1953 3136 2496 2501 2531 2461 2999 2664 2664 2613 2818 3063 2466 2740 2766 2612 2756 2243 2594 2533 2656 2367 2538 2834 2515 2510 2715 2371 2677 2166 2250 2564 2598 2378 2316 2481 2372 2195 2341 2273 2767 2861 2035 2484 2587 2927 2695 2742 2738 2771 2605 2987 2323 2672 3419 3884 3205 3492 3237 3775 2691 3089 3138 3560 3549 3283 4014 3281 3532 3537 3116 3646 3478 3444 3286 3411 3615 3060 3343 3013 2658 3123 3020 2954 2856 2761 2950 3005 2893 2482 2458 3004 2631 2644 2653 2516 2972 2505 2662 2446 2003 2852 2388 2422 2471 2444 2317 2384 2132 2248 1959 2357 2679 2508 2651 2281 2317 2381 2239 2202 2400 2395 2292 2306 2092 1863 2403 2467 2206 2089 2019 1983 2148 2360 2535 2074 2135 2127 2209 2254 2189 2792 3502 2256 2394 2665 3130 2476 2578 2648 3196 2241 2122 2485 2820 2307 2321 2648 3162 3708 3218 3192 2929 2380 2698 3755 3623 3327 3224 3083 2906 3404 3076 3031 SD 99 148 274 273 304 172 221 278 179 102 81 236 236 113 84 121 73 137 240 259 267 528 401 487 239 259 245 76 284 109 119 395 142 280 312 355 345 230 233 204 108 252 217 348 232 111 285 82 281 163 225 113 207 126 280 397 518 283 Time .....(hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63 6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 fs Cr3Qf:5 Croii'J { -F - ; ce 3 :3. PFOS VCOa(ml/h/kg mouse) Average VC02 cage 0104 caoe0108 cape 0204 cage 0208 (ml/h/kg mouse) 3618 3732 3829 3220 3550 3414 3149 3553 3346 3365 3077 3062 3321 2963 3106 3331 3294 3219 3015 3215 3459 3188 3324 2801 3193 3301 2650 2970 2656 2894 3344 2303 2875 2554 2769 3167 2783 2760 2326 2759 2754 2270 2407 2550 2495 2745 2591 2365 2139 2460 3329 2992 2511 2371 2801 3154 2288 2097 2433 2493 2765 2771 2441 2405 2595 2658 2817 2364 2486 2576 3248 2519 2387 2668 2705 3643 3328 2734 2974 3170 3711 3457 3256 3206 3408 3410 3409 3637 3476 3483 3586 3714 3225 3340 3466 3324 3143 3102 2929 3124 3436 3095 3183 2900 3153 3213 2431 3022 2895 2890 3040 2809 3004 2693 2887 3259 2784 2951 2688 2921 3172 2333 2737 2351 2648 3179 2503 2817 2633 2783 3080 2433 2362 2717 2648 2992 2068 2449 2838 2587 2701 2306 2399 2627 2508 : 2828 2532 2224 2310 2474 2695 2234 2681 2499 2527 2775 2233 2426 2409 2461 2741 2411 2500 2573 2556 2766 2411 2616 2534 2582 3454 2739 3044 2867 3026 3931 3266 3167 3104 3367 3393 3634 3407 3247 3420 3656 3495 3236 3014 3350 3109 3309 3021 3121 3140 3360 2940 2868 2811 2995 , 3313 2510 2896 2675 2849 3183 2708 2683 2661 2809 3210 2732 2631 2576 2787 3309 2775 2552 2529 2791 3020 2472 2453 2522 2617 2882 2549 2424 2522 2594 2636 2062 2504 2233 2359 2675 2302 2208 2333 2379 2419 2724 2040 2379 2197 2060 2131 2004 2197 2291 2533 2023 2195 1971 2180 2450 2344 2016 2234 2261 2505 2242 1969 2016 2183 3034 3260 2294 3089 2265 2531 2426 2702 2505 2895 3398 2900 2924 3037 3065 3198 3204 3044 3244 3172 3051 3148 2734 3064 2999 SD 226 168 152 141 284 310 449 344 207 265 441 452 200 201 380 399 230 107 224 162 223 333 165 251 396 294 326 414 186 270 215 227 140 149 312 382 160 283 121 249 348 250 289 363 270 199 260 204 161 332 254 185 246 360 337 230 88 182 Page A45 of A172 Appendix to TNQ-report of 3M-02 Final version Time group 1 vs 3 nini p V C 0 2, p-values Mann Whitney U-test group 1 vs 4 nlnl P group 1 vs 6 nInI p group 3 vs 4 group 3 vs 6 group 4 vs 6 nInI p nI n I p n|n| p A 0.057 4 4 0.343 4 0.200 4 4 0.886 4 0.343 4 4 0.886 mmm4 4 4 0.343 4 0.686 4 4 0.486 4 0.886 4 4 0.486 4 0.686 4 4 0.686 4 0.200 4 4 1.000 4 1.000 4 4 0.886 4 1.000 4 4 0.486 4 0.686 4 4 0.686 4 0.486 4 4 0.343 4 0.686 4 4 0.686 4 0.343 4 4 0.486 4 0.686 4 4 0.686 4 0.686 4 4 0.486 A 0.486 4 4 0.343 mi4 m 4 4 0.686 4 0.057 4 4 1.000 0.200 4 4 0.686 4 0.114 4 4 1.000 4 0.486 4 4 1.000 4 0.114 4 4 0.686 4 0.114 4 4 0.200 4 0.886 4 4 0.886 4 0.886 4 4 0.886 4 0.486 4 4 1.000 4 1.000 4 4 1.000 4 1.000 4 4 0.486 4 0.886 4 4 0.886 4 0.686 4 4 0.200 4 1.000 4 4 1.000 4 0.343 4 4 0.114 4 1.000 4 4 0.686 A 0.886 4 4 0.886 4 0.886 4 4 0.886 4 0.029 4 4 0.886 4 0.057 4 4 0.886 - 0.114 4 4 0.343 - 0.486 4 4 0.886 0.686 4 4 0.686 0.200 4 4 0.200 0.886 4 4 0.486 0.200 4 4 0.200 4 0.686 4 4 0.343 4 0.114 4 4 0.114 4 0.486 4 4 1.000 4 0.886 4 4 0.886 4 0.114 4 4 1.000 4 0.343 4 4 0.686 0.486 4 4 0.886 4 0.114 4 4 0.686 4 0.886 4 4 0.486 0.343 4 4 0.343 4 0.486 4 4 0.343 0.486 4 4 0.200 4 0.057 4 4 1.000 4 0.114 4 4 1.000 04-12-2006 p<0.05 f5 Page A46 of A172 Appendix to TNQ-report of 3M-02 Final version 04-12-2006 Respiratory exchange ratio Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24,9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Respiratory exchange ratio caste 0101 cage 0105 cage 0201 cage 0205 0.960 0.661 1.015 0.895 1.025 0.902 1.037 0.934 0.997 0.900 0.994 0.931 0.971 0.854 0.962 0.909 1.014 0.848 0.906 0.884 0.966 0.933 0.975 0.885 0.939 0.841 1.000 0.892 0.981 0.839 0.976 0.883 1.008 0.890 0.926 0.845 0.962 0.817 0.917 0.837 0.969 0.824 0.905 0.815 0.896 0.827 0.936 0.792 0.906 0.821 0.895 0.803 0.877 0.824 0.966 0.843 0.852 0.876 0.867 0.862 0.908 0.878 0.890 0.857 0.941 0.886 0.960 0.785 0.923 0.877 0.936 0.853 0.965 0.823 1.007 0.877 0.963 0.802 0.995 0.866 0.961 0.784 0.977 0.903 0.926 0.805 0.952 0.856 0.948 0.765 0.876 0.747 0.889 0.809 0.881 0.797 0.879 0.780 0.907 0.870 0.882 0.764 0.894 0.821 0.985 0.920 0.998 0.865 1.030 0.936 1.040 0.881 1.027 0.916 1.012 0.856 0.972 0.882 0.947 0.827 0.962 0.890 0.946 0.814 0.930 0.892 0.985 0.863 0.940 0.882 0.959 0.845 0.905 0.920 0.941 0.908 0.913 0.916 0.968 0.915 0.881 0.886 0.881 0.856 0.907 0.888 0.964 0.901 0.919 0.870 0.981 0.910 0.892 0.870 0.923 0.871 0.832 0.827 0.975 0.812 0.907 0.816 0.943 0.828 0.916 0.865 0.956 0.813 0.910 0.831 0.888 0.781 0.903 0.817 0.899 0.787 0.964 0.763 0.931 0.802 0.963 0.861 0.875 0.766 0.896 0.774 0.830 0.772 0.933 0.784 0.817 0.759 0.890 0.745 0.810 0.783 0.876 0.794 0.857 0.721 0.811 0.758 0.907 0.772 0.777 0.839 0.926 0.785 0.852 0.775 0.903 0.830 0.887 0.901 0.912 0.814 0.855 0.909 0.690 0.831 0.891 0.921 0.931 0.854 0.870 0.873 0.930 0.866 0.888 0.844 0.872 0.864 Average RER 0.933 0.974 0.955 0.924 0.913 0.940 0.918 0.920 0.917 0.883 0.878 0.863 0.856 0.878 0.864 0.883 0.893 0.897 0.918 0.906 0.906 0.885 0.834 0.844 0.859 0.840 0.942 0.972 0.953 0.907 0.903 0.917 0.906 0.918 0.928 0.876 0.915 0.920 0.889 0.861 0.874 0.887 0.853 0.851 0.865 0.867 0.818 0.823 0.807 0.812 0.812 0.832 0 .8 4 0 0.878 0.871 0.899 0.885 0.867 SD 0.069 0.067 0.048 0.054 0.072 0.041 0.068 0.070 0.069 0.068 0.073 0.065 0.052 0.063 0.010 0.022 0.079 0.039 0.083 0.089 0.087 0.067 0.095 0.047 0.055 0.060 0.062 0.076 0.081 0.066 0.067 0.053 0.052 0.016 0.027 0.013 0.033 0.046 0.025 0.076 0.062 0.062 0.058 0.059 0.097 0.080 0.059 0.077 0.062 0.070 0.067 0.069 0.053 0.044 0.035 0.035 0.030 0.018 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Respiratory exchange ratio cage 0102 cage 0106 cage 0202 cage 0206 0.937 0.894 0.893 0.878 0.950 0.919 0.987 0.903 0.959 0.882 0.972 0.887 0.931 0.884 0.959 0.856 0.944 0.910 0.909 0.842 0.922 0.889 0.932 0.869 0.920 0.600 0.928 0.695 0.923 0.867 0.867 0.805 0.946 0.831 0.908 0.851 0.927 0.808 0.874 0.841 0.836 0.832 0.863 0.807 0.831 0.804 0.916 0.803 0.855 0.863 0.921 0.763 0.833 0.858 0.847 0.780 0.858 0.846 0.874 0.849 0.834 0.878 0.913 0.871 0.894 0.812 0.895 0.818 0.901 0.887 0.856 0.809 0.943 0.982 0.875 0.893 0.858 0.771 0.975 0.855 0.851 0.743 0.896 0.855 0.877 0.783 0.862 0.859 0.871 0.744 0.867 0.823 0.850 0.779 0.842 0.830 0.837 0.802 0.835 0.801 0.922 0.765 0.852 0.871 0.994 0.868 0.960 0.929 0.988 0.870 0.957 0.917 0.998 0.920 0.955 0.904 0.949 0.901 0.864 0.811 0.916 0.931 0.829 0.766 0.912 0.842 0.825 0.824 0.912 0.851 0.833 0.821 0.906 0.854 0.883 0.919 0.900 0.888 0.893 0.861 0.890 0.902 0.902 0.809 0.929 0.892 0.882 0.845 0.958 0.928 0.907 0.895 0.892 0.873 0.893 0.808 0.938 0.875 0.853 0.840 0.930 0.855 0.814 0.809 0.864 0.826 0.829 0.812 0.870 0.829 0.832 0.790 0.815 0.800 0.811 0.783 0.922 0.786 0.840 0.784 0.884 0.858 0.880 0.803 0.809 0.833 0.860 0.854 0.861 0.863 0.746 0.801 0.793 0.877 0.770 0.719 0.759 0.822 0.778 0.790 0.783 0.814 0.789 0.760 0.754 0.758 0.811 0.767 0.794 0.787 0.894 0.844 0.873 0.867 0.841 0.794 0.862 0.835 0.830 0.609 0.879 0.856 0.875 0.854 0.892 0.821 0.826 0.826 0.893 0.826 0.850 0.853 Avenge RER 0.900 0.940 0.925 0.908 0.901 0.903 0.886 0.866 0.884 0.862 0.834 0.839 0.851 0.830 0.857 0.874 0.855 0.894 0.892 0.865 0.837 0.845 0.826 0.825 0.819 0.852 0.938 0.933 0.944 0.881 0.860 0.851 0.854 0.895 0.881 0.876 0.887 0.912 0.876 0.877 0.852 0.833 0.830 0.802 0.833 0.856 0.819 0.838 0.790 0.787 0.787 0.772 0.828 0.821 0.858 0.866 0.866 0.831 SO 0.025 0.037 0.047 0.046 0.042 0.029 0.059 0.048 0.053 0.051 0.023 0.053 0.065 0.034 0.013 0.032 0.046 0.037 0.071 0.084 0.065 0.042 0.059 0.032 0.020 0.065 0.053 0.051 0.042 0.059 0.077 0.041 0.040 0.010 0.030 0.045 0.034 0.031 0.051 0.044 0.056 0.022 0.033 0.014 0.065 0.037 0.054 0.027 0.066 0.027 0.022 0.026 0.023 0.039 0.036 0.013 0.033 0.014 Page A47 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 46.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Respiratory exchange ratio cage 0103 cage 0107 cage 0203 cage 0207 0.927 0.918 0.932 0.913 0.944 0.925 0.942 0.944 0.889 0.920 0.938 0.922 0.913 0.905 0.907 0.894 0.885 0.881 0.896 0.920 0.858 0.889 0.876 0.895 0.848 0.843 0.869 0.852 0.901 0.817 0.861 0.860 0.862 0.791 0.795 0.802 0.851 0.807 0.823 0.884 0.820 0.918 0.843 0.837 0.810 0.952 0.803 0.884 0.919 0.943 0.842 0.839 0.863 0.905 0.824 0.857 0.845 0.880 0.802 0.845 0.868 0.787 0.879 0.888 0.829 0.828 0.875 0.871 0.855 0.912 0.912 0.899 0.922 0.968 0.948 0.926 0.933 0.959 0.960 0.934 0.915 0.919 0.963 0.932 0.815 0.912 0.930 0.916 0.795 0.872 0.882 0.833 0.813 0.806 0.870 0.812 0.794 0.851 0.896 0.801 0.866 0.938 0.666 0.749 0.942 0.939 0.856 0.845 0.890 0.945 0.812 0.929 0.903 0.968 0.843 0.867 0.812 0.919 0.795 0.821 0.781 0.925 0.796 0.794 0.813 0.923 0.829 0.754 0.840 0.892 0.884 0.857 0.860 0.910 0.915 0.861 0.925 0.913 0.924 0.871 0.922 0.882 0.921 0.839 0.939 0.890 0.922 0.885 0.964 0.919 0.931 0.926 0.945 0.958 0.940 0.922 0.900 0.909 0.840 0.908 0.863 0.698 0.830 0.893 0.819 0.819 0.803 0.862 0.814 0.801 0.803 0.792 0.823 0.777 0.829 0.810 0.820 0.803 0.828 0.839 0.809 0.786 0.834 0.849 0.801 0.777 0.795 0.838 0.817 0.811 0.745 0.832 0.802 0.754 0.781 0.798 0.753 0.785 0.806 0.771 0.754 0.786 0.820 0.796 0.789 0.769 0.819 0.803 0.763 0.802 0.846 0.809 0.862 0.924 0.809 0.781 0.804 0.841 0.850 0.822 0.939 0.789 0.877 0.846 0.955 0.799 0.878 0.888 0.936 0.836 0.841 0.660 Average RER 0.922 0.939 0.917 0.905 0.895 0.880 0.853 0.860 0.812 0.841 0.854 0.862 0.886 0.862 0.843 0.855 0.851 0.894 0.941 0.947 0.932 0.893 0.846 0.825 0.836 0.855 0.895 0.894 0.895 0.837 0.824 0.830 0.868 0.887 0.908 0.891 0.909 0.935 0.941 0.889 0.871 0.826 0.803 0.810 0.822 0.819 0.803 0.801 0.786 0.777 0.789 0.795 0.805 0.831 0.842 0.863 0.880 0.868 SD 0.008 0.009 0.020 0.008 0.018 0.016 0.012 0.034 0.034 0.034 0.044 0.070 0.053 0.033 0.032 0.046 0.026 0.027 0.021 0.015 0.022 0.053 0.040 0.030 0.048 0.078 0.052 0.059 0.055 0.056 0.068 0.070 0.024 0.030 0.025 0.040 0.026 0.020 0.015 0.033 0.031 0.025 0.009 0.023 0.015 0.028 0.026 0.039 0.025 0.019 0.027 0.021 0.034 0.029 0.060 0.063 0.064 0.047 Time (hr) 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 eiahciic ^.iqos group HF + Q0C3 '-i. PROS Respiratory exchanoe rabo | Average cage 0104 cage 0108 cage 0204 cage 0208 RER 0.920 0.863 0.914 0.888 0.896 0.903 0.790 0.935 0.916 0.886 0.923 0.802 0.903 0.878 0.876 0.900 0.871 0.899 0.877 0.887 0.894 0.874 0.911 0.891 0.892 0.909 0.821 0.887 0.850 0.867 0.888 0.741 0.893 0.799 0.830 0.882 0.796 0.848 0.774 0.825 0.826 0.782 0.777 0.839 0.806 0.855 0.817 0.787 0.771 0.808 0.891 0.850 0.782 0.814 0.834 0.853 0.772 0.718 0.805 0.787 0.835 0.895 0.828 0.785 0.836 0.806 0.845 0.793 0.818 0.815 0.868 0.784 0.771 0.830 0.813 0.892 0.828 0.814 0.875 0.852 0.851 0.871 0.804 0.848 0.844 0.908 0.861 0.879 0.886 0.888 0.911 0.914 0.894 0.885 0.901 0.886 0.900 0.909 0.848 0.885 I 0.868 0.840 0.912 0.862 0.871 0.843 0.764 0.888 0.827 0.830 0.776 0.819 0.836 0.812 0.811 0.836 0.812 0.865 0.830 0.836 0.824 0.756 0.828 0.761 0.792 0.890 0.825 0.847 0.839 0.850 0.929 0.807 0.801 0.869 0.852 0.916 0.759 0.828 0.902 0.851 0.867 0.813 0.807 I 0.871 0.839 0.898 0.784 0.793 0.790 0.816 0.808 0.787 0.846 0.782 0.806 0.816 0.827 0.785 0.820 0.830 0.804 0.825 0.834 0.814 0.821 0.836 0.826 0.885 0.822 0.842 0.896 0.827 0.859 0.883 0.866 0.890 0.885 0.905 0.864 0.909 0.901 0.860 0.891 0.897 0.857 0.836 0.837 0.868 0.880 0.885 0.829 0.860 0.885 0.852 0.860 0.813 0.861 0.841 0.859 0.841 0.835 0.852 0.846 0.792 0.795 0.817 0.799 0.743 0.779 0.784 0.768 0.750 0.802 0.790 0.798 0.855 0.841 0.834 0.783 0.735 0.762 0.745 0.784 0.792 0.793 0.782 0.803 0.806 0.795 0.797 0.838 0.833 0.741 0.776 0.813 0.816 0.772 0.763 0.791 0.797 0.820 0.833 0.787 0.773 0.809 0.850 0.868 0.885 0.876 0.814 0.807 0.811 0.756 0.792 0.782 0.786 0.836 0.871 0.893 0.869 0.753 0.715 0.766 0.757 0.715 0.755 0.788 0.835 0.878 0.811 0.749 0.736 0.709 0.733 0.722 0.775 0.825 0.825 0.846 0.832 0.782 0.774 0.755 0.764 0.757 0.792 0.830 0.854 0.873 0.832 SD 0.026 0.066 0.053 0.015 0.015 0.039 0.074 0.049 0.031 0.037 0.047 0.057 0.045 0.022 0.044 0.037 0.028 0.013 0.014 0.027 0.030 0.051 0.025 0.022 0.039 0.028 0.060 0.073 0.034 0.055 0.029 0.020 0.013 0.029 0.030 0.015 0.031 0.032 0.023 0.021 0.023 0.047 0.042 0.061 0.034 0.011 0.043 0.033 0.037 0.057 0.033 0.025 0.046 0.041 0.033 0.028 0.020 0.027 Page A48 of A172 Appendix to TNO-report of 3M-02 Final version p. 84 04-12-2006 Respiratory e x ch a n g e ratio, p-values M ann W hitney U-test Time group 1 vs 3 group 1 vs 4 1 1 v s 6 grou p 3 v s 4 grou p 3 v s 6 | group 4 vs 6 1 M 6.1 7.4 8.6 9.9 11.11 1241 1.000 _____ n t n | p 0.486 0.686 0.886 0.343 0.343 I 0.666 0.486 0.886 0.200 nInI 0.686 H H T 0.343 0.343 1.000 0.114 1.000 0.343 1.000 1.000 0.686 0.114 0.4861 P [n ] n| p 0.886 4 g 0.200 0.343 4 H 0.057 0.343 4 9 0.343 0.686 4 9 0.114 0.686 4 9 0.886 0,200 4 4 F iR a 3 6 1 14.91 TT 17.4 136| 19.9 I 21.1 ] 22.' 23j 24.! 26.11 274| 0.686 0.343 0.686 0.886 0.686 0.886 1.000 0.486 0.343 0.886 0.686 1.000 0.886 0.486 0.200 4 0.343 * 0.343 4 0.057 0.486 4 1.000 4 1.000 4 0.343 4 0.886 4 0.343 4 0.486 4 0.343 4 0.886 4 0.886 4 1.000 4 0.886 0.200 0.114 M S i 0.200 0.343 0,200 0.886 0,114 0.200 0.343 0.343 11..000000 0.886 0.486 0.3431 0.686 0.114 0.6861 0.486 0.886 0.886 0.343 0.343 011...8000800601 0.343 0.343 0.0291 0.114 0.343 0.057 01..200000 0.486 0.686 0.486 0 .2 0 0 0.486 1 0.686 0.886 0.686 0.686 0.486 4 g 1.000 4 9 0.343 4 9 0.686 9 0.343 d 9 0.686 4 9 0.200 4 9 0.114 4 9 0.057 9 0.343 4 9 1000 4 4 E OE 3 A B 0.486 4 B 0.029 d B 0.029 i B n irw o 1.0000.686 4 1.000 0.343 0.2001 0.686 4 n 0.200 33.6 0.686 4 0.886 0.4861 0.486 4 a 0.343 34.9 0.686 4 0.886 1.000 0.886 1.000 4 H 0.486 I 1.00036. 0.343 4 0.686 0.114 0.886 0.343 4 H 0.343 37, 0.886 4 1.000 0.886 0-686 4 B 0.686 33 1.000 4 0.343 0.200 0.343 0.200 4 H 0.343 39 0.486 4 0.343 0.114 0.486 0.114 4 H 0.486 il 0.886 4 0.343 0.114 0.200 | 0.029 4 a 0.343 42 0.686 4 0.114 0.114 0.486 0.114 4 43 44 46. 47. 43 49 51 52 53 54 53 57. 53 59 61 62 63 64 63 67, 63 69.9 1 71 JJ 72.41 73.6] 7491 76.11 77.' 0.486 0.114 0.200 0.057 0900 0.343 0.343 0.686 0.886 0.343 0.486 0.200 0.686 0.200 0.686 1.000 1.000 0.486 0.686 0.486 1.000 0.114 0.886 0.114 0.886 0.343 0.686 0.057 4 0.114 4 0.114 4 0.486 4 0.343 4 0.686 4 0.486 4 0.886 4 0.343 4 0.057 4 0.343 4 1.000 4 0.200 4 0.343 4 0.486 4 1.000 4 0.343 4 0.886 4 0.886 4 0.686 4 0.486 4 0.886 4 0.686 4 0.343 4 0.114 4 0.486 4 0.486 4 0.686 4 , 0.486 0.057 0.886 0.343 0.200 0.114 0.686 0.114 0.057 0.200 0.343 0.343 0.343 0.686 0.866 0.886 0.486 0.200 0.114 0.114 0.057 0.200 0.114 0.886 0.114 0-686 I 0.686 0.200 0.057 0.686 0.486 0.486 0.343 0.686 1.000 0.200 0.486 0.200 0.8861 0.200 0.686 0.686 0.6861 01..808060 0.1141 0.486 0.343 0.200 0.057 0.686 0.486 0.886 0.686 0.486 0.343 0.114 0.200 0.343 1.000 0.343 0.114 I 0.486 0.114 1.000 0.886 0.200 0.886 0.057 0.200 0.486 0.686 0.886 0.886 4 H 1.000 4 H 1.000 4 4 fiX fr a 4 a 0.114 4 H 0.114 4 g 0.486 4 g 0.343 4 B 0.029 4 4 1*^751 4 4 ia w .n i 4 B 0.057 4 B 0-200 4 B 1.000 4 g 1.000 4 g 0.343 4 B 0.200 4 B 0.686 4 B 1000 4 B 0.886 4 B 1.000 4 B 0.343 4 ;CEa 4 g 0.200 4 B 0.200 4 B 1000 4 B 0.886 4 4 I 3 4 H 0 .2 0 0 ] I P<0-05 1 Page A49 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 D6. Energy expenditure T itle (hr) 6.1 7.4 9.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31,1 32.4 33.6 34.9 36.1 3>.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 .9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Energy expenditure {kcaWVkq mouse} case 0101 cage 0105 cage 0201 cage 0205 1526 17.60 18.09 15.65 14.77 18.58 16.69 14.83 14.06 1722 15.46 15.02 15.29 1723 18.03 14.38 13.87 15.56 1421 15.73 14.09 14.71 16.01 14.24 13.11 15.06 13.84 13.08 13.48 13.36 13.69 1327 12.13 13.82 13.73 13.00 14.07 13.01 14.23 13.37 15.59 13.78 12.86 13.16 13.62 14.11 14.61 14.87 12.64 14.44 12.93 1221 13.44 13.08 14.15 14.04 14.61 14.60 14.57 13.58 18.76 18.08 18.68 19.18 14.71 16.46 17.67 18.47 16.80 1725 17.38 14.72 14.60 15.94 17.12 17.36 14.85 15.69 15.70 14.61 14.90 15.61 16.65 14.99 13.70 1520 16.48 15.57 13.66 15.14 16.64 13.65 15.51 14.87 15.70 13.65 13.93 14.80 13.31 13.10 13.90 14.00 16.10 13.12 13.43 12.68 " m 2 " 12.34 12.20 13.38 14,13 13.03 13.65 1320 1321 11.93 11.75 13.71 1331 13.69 12.71 1229 13.16 12.74 12.64 13.18 14.44 12.48 13.72 14.15 14.99 13.38 14.46 14.15 13.45 13.61 17.69 17.46 18.78 16.77 16.95 16.08 18.41 16.32 16.37 17.06 17.66 15.18 14.77 16.94 16.72 1527 14.41 15.64 15.92 1525 14.84 15.90 1622 14.90 13.10 15.64 15.87 13.62 13.93 15.75 1627 14.08 13.93 13.92 16.08 14.73 14.30 15.21 (5.35 12.69 13.41 13.41 1326 12.72 11.94 14.05 13.54 11.28 12.07 12.94 1422 12.19 13.53 1228 14.41 11.88 11.78 10.77 1121 12.25 12.67 12.95 13.74 11.51 11.52 12.61 12.64 12.73 12.09 12.89 12.94 11.99 12.71 11.98 13.86 13.18 1529 1524 16.61 15.93 16.15 1721 1920 16.67 16.12 16.43 17.30 16.02 1428 15.90 16.75 1424 14.01 1621 16.58 15.16 Average energy expenditure fkcalftVkg mouse) 16.65 1622 15.44 1623 14.92 14.76 13.77 13.50 13.17 13.67 13.85 14.30 13.11 13.68 14.34 18.67 16.83 16.54 1625 1521 15.54 1526 14.82 14.93 1X78 1428 13.18 13.19 1X02 1327 1X75 1X19 14.06 1X93 17.67 1X94 16.57 15.95 15.33 15.46 14.56 1X01 14,67 14.39 13.35 1X70 1X86 1X02 11.58 1X72 1X37 12.48 1X94 1X84 17.31 16.47 1529 15.49 SO 1.40 1.81 1.32 1.69 0.88 0.86 0.93 027 0.78 0.58 1.22 0.56 0.95 0.51 0.50 0.45 1.64 1.24 1.27 0.57 0.80 1.16 1.38 023 0.7$ 1.28 0.84 0.80 0.75 1.01 0.32 0.89 0.70 0.48 0.84 105 1.06 1.05 0.66 0.70 1.40 1.18 1.02 1.22 0.47 1.31 0.99 1.16 0.62 0,92 0.57 0.51 0.80 0.57 1.33 0.58 1.24 1.16 Time w 6.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 38.1 37.4 38.6 39.9 41.1 42.4 43.6 44.9 46.1 47.4 48.6 49.9 S1.1 S2.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Energy expenditure (kcaWVkg mouse) cage 0102 cage 0106 cage 0202 cage 0206 17.43 16.55 15.54 17X0 17.09 16.86 16.94 17.80 15.39 15.70 17X9 16.16 15.91 14.45 15.57 1X99 16.34 15.43 18.84 15.42 14.64 15.03 1X85 14.16 15.19 12.93 15.35 15.09 13.95 13.93 15.14 12X7 14.44 1X92 1X41 12X2 14.01 12.76 17X2 13.38 14.98 14.58 15.00 12X1 12.72 13.02 15.40 12.07 13.81 1X65 16.90 13.71 13.46 14.25 15.08 13.72 15.62 12.57 17X0 13X6 18.75 18.37 20X2 17.56 18.06 19.16 19X7 18.43 16.48 15.80 17.65 17.88 16.92 12.95 16.88 15.09 15.90 1X54 17.00 16.45 14.09 12X1 17.31 15X3 14.12 13.77 17X6 14.77 14.78 1X74 16X4 13.97 15.12 1X05 1X89 15.06 14.52 14.64 1X12 13X2 15.83 1X02 16X6 14.16 13.76 1X26 15.82 12.58 13.81 1X37 15.86 1X21 13.39 1X95 15.06 12X2 13.12 10.84 1X01 15.10 1X64 11.69 15.73 13.03 13.95 1X08 16X1 13X2 1X47 1X78 15.51 12.95 15.18 1X38 16.4$ 13.52 17.57 15.45 18.91 17.72 19.09 17.24 18.96 18.82 17.30 16.92 17.99 17.07 16.05 14.75 16X7 16.18 1X77 13.77 16.86 15X3 15.50 14.08 17.30 15.96 15.48 14.73 16.46 15.33 14.11 13.92 16.45 15.71 15X4 15.13 17.39 15.66 14.87 13.68 16.69 14.54 13.37 15.05 14.93 12.60 12.14 1X59 14.17 13.05 11.67 1X16 14.32 11X7 11.45 11.64 15.69 13.95 12.32 11.23 13.41 11X5 11.54 11X1 14.36 13.65 11.90 1X82 13.42 11.92 11.77 11.55 13.71 11X2 13.05 11.30 15.66 13.56 15.71 14.02 16.76 15.30 17.40 1X88 19.87 17X0 15.92 16.23 17.67 16X7 15.41 14.16 16.82 16.69 14.65 14.28 17.27 16X9 Average energy expenditure fkcaVh/kg mouse) 1X68 17.17 1X13 15.73 16.51 15.17 14.64 13.82 14.32 14.34 14.34 13X0 14.52 14.13 1X76 1X80 1X88 16.95 15.46 1X47 14.71 15.00 14X3 1X03 14.62 1X60 13.61 13.57 13.48 13.77 13X2 13.86 13.68 1X39 17.41 18X3 17.32 1X81 1X66 1X71 1X50 1X05 15.91 14.94 13.99 13X4 1X60 13.18 12.05 12.69 12X2 12X1 13.44 1X44 17.59 16.68 15.77 15.62 SD 0.85 0.43 0.83 1.05 1.61 1.17 1.14 1.18 1X1 1.99 1.04 1X5 1.59 0.72 2.12 1.25 0.80 0.96 1.88 0.45 2.13 1.62 1.47 1.57 1.19 1.95 1X1 1.70 1.11 2.29 1.68 1.74 1X5 1,79 1.44 0X7 0.48 0.71 1.46 1.33 0.72 173 1.04 1.27 1.20 0.86 1.40 2.02 1.04 1.55 0.74 1.00 1.88 113 1.67 0.77 1.25 1.40 09 PageA50of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Time (hr) 8.1 7.4 8.6 9.9 11.1 12.4 13.6 14.9 16.1 17.4 18.6 19.9 21.1 22.4 23.6 24.9 28.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 37.4 386 39.9 41.1 42.4 43.6 449 46.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Energy expenditure (kcaJ/h/kq mouse) cage 0103 cage 0107 cage 0203 cage 0207 17.72 18.32 18.90 18.10 17.11 17.10 18.69 17.78 16.08 17.64 18.56 15.91 16.29 16.21 18.34 14.84 15.79 14.83 18.65 18.03 15.90 16.76 18.05 16.46 15.81 13.50 15.81 14.17 15.50 14.04 16.75 14.00 15.27 16.11 15.26 13.82 15.08 15.83 15.47 13.59 14.56 14.33 14.50 14.54 14.82 15.78 15.58 14.07 15.26 14.93 14.36 13.74 14.90 14.62 15.33 16.29 15.70 15.43 15.87 14.86 19.72 21.15 18.58 17.89 20.61 20.83 20.66 20.08 18.46 19.15 18.98 18.45 17.40 16.86 19.60 18.59 15.63 17.63 18.26 16.07 16.78 15.69 18.33 15.98 15.19 14.09 19.84 16.07 15.21 15.71 19.30 15.97 14,31 11.67 17.68 147 15.18 14.05 16.54 15.91 14.73 15.03 17.38 15.56 14.63 13.86 15.71 12.89 13.96 13.36 14.92 14.30 14.49 14.67 15.65 12.34 14.01 13.51 14.35 13.17 14.08 13.28 14.29 13.21 13.4? 14.84 16.71 12.78 14.88 16.08 15.00 15.51 15.64 14.14 16.17 13.12 18.28 17.34 17.39 15.11 20.86 19.42 20.34 17.84 18.77 18.11 21.60 18.16 18.37 16.76 19.49 18.59 17.30 17.80 19.21 16.43 16.46 14.44 18.06 16.42 16.08 15.13 17.28 16.52 14.64 14.47 18.02 14.88 15.67 15.08 17.87 15.07 14.27 12.31 18.74 14.18 14.48 12.77 15.72 13.40 14.56 13.63 14.62 13.60 13.88 12.02 15.96 12.96 13.99 13.95 14.50 12.77 12.49 11.40 15.11 14.61 13.11 11.93 14.36 12.75 12.66 12.96 14.99 12.77 13.64 13.88 14.56 13.32 13.62 14.26 14.77 12.59 15.79 15.84 15.90 14.20 18.77 19.12 18.49 16.54 19.64 17.74 21.04 18.51 16.81 14.27 20.24 17.03 16.94 15.58 19.19 16.40 Average energy expenditure (keaVh/ka mouse) 1846 17.67 17.05 16.42 16.33 18.79 14.82 15.07 15.12 14.99 1448 15.06 14.57 1538 1546 19.33 20.50 18.76 18.16 1630 1539 1639 16.55 14.61 1542 15.67 1437 14.14 1439 13.76 13.71 1445 1537 14.77 17.03 19.62 19.16 1830 17.68 1635 1635 15.50 15.92 1437 14.09 14.11 13.70 13.80 1340 13.04 1335 1335 1331 1543 1833 19.23 17.09 17.03 SD 0.49 0.75 137 1.44 1.64 0.91 1.17 132 0.95 0.99 0.11 0.78 0.67 0.73 044 1.43 038 036 131 135 1.18 2.50 136 246 1.07 1.18 130 0.65 1.39 0.52 0.55 1.73 055 1,40 135 133 1.65 1.14 1.16 148 0.90 1.69 1.33 132 130 0.56 1.69 0.73 1.75 1.01 1.10 0.53 034 032 1.16 143 245 1.55 Time 6.1 74 8.6 9.9 11.1 12.4 13.8 14.9 16.1 174 18.6 19.9 21.1 22.4 23.6 24.9 26.1 27.4 28.6 29.9 31.1 32.4 33.6 34.9 36.1 374 38.6 39.9 41.1 42.4 43.6 44.9 48.1 47.4 48.6 49.9 51.1 52.4 53.6 54.9 56.1 57.4 58.6 59.9 61.1 62.4 63.6 64.9 66.1 67.4 68.6 69.9 71.1 72.4 73.6 74.9 76.1 77.4 Energy expenditure (kcatfhflm mouse) cage 0104 cage 0108 cetre 0204 cage 0208 19.47 21.08 19.61 17.81 18.62 19.06 18.86 18.04 16.51 18.30 18.11 16.54 18.20 18.49 17.62 16.84 19.01 17.84 18.00 15.43 17.93 15.57 16.43 15.19 1849 14.69 1583 15.23 17.61 18.67 15.79 14.34 16.12 13.87 14.75 14.73 15.64 15.29 14.35 13.23 18.32 17.05 15.27 13.96 17.91 14.12 13.69 14.47 16.01 1554 14.26 14.58 15.85 16.13 14.28 14 54 18.23 15.33 14.68 15.49 20.12 19.45 16.14 16.60 21.17 19.41 19.46 18.32 18.52 18.96 20.24 19.23 19.45 20.08 17.71 18.51 18.39 17.21 16.81 16.76 1929 17.83 17,23 16.41 18.53 15.12 16.72 16.89 18.69 16.53 17.36 15.99 18.84 16.51 16.59 15.65 18.59 14,60 15.98 14.62 17.62 14.68 16.18 15.30 16.44 14.48 14.14 15.27 16.15 12.94 14.29 15.48 15.21 13.64 14.29 14.75 15.50 15.37 13.42 14.02 15.94 13.52 15.32 15.22 16.36 13.59 14.18 14.09 16.01 14.15 14.92 14.94 16.00 14.11 14.52 14.85 18.97 15.99 1728 15.91 21.66 18.42 17.93 17.63 18.81 19.72 18.76 18.78 19.91 19.11 17.76 17.42 18.13 18.31 17.13 17.61 19.04 16.79 16.98 1620 19.21 15.17 17.38 16-23 18.16 16.35 17.05 16.49 18.42 16.12 16.12 1854 18.84 18.11 16.36 16.05 17.44 15.08 15.28 15.33 17.66 1552 14.46 1552 15.27 13.11 14.82 13.76 15.52 14.03 13.04 14.49 14.25 12.15 13.77 13.48 15.72 14 09 13.50 12.84 15.34 12.66 13.55 13.02 15.08 14.14 12.63 1455 14.87 13.67 12.91 13.09 17.36 13.95 14.19 14.87 18.36 17.87 15.30 15.80 18.82 16.27 16.93 17.77 17.86 17.62 16.97 18.62 18.01 17.66 1650 17.84 Average energy expenditure (kcaUhtoa mouse) 19,49 18.85 1756 17.79 17.57 1658 16.06 16.10 14.87 14.63 16.15 15.05 15.02 1550 15.93 18.08 19.59 1954 1654 1759 17.69 16.81 17.14 16.90 15.95 15.9 15.06 14.72 14.47 14.58 15.00 14,56 15.00 14.87 17.03 18.91 19.02 18.55 17.79 1755 17.01 17.01 16.73 16.84 15.78 15.64 1454 1457 13.41 14.04 13.64 14.02 13.64 15.09 16.83 17.45 17.76 17.43 SD 1.34 0.44 0.97 0.73 1.52 152 1.68 1.39 0.93 1.08 1.92 1.94 0.76 0.93 1.57 2.00 1.18 0.73 1.04 0.76 152 1.39 1.18 156 1.88 157 1.02 1.41 0.67 1.02 1.04 153 0.76 0.81 1.43 1.86 0.47 1.16 0.53 1.24 1.72 0.82 1.13 1.34 1.11 159 0.98 1.03 0.90 153 1.19 1.02 0.89 1.56 1.51 1.10 0.68 0.63 W PageA51 of A172 Appendix to TNO-report of 3M-02 Final version Energy expenditure, p-values Mann Whitney U-test group 1 vs 4 group 1 vs 6 group 3 vs 4 group 3 vs 6 group 4 vs 6 s s linl n| p n I n p n| n| p n I n I p nln O . 29 4 4 0.200 4 | 4 - 0.114 - ! 0.114 4 4 0.686 A I 0.057 4 4 0.200 4 0.343 4 4 0.486 4 ' 0.200 4 4 0.343 4 0.343 4 4 0.343 . ^ 0.057 4 4 0.343 7 4 0.686 4 4 0.886 4 0.686 4 4 0.686 4 0.200 4 4 0.343 4 0.200 4 4 0.686 4 0.343 4 4 0.686 4 0.114 4 4 0.686 4 0.686 4 4 0.886 4 0.686 4 4 0.486 4 0.486 4 4 0.343 4 0 2 0 1 4 4 0.343 0.6864 0 2 9 *1 4 4 0.486 4 0.114 4 4 4 0.114 4 4 0.343 4 0.200 4 4 0.686 4 0.057 4 4 0.343 4 0.200 : 4 4 0.200 4 0.486 4 4 0.886 4 0.486 4 4 0.886 4 0.200 4 4 0.486 4 0.343 4 4 0.686 4 0 . 2 0 0 1 4 4 1.000 4 0.686 4 4 0.343 4 0.343 4 4 0.114 4 0.343 4 4 0.886 4 0.200 4 4 0.486 4 0 886 4 4 1.000 0.6864 0.886 4 4 0.886 4 0.886 4 4 4 0.029 4 4 0.486 4 0.029 4 4 0.886 _ 4 0 2 9 4 4 4 0.886 4 0.200 4 4 0.486 4 0.343 4 4 0.486 0.057 4 4 0.200 4 0.200 4 4 0.200 4 0.200 4 4 0.210 4 0.029 4 4 0.200 -+ 0.029 4 4 0.114 4 0.200 4 4 0.686 4 4 0.486 4 4 0.486 _ 4 0.114 4 4 0.886 4 0.486 4 4 0.343 4 0.200 4 4 0.486 4 0.057 4 4 0.686 4 0.886 4 4 1.000 4 0.686 4 4 0.486 4 0.686 4 4 0.114 4 0.200 4 4 0.200 H 0.029 4 4 0.486 ^ 4 0.114 4 4 0.686 04-12-2006 p<0.05 Page A52of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Appendix E. Fecal bile acid, sterol and fatty acid measurements E1. Fecal weight measurements, bodyweight and food intake measurements Bodyweight and food intake during collecting feces Mouse# I I I I I I I | I I I I I I I I I I I I I I l I I I 5- i . .fi**-! a 1 1 I I I I-W W ! ,i 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 cage# 1 1 2 2 3 3 4 5 5 5 6 6 6 7 8 8 8 9 9 10 11 12 13 13 14 15 15 15 16 17 18 18 19 19 19 19 20 20 21 21 22 22 22 22 23 24 25 25 26 27 28 29 29 30 30 31 31 31 31 32 33 34 35 36 36 37 38 38 39 39 eartag LR 01 12 00 20 00 02 11 00 02 12 10 21 02 01 10 01 20 00 21 0 0 02 01 1 0 01 20 2 02 0 0 02 00 10 0 02 00 1 00 0 0 12 12 20 0 00 12 01 10 12 10 02 00 01 02 22 2 20 10 02 00 12 00 00 20 10 01 collection point 1:2 days faces collection start collection point 1 end collection point 1 bodyweight (g) food on cage (g) bodyweight (g) food off cage (g) 33.9 33.9 31.2 49.7 30.5 38.9 30.4 31.0 28.1 51.9 28.2 39.5 31.1 30.6 29.5 47.4 29.4 35.8 26.7 26.9 27.2 20.5 30.4 30.6 30.6 31.0 27.2 61.6 27.3 44.8 33.0 33.1 29.8 30.0 312 68.5 32.0 48.6 32.8 31.7 33.1 24.8 30.8 31.0 30.1 29.8 32.8 68.0 32.8 47.2 26.5 26.6 26.9 42.3 28.6 31.8 29.0 29.2 28.6 23.4 27.8 32.1 28.3 26.2 28.8 32.9 28.9 27.2 32.2 32.3 28.8 44.9 28.9 30.3 25.8 31.2 26.3 25.2 28.7 28.5 30.1 30.1 25.0 77.5 24.7 61.0 29.8 23.1 29.7 17.1 27.0 26.3 27.0 20.0 30.3 30.2 29.3 48.3 29.2 36.3 29.2 28.9 30.9 30.7 31.0 30.7 25.6 90.1 25.8 68.4 27.0 26.7 30.2 43.1 30.2 31.0 27.7 27.2 27.7 47.9 27.7 36.0 28.9 30.0 25.2 25.3 27.6 27.8 28.5 90.3 28.1 68.8 28.6 37.8 29.7 30.8 28.5 27.2 28.6 21.1 30.3 30.1 29.3 50.7 29.9 37.2 27.3 27.7 27.2 22.1 27.7 27.1 28.7 20.2 28.4 31.9 28.5 26.2 31.3 31.5 27.3 52.3 27.2 40.4 30.2 30.0 28.8 42.9 28.4 31.2 25.6 25.4 27.6 27.8 29.1 29.1 30.3 88.0 30.7 67.7 26.7 30.5 26.7 25.0 28.2 29.1 27.7 23.4 27.3 31.2 26.7 25.5 269 34.7 26.6 29.3 289 28.5 27.4 212 28.2 9.4 26.1 26.4 26.1 21.3 26.0 26.2 26.7 53.0 26.9 42.5 23.6 23.0 25.1 43.8 24.6 32.4 Average bodyweight {g) 33.90 30.85 30.70 28.15 30.85 29.45 26.95 30.50 30.80 27.25 33.05 29.90 31.60 32.95 30.90 29.95 32.80 26.55 26.75 28.90 28.05 28.85 32.25 28.85 26.05 28.60 30.10 24.85 29.75 27.00 30.25 29.25 29.05 30.80 30.85 25.70 26.85 30.20 27.45 27.70 29.45 25.25 27.70 28.30 29.15 28.55 30.20 29.60 27.25 28.20 28.45 31.40 2725 30.10 28.60 25.50 27.70 29.10 30.50 26.70 27.95 27.00 26.40 28.35 27.80 26.10 26.10 26.80 23.30 24.85 Food intake (g) coB. point 1 10.8 12.4 11.6 6.4 16.8 19.9 6.9 20.8 10.5 5.8 5.9 5.7 14.6 6.0 16.5 6.0 6.3 12.0 21.7 12.1 11.9 21.5 7.0 6.1 13.5 5.6 6.9 5.7 11.9 11.7 20.3 5.5 5.7 5.7 5.4 11.8 5.1 10.5 11.4 Page A53 of A172 p. 89 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Mouse# I1 I2 3 4 5 I6 I7 8 I9 10 I 11 12 13 I 14 15 I 16 I 17 I 18 I 19 I 20 I 21 22 I 23 I 24 I 25 I 26 I 27 I 28 1 29 1 30 I 31 I 32 I 33 1 I I 1 1 1 1 S ':-' 1 5 i i1 I -.g ^ 5 * 5'?.\ .5 I I I I I I 1 1 I I 1 1 9 7 34 35 36 37 36 39 40 41 42 43 44 45 46 47 48 49 so 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 -- 11. 67 68 69 70 cage# 1 1 2 2 3 3 4 5 5 5 6 6 6 7 8 8 8 9 9 10 11 12 13 13 14 15 15 15 16 17 18 18 19 19 19 19 20 20 21 21 22 22 22 22 23 24 25 25 26 27 28 29 29 30 30 31 31 31 31 32 33 34 35 36 36 37 38 38 39 39 eartag LR 01 12 00 20 00 02 11 00 02 12 10 21 02 01 10 01 20 00 21 10 01 02 01 11 10 0 20 12 02 10 10 02 00 10 01 02 00 11 00 1 10 01 11 12 12 20 01 11 00 12 01 10 12 10 02 00 01 02 22 2 20 10 02 00 12 00 00 20 10 01 collection point 2; 3 days feces collection start collection point 2 end collection point 2 bodyweight (g) food on cage (g) bodyweight (g) food off cage (g) 33.9 32.9 30.5 38.9 30.1 22.1 31 30 28.2 39.5 28.3 19.5 30.6 292 29.4 35.8 30.4 16.8 27.2 20.5 26.3 10.7 30.6 30 31 30.5 27.3 44.8 27 19.2 33.1 32.2 30 30 32 48.6 31.2 19.8 33.1 24.8 33.6 12.4 31 30.1 29.8 30 32.8 47.2 32.5 14.2 26.6 26.3 26.6 31.8 26.9 13.6 28.8 23.4 29 13.4 28.3 26.2 28.4 15.7 28.9 27.2 29.2 16.2 32.3 32.3 28.9 30.3 292 7.1 26.3 25.2 26.1 15.6 28.5 28.8 30.1 29.8 24.7 61.0 25.1 32.7 29.7 17.1 29.4 8.0 27 20.0 27.6 9.4 30.2 29.6 29.2 36.3 28.7 16.5 28.9 29.6 30.7 30.5 30.7 30.7 25.8 68.4 262 33.2 26.7 26.9 30.2 31.0 30.1 11.1 27.2 27.2 27.7 36.0 27.7 17.7 30 29.1 25.3 25.5 27.8 28.6 28.1 68.8 28.8 32.3 29.7 30.8 30.1 20.1 28.6 21.1 28.6 10.6 30.1 30.1 29.9 37.2 29.7 15.5 27.2 22.1 27 12.5 28.7 202 28.4 10.2 28.5 262 28.7 16.6 31.5 31.4 27.2 40.4 27 20.3 30 29.7 28.4 312 28 12.3 25.4 25.3 27.8 26.9 29.1 28.8 30.7 67.7 30.9 36.7 26.7 25.0 26.3 16.0 27.7 23.4 29.1 12.7 26.7 25.5 27.8 16.3 26.6 29.3 26.8 20.0 28.5 28.9 28.2 56.1 27.6 38.8 26.1 21.3 26.2 12.1 26.2 26.7 26.9 42.5 27.2 24.9 23 23.6 24.6 32.4 25.7 15.0 Average bodyweight (g) 33.40 30.30 30.50 28.25 29.90 29.90 26.75 30.30 30.75 27.15 32.65 30.00 31.60 33.35 30.55 29.90 32.65 26.45 26.75 28.90 28.35 29.05 32.30 29.05 26.20 26.65 29.95 24.90 29.55 27.30 29.90 28.95 29.25 30.60 30.70 26.00 26.80 30.15 2720 27.70 29.55 25.40 28.20 28.45 29.90 28.60 30.10 29.80 27.10 28.55 28.60 31.45 27.10 29.85 2820 25.35 27.35 28.95 30.80 26.50 28.40 27.25 26.70 28.70 27.90 26.15 26.45 27.05 23.30 25.15 Food intake (g) cofl. point 2 16.8 20.0 19.0 9.8 25.6 28.8 12.4 33.0 18.2 10.0 10.5 11.0 23.2 9.6 28-3 9.1 10.6 19.8 35.2 19.9 18.3 36.5 10.7 10.5 21.7 9.6 10.0 9.6 20.1 18.9 31.0 9.0 10.7 9.2 9.3 17.3 9.2 17.6 17.4 Page A54 of A172 n Appendix to TNO-report of 3M-02 Final version 04-12-2006 Group# Mouse# ' *S y*. " 5. 5 S'. . 5. 5 ' ' i. '5 .5 J13S. r' r- 7*- . 7 78 .7 * Ti 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 36 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 cage# 1 1 2 2 3 3 4 5 5 5 6 6 6 7 8 8 8 9 9 10 11 12 13 13 14 15 15 15 16 17 18 18 19 19 19 19 20 20 21 21 22 22 22 22 23 24 25 25 26 27 28 29 29 30 30 31 31 31 31 32 33 34 35 36 36 37 38 38 39 39 eartaa LR 01 12 00 20 00 02 11 00 02 12 10 21 02 01 10 01 20 00 21 10 01 02 01 10 0 20 2 02 10 10 02 00 10 01 02 00 11 00 11 10 01 11 12 12 20 01 11 00 12 01 10 12 10 02 00 01 02 22 21 20 10 02 00 2 00 00 20 0 0 collection point 3; 2 days feces coBection start collection pant 3 end collection point 3 bodyweigtit (g) food on cage (g) bodyweight (g) food off cage (g) 32.9 33.2 30.1 78.3 30.2 68.3 30 30.2 28.3 73.4 28.4 63.2 29.2 31.1 30.4 67.0 29.6 56.6 26.3 38.3 26.5 33.4 30 30.8 30.5 30.7 27 94.8 27.1 81.0 32.2 32.8 30 30.0 31.2 97.1 31.8 80.4 33.6 42.0 33.1 36.8 30.1 30.3 30 30.9 32.5 85.6 32.7 67.2 26.3 25.9 26.9 60.9 26.5 52.8 29 46.3 29.1 41.2 28.4 39.9 29.1 34.3 29.2 40.9 29.2 35.5 32.3 32.5 29.2 56.5 29.3 43.8 26.1 46.1 26.4 41.0 28.8 29.1 29.8 30.2 25.1 114.5 25.4 100.9 29.4 34.9 29.6 29.9 27.6 37.9 27.8 33.3 29.6 29.9 28.7 61.5 29.2 50.2 29.6 29.8 30.5 30.5 30.7 31.1 262 130.6 26.5 111.9 26.9 28.0 30.1 70.0 30.9 58.0 27.2 27.5 27.7 67.9 27.5 57.3 29.1 29.8 25.5 25.6 28.6 29.2 28.8 138.4 29.1 119.7 30.1 52.3 29.6 47.7 28.6 37.3 28.6 31.9 30.1 30.6 29.7 74.9 30.2 52.3 27 39.9 27.3 35.1 28.4 35.4 28.6 30.3 28.7 44.3 28.7 38.8 31.4 32.1 27 66.8 27.4 55.8 29.7 30.2 28 60.0 28.5 49.9 25.3 25.6 26.9 27.2 28.8 28.6 30.9 142.0 30.6 124.9 26.3 46.7 26*6 41.8 29.1 35.7 28.5 30.7 27.8 42.4 27.8 37.5 26.8 46.1 27.0 40.3 28.9 28.7 27.6 90.6 28.2 81.1 26.2 39.7 27.1 34.2 26.7 26.7 27.2 76.5 27.3 66.5 23.6 23.8 25.7 65.9 25.4 56.1 Average bodyweight (g) 33.05 30.15 30.10 28.35 30.15 30.00 26.40 30.40 30.60 27.05 32.50 30.00 31.50 33.35 30.20 30.45 32.60 26.10 26.70 29.05 28.75 29.20 32.40 29.25 26.25 28.95 30.00 25.25 29.50 27.70 29.75 28.95 29.70 30.50 30.90 26.35 27.45 30.50 27.35 27.60 29.45 25.55 26.90 28.95 29.85 28.60 30.35 29.95 27.15 28.50 28.70 31.75 27.20 29.95 28.25 25.45 27.05 28.70 30.75 26.45 28,80 27.80 26.90 28.80 27.90 26.65 26.70 27.25 23.70 25.55 Food intake (g) coll, point 3 10.0 102 10.4 4.9 13.8 16.7 5.2 18.4 8.1 5.1 5.6 5.4 12.7 5.1 13.6 5.0 4.6 11.3 18.7 12.0 10.6 18.7 4.6 5.4 22.6 4.8 5.1 5.5 11.0 10.1 17.1 4.9 5.0 4,9 5.8 9.5 5.5 10.0 9.8 Page A55 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Fecal weight Group j cage# 2 2 2 2 2 4 4 4 4 -9 * * - ' . - - S : " - - ':- S'-dPt-rtf 6 n 6 6 0 7 7' - 72". 7' *?'7> 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Feces collection point 1; 2 days collection number of Tube empty Tube full Tube full after freeze drying mice 2 2 (9) 6.6630 6.6490 (9) 8.3248 8.6605 (9) 8.0444 8.3426 2 6.6221 8.6309 1 6.6808 7.664 8.3229 7.4697 3 6.6712 9.4474 8.9658 3 6.6634 9.8825 9.4253 1 6.6794 7.8244 3 6.6207 10.2937 7.6266 9.7316 2 6.6605 8.2584 1 6.6630 7.5596 8.0008 7.4162 1 6.6697 7.5244 1 6.6594 7.5056 2 6.6379 9.0681 7.3942 7.3806 8.6519 1 6.6624 7.5339 7.4049 3 6.6568 9.433 8.9562 1 6.6607 7.5433 1 6.6312 7.6683 7.3604 7.5344 2 6.6765 8.6953 8.3655 4 6.6311 10.3697 9.7743 2 6.6493 8.6134 2 6.6603 8.6425 8.2667 8.2974 4 6.6918 10.4134 9.8868 1 6.6683 7.7517 7.5567 1 6.7028 7.6752 7.5296 2 6.6868 9.2271 8.8008 1 6.6825 7.5842 7.456 1 6.6889 7.8388 7.6669 1 6.6648 7.6251 2 6.6617 8.6518 7.4945 8.3356 2 6.6503 8.6434 8.3383 4 6.6110 9.8507 9.3193 1 6.6541 7.4635 7.3215 1 6.6615 7.3485 7.2406 1 6.6589 7.5622 7.3939 1 6.6820 7.476 7.3376 2 6.6335 8.4094 1 6.6727 7.4706 8.0542 7.3318 2 6.6890 8.3375 2 6.6706 8.2396 8.054 7.9761 freeze dried feces (g) 1.3814 1.6936 1.7008 0.7889 2.2946 2.7619 0.9472 3.1109 1.3403 0.7532 0.7245 0.7212 2.0140 0.7425 2.2994 0.6997 0.9032 1.6890 3.1432 1.6174 1.6371 3.1950 0.8884 0.8268 2.1140 0.7735 0.9780 0.8297 1.6739 1.6880 2.7083 0.6674 0.5791 0.7350 0.6556 1.4207 0.6591 1.3650 1.3055 Page A56of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Group cage# 2 2 2 2 4. 4 4 4 *> N '5 r v Sir'1. ; P' 8 6 6 6 .-7 -7 1 W yr. -5- V7V'" ' ` 7' ' 7tt- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Feces collection point 2; 3 days collection number of Tube empty Tube full Tube full after freeze drying mice 2 (g) 6.6728 (9) 9.043 (9) 8.654 2 6.6313 9.7853 2 6.6987 9.5813 9.3421 9.188 1 6.6499 8.1391 3 6.6805 10.6433 7.9395 10.0911 3 6.6610 11.3687 1 6.6450 8.426 10.736 8.1553 3 6.6479 12.3927 11.5086 2 6.6676 9.2172 8.8482 1 6.6137 8.0501 7.8405 1 6.6564 8.1514 7.945 1 6.6806 8.3368 2 6.6554 10.2976 8.1005 9.7616 1 6.6905 8.1358 7.9299 3 6.6709 11.0804 10.4427 1 6.6500 8.0103 7.7806 1 6.6622 8.2678 8.0649 2 6.6790 9.8571 9.3923 4 6.6452 12.0852 2 6.6370 9.933 2 6.6464 9.514 11.2859 9.4414 9.0495 4 6.6068 12.3762 11.578 1 6.6411 8.2187 7.9954 1 6.6557 8.1263 7.9237 2 6.6822 10.3758 9.8975 1 6.6134 8.0183 7.792 1 6.6799 8.1596 7.914 1 6.6762 8.1471 7.9636 2 6.6989 9.824 9.3799 2 6.6732 9.5294 9.1304 4 6.6621 11.3048 10.6299 1 6.6310 7.9252 7.7404 1 6.6902 8.1846 7.96 1 6.6712 7.9152 7.7309 1 6.6337 7.9533 2 6.6633 9.2718 1 6.6324 7.8893 7.7519 8.8526 7.7077 2 6.6776 9.1802 2 6.6697 9.253 8.8063 8.8469 freeze dried feces (g) 1.9812 2.7108 2.4893 1.2896 3.4106 4.0750 1.5103 4.8607 2.1806 1.2268 1.2886 1.4199 3.1062 1.2394 3.7718 1.1306 1.4027 2.7133 4.6407 2.8044 2.4031 4.9712 1.3543 1.2680 3.2153 1.1786 1.2341 1.2874 2.6810 2.4572 3.9678 1.1094 1.2698 1.0597 1.1182 2.1893 1.0753 2.1287 2.1772 Page A57 of A172 Appendix to TNQ-report of 3M-02 Final version 04-12-2006 Group 2 2 2 2 2 4 4 4 4 ip - 5 5 ;:- 6 6 6 6 6 -7 1 .7 ? 7 7 7 . cage# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Feces collection point 3; 2 days collection num ber of Tube empty Tube full Tube full after freeze drying mice 2 (9) 6.6429 (g ) 8.5821 (g ) 8.2399 2 6.6725 8.9822 8.6509 2 6.6831 8.9939 8.653 1 6.6645 7.7309 7.5727 3 6.6432 9.9317 9.4021 3 6.6844 10.106 1 6.6652 7.8515 9.6123 7.6749 3 6.6453 10.6479 10.0341 2 6.6397 8.3585 1 6.6569 7.7971 8.1165 7.6376 1 6.6860 7.7179 7.5628 1 6.6590 7.7398 7.58 2 6.6605 9.3189 1 6.6334 7.6444 8.8877 7.4947 3 6.6582 9.6295 9.1847 1 6.6145 7.6351 1 6.6692 7.7894 7.4819 7.631 2 6.6935 9.0966 8.7345 4 6.6306 10.6276 10.077 2 6.6585 9.1542 8.7793 2 6.6901 8.8739 8.5242 4 6.6840 10.9177 10.2953 1 6.6687 7.6866 7.5361 1 6.6635 7.8102 7.6563 2 6.6833 9.43 9.0427 1 6.6333 7.6942 7.5479 1 6.6752 7.7479 7.5966 1 6.6351 7.7734 7.6365 2 6.6633 9.0885 8.5695 2 6.6611 8,8705 8.5556 4 6.6657 10.3688 9.9106 1 6.6786 7.7434 7.5484 1 6.6860 7.6314 7.485 1 6.6887 7.5625 7.393 1 6.6783 7.9028 7.6867 2 6.6551 8.561 8.2858 1 6.6481 7.617 7.4543 2 6.6522 8.6462 2 6.6641 8.7084 8.3568 8.3702 freeze dried feces (g) 1.5970 1.9784 1.9699 0.9082 2.7589 2.9279 1.0097 3.3888 1.4768 0.9807 0.8768 0.9210 2.2272 0.8613 2.5265 0.8674 0.9618 2.0410 3.4464 2.1208 1.8341 3.6113 0.8674 0.9928 2.3594 0.9146 0.9214 1.0014 1.9062 1.8945 3.2449 0.8698 0.7990 0.7043 1.0084 1.6307 0.8062 1.7046 1.7061 MS V * Page A58 of A172 Appendix to TNO-report of 3M-02 Final version Feces weight, bodyweight and food intake GC samples GC-1 Feces collection 3M-02 week 6; collection point 1,2 days collection AMainyAwjl feces collected Bodyweight Food intake (9) (9) (9) 1.2 3.075 123.6 23.2 i 3'4 5 6 7.8 2.4897 2.2946 2.7619 4.0581 87.25 88.55 94.55 126.6 18 16.8 19.9 27.7 I 9,10 2,0935 82.2 16.3 11.12,13 3.4597 118 26.2 I 15 I 14,16,17 18 I 19 I 20,21 2.2994 2.3454 1.689 3.1432 3.2545 83.55 82.8 59.5 116.4 112.2 16.5 18.3 12 21.7 24 22 3.195 110.7 21.5 24,25 2.9408 88.35 19.6 I 23,2627 2.6399 84.6 19.5 I 28,29 31 I 30,32 2.5036 2.7083 2.3554 87.1 112.8 85.4 17.6 20.3 17.2 33,34,35 1.9697 81.35 16.8 I 36,37 2.0798 82.25 16.9 I 38,39 2.6705 101.05 21.9 GC-2 code 2:1-1 2:1-2 2:1-3 2:2-4 2:2-5 2:2-6 2:3-7 2:3-8 2:3-9 2:4-10 2:4-11 2:4-12 2:5-13 2:5-14 2:5-15 2:6-16 2:6-17 2:6-18 2:7-19 2:7-20 2:7-21 Feces collection 3M-02 week 6; collection point 2 ,3 days collection Group cage# feces collected Bodyweight Food intake (9) (9) (9) 1,2 4.692 122.45 36.8 3,4 3.7789 86.55 28.8 5 3.4106 88.2 25.6 6 4.075 94.25 28.8 7,8 6.371 126.45 45.4 9,10 3.4074 82.1 28.2 11,12,13 5.8147 118.75 44.7 15 3.7718 83.5 28.3 14,16,17 3.7727 83.05 29.3 18 2.7133 58.85 19.8 19 4.6407 116.55 35.2 20,21 5.2075 111.85 38.2 22 4.9712 111.6 36.5 5 .' 24,25 4,4833 88.5 32.2 23,26,27 3.767 85.55 30.3 28,29 3.9684 87.15 29.7 31 3.9678 112.45 31 30,32 . .*.7 i1 33.34,35 - -,'j f . 36,37 7 38,39 3.5666 3.4477 3.2646 4.3059 84.55 82,35 82.75 101.95 27.9 29.2 26.5 35 GC-3 code 3:1-1 3:1-2 3:1-3 3:2-4 3:2-5 3:2-6 3:3-7 3:3-8 3:3-9 3:4-10 3.4-11 3:4-12 3:5-13 3:5-14 3:5-15 3:6-16 3:6-17 3:6-18 3:7-19 3:7-20 3:7-21 Feces colection 3M-02 week 6; collection point 3 ,2 days collection Group cage# feces collected Bodyweight Food intake (9) (9) 1,2 3.5754 121.65 (9) 20.2 3.4 2.8781 86.55 15.3 5 2.7589 88.05 13,8 2 6 2.9279 94 16.7 2 7,8 4.3985 9,10 2.4575 126.6 81.85 23.6 13.2 11,12,13 4.025 119.6 23.7 15 2.5265 84.2 13.6 14,16,17 2.6905 83.45 14.7 4 18 2.041 58.7 11.3 A 19 3.4464 117.45 18.7 4 20,21 3.9549 112.9 22.6 .` 5- 22 3.6113 112.85 18.7 24.25 23,26,27 3.3522 2.7034 88.9 85.5 28 14.5 28.29 2.9076 87.65 16.5 6 31 3.2449 30,32 2.7643 111.95 84.65 17.1 15 7 i] 33,34,35 2.5117 835 15,7 7 , 36.37 7 38,39 2.4369 3.4107 83.35 103.2 15 19.8 04-12-2006 Page A59 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 E2. Fecal bile acid measurements weight feces a-muricholate deoxycholate cholate lithocholate B-muricholate w-muricholate hyodeo/urso 5.8 area 770 2880 837 1412 3635 3854 411 mg response factor 1.38 1 1 1 1.08 1 1 area corrected for response factor 1062.6 2880 979 1412 3925.8 5704 411 % 6.5 17.6 6.0 8.6 24.0 34.8 2.5 pg bile acid ig bile acid nmol bile acid/ mg feces mg feces 0.447 1.211 77.1 208.9 0.189 0.532 0.412 71.0 0.174 0.594 102.4 0.272 1.651 284.7 0.697 2.399 0.173 413.6 29.8 1.012 0.076 total | 13799 | | 16375 | 100 | 7 | 1187 j 3.0 | nor-hyodeoxy f 23775 | 1 | 23775 j 23775 weight feces 6 mg a-muricholate deoxycholate cholate lithocholate B-muricholate w-muricholate hyodeo/urso area 481 1551 1143 2203 5717 261 response factor 1.38 1 1 1 1.08 1 1 area corrected for response factor 663.78 1551 1337 2203 2399.76 8461 261 % 3.9 9.2 7.9 13.1 14.2 50.1 1.5 pg bile acid lg bile acid nmol bile acid/ mg feces mo feces 0.276 46.0 0.113 0.645 107.4 0.274 0.556 92.6 0.227 0.916 152.6 0.405 0.997 166.2 0.407 3.517 0.108 586.1 18.1 1.435 0.046 total I 13578 | | 16877 | 100 | 7 | 1169 | 2.9 nor-hyodeoxy I 24059 | 1 | 24059 ~~1 24059 weight feces 5.5 mg a-muricholate deoxycholate cholate lithocholate B-muricholate w-muricholate hyodeo/urso area 304 1047 535 1334 1317 3031 192 response area corrected factor for response factor 1.38 1 1 1 1.08 1 1 419.52 1047 626 1334 1422.36 4486 192 % 4.4 11.0 6.6 14.0 14.9 47.1 2.0 pg bile acid tg bile acid nmol bile acid/ mg feces mg feces 0.269 0.672 48.9 122.1 0.120 0.311 0.401 0.856 73.0 155.6 0.179 0.413 0.912 2.877 0.123 165.9 523.1 22.4 0.406 1.280 0.057 total j 7760 I I 9527 | 100 | 6 | 1111.... \ 2.8 | nor-hyodeoxy 1 15591 j 1 | 15591 | 15591 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Y? Page A60of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 weight feces 7.2 area a-muricholate deoxycholate cholate lithocholate S-muricholate w-muricholate hyodeo/urso 314 688 213 335 803 507 mg response area corrected factor for response factor 1.38 433.32 1 688 1 249 1 335 1.08 867.24 1 750 10 % pg bile acid ig bile acid nmol bile acid/ mg feces 13.0 0.940 130.5 0.319 20.7 1.492 207.2 0.528 7.5 0.540 75.0 0.184 10.1 0.726 100.9 0.268 26.1 1.880 261.2 0.639 22.6 1.627 226.0 0.553 0.0 0.000 0.0 0.000 total I 2860.. '1 I 3323 | 100 | 7 | 1001 | 2.5 nor-hyodeoxy | 4612 I 1 I 4612 | 4612 weight feces 7.2 mg a-muricholate deoxycholate cholate lithocholate R-muricholate w-muricholate hyodeo/urso area 1014 2554 2363 2730 4506 9183 213 response area corrected factor for response factor 1.38 1399.32 1 2554 1 2765 1 2730 1.08 4866.48 1 13591 1 213 % pg bile add ig bile add nmol bile add/ mg feces mg feces 5.0 0.472 65.5 0.160 9.1 0.861 119.6 0.305 9.8 0.932 129.4 0.317 9.7 0.920 127.8 0.339 17.3 1.640 227.8 0.558 48.3 4.581 636.3 1.557 0.8 0.072 10.0 0.025 total I 22563 | | 28118 | 100 | 9 | 1316 | T f nor-hyodeoxy | 29667 | 1 | 29667 | 29667 weightfeces 5.1 mg a-muricholate deoxycholate cholate lithocholate S-muricholate w-muricholate hyodeo/urso area 1430 3201 3202 808 5434 7559 255 response area corrected factor for response factor 1.38 1973.4 1 3201 1 3746 1 808 1.08 5868.72 1 11187 1 255 % pg bile acid )g bile add nmol bile add/ mg feces mg feces 7.3 0.452 88.6 0.217 11.8 0.733 143.6 0.366 13.9 0.857 168.1 0.411 3.0 0.185 36.3 0.096 21.7 1.343 263.3 0.645 41.4 2.560 502.0 1.229 0.9 0.058 11.4 0.029 total I 21889 | | 27040 | 100 | 6 | 1213 | 3.0 | nor-hyodeoxy | 43696 | 1 | 43696 | 43696 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Page A61 of A172 / Appendix to TNO-report of 3M-02 Final version 04-12-2006 w eight fe c e s 7 mg a-m u rich olate d eo xych o late ch o late lith o c h o la te 6 -m uricholate w -m uricholate hyodeo/urso area 755 6854 1031 1211 6883 6151 349 resp o n se fa cor 1 .3 8 1 1 1 1 .0 8 1 1 area corrected fo r resp o n se facto r 1 0 4 1 .9 6854 1206 1211 7 4 3 3 .6 4 9103 349 % 3 .8 2 5 .2 4 .4 4 .5 2 7 .3 3 3 .5 1 .3 p g bile acid >g bile acid nm ol bile add/ m g fe ce s m g fe ce s 0.3 1 9 2 .1 0 1 4 5 .6 3 0 0 .1 0.1 1 2 0.7 6 4 0 .3 7 0 5 2 .8 0.1 2 9 0 .3 7 1 5 3 .0 0 .1 4 1 2.2 7 8 325 .5 0.7 9 7 2.7 9 0 398 .6 0.9 7 6 0.1 0 7 1 5 .3 0 .0 3 9 to ta l l 23234 | | 27199 | 100 < 8 | 1191 | 3 .0 nor-hyodeoxy | 32626 | 1 | 32626 1 32626 w eight fe c e s 6 .6 mg a-m uricholate d eo xych o late ch o late lith o c h o la te 6-m uricholate w -m uricholate hyodeo/urso area 475 1558 748 1489 1894 3897 198 response facto r 1 .3 8 1 1 1 1 .0 8 1 1 are a corrected for resp o n se facto r 6 5 5 .5 1558 875 1489 2 045.52 5768 198 % 5 .2 1 2 .4 7 .0 1 1 .8 1 6 .2 4 5 .8 1 .6 p g bile a d d >g bile a d d nm ol b ile a d d / m g fe ce s m g fe ce s 0.3 2 5 0.771 0 .4 3 3 0.7 3 7 1 .0 1 3 4 9 .2 1 1 6 .9 6 5 .7 1 1 1 .7 1 5 3 .5 0.1 2 0 0.2 9 8 0 .1 6 1 0.2 9 7 0.3 7 6 2.8 5 6 0 .0 9 8 4 3 2 .7 1 4 .9 1 .0 5 9 0.0 3 8 to ta l j 10259 | I 12589 j 100 | 6 | 944 | 2 .3 nor-hyodeoxy j 20195 | 1 | 20195 | 20195 w eight fe c e s 6 .1 mg area a-m u rich olate d eo xych o late ch o late lith o c h o la te B -m uricholate w -m uricholate hyodeo/urso 750 1906 1166 974 3492 4213 396 resp o n se facto r 1 .3 8 1 1 1 1 .0 8 1 1 area corrected for resp o n se facto r 1035 1906 1364 974 3 7 7 1 .3 6 6235 396 % 6 .6 1 2 .2 8 .7 6 .2 2 4 .0 3 9 .8 2 .5 p g bile a d d ig bile acid nm ol bile add/ m g fe ce s m g fe ce s 0.4 2 9 0 .7 9 1 7 0 .4 1 2 9 .6 0 .1 7 2 0 .3 3 0 0.5 6 6 0 .4 0 4 9 2 .8 6 6 .2 0 .2 2 7 0 .1 7 6 1 .5 6 5 256 .5 0.6 2 8 2 .5 8 7 0 .1 6 4 4 2 4 .1 2 6 .9 1 .0 3 8 0.0 6 9 total f " '12897* | | 15682 | 100 |~ 7 | " 67 | " T nor-hyodeoxy | 24103 | 1 1 24103 | 24103 a-m uri deoxy ch o late lith o c h o l b-m uri w -m uri hyodeoxy/urso 4 0 8 .6 392 .6 4 0 8 .6 376 .6 4 0 8 .6 4 0 8 .6 3 9 2 .6 /O ! Page A62of A172 Appendix to TNO-report of 3M-02 Final version 1:2-4 weight feces 6.3 mg area response area corrected % pg bile acid ig bile acid nmol bile add/ factor for response factor mg feces mg feces a-muricholate deoxycholate 133 741 1.38 1 183.54 741 3.3 0.211 33.5 13.3 0.853 135.4 0.082 0.345 cholate 488 1 571 10.3 0.657 104.3 0.255 lithocholate 509 1 509 9.2 0.586 93.0 0.247 6-muricholate w-muricholate hyodeo/urso 758 1756 133 1.08 1 1 818.64 2599 133 14.7 0.942 149.6 46.8 2.991 474.8 2.4 0.153 24.3 0.366 1.162 0.062 total I 4518 I I 5555 | 100 | 6 | 1015 | 2.5 nor-hyodeoxy| 8688 | 1 I 8688 1 8688 1:2-5 weight feces 6.4 mg a-muricholate deoxycholate cholate lithocholate E-muricholate w-muricholate hyodeo/urso area 385 1785 1437 1980 5071 4256 151 response area corrected factor for response factor 1.38 531.3 1 1785 1 1681 1 1980 1.08 5476.68 1 6299 1 151 % pg bile add ig bile acid nmol bile acid/ mg feces mgfeces 3.0 0.208 32.5 0.080 10.0 0.699 109.3 0.278 9.4 0.659 102.9 0.252 11.1 0.776 121.2 0.322 30.6 2.146 335.3 35.2 2.468 385.6 0.821 0.944 0.8 0.059 9.2 0.024 tota! I 15065 | | 17904 | 100 | 7 | 1096 | 2.7 nor-hyodeoxyI 25522 t 1 | 25522 | 25522 weightfeces 5.3 mg a-muricholate deoxycholate cholate lithocholate C>-muricholate w-muricholate hyodeo/urso area 696 3055 1508 3344 2771 7511 363 response area corrected factor for response factor % pg bile acid tg bile acid nmol bile acid/ mg feces mg feces 1.38 960.48 4.1 0.324 61.2 0.150 1 3055 12.9 1.032 194.7 0.496 1 1764 7.5 0.596 112.5 0.275 1 3344 14.2 1.130 213.2 0.566 1.08 2992.68 12.7 1.011 190.8 0.467 1 11116 47.1 3.756 708.6 1.734 1 363 1.5 0.123 23.1 0.059 total I 19248 | | 23596 | 100 | 8 | 1504 I 3.7 nor-hyodeoxy| 29599 | 1 | 29599 | 29599 amuri deoxy cholate iithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 04-12-2006 /ox Page A63 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 weightfeces 7.7 mg area response area corrected factor for response factor % pg bile add g bile acid nmd bile add/ mg feces mg feces a-muricholate deoxycholate cholate lithochoiate 564 1799 924 1298 1.38 1 1 1 778.32 1799 1081 1298 6.1 0.518 67.3 14.1 1.198 155.6 8.4 0.720 93.5 10.1 0.864 112.2 0.165 0.396 0.229 0.298 B-murichdate w-muricholate hyodeo/urso 1925 3892 1.08 1 1 2079 5760 0 16.2 1.384 179.8 45.0 3.836 498.1 0.0 0.000 0.0 0.440 1.219 0.000 total I 10402 I I 12796 | 100 | 9 | 1107 | 2.7 | nor-hyodeoxy f 15018 | 1 I 15018 | 15018 weightfeces 6.6 mg a-murichdate deoxycholate cholate lithochoiate B-muricholate w-muricholate hyodeo/urso area 521 1246 771 1507 3321 3749 322 response area corrected factor for responsefactor 1.38 718.98 1 1246 1 902 1 1507 1.08 3586.68 1 5549 1 322 % pg bile acid ig bile acid nmd bile add/ mg feces mgfeces 5.2 0.391 59.2 0.145 9.0 0.677 102.6 0.261 6.5 0.490 74.3 0.182 10.9 0.819 124.1 0.330 25.9 1.949 295.3 0.723 40.1 3.016 456.9 2.3 0.175 26.5 1.118 0.068 total | 11437 I I 13831 I 100 | 8 j 1139 1 2.8 1 nor-hyodeoxy | 18400 | 1 | 18400 1 18400 weight feces a-muricholate deoxycholate cholate lithochoiate 8-muricholate w-muricholate hyodeo/urso area 1341 4170 1935 3343 3407 9002 474 response area corrected % pg bile add ig bile acid nmd bile add/ factor for response factor mg feces mg feces 1.38 1850.58 6.4 0.624 97.4 0.238 1 4170 14.3 1.405 219.6 0.559 1 2264 7.8 0.763 119.2 0.292 1 3343 11.5 1.127 176.0 0.467 1.08 3679.56 12.6 1.240 193.7 0.474 1 13323 45.8 4.490 701.5 1.717 1 474 1.6 0.160 25.0 0.064 tota! I 23672 | | 29104 | 100 | 10 | 1532 | .......3.8 nor-hyodeoxy I 29674 | 1 | 29674 | 29674 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Page A64ofA172 Appendix to TNO-report of 3M-02____________ Final version p. 100 04-12-2006 weight feces a-muricholate deoxycholate cholate lithocholate S-muricholate w-muricholate hyodeo/urso 6.4 area 469 1577 949 839 2511 4030 280 mg response area corrected factor for response factor 1.38 647.22 1 1577 1 1110 1 839 1.08 2711.88 1 5964 1 280 % Mgbile acid k)bile acid nmol bile acid/ mg feces mg feces 4.9 0.389 60.7 0.149 12.0 0.947 148.0 0.377 8.5 0.667 104.2 0.255 6.4 0.504 78.7 0.209 20.7 1.629 254.5 0.623 45.4 3.583 559.8 1.370 2.1 0.168 26.3 0.067 total | 10655 | I 13130 | 100 | 8 | 1232 | 3,0 nor-hyodeoxy | 16647 | 1 | 16647 | 16647 weightfeces 6.5 mg a-murichoiate deoxycholate cholate lithocholate S-muricholate w-muricholate hyodeo/urso area 438 1197 858 906 3408 2988 364 response area corrected factor for response factor 1.38 604.44 1 1197 1 1004 1 906 1.08 3680.64 1 4422 1 364 % pg bile acid ig bile acid nmol bile acid/ mg feces mg feces 5.0 0.321 49.3 0.121 9.8 0.635 97.7 0.249 8.2 0.532 81.9 7.4 0.481 73.9 30.2 1.952 300.3 0.200 0.196 0.735 36.3 2.345 360.8 3.0 0.193 29.7 0.883 0.076 total | 10159 I' j 12178 | 100 | 6 1 994 | 2.5 nor-hyodeoxyI 18855 | 1 | 18855 1 18855 weight feces a-murichotate deoxycholate cholate lithocholate S-muricholate w-muricholate hyodeo/urso area 871 2878 1791 1999 2933 6248 527 response area corrected % pg bile acid ig bile acid nmol bile acid/ factor for response factor mgfeces mg feces 1.38 1201.98 5.7 0.458 83.4 0.204 1 2878 13.6 1.098 199.6 0.508 1 2095 9.9 0.799 145.3 0.356 1 1999 9.5 0.762 138.6 0.368 1.08 3167.64 15.0 1.208 219.7 0.538 1 9247 43.8 3.527 641.2 1.569 1 527 2.5 0.201 36.5 0.093 total | 17247 | | 21116 | 100 | 8 j 1464 | ""SIT nor-hyodeoxy | 26219 j 1 | 26219 | 26219 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Page A65 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 weight feces a-muricholate deoxycholate cholate lithochotate 6-muricholate w-muricholate hyodeo/urso 5.8 area 463 2160 560 2503 3081 5475 269 mg response area corrected factor for response factor 1.38 638.94 1 2160 1 655 1 2503 1.08 3327.48 1 8103 1 269 % pg bile acid tg bile acid nmol bile acid/ mg feces mg feces 3.6 0.283 48.7 12.2 0.956 164.8 0.119 0.420 3.7 0.290 50.0 0.122 14.2 1.108 191.0 0.507 18.8 1.472 253.9 45.9 3.586 618.2 1.5 0.119 20.5 0.621 1.513 0.052 total I 14511 I I nor-hyodeoxyj 22598 \ 1 | 17657 22598 I 100 I 8 | 1347 | | 22598 84 weight feces 5.4 mg a-muricholate deoxycholate cholate lithocholate S-muricholate w-muricholate hyodeo/urso area 258 1480 573 1667 1531 3397 212 response area corrected factor for response factor 1.38 356.04 1 1480 1 670 1 1667 1.08 1653.48 1 5028 1 212 % pg bile acid tg bile acid nmol bile acid/ mgfeces mg feces 3.2 0.226 41.8 13.4 0.939 173.9 0.102 0.443 6.1 0.425 78.8 0.193 15.1 1.058 195.9 14.9 1.049 194.3 0.520 0.476 45.4 3.190 590.8 1.446 1.9 0.135 24.9 0.063 total | 9118 | | 11066 | 100 j 7 | 1300 j 3.2 nor-hyodeoxy | 15759 l 1 1 15759 j 15759 weight feces 6.4 mg a-muricholate deoxycholate cholate lithocholate 6-muricholate w-muricholate hyodeo/urso area 483 1604 840 2300 2706 3466 response area corrected factor for response factor % pg bile add g bile acid nmol bile acid/ mg feces mg feces 1.38 666.54 4.9 0.414 64.7 0.158 1 1604 11.8 0.997 155.8 0.397 1 983 7.2 0.611 95.5 0.234 1 2300 16.9 1.430 223.4 0.593 1.08 2922.48 21.5 1.817 283.8 0.695 1 5130 37.7 3.189 498.2 1.219 1 0 0.0 0.000 0.0 0.000 total I 11399 I I 13606 | 100 | 8 | 1321 I 83 nor-hyodeoxy 1 16088 | 1 | 16088 | 16088 a-muri deoxy cholate Irthochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Page A66 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 area response area corrected % pg bile acid ig bile add nmol bile acid/ factor for responsefactor mg feces mgfeces a-muricholate 213 deoxychotate 1811 1.38 1 293.94 1811 2.1 0.139 27.9 12.7 0.858 171.6 0.068 0.437 cholate 1070 1 1252 8.7 0.593 118.7 0.290 lithocholate 2080 1 2080 14.5 0.986 197.1 0.523 B-muricholate 2392 1.08 2583.36 18.0 1.224 244.8 0.599 w-muricholate 4005 1 5927 41.4 2.809 561.8 1.375 hyodeo/urso 367 1 367 2.6 0.174 34.8 0.089 total l 11938 | l 14315 | 100 | 7 | 1357 | 3.4 nor-hyodeoxy| 21102 | 1 | 21102 1 21102 weightfeces 7.3 mg area response area corrected % pg bileacid >gbileadd nmol bile acid/ factor for responsefactor mgfeces mg feces a-muricholate 222 1.38 306.36 4.1 0.300 41.0 0.100 deoxychdate 790 1 790 10.6 0.772 105.8 0.269 cholate 602 1 704 9.4 0.689 94.3 0.231 lithocholate a-murlcholate w-muricholate hyodeo/urso 1050 1101 2234 135 1 1.08 1 1 1050 1189.08 3306 135 14.0 1.026 140.6 15.9 1.162 159.2 44.2 3.232 442.8 1.8 0.132 18.1 0.373 0.390 1.084 0.046 total | 6134 | | 7481 | 100 | 7 | 1002 | 2.5 | nor-hyodeoxyI 10229 | 1 I 10229 | 10229 a-muricholate deoxycholate cholate lithocholate G-muricholate w-muricholate hyodeo/urso area 887 2496 1947 482 4734 4874 response area corrected factor for responsefactor 1.38 1224.06 1 2496 1 2278 1 482 1.08 5112.72 1 7214 10 % 6.5 13.3 12.1 2.6 27.2 38.4 0.0 total | 15420 | l 18806 | 100 pgbileacid ig bile acid nmol bile acid/ mgfeces mgfeces 0.333 62.9 0.154 0.679 128.2 0.326 0.620 117.0 0.286 0.131 24.8 0.066 1.391 262.5 0.643 1.963 370.4 0.907 0.000 0.0 0.000 5 |... 966 | ~ 2~4 nor-hyodeoxy| 36744 I 1 | 36744 | 36744 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Page A67 of A172 Appendix to TNO-report of 3M-02 Final version weightfeces 8.2 mg area response area corrected % pg bileacid g bile acid nmol bile acid/ factor for response factor mgfeces mgfeces a-muricholate 727 deoxycholate 3031 1.38 1 1003.26 3031 4.9 0.379 46.3 14.7 1.146 139.8 0.113 0.356 cholate 1265 1 1480 7.2 0.560 68.3 0.167 lithocholate 1974 1 1974 9.6 0.747 91.0 0.242 IJ-muricholate 4421 1.08 4774.68 23.2 1.806 220.2 0.539 w-muricholate 5331 1 7890 38.4 2.984 363.9 0.891 hyodeo/urso 401 1 401 2.0 0.152 18.5 0.047 total I 17150 I I 20554 | 100 j 8 | 948 | 2.4 nor-hyodeoxy| 26440 | 1 | 26440 ~ | 26440 weightfeces 6.4 mg area response area corrected factor for responsefactor % pg bile acid g bile acid nmol bile acid/ mgfeces mgfeces a-muricholate 541 1.38 746.58 4.7 0.264 41.3 0.101 deoxycholate 1842 1 1842 11.6 0.652 101.8 0.259 cholate 998 1 1168 7.4 0.413 64.5 0.158 lithocholate 1904 1 1904 12.0 0.674 105.2 0.279 3-muricholate w-muricholate hyodeo/urso 2601 4796 270 1.08 1 1 2809.08 7098 270 17.7 0.994 155.3 44.8 2.511 392.3 1.7 0.096 14.9 0.380 0.960 0.038 total I 12952 I I 15837 | 100 | 6 | 875 | 2.2 nor-hyodeoxy[ 28268 | 1 | 28268 | 28268 weightfeces 7 mg area response area corrected % pg bile acid g bile acid nmol bileacid1 factor for responsefactor mgfeces mgfeces a-muricholate 612 1.38 844.56 4.8 0.381 54.5 0.133 deoxycholate cholate lithocholate B-muricholate w-muricholate hyodeo/urso 2141 1127 2027 3995 4490 224 1 1 1 1.08 1 1 2141 1319 2027 4314.6 6645 224 12.2 0.967 138.1 7.5 0.595 85.0 11.6 0.915 130.7 24.6 1.948 278.3 37.9 3.000 428.6 1.3 0.101 14.4 0.352 0.208 0.347 0.681 1.049 0.037 total I 14616 I I 17515 | 100 | 8 "| 1130 | 2.8 nor-hyodeoxy| 22149 | 1 \ 22149 | 22149 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 04-12-2006 /07 Page A68ofA172 Appendix to TNO-report of 3M-02____________ Final version p. 104 04-12-2006 weight feces 5.1 mg area response area corrected % pg bile acid ng bile add/ nmol bile add/ factor for responsefactor mgfeces mgfeces a-muricholate 103 deoxycholate 354 1.38 1 142.14 354 4.0 0.114 22.3 9.9 0.283 55.6 0.055 0.142 cholate 286 1 335 9.3 0.268 52.5 0.129 lithocholate 394 1 394 11.0 0.315 61.8 0.164 6-muricholate 349 1.08 376.92 10.5 0.302 59.2 0.145 w-murichoiate 1185 hyodeo/urso 235 1 1 1754 48.8 1.404 275.2 0.674 235 6.5 0.188 36.9 0.094 total I 2906 I I 3590 j 100 | 3 | 563 | 1.4 nor-hyodeoxy) 12494 | 1 | 12494 ~| 12494 1:4-11 weight feces 4.8 mg area response area corrected % pg bile acid ng bile add/ nmol bile add/ factor for response factor mg feces mgfeces a-muricholate 276 1.38 380.88 12.2 0.454 94.6 0.231 deoxycholate 396 1 396 12.6 0.472 98.3 0.250 cholate 184 1 215 6.9 0.257 53.5 0.131 lithocholate 214 1 214 6.8 0.255 53.1 0.141 R-muricholate w-muricholate hyodeo/urso 555 896 1.08 1 1 599.4 1326 0 19.1 0.715 42.3 1.581 0.0 0.000 148.9 329.3 0.0 0.364 0.806 0.000 total I 2521 I I 3132 | 100 | 4 | 778 | 1,9 nor-hyodeoxy| 8389 | 1 j 8389 I 8389 1:4-12 weight feces 6 mg a-muricholate deoxycholate cholate lithocholate B-muricholate w-muricholate hyodeo/urso area 320 852 475 954 954 2059 response area corrected factor for response factor 1.38 441.6 1 852 1 556 1 954 1.08 1030.32 1 3047 10 % pg bile acid ng bile add/ nmol bile acid/ mg feces mgfeces 6.4 0.390 65.1 0.159 12.4 0.753 8.1 0.491 125.5 81.9 0.320 0.200 13.9 0.843 140.6 0.373 15.0 0.911 44.3 2.694 151.8 449.0 0.372 1.099 0.0 0.000 0.0 0.000 total I 5614 I I 6881 | 100 | "" 6 | 1014 | 2.5 nor-hyodeoxyI 11311 | 1 | 11311 ***| 11311 a-muri deoxy cholate llthochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Page A69 of A172 Appendix to TNO-report of 3M-02____________ Final version p. 105 04-12-2006 weightfeces 6.2 mg a-muricholate deoxycholate cholate lithocholate -muricholate w-muricholate hyodeo/urso area 361 1051 1247 1420 979 2197 response area corrected factor for responsefactor 1.38 498.18 1 1051 1 1459 1 1420 1.08 1057.32 1 3252 10 % pg bile acid ngbile acid/ nmol bile acid/ mg feces mg feces 5.7 0.258 41.6 0.102 12.0 0.544 87.7 0.223 16.7 0.755 121.7 0.298 16.3 0.735 118.5 0.315 12.1 0.547 88.2 0.216 37.2 1.682 271.3 0.664 0.0 0.000 0.0 0.000 total | 7255 | j 8737 | 100 | 5 | 729 1 1.8 nor-hyodeoxy| 19330 | 1 j 19330 " ] 19330 weight feces a-muricholate deoxycholate cholate lithocholate -muricholate w-muricholate hyodeo/urso area 842 1126 866 1217 2527 3746 481 response area corrected % pg bile acid ng bile acid/ nmol bile acid/ factor for response factor mg feces mg feces 1.38 1161.96 8.8 0.510 77.2 0.189 1 1126 8.5 0.494 74.8 0.191 1 1013 7.6 0.444 67.3 0.165 1 1217 9.2 0.534 80.9 0.215 1.08 2729.16 20.6 1.197 181.4 0.444 1 5544 41.8 2.432 368.4 0.902 1 481 3.6 0.211 32.0 0.081 total I 10805 I I 13272 | 100 | 6 " | 882 | ~ " 2.2 nor-hyodeoxyI 22800 | 1 | 22800 | 22800 2:4-12 weight feces 7.6 mg a-muricholate deoxycholate cholate lithocholate -muricholate w-muricholate hyodeo/urso area 543 902 1180 1019 1622 2624 206 response area corrected factor for response factor 1.38 749.34 1 902 1 1381 1 1019 1.08 1751.76 1 3884 1 206 % pg bile acid ngbile acid/ nmol bile acid/ mgfeces mg feces 7.6 0.555 73.0 0.179 9.1 0.668 87.9 0.224 14.0 1.023 134.5 0.329 10.3 0.755 99.3 0.264 17.7 1.297 170.7 0.418 39.3 2.876 2.1 0.153 378.5 20.1 0.926 0.051 total | 8096 | | 9892 I 100 | 7 | 964 | 2.4* nor-hyodeoxy| 13502 | 1 | 13502 1 13502 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Page A70of A172 Appendix to TNO-report of 3M-02____________ Final version p. 106 04-12-2006 3:4-10 weight feces 8.1 mg area response area corrected factor for responsefactor % pg bile acid ig bile acid nmol bile acid/ mgfeces mgfeces a-muricholate deoxycholate 374 928 1.38 1 516.12 928 6.4 0.307 38.0 11.5 0.553 68.2 0.093 0.174 cholate 737 1 862 10.7 0.514 63.4 0.155 lithocholate 749 1 749 9.3 0.446 55.1 0.146 6-murichoiate 1189 1.08 1284.12 15.9 0.765 94.4 0.231 w-murichoiate 2355 hyodeo/urso 244 1 1 3485 43.2 2.076 256.3 0.627 244 3.0 0.145 17.9 0.046 total | 6576 I j 8069 | 100 | 5 | 593 | 1,5 nor-hyodeoxy| 16790 | 1 | 16790 | 16790 3:4-11 weightfeces 8.3 mg a-muricholate deoxycholate cholate lithocholate 6-muricholate w-muricholate hyodeo/urso area 474 1184 335 668 2030 2413 257 response area corrected factor for responsefactor 1.38 654.12 1 1184 1 392 1 668 1.08 2192.4 1 3571 1 257 % pg bile acid rgbile acid nmol bile add/ mg feces mgfeces 7.3 0.475 57.2 ...... 0.'l40 13.3 0.860 103.6 0.264 4.4 0.285 34.3 0.084 7.5 0.485 58.4 24.6 1.592 191.8 0.155 0.469 40.0 2.593 312.5 2.9 0.187 22.5 0.765 0.057 total | 7361 j j 8919 | 100 | 6 | 780 | 1,9 nor-hyodeoxy| 13770 | 1 | 13770 *1 13770 weight feces 6.6 mg area response area corrected % Mgbile acid ig bile acid nmol bile add/ factor forresponse factor mgfeces mgfeces a-muricholate 436 1.38 601.68 6.8 0.394 59.7 0.146 deoxycholate 831 1 831 9.4 0.544 82.4 0.210 cholate 1182 1 1383 15.7 0.905 137.1 0.336 lithocholate 693 1 693 7.9 0.454 68.7 0.182 B-muricholate 1774 1.08 1915.92 21.8 1.254 190.0 0.465 w-muricholate 2161 1 3198 36.4 2.093 317.1 0.776 hyodeo/urso 172 1 172 2.0 0.113 17.1 0.043 total 1 7249 | | 8795 1 100 | 6 ~T...'872 | 2.2 1 nor-hyodeoxy| 15281 | 1 | 15281 *~l 15281 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 // Page A71 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 weight feces 5.1 mg a-muricholate deoxychotate cholate lithocholate 6-muricholate w-muricholate hyodeo/urso area 279 1398 233 1493 1698 1962 138 response area corrected factor for response factor 1.38 385.02 1 1398 1 273 1 1493 1.08 1833.84 1 2904 1 138 % pg bile acid ng bile acid/ nmol bile acid/ mg feces mg feces 4.6 0.194 16.6 0.706 38.1 138.4 0.093 0.352 3.2 0.138 27.0 0.066 17.7 0.754 147.8 0.392 21.8 0.926 181.5 0.444 34.5 1.466 287.4 1.6 0.070 13.7 0.703 0.035 total I 7201 \ I 8424 | 100 | 4 | 834 | 2.1 nor-hyodeoxy| 19809 | 1 j 19809 ~ | 19809 weight feces 5.9 mg area response area corrected % pg bile acid ng bile acid/ nmol bile acid/ factor for response factor mg feces mg feces a-muricholate 273 1.38 376.74 4.5 0.202 34.2 0.084 deoxycholate 1027 1 1027 12.2 0.550 93.3 0.238 cholate 1007 1 1178 14.0 0.631 107.0 0.262 lithocholate 1318 1 1318 15.7 0.706 119.7 0.318 B-muricholate 1369 1.08 1478.52 17.6 0.792 134.3 0.329 w-muricholate 1924 1 2848 33.8 1.526 258.6 0.633 hyodeo/urso 193 1 193 2.3 0.103 17.5 0.045 tota! | 7111 \ | 8419 | 100 | 5 I 764 | T? nor-hyodeoxy| 18666 | 1 | 18666 | 18666 weightfeces 6.6 mg a-muricholate deoxycholate cholate lithocholate B-muricholate w-muricholate hyodeo/urso area 201 976 457 1197 374 2027 response area corrected factor for response factor 1.38 277.38 1 976 1 535 1 1197 1.08 403.92 1 3000 10 % pg bile acid ng bile acid/ nmol bile acid/ mgfeces mgfeces 4.3 0.239 36.3 0.089 15.3 0.842 127.6 0.325 8.4 0.461 69.9 0.171 18.7 1.033 6.3 0.349 47.0 2.589 0.0 0.000 156.5 52.8 392.3 0.0 0.416 0.129 0.960 0.000 tota! j 5232 I I 6389 | 100 | 6 | 836 | 2.1 nor-hyodeoxy | 11586 | 1 | 11586 1 11586 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Page A72 of A172 Appendix to TNO-reoort of 3M-02____________ Final version p. 108 04-12-2006 weight feces 6.9 mg a-muricholate deoxycholaie cholate lithocholate S-muricholate w-muricholate hyodeo/urso area 170 421 188 1014 816 778 196 response area corrected factor for response factor 1.38 234.6 1 421 1 220 1 1014 1.08 881.28 1 1151 1 196 % pg bile acid ng bile acid/ nmol bile acid/ mgfeces mgfeces 5.7 0.289 41.9 0.103 10.2 0.519 75.2 0.191 5.3 0.271 39.3 0.096 24.6 1.249 181.0 0.481 21.4 1.086 157.3 0.385 28.0 1.418 205.6 0.503 4.8 0.241 35.0 0.089 total j 3583 | 1 4118 1 100 | 5 | 735 | 1,6 nor-hyodeoxy| 8118 I 1 I 8118 | 8118 weight feces 7.1 mg area a-muricholate deoxycholate cholate lithocholate S-muricholate w-muricholate hyodeo/urso 488 1599 1339 1652 2345 2835 357 response area oorrected % pg bile acid ng bile acid/ nmol bile acid/ factor for response factor mg feces mg feces 1.38 673.44 5.4 0.342 48.1 0.118 1 1599 12.7 0.812 114.3 0.291 1 1567 12.5 0.795 112.0 0.274 1 1652 13.1 0.838 118.1 0.314 1.08 2532.6 20.1 1.285 181.0 0.443 1 4196 33.4 2.130 299.9 0.734 1 357 2.8 0.181 25.5 0.065 total I 10615 | [ 12576 | 100 I 6 899 | 2.2 nor-hyodeoxy|_ _ 1 9 7 0 2 _ |1 _ _ J i I . 2:5:15- weight feces 5.4 : mg 19702 I 19702 SZSEjaEE a-muricholate deoxycholate cholate lithocholate S-muricholate w-muricholate hyodeo/urso area 1147 3831 1371 3141 2207 7603 531 response area corrected % pg bile acid ng bile acid/ nmol bile acid1 factor for response factor mg feces mgfeces 1.38 1582.86 6.5 0.461 85.3 0.209 1 3831 15.7 1.115 206.5 0.526 1 1604 6.6 0.467 86.5 0.212 1 3141 12.9 0.914 169.3 0.450 1.08 2383.56 9.8 0.694 128.5 0.314 1 11252 46.3 3.275 606.5 1.484 1 531 2.2 0.155 28.6 0.073 total | 19831 j I 24326 | 100 | 7 | 1311 | .....I ? nor-hyodeoxy | 34357 | 1 | 34357 | 34357 a-muri deoxy cholate iithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 "J - Page A73of A172 Appendix to TNO-reoort of 3M-02____________ Final version { ,,, 3 ^ - 1 3 ^ : weight feces 7.9 mg area response area corrected % pg bileacid ng bile add/ nmol bile acid/ factor for responsefactor mgfeces mgfeces a-muricholate 608 deoxycholate 1723 1.38 1 839.04 1723 6.4 0.282 13.2 0.580 35.7 73.4 0.087 0.187 cholate 925 1 1082 8.3 0.364 46.1 0.113 lithocholate 1896 1 1896 14.5 0.638 80.7 0.214 a-muricholate 2481 1.08 2679.48 20.5 0.901 114.1 0.279 w-muricholate 3000 hyodeo/urso 400 1 1 4440 34.0 1.493 189.0 0.463 400 3.1 0.135 17.0 0.043 total I 11033 | | 13060 | 100 | 4 | 556 | 1,4 ~| nor-hyodeoxy| 29729 j 1 | 29729 ~ | 29729 weight feces 8.1 mg area response area corrected % pg bile add ng bile acid/ nmol bile acid/ factor for responsefactor mgfeces mgfeces a-muricholate 391 1.38 539.58 5.7 0.264 32.6 0.080 deoxycholate 1007 1 1007 10.7 0.493 60.9 0.155 cholate 1069 1 1251 13.2 0.613 75.7 0.185 lithocholate 799 1 799 8.5 0.391 48.3 0.128 a-muricholate 1999 1.08 2158.92 22.8 1.058 130.6 0.320 w-muricholate 2126 1 3146 33.3 1.542 190.3 0.466 hyodeo/urso 548 1 548 5.8 0.268 33.1 0.084 total | 7939 | | 9450 t 100 j 5 | 572 l........ 14 1 nor-hyodeoxy| 20411 | 1 | 20411 ~1 20411 I -3S-15 weightfeces . 'V 7.3 mg " ~l area response area corrected % pg bile acid ngbile add/ nmol bile add/ factor forresponse factor mgfeces mgfeces a-muricholate 908 1.38 1253.04 5.7 0.363 49.8 0.122 deoxycholate 3513 1 3513 16.0 1.019 139.5 0.355 cholate 1190 1 1392 6.4 0.404 55.3 0.135 lithocholate 2079 1 2079 9.5 0.603 82.6 0-219 B-muricholate 2885 1.08 3115.8 14.2 0.903 123.7 0.303 w-muricholate 6815 1 10086 46.0 2.924 400.6 0.980 hyodeo/urso 481 1 481 2.2 0.139 19.1 0.049 total | 17871 | | 21920 | 100 | 6 |.. 871 I...... 2.2 nor-hyodeoxy| 34491 | 1 | 34491 | 34491 P- 109 04-12-2006 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 //3 Page A74of A172 Appendix to TNO-report of 3M-02____________ Final version weightfeces 5,8 mg area response area corrected % pg bileacid ng bileacid/ nmol bileacid/ factor for response factor mgfeces mgfeces a-muricholate 222 1.38 306.36 5.9 0.188 32.4 0.079 deoxycholate 654 1 654 12.6 0.401 69.1 0.176 cholate 724 1 847 16.3 0.519 89.5 0.219 lithocholate 565 1 565 10.9 0.346 59.7 0.159 B-muricholate 752 1.08 812.16 15.6 0.498 85.8 0.210 w-muricholate 1281 1 1896 36.4 1.162 200.4 0.490 hyodeo/urso 123 1 123 2.4 0.075 13.0 0.033 total | 4321 | j 5203 | 100 | 3 | 550 I 1.4 nor-hyodeoxy| 16312 | 1 | 16312 ~1 16312 7 weight feces 6.2 mg area response area corrected % pg bileacid ngbileacid/ nmol bile acid/ factor forresponsefactor mgfeces mgfeces a-muricholate 199 1.38 274.62 7.0 0.250 40.3 0.099 deoxycholate 543 1 543 13.8 0.494 79.7 0.203 cholate 411 1 481 12.2 0.438 70.6 0.173 lithocholate 518 1 518 13.2 0.472 76.1 0.202 R-muricholate 383 1.08 413.64 10.5 0.377 60.7 0.149 w-muricholate 1077 1 1594 40.5 1.451 234.1 0.573 hyodeo/urso 111 1 111 2.8 0.101 16.3 0.042 tota! | 3242 | | 3935 | 100 | 4 | 578 I 1.4 nor-hyodeoxyI 10983 | 1 | 10983 | 10983 area response area corrected % pg bile acid ngbileacid/ nmol bile acid/ factor for responsefactor mgfeces mgfeces a-muricholate 117 1.38 161.46 2.8 0.120 19.0 0.047 deoxycholate 650 1 650 11.3 0.483 76.7 0.195 cholate 962 1 1126 19.5 0.837 132.8 0.325 lithocholate 598 1 598 10.4 0.444 70.5 0.187 S-muricholate w-muricholate hyodeo/urso 677 1575 174 1.08 1 1 731.16 2331 174 12.7 0.543 40.4 1.732 3.0 0.129 86.3 275.0 20.5 0.211 0.673 0.052 total | 4753 | | 5771 | 100 | 4 | 681 | 1,7 nor-hyodeoxy| 13455 | 1 | 13455 | 13455 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 p. 110 04-12-2006 //f Page A75of A172 Appendix to TNO-report of 3M-02____________ Final version p. 111 04-12-2006 2 :6 -1 6 weightfeces a-muricholate deoxycholate cholate lithocholate B-muricholate w-muricholate hyodeo/urso 5.6 area 215 821 747 598 840 1244 226 mg response area corrected factor forresponsefactor 1.38 296.7 1 821 1 874 1 598 1.08 907.2 1 1841 1 226 % pgbile acid ngbile acid/ nmol bile acid/ mg feces mg feces 5.3 0.169 14.8 0.469 30.2 83.7 0.074 0.213 15.7 0.499 89.1 0.218 10.7 0.341 60.9 0.162 16.3 0.518 33.1 1.051 4.1 0.129 92.5 187.6 23.0 0.226 0.459 0.059 total I 4691 I I 5564 I 100 I 3 | 567 | 1.4 | nor-hyodeoxy| 17522 | 1 | 17522 1 17522 2 :6 -1 7 weight feces 5.5 mg area a-muricholate deoxycholate cholate lithocholate B-muricholate w-muricholate hyodeo/urso 196 966 220 486 177 733 response area corrected % pgbile acid ng bileacid/ nmol bile acid/ factor forresponsefactor mg feces mg feces 1.38 270.48 8.3 0.241 43.9 0.107 1 966 29.7 0.862 156.7 0.399 1 257 7.9 0.230 41.8 0.102 1 486 14.9 0.434 78.8 0.209 1.08 191.16 5.9 0.171 31.0 0.076 1 1085 33.3 0.968 176.0 0.431 1 0 0.0 0.000 0.0 0.000 total I 2778 i I 3256 | 100 j 3 | 528 | 1.3 ~l nor-hyodeoxyT 11207 I 1 I 11207 | 11207 weightfeces 7.4 mg area response area corrected % ggbile acid ng bileacid/ nmol bile acid/ factor forresponse factor mg feces mgfeces a-muricholate 553 1.38 763.14 6.3 0.365 49.3 0.121 deoxycholate 1729 1 1729 14.3 0.827 111.8 0.285 cholate 1319 1 1543 12.8 0.738 99.8 0.244 lithocholate 1154 1 1154 9.6 0.552 74.6 0.198 B-muricholate 1500 1.08 1620 13.4 0.775 104.7 0.256 w-muricholate 3330 hyodeo/urso 318 1 1 4928 40.9 2.358 318.6 0.780 318 2.6 0.152 20.6 0.052 total I 9903 | | 12056 | 100 | 6 | 779 | 1.9 ~1 nor-hyodeoxyI 20902 | 1 | 20902 | 20902 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 //T Page A76 of A172 Appendix to TNO-report of 3M-Q2 Final version 04-12-2006 weight feces a-m uricholate deoxycholate etiolate lithocholate ti-m uricholate w -m uricholate hyodeo/urso 7.4 area 621 1219 1628 685 1439 1866 253 mg response factor 1.38 1 1 1 1.08 1 1 area corrected fo r response factor 856.98 1219 1905 685 1554.12 2762 253 % 9.3 13.2 20.6 7.4 16.8 29.9 2.7 pg bile acid ng bile acid/ nmol bile add/ mg feces mg feces 0.369 0.525 49.9 70.9 0.122 0.181 0.820 110.8 0.271 0.295 39.9 0.106 0.669 90.4 0.221 1.189 160.7 0.393 0.109 14.7 0.038 total I 7711 I I 9235 I 100 I 4 | 537 | 1.3 nor-hyodeoxyl 23222 I 1 | 23222 | 23222 7 weight feces 8.1 mg a-m uricholate deoxycholate cholate lithocholate B-m uricholate w -m uricholate hyodeo/urso area 829 1831 1171 653 1120 4227 295 response factor 1.38 1 1 1 1.08 1 1 area corrected fo r response fa d o r 1144.02 1831 1370 653 1209.6 6256 295 % pg bile acid ng bile acid/ nmol bile acid/ mg feces mg feces 9.0 0.427 52.7 0.129 14.4 0.683 84.3 0.215 10.7 0.511 63.1 0.154 5.1 0.243 30.1 0.080 9.5 0.451 55.7 0.136 49.0 2.3 2.332 0.110 288.0 13.6 0.705 0.035 total | 10126 | | 12759 | 100 | 5 | 587 | 1.5 nof-hyodeoxy I 26821 I 1 I 26821 ~ | 26821 weight feces 7.7 mg a-m uricholate deoxycholate cholate lithocholate B -m uricholate w -m uricholate hyodeo/urso area 441 1194 995 999 934 1799 response factor 1.38 1 1 1 1.08 1 1 area corrected for response factor 608.58 1194 1164 999 1008.72 2663 0 % 8.0 15.6 15.2 13.1 13.2 34.9 0.0 pg bile add ng bile acid/ nmol bite add/ mg feces mg feces 0.253 0.497 32.9 64.5 0.080 0.164 0.484 0.416 62.9 54.0 0.154 0.143 0.420 1.108 54.5 143.9 0.133 0.352 0.000 0.0 0.000 total j 6362 ) | 7637 | 100 | 3 | 413 | 1. nor-hyodeoxyl 24030 | 1 | 24030 | 24030 a-m uri deoxy cholate lithochol b-m uri w -m uri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Page A77 of A172 // Appendix to TNO-report of 3M-02____________ Final version p. 113 04-12-2006 area response area corrected % pgbile acid ng bile acid/ nmol bile acid/ factor for response factor mgfeces mgfeces a-muricholate 493 deoxycholate 1997 1.38 1 680.34 1997 8.4 0.556 106.9 24.7 1.631 313.7 0.262 0.799 cholate 1424 1 1666 20.6 1.361 261.7 0.641 lithocholate 376 1 376 4.6 0.307 59.1 0.157 B-muricholate 1518 1,08 1639.44 20.2 1.339 257.6 0.630 w-muricholate 1175 1 1739 21.5 1.421 273.2 0.669 hyodeo/urso 1 0 0.0 0.000 0.0 0.000 total j 6983 | nor-hyodeoxyj 12241 | | 1| 8098 12241 | 100 | | 12241 7 | 1272 | 3.2 ~ | weightfeces 5.8 mg area response area corrected % pg bile acid ng bileadd/ nmol bileacid/ factor for response factor mg feces mgfeces a-muricholate 602 1.38 830.76 15.4 0.725 125.0 0.306 . deoxycholate cholate 895 817 1 1 895 16.5 0.781 134.7 0.343 956 17.7 0.834 143.8 0.352 lithocholate 302 1 302 5.6 0.264 45.4 0.121 B-muricholate w-muricholate hyodeo/urso 659 977 267 1.08 1 1 711.72 1446 267 13.2 0.621 26.7 1.262 4.9 0.233 107.1 217.6 40.2 0.262 0.533 0.102 totai | 4519 | | 5408 | 100 j 5 | 814 | 2.0 ~~| nor-hyodeoxy| 11457 | 1 | 11457 | 11457 | 1 .7 -2 * weigmreces .* 5.4 ' ,r ; mg t v ,- ' - V '- '.'iy - f t area response area corrected % pg bile acid ng bile acid/ nmol bile acid/ factor for response factor mgfeces mgfeces a-muricholate 512 1.38 706.56 10.0 0.525 97.3 0.238 deoxycholate cholate lithocholate 1419 1545 649 1 1 1 1419 20.0 1.055 195.3 0.498 1808 25.5 1.344 248.8 0.609 649 9.1 0.482 89.3 0.237 B-muricholate w-muricholate hyodeo/urso 893 912 204 1.08 1 1 964.44 1350 204 13.6 0.717 19.0 1.003 2.9 0.152 132.8 185.8 28.1 0.325 0.455 0.072 total | 6134 | | 7100 | 100 I 5 | 977 | 2A nor-hyodeoxy| 13452 | 1 1 13452 | 13452 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 //7 Page A78of A172 Appendix to TNO-report of 3M-02____________ Final version p. 114 04-12-2006 C14:0 016:0 C16:1 C 18fl C18:1 C18:2 C18:3 area % M9 fatty ng fatty add nmol fatty add/ add mg feces mg feces 26211 1.5 10.5 1538 6.73 674622 39.1 269.2 39589 154.40 5831 907134 0.3 52.5 2.3 362.0 342 53233 1.35 187.11 95082 5.5 37.9 5580 19.75 4891 0.3 2.0 287 1.02 12523 0.7 5.0 735 2.64 iota! I 1726294 | 100 I 688.9 I 101304 | 373.01 I C15:0 I 200479 | weight feces 5.3 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area 12309 339628 3302 368609 45053 2365 4243 % Mg fatty ng fatty add nmol fatty add/ add mg feces mg feces 1.6 10.3 1940 8.49 43.8 283.7 53533 208.79 0.4 2.8 520 2.05 47.5 307.9 58101 204.22 5.8 37.6 7101 25.14 0.3 2.0 373 1.33 0.5 3.5 669 2.40 to ta l I 775509 I 100 I 647.9 I 122237 | 452.42 | C15:0 I 95763 | 1:6*16 weight feces 11.7 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % M9 fatty ng fatty add nmol fatty add/ add mg feces mg feces 20396 1.2 12.5 1070 4.69 664144 40.5 407.7 34649 135.92 5241 0.3 32 275 1.08 654148 52.1 524.4 44819 157.54 80539 4.9 49.4 4226 14.96 5034 0.3 3.1 264 0.94 11097 0.7 6.8 582 2.09 total | 1640599 | 100 | 1007.2 | 86086 I 317.21 | C15:0 I 130309 | 1:6-17 weight feces 8.9 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % M9 fatty ng fatty add nmol fatty add/ add mg feces mg feces 12220 1.4 8.4 939 4.11 366294 42.7 250.4 28137 109.74 3077 0.4 2.1 236 0.93 427401 49.9 292.2 32831 115.40 40324 4.7 27.6 3098 10.96 2415 0.3 1.7 186 0.66 5166 0.6 3.5 397 1.42 total | 856897 | 100 | 585.8 | 6S824 I 243.23 | C15:0 I 117016 I weight feces 8.1 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % M9 fatty ng fatty acid nmol fatty add/ add mg feces mg feces 4597 1.7 4.7 584 2.56 119586 44.4 123.1 15199 59.28 872 0.3 0.9 111 0.44 128120 47.6 131.9 16283 57.23 13678 5.1 14.1 1738 6.15 939 0.3 1.0 119 0.43 1419 0.5 1.5 180 0.65 total j 269211 I 100 | 277.1 | 3421S | 126.73 | C15: r ... 777..... | weight feces 10.8 mg C14:0 C16:0 C16:1 C18:0 C18-.1 C18:2 C18;3 area % M9 fatty rgfatty add nmol fatty acid/ add mg feces mg feces 17671 1.4 10.4 962 4.21 535579 41.3 315.0 29168 113.76 4357 0.3 2.6 237 0.93 659139 50.8 387.7 35897 126.18 67882 5.2 39.9 3697 13.09 4052 0.3 2.4 221 0.79 8388 0.6 4.9 457 1.64 total | 1297068 I 100 | 762.9 | 70639 | 260.59 I C15:0 I 136015 I Page A114 of A172 Appendix to TNO-rePort of 3M-02____________ Final version p. 115 04-12-2006 weight feces 12.1 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % N9 fatty ng fatty acid nmol fatty acid/ add mg feces mg feces 16368 1.4 12.4 1028 4.50 492848 43.0 374.7 30966 120.77 3736 0.3 2.8 235 0.92 567463 49.5 431.4 35654 125.32 55948 4.9 42.5 3515 12.44 3689 0.3 2.8 232 0.83 6084 0.5 4.6 382 1.37 total | 1146136 | 100 | 871.4 | 72013 | 266.16 | C15:0 | 105228 | 3:6-16 weight feces 9.6 mg C14:0 016:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % Mg fatty ng fatty add nmol fatty add/ acid mg feces mg feces 11600 1.2 10.6 1109 4.86 381872 39.5 350.5 36507 142.38 3071 0.3 2.8 294 1.15 510655 52.8 468.7 48818 171.59 50174 5.2 46.0 4797 16.98 2963 0.3 2.7 283 1.01 6403 0.7 5.9 612 2.20 total | 966738 | 100 | 8872 | 92420 | 340.17 | c 15: | 87169 I weight feces 8.5 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % Mg fatty ng fatty add nmol fatty add/ add mg feces mg feces 7619 22 7.2 846 3.70 165470 48.1 156.1 18369 71.64 1010 0.3 1.0 112 0.44 151890 44.1 143.3 16862 59.27 15484 4.5 14.6 1719 6.08 1040 0.3 1.0 115 0.41 1620 0.5 1.5 180 0.65 tota! C15:0 | 344133 I 100 I 324.7 | 38204 | | 84780 | 142.20 | weight feces 7.6 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % M9 fatty ng fatty add nmol fatty add/ add mg feces mg feces 6657 1.8 6.5 849 3.72 174284 453 168.9 86.67 1200 0.3 1.2 153 0.60 175484 46.1 170.0 22375 78.65 19588 5.2 19.0 2498 8.84 1351 0.4 1.3 172 0.61 1768 0.5 1.7 225 0.81 total | 380332 | 100 | 368.6 I 48494 | .. 179,90 I C15:0 I 62557 | weight feces 10.2 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % Mg fatty ng fatty add nmol fatty acid/ add mg feces mg feces 13778 1.8 9.5 928 4.06 361552 46.5 248.5 24363 95.02 2752 0.4 1.9 185 0.73 355381 45.7 244.3 23948 84.17 38817 5.0 26.7 2616 9.26 2125 0.3 1.5 143 0.51 3865 0.5 2.7 260 0.94 tota! | 778270 | 100 | 534.9 | 52444 | 194.69 | 015:0 | 116392 | 3 : 6-18 weight feces 8.1 mg C14:0 016:0 C16:1 C18:0 C18:1 018:2 018:3 area % Mg fatty ng fatty add nmol fatty add/ add mg feces mg feces 8700 1.3 8.3 1022 4.47 277658 41.6 264.1 32606 127.17 1872 0.3 1.8 220 0.86 334779 50.4 318.4 39313 138.18 34667 5.2 33.0 4071 14.41 1792 0.3 1.7 210 0.75 4339 0.7 4.1 510 1.83 total I 663807 | 100 | 631.4 | 77952 | ..768.. | C15:0 I 84105 | /// Page A115of A172 Appendix to TNO-report of 3M-02____________ Final version p. 116 04-12-2006 area % pg fatty ng fatty acid nmcrtfatty add/ add mg feces mg feces C14:0 9334 2.2 6.0 557 2.44 C16:0 205542 47.5 131.2 12263 47.83 C16:1 1575 0,4 1.0 94 0.37 018:0 189750 43.8 121.1 11321 39.79 C18:1 23168 5.4 14.8 1382 4.89 C182 1823 0.4 12 109 0.39 C18S3 1777 0.4 1.1 106 0.38 tota! I 432969 I 100 | 276.4 I 25831 I 96.09 | C15.0 I 125320 I 014:0 016:0 C16:1 018:0 C18:1 C16:2 018:3 area 10569 262993 1907 260336 26561 1861 2435 % pg fatty ng fatty add nmol fatty add/ acid mg feces mg feces 1.9 8.5 689 3.02 46.4 212.5 17137 66.84 0.3 1.5 124 0.49 45.9 210.3 16963 59.63 4.7 21.5 1731 6.13 0.3 1.5 121 0.43 0.4 2.0 159 0.57 total I 566662 I 100 | 457.9 I 36924 | 137.09 ~ | C15:0 I 99012 I weight feces 014:0 C16:0 016:1 C18.-0 C18-.1 018:2 C18:3 area 5300 116241 798 106821 14034 1332 1059 % pg fatty ng fatty add nmol fatty add/ add mg feces mg feces 2.2 3.9 545 2.39 47.3 86.1 11953 46.62 0.3 0.6 82 0.32 43.5 79.1 10984 38.61 5.7 10.4 1443 5.11 0.5 1.0 137 0.49 0.4 0.8 109 0.39 total I 245585 I 100 I 181.8 I 25254... F 93.92 | C15:0 I 108053 I I 1:7- weight feces 10 area % pg fatty ng fatty add nmol fatty acid/ add mg feces mg feces C14:0 9522 1.5 6.2 622 2.72 016:0 270616 42.3 176.8 17684 68.97 C16:1 2338 0.4 1.5 153 0.60 C18:0 313018 49.0 204.6 20455 71.90 C18:1 37006 5.8 24.2 2418 8.56 C18:2 3195 0.5 2.1 209 0.74 C18.-3 3428 0.5 2.2 224 0.80 total I 639123 | 100 | 417.7 I 41766 | 154.30 | C15:0 1 122421 I C14:0 C16:0 C16:1 C18:0 018:1 C18:2 C18:3 area 4357 90363 573 77962 9578 799 815 % pg fatty ng fatty add nmd fatty add/ add mg feces mg feces 2.4 3.7 608 2.66 49.0 75.7 12617 49.21 0.3 0.5 80 0.31 42.3 65.3 10886 38.26 5.2 8.0 1337 4.73 0.4 0.7 112 0.40 0.4 0.7 114 0.41 total I 184447 | 100 | 154.5 I 25754 | 95.99----- 1 C15:0 | 95490 | area C14:Q C16:0 C16:1 C18:0 C18:1 C18:2 C18.-3 7650 212450 1476 217370 22360 1545 2300 total I 465151 | C15:0 I 109572 | % pg fatty ng fatty add nmol fatty add/ add mg feces mg feces 1.6 5.6 642 45.7 155.1 17829 2.81 69.54 0.3 1.1 124 46.7 158.7 18242 0.49 64.12 4.8 16.3 1876 6.64 0.3 1.1 130 0.5 1.7 193 0.46 0.69 100 | 339.6 I 39036 I.......447S /0 Page A116of A172 Appendix to TNO-report of 3M-02 Final version weight feces 5.8 mg C14;0 016:0 C16:t C18:0 C18:1 C18:2 C18:3 area % M9 fatty ng fatty acid nmol fatty acid/ add mg feces mg feces 4977 1.9 4.8 822 3.60 125240 46.9 120.0 20682 80.66 723 0.3 0.7 119 0.47 123021 46.0 117.8 20316 71.41 11441 4.3 11.0 1889 6.69 691 0.3 0.7 114 0.41 1184 0.4 1.1 196 0.70 toW I 267277 I 100 | 256.0 I 4413 | 163.94 | C15:0 I 83524 | I .3:7-20 weight feces 7.7 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % M9 fety ng fatty acid nmol fatty add/ acid mg feces mg feces 5155 1.9 43 548 2.40 125067 46.5 102.5 13306 51.90 811 0.3 0.7 86 0.34 121012 45.0 99.1 12875 45.25 14616 5.4 12.0 1555 5.50 1143 0.4 0.9 122 0.43 1266 0.5 1.0 135 0.48 tota! I 269070 | 100 | 220.4 | 28627 | 106.31 | C15:0 I 97655 | weight feces 17 mg C14:0 C16:0 C16:1 C18:0 C18:1 018:2 018:3 area % pg fatty ng fatty acid nmol fatty add/ add mg feces mg feces 11944 1.3 10.3 603 2.64 374593 41.5 321.7 18926 73.82 3142 460518 0.3 51.0 2.7 395.5 159 23268 0.62 81.78 44662 4.9 38.4 2257 7.99 3197 0.4 2.7 162 0.58 5430 0.6 4.7 274 0.99 total | 903486 | 100 | 776.0 | 45648 | 168.41 | C15:0 | 93140 | p. 117 04-12-2006 Page A117of A172 Appendix to TNO-report of 3M-02 SS S8 Sg I? I* S it 3 8 % II 8 ss 32 8 Final version 8 n a 8 ss O s 2 3 SC s s> s p. 118 04-12-2006 u Page A118 of A172 Appendix to TNO-report of 3M-02____________ Final version p. 119 04-12-2006 P3 Page A119 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Fatty acid balance cage# 1.2 3.4 5 I 1,2 I 3,4 I5 u I 3.4 I5 GC sample# 1:1-1 1:1-2 1:1-3 2:1-1 2:1-2 2:1-3 3:1-1 3:1-2 3:1-3 total tottyadds (pmotfg feoes} 241.33 29224 345.18 208.40 246.11 326.56 304.96 310.59 290.07 I6 I 7.8 I 9,10 I6 7.6 I 9,10 I6 I 7.8 I 9.10 1:2-4 1:2-5 1:2-6 2:2-4 2 2 -5 2 2 -6 32-4 32-5 32-6 454.92 546.64 155.01 364.58 450.12 112.97 372.45 428.65 153.80 I 11.12,13 I 15 I 14,16,17 I 11.12.13 I 15 14.16.17 1 11,12,13 I 15 I 14,16.17 1:3-7 1S-8 1:3-9 2:3-7 2:3-8 25-9 3:3-7 3:3-8 3:3-9 183.38 284.77 334.26 175.80 30629 243.12 160.36 221.78 351.76 I 16 19 I 20,21 I 16 I 19 I 20,21 1 16 I 19 I 20,21 1:4-10 1:4-11 1:4-12 2:4-10 2:4-11 2:4-12 3:4-10 3:4-11 3:4-12 -SJL. AVERAGr 22 24.25 23.26,27 22 24,25 23,26,27 22 24.25 23,26,27 j 28,29 I 31 1 30,32 1 28.29 1 31 1 30.32 1 28.29 1 31 1 30,32 1:5-13 15-14 1:5-15 2:5-13 2:5-14 2:5-15 3:5-13 3:5-14 3:5-15 1:6-16 1:6-17 1:6-18 2:6-16 2:6-17 2:6-18 3:6-18 3:6-17 3:6-16 367.06 238.78 351.76 353.44 296.84 289.63 305.94 351.28 410.51 29181 499.96 19427 327.35 460.38 113.53 373.01 452.42 126.73 317.21 243.23 260.59 266.16 142.20 194.69 340.17 179.90 287.68 33,34.35 36.37 38,39 33,34.35 36.37 38,39 33,34.35 36.37 38,39 1:7-19 1:7-20 1:7-21 2:7-19 2:7-20 2:7-21 3:7-19 3:7-20 3:7-21 96.09 93.92 154.30 137.09 95.99 144.75 163.94 106.31 168.41 total fee (9) 3.075 2.4897 2.2946 4.692 3.7789 3,4106 3.5754 2.8781 2.7589 2.7619 4.0581 2.0935 4.075 6.371 3.4074 2.9279 4.3985 2.4575 3.4597 22994 2.3454 5.8147 3.7718 3.7727 4.025 2.5265 2.6905 1689 3.1432 3.2545 2.7133 4.6407 5.2075 2.041 3.4464 3.9549 3.195 2.9408 2.6399 4.9712 4.4833 3767 3.6113 3.3522 2.7034 2.5036 2.7083 2.3554 3.9684 3.9678 3.5666 2.9076 32449 2.7643 1.9697 2.0798 2.6705 3.4477 32646 4.3059 2.5117 2.4369 3.4107 average weight days (9) 123.6 8725 88.55 122.45 2 2 2 3 66.55 882 3 3 121.65 86.55 88.05 2 2 2 94.55 126.6 822 94.25 126.45 82.1 94 126.6 81.85 2 2 2 3 3 3 2 2 2 h 83.55 82.8 118.75 83.5 83.05 119.6 84.2 83.45 59.5 116.4 1122 58.85 116.55 111.85 58.7 117.45 112.9 2 2 2 3 3 3 2 2 2 2 2 2 3 3 3 2 2 2 110.7 88.35 84.6 1116 88.5 85.55 112.85 88.9 85.5 2 2 2 3 3 3 2 2 2 87.1 112.8 85.4 87.15 112.45 84.55 87.65 111.95 84.65 2 2 2 3 3 3 2 2 2 81.35 82.25 101.05 82.35 82.75 101.95 83.5 83.35 1032 2 2 2 3 3 3 2 2 2 fatty acids excretion (ymoi/100 gr mousefttey) 300.20 416.95 447.24 266.18 358.19 420.92 448.15 516.42 454.44 403.19 80.09 664.43 876.12 197.40 525.43 755.96 156.29 580.05 744.63 230.89 525.69 268.58 268.83 39186 473.41 286.94 46420 368.14 269.83 332.70 567.05 380.33 103.97 520.97 322.39 510.16 543.18 393.97 449.48 531.88 515.39 719.00 500.71 110.09 421.11 832.08 303.10 486.06 777.41 166.63 596.83 852.97 200.35 515.17 265.81 455.90 292.00 359.37 403.99 167.25 273.76 56422 260.72 469.72 360.77 124.47 116.33 118.75 203.89 191.32 126.23 203.79 246.56 155.41 278.30 18229 57.68 food intake la) 232 18 16.8 36.8 28.8 25.6 202 15.3 13.8 19.9 27.7 16.3 28.6 45.4 282 16.7 23.6 132 262 16.5 18.3 44.7 28.3 29.3 23.7 13.6 14.7 12 21.7 24 19.8 35.2 38.2 113 18.7 22.6 21.5 19.6 19.5 36.5 32.2 30.3 18.7 28 14.5 17.6 20.3 172 29.7 31 27.9 16.5 17.1 15 16.8 16.9 219 292 26.5 35 15.7 15 19.8 fatty acids uptake (pmot/100 gr mouse/day) 528322 5606.79 5340.11 5639.32 6244.01 5446.40 4673.78 4975.69 4411.43 5924.08 6158.51 558142 5733.89 6737.13 6445.31 5000.55 5246.96 453925 6249.55 5558.62 8220.85 7063.37 6359.72 6620.12 5577.59 4546.28 4958.16 5676.67 5247.30 6020.71 6313.30 566720 6408.63 5418.38 448144 5634.35 5466.64 6244.23 6487.74 6137.15 6827.33 6646.00 4664.12 8865.14 4773.44 5687.53 5065.43 5668.91 6394.80 5172.97 6191.97 5298.60 4299.33 4987.62 5812.74 5783.36 6100.10 6653.61 6009.19 6441.97 529226 5065.41 540026 cholesterol balance (pmot/100 gr mousetfay) 4983.02 5389.84 4892.87 5373.15 5885.82 5025.48 4225.84 4459.27 3956.98 491023 61193 5259.65 5282.39 5384.03 5208.45 596117 6289.03 4420.50 4502.33 4308.36 5181.77 682.54 5980.71 5166.76 5747.45 6776.43 5895.52 625198 5307.76 4213.57 4391.11 5525.70 84188 5155.70 4924.91 5510.55 5770.13 527322 5959.15 4886.51 3966.05 4915.34 515128 587.51 5045.52 5412.15 6184.64 5651.09 6049.92 6479.38 406729 8012.16 4573.08 5719.47 1159.56 5231.63 4773.43 5309.54 5990.81 5005.72 5918.21 4734.38 4038.62 4517.90 5057.81 634.70 5696.42 5664.61 5896.21 6462.29 5882.96 6238.18 5045.71 4910.00 5121.96 5657.59 537.83 molw C14:0 C16:0 016:1 C18:0 C18:1 C18:2 C18:3 average 228.4 256.4 254.4 284.5 282.5 280.5 278.5 MYSTRIC ACID PALMITIC ACID PALMITOLEIC ACID STEARIC ACID OLEIC ACID UOLEIC ACO LINOLENIC ACID 266.46 g/mol diet; 15 % fat= 0563 mmol per g diet Page A120 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Page A121 of A172 Appendix to TNO-report of 3M-02 Final version Appendix F. Intestinal cholesterol absorption Cholesterol absorption Oral gavage mbc: 14 C dpm average: 76083.3 76547.5 75205.8 74174.5 73763.5 75669.7 I 75241 " 1 3Hdpm average: 92065.2 92196.7 90472.4 88575.4 92526.8 92548.9 t 91398 ] 14C/3H dosing: I 0.823] i Mouse# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 14C dpm feces 7900.00 9070.58 8759.79 7943.46 9442.29 9027.49 9996.01 10914.90 11495.20 8402.11 8966.49 9560.47 10754.70 11858.50 12382.30 8918.47 9795.44 9570.76 11342.40 10247.10 11420.10 10160.50 11533.30 11375.70 9360.62 9242.62 9409.35 10257.10 11572.10 11790.60 7215.69 6854.01 8420.46 7620.85 7592.86 7701.77 10718.20 9765.43 11045.10 8973.56 10118.90 10188.80 11415.30 11521.70 9926.39 9713.21 10522.10 10357.80 10622.10 10309.40 9363.58 9289.29 8492.80 9169.78 7720.92 8438.85 8631.31 10252.90 9662.34 10589.00 3H dpm feces I 1 % cholesterol absorption | 17857.0 0.442 20224.1 0.449 19669.7 0.445 AVERAGE 0.445 45.89 1 17369-9 0.457 20044.0 19314.7 0.471 0.467 AVRAE' 0.465 43.48 1 16226.5 0.616 17696.9 0.617 19154.0 0.600 AVERAGE 0.611 25.78 1 15319.6 0.548 16793.3 16635.3 0.534 0.575 AVERAGE 0.552 32.90 1 19111.4 0.563 20818.2 0.570 21352.2 0.580 AVERAGE 0.571 30.67 1 19316.9 21221.6 0.462 0.462 20766 7 0.461 A v ilW 0A61 4^95 1 19935.8 0.569 17921.2 19737.7 0.572 0.579 AVERAGE 5353 '30.38 ' ' 1 22122.3 0.459 24951.5 0.462 24849.9 0.458 AVERAGE 0.460 44.15 1 18676.0 0.501 18523 6 0.499 18338.6 0.513 A 'v ft 0.504 38.73 1 18752.4 0.547 20953.2 0.552 21592.7 0.546 AVERAGE 0.548 33.38 1 13209.8 0.546 12708.1 14817.4 0.539 0.568 ^R X g " 3 33.03 1 13562.2 13608.7 0.562 0.558 13685.9 0.563 AVERAGE 0.561 31.87 1 22219.9 0.482 20159.2 0.484 22642.2 0.488 AVERAGE 0.485 41.10 1 15531.3 0.578 17347.9 0.583 P W JT17603.2 0.579 -- T555-- -------------- 5 3 ^ -------------1 21059.0 0.542 21372.1 0.539 19181.1 AVERAGE 0o.i518 35.27 1 17538.7 0.554 18995.2 18714.0 0.554 0553 AVERAGE 0.554 2.73 1 17065.5 0.622 16759.2 0.615 15186.6 0.617 VS" 0.618 24.92 1 18158.2 0512 16878.2 0.503 18096.8 AVERAGE o05io0>7 38.39 1 15528.4 0.497 17238. 17527.8 0.490 0.492 A v ta 0.493 40.11 1 18655.8 0.550 17086.2 0.566 19019.0 0.557 ' V I'iU . 04-12-2006 Page A122ofA172 Appendix to TNO-reoort of 3M-02 Final version i M ouse# 21 14C d p m fe c e s 8 6 3 2 .7 6 7 7 5 7 .7 8 8 7 3 2 .1 2 I 22 9 3 1 7 .5 8 1 0 3 4 9 .1 0 1 0 9 9 5 .4 0 i 23 I 24 I 25 1 2 7 2 6 .8 0 1 1 9 2 2 .7 0 1 0 8 2 4 .4 0 1 1 8 0 3 .8 0 1 1 5 0 9 .6 0 1 0 4 4 6 .2 0 9 8 2 4 .3 3 1 0 5 3 6 .1 0 1 0 8 1 9 .0 0 i 26 I 27 i 28 9 9 4 4 .1 7 9896 95 9 0 2 4 .0 8 1 1 0 1 2 .1 0 1 2 3 4 5 .1 0 1 2 6 1 1 .6 0 1 0 5 3 9 .2 0 1 1 9 9 7 .9 0 1 1 7 3 3 .2 0 1 29 i 30 8 4 6 7 .1 7 8 6 1 5 .2 3 779176 1 0 6 1 1 .2 0 1 1 6 6 9 .6 0 1 1 7 0 4 .2 0 i 31 I 32 6 7 9 7 .4 1 8 4 7 9 .3 6 8 0 4 2 .2 5 9 6 6 7 .2 2 1 0 3 3 2 .0 0 1 0 2 9 0 .4 0 I 33 I 34 i 35 i 36 i 37 8 2 7 4 .8 7 9 0 4 3 .4 6 8 3 5 9 .9 5 8 3 3 7 .8 7 8 9 7 8 .7 2 9 2 9 0 .9 8 9 2 6 0 .7 3 8 9 2 6 .6 9 8 2 1 6 .5 7 7 3 8 8 .6 5 780 1 .0 1 827 2 .1 1 8 7 7 5 .4 7 8 6 2 7 .5 0 7 9 3 5 .4 4 1 38 8 2 6 5 .7 2 767 2 .7 1 7 3 7 2 .1 5 1 39 l 40 1 41 6 5 1 2 .9 6 7 5 6 5 .0 0 7 4 4 4 .8 1 8 5 6 9 .4 0 9 5 4 0 .7 6 9 5 2 3 .5 9 8 3 3 0 .0 2 9 4 1 2 .7 3 9 4 2 7 .1 8 I_________ 42 9 6 0 7 .5 1 9 8 8 9 .2 5 957726 I 43 1 0 9 9 0 .9 0 9 6 5 1 .9 9 11648 90 i 44 t 45 1__________ 9 1 2 0 .8 2 1088720 1 1 0 2 3 .6 0 7 5 5 9 .6 3 8 8 3 2 .9 0 9 3 6 9 .6 2 3 H d p m fe c e * 1 4 /3 H fe c e * % c h o le s te ro l a b s o rp tio n i 1 8 2 6 3 .4 0 .4 7 3 1 6 5 4 3 .7 1 8 1 5 1 .9 0 .4 6 9 0.4 8 1 AV A a 0 .4 7 4 4239 I 187972 207502 213982 0 .4 9 6 0 .4 9 9 0 .5 1 4 AVERAGE 0 .5 0 3 3893 | 2 1 9 2 2 .9 0.5 6 1 2 0 8 5 9 .7 0 .5 7 2 1 9 0 7 3 .0 0 .5 6 8 VERSE 0 .5 7 3 3 .} | 2 0 5 3 1 .4 5373 1 9 9 3 0 .8 A1^7fi9ft2A6.4S 0 .5 7 7 0 .5 8 3 0 .5 7 8 2 9 .7 4 I 1 7 8 2 3 .8 0 .5 5 1 1 9 6 3 8 .0 0 .5 3 7 1 9 7 1 7 .7 0 .5 4 9 AVERAGE 0 .5 4 5 3 3 .7 4 | 1 7 8 6 1 .6 1 7 8 3 0 .0 162512 0 .5 5 7 0 .5 5 5 0 .5 5 5 AVERAGE 0 .5 5 6 3 2 .5 0 | 2 0 5 3 0 .5 0 .5 3 6 2 2 8 2 6 .4 2 3 7 4 0 .9 0.5 4 1 0.5 3 1 AVERAGE 5 !S 3 6 3 4 .8 7 | 1 8 1 3 8 .3 0 .5 5 1 2 1 6 4 0 .1 0 .5 4 9 -- S.35521335.4 AVERAGE 0 .5 5 0 ...... T5 I 1 9 7 1 3 .7 1 9 8 3 7 .0 0 .4 3 0 0 .4 3 4 1 8 3 4 3 .0 0 .4 2 5 A V R A JuE 0 .4 3 0 4 7 .8 1 1 6 1 1 8 .3 0 ,6 5 8 175652 1 7 9 9 5 .9 0 .6 6 4 0 .6 5 0 AVERAGE 0 .6 5 8 2 0 .1 1 | 1 5 0 9 0 .2 0 .4 5 0 1 8 3 6 3 .5 176552 0 .4 6 2 0 .4 5 6 AVERAGE 0 4 6 4 4 .6 2 I 1 8 2 9 9 .0 0 .5 2 8 1 9 5 2 5 .1 1 9 4 1 6 .7 0 .5 2 9 0 .5 3 0 AVRAGE 0 .5 2 9 3 5 .7 2 I 1 8 9 6 5 .3 0 .4 8 9 1 8 5 5 5 .B 0 .4 8 7 1 7 0 6 4 .5 0 .4 9 0 AVERAGE 0 .4 8 9 4 0 .6 3 | 1 6 9 3 8 .5 0452 1 8 1 4 2 .5 0 .4 9 5 1 8 5 0 0 .1 0 .5 0 2 AVERAGE 0 .4 9 6 3 9 .6 9 | 2 1 6 1 8 .5 2 0 6 5 5 .8 1 9 3 0 1 .2 0 .4 2 8 0 .4 3 2 0 .4 2 6 AVERAGE 0 .4 2 9 4 .9 | 1 7 1 0 1 .3 0 .4 3 2 1 8 0 8 2 .3 1 9 1 2 8 .3 0.4 3 1 0 .4 3 2 AVERAGE 0432 4 7 .5 3 | 1 8 3 3 1 .8 0 .4 7 9 181 7 0 .1 0 .4 7 5 1 6 9 4 4 .8 0 .4 6 8 AVERAGE 0 .4 7 4 4 2 .4 3 i 1 7 1 6 6 .9 1 6 0 9 9 .9 0 .4 8 1 0 .4 7 7 1 5 5 3 3 .3 0 .4 7 5 AVERAGE 0 .4 7 8 4 1 .9 9 | 1 3 4 2 4 .4 0485 1 5 5 1 0 .5 1 5 2 9 8 .9 AVRAE 0 .4 8 8 0 .4 8 7 0 .4 8 7 S53S | 1 7 5 1 4 .8 0 .4 8 9 1 9 5 1 4 .6 1 9 8 1 4 .4 0 .4 8 9 0.4 8 1 AVERAGE 0 .4 8 6 4 0 .9 3 | 1 6 0 0 6 .8 1 7 7 3 7 .5 175505 0 .5 2 0 0.5 3 1 0 .5 3 7 AVERAGE 0 .5 2 9 3 5 .6 9 | 1 9 6 2 4 .7 1 9 9 4 1 .0 1 9 5 8 0 .0 0 .4 9 0 0 .4 9 6 0 .4 9 4 AVERAGE 0 .4 9 3 4 0 ,0 8 1 2 0 0 0 9 .6 1 7 4 2 2 .8 0 .5 4 9 0 .5 5 4 2 0 4 4 5 .0 0 .5 7 0 A V E R A G E --------0 .5 5 8 3 2 .2 6 | 1 7 7 4 7 .4 2 0 8 7 8 .9 0 .5 1 4 0 .5 2 1 2 1 2 9 4 .1 AVERAGE 0 .5 1 8 0 .5 1 8 3 7 .1 2 | 1 4 3 1 4 .6 1 6 6 0 4 .8 0 .5 2 8 0532 1 7 2 0 3 .4 0545 AVERAGE 0 .5 3 5 3502 | 04-12-2006 Page A123of A172 Appendix to TNO-report of 3M-02 Final version ) Mouse 46 I 47 I 48 i 49 I SO I 51 I 52 I 53 I 54 i 55 i 56 I 57 i 58 I 59 i 60 ! 61 i 62 i 63 I 64 I 65 I 66 I 67 I 68 i 69 I 70 i 14C dpm tecas 3H dpm feces 14/3H feces % cholesterol absorption 13292.00 23039.1 0.577 12025.90 8154.44 20891.1 14093.8 0.576 0.579 AVERAGE 0.577 29.90 7073.99 11977.8 0.591 830355 8312.14 13879.6 13866.1 0.598 0.599 AVRAG" .596 ------------ w m ----------- 8105.89 14355.8 0.565 9177.91 161422 0.569 8404.42 14482.0 AVRAG 0.580 1H 35!E 9083.21 16293.9 0.557 10025.30 17848.5 0.562 10551.50 18335.8 0.575 AVERAG 0.565 31.38 7998.17 17522.8 0456 9589.24 20188.8 0.475 9485.67 20422.8 0.464 AVERAGE 0.485 43.48 8469.79 18261.6 0.464 9318.33 19807.5 0470 9497.10 20101.5 0.472 AVERAGE 0.469 43.04 861382 19823.6 0.435 869927 19531.5 0.445 7646.35 17499.6 AV&AE '00343379 46.66 10299.70 20630.3 0499 9010 87 17880.3 0.504 10286.90 20622.3 0.499 AVERAGE 0.501 39.18 8794.09 18229.9 0.482 9327.04 19419.7 0.480 9893 72 20500.1 AVERAGE 0.483 0482 3 5 12083.00 22066.1 0.548 11744.30 21734.5 0.540 10287.20 19073.9 AVERAGE 0S.!5S339 34.11 7269.37 16991 1 0.428 8544.20 19484.0 0439 8283.26 19099.1 0.434 AVRAE 47.36 790968 182242 0.434 7041.53 16492.5 0.427 8328.59 19376.2 0430 AVERAGE 0.430 47.73 7120.23 17677.8 0.403 8148.99 19926.7 0.409 8204.53 19132.5 0.429 AVERAGE 0.413 49.77 897584 20407.6 0.440 9015.56 19983.1 0.451 7523.90 ~ S717E5R63S.54T* 05.343258 46.58 7587.01 17288.6 0439 8719.17 19191.4 0.454 8811.04 195132 0.452 AVERAGE 0M 8 45.55 7582.08 18143.7 TO 6218.83 8867.68 20142.1 21451^ 0.408 0413 avefU e .41 49.82 8808.83 21295.7 0414 8584.87 20799.9 0.413 7447.43 18297.0 0.407 AVERAGE 0.411 50.06 7758.45 17723.8 0.438 6896,60 15907.7 0.434 7801.31 18207.4 0.428 AVERAGE 0.433 47.37 8952.17 21000.2 0.426 10058.20 23550.7 0.427 10221.10 23744.5 0.430 AVERAGE 0.428 48 02 817180 18430.1 0.443 7457.41 17029.8 0436 853829 192602 0.443 avS age 0.442 46.36 8381.63 203819 0411 789Q.69 19487.6 0.405 7086.95 17369.0 0.408 AVERAGE 0.408 50.43 980020 234282 0.418 11238.90 26990.4 0416 11334.00 26923.6 0421 AVERAGE 0.419 49.16 6081.05 14094.2 0.431 6823.98 15200.4 0.449 6967.67 -- 1v6B0&00.0E 0435 0.439 46.72 7436.52 182411 0.408 8308.62 20465.5 0.406 8710.60 21554.3 0.404 AVERAGE 0406 5069 6595.56 21140.4 0.407 8773.69 213902 0.410 7650.67 18677.5 0410 AVERAGE 0.409 5034 04-12-2006 Page A124of A172 Appendix to TNO-report of 3M-02 Group# | Mouse# 1 2 3 4 S 6 7 8 9 10 11 2 12 13 14 15 2 16 2 17 18 19 20 21 22 23 24 25 26 27 28 29 30 4 4 4 4 4 4 4 4 .3 4 ' AVERAGE SO 5 5 5 5 .5 . ' 5 * s5 5. 0 AVhKAiX sD 6 0 '.3 ,2 G Q AVERAGE SD * 7" 7 / r 7 7 -7 7 7 AVERAGE SD 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 % cholesterol absorption 45.89 43.48 25.78 32.90 30.67 43.95 30.38 44.15 38.73 33.38 36.9 7.2 33.03 31.87 41.10 29.56 35.27 32.73 24.92 38.39 40.11 32.30 $3. 5.0 42.39 38.93 30.37 29.74 33.74 32.50 34.87 33.19 47.82 20.11 34.4 7.5 44.62 35.72 40.63 39.69 47.92 47.53 42.43 41.99 40.90 40.93 42.2 3.7 35.69 40.08 32.26 37.12 35.02 29.90 27.59 30.62 31.38 43.48 34.3 4.9 43.04 46.68 39.18 41.48 34.11 47.36 47.73 49.77 46.58 45.55 44.1 4.7 49.82 50.06 47.37 48.02 46.36 50.43 49.16 46.72 50.69 50.34 48.9 1.6 Final version 04-12-2006 Page A125 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 week 8/9 week 8/9 week 8/9 n I n I p~ group 2 vs4 nI nI p Intestinal cholesterol absorption, p values M ann-W hitney U-lest group 1vs 6 group 1vs 7 nInI p n| n| 0.143 n I n I__ E__ n I n I P 53 H h i . 0.015 group2 vs 5 I group 3 vs 4 I group 3 vs 5 nI n group 2 vs 3 n I n I 0.7p96 I group 3 vs6 I I group 3 vs 7 group4 vs 5 group4 vs 6 group4 vs 7 group 5 vs 6 group 5 vs 7 group6 vs 7 0.003 lpc0.05| Page A126 of A172 Appendix to TNO-report of 3M-02 Final version p. 127 04-12-2006 Appendix G. Liver weight and perigonadal fat weight and lipid contents liver G1. Liver weight and perigonadal fat weight group Mouse# 10 eartag bodyweight L R (9)___ 34.5 31.9 30.4 28.9 32.4 30.1 28.4 32.8 32 28 35.2 12 30.3 34 34.9 IS 30.4 16 31.3 32.9 18 29.2 19 28.8 20 2212 23 33.6 24 29.8 25 27.7 30.9 27 31.2 28 26.3 32.3 29.6 31 32 28.5 33 29.1 34 29.8 35 31.1 36 26.4 37 27.6 38 30.1 39 27.8 28.3 AVfcKAGfc 42 44 46 47 48 49 50 51 52 53 54 55 56 58 59 31.8 25.9 29.6 29.2 29.6 30.2 31.9 30.1 29.2 28 25.9 27.5 26.9 25.4 26.6 27.8 ; -7 AVEKAG SD - 61 62 63 64 6656 68 69 29.9 30 28.7 30.3 29.4 28.3 28.1 28 25 28 perigonadal fat weight (g) 1.06 0.72 0,68 0.39 0.81 0.57 0.63 0.94 0.89 0.48 0.21 1.16 0.53 0.92 0.78 0.55 0.50 0.74 0.92 Q.74 0.23 0.77 0.74 0.59 0.37 0.42 0,64 0.75 1.05 0.23 0.24 0.26 0.42 0.45 0.39 0.23 0.30 0.34 0.32 0.34 0.08 0.82 0.54 0.39 0.51 0.52 0.86 0.47 0.58 0.16 0.23 0.34 0.14 0.19 0.30 0.41 0.37 0.21 0.28 0.09 0.34 0.38 0.30 0,32 0.29 0.33 0.03 liver weight liver weight l i l as % ofbw(%) 2.19 6.35 6,27 1.66 5.46 1.41 4.88 1.68 5.19 1.7 5.65 1.61 5.67 1.77 5.40 1.68 5.25 1.49 5.32 1.72 5.54 0.46 2.77 7.87 1.17 1.88 5.53 725 1.68 5.53 1.23 3.93 1.35 4.10 6.30 1.53 5.31 1.98 6.35 5.60 0.53 1.38 6.45 7.19 21..2678 6.76 5.64 1.56 5.63 2.06 1.85 5.93 2.42 2.14 1.97 0.31 0.71 3.46 12.23 3.68 3.69 1122..9618 3.93 13.19 4.23 1.94 3.62 13.12 3.9 12.96 4.03 14.50 3.39 11.98 12.45 0.63 1.92 1.9 5.97 1.71 6.60 8.38 1.69 5.79 7.60 2.46 4.61 6.25 1.82 6.23 2.14 7.23 1.98 0.34 1.16 3.9 13.93 4.87 15.61 4.1 15.83 4.17 15.16 4.13 15.35 3.48 13.70 3.13 11.77 14.78 4.74 4.33 16.40 4.10 14.83 0.52 1.37 4.29 14.35 4.33 13.52 4.28 3.85 13,60 3.83 13.68 13.32 3.92 14.00 3.98 13.91 0.30 0.37 Page A127of A172 /3I Appendix to TNO-reoort of 3M-02____________ Final version Livefweight; perigonadal fat weight; Itverweight as % of boctyweight, p values Mantv-Whitney U-test p. 128 04-12-2006 /3(yL Page A128ofA172 Appendix to TNO-report of 3M-02____________ Final version G2. Lipid contents liver Liverhomogenates; protein concentration mouse 1 13 21 31 45 51 61 2 14 22 32 46 52 62 3 17 23 33 41 53 63 4 11 24 34 42 54 64 6 12 25 35 43 55 65 5 15 26 36 44 56 66 7 16 27 37 47 57 67 8 18 28 38 48 58 68 9 19 29 39 49 59 69 10 20 40 60 30 50 70 measured prot cone(mq/ml) well 1 well 2 0.551 0.56 0.292 0.301 0.701 0.686 1.058 1.098 0.384 0.389 0.468 0.46 1.208 1.279 0.468 0.46 0.479 0.468 0.596 0.587 1.011 0.949 0.707 0.692 1.406 1.44 1.185 1.185 0.35 0.341 0.59 0.579 0.768 0.772 0.622 0.607 0.484 0.494 1.022 1.091 0.942 0.945 0.75 0.734 0.734 0.71 0.508 0.516 0.747 0.722 0.407 0.407 1.054 1.047 0.778 0.765 0.489 0.468 0.185 0.187 0.263 0.274 0.822 0.829 0.546 0.573 1.251 1.231 1.627 1.608 0.524 0.529 0.579 0.579 0.533 0.541 0.296 0.321 0.758 0.749 0.707 0.71 0.453 0.455 0.468 0.478 0.433 0.425 0.438 0.438 1.099 1.151 0.13 0.13 0.93 0.934 1.953 2.04 0.428 0.415 0.36 0.351 0.463 0.458 1,124 1.147 0.481 0.465 0.372 0.363 0.779 0.801 0.465 0.478 0.568 0.554 0.776 0.755 0.173 0.169 0.62 0.612 2.018 2.035 0.692 0.674 0.321 0.314 0.341 0.341 0.239 0.235 0.355 0.348 0.175 0.177 0.743 0.728 1.444 1.462 dilution Proteincone (mg/ml) 20 11.11 20 5.93 20 13.87 20 21.56 20 7.73 20 9.28 20 24.87 20 9.28 20 9.47 20 11.83 20 19.60 20 13.99 20 28.46 20 23.70 20 6.91 20 11.69 20 15.40 20 12.29 20 9.78 20 21.13 20 18.87 20 14.84 20 14.44 20 10.24 20 14.69 20 8.14 20 21.01 20 15.43 20 9.57 20 3.72 20 5.37 20 16.51 20 11.19 20 24.82 20 32.35 20 10.53 20 11.58 20 10.74 20 6.17 20 15.07 20 14.17 20 9.08 20 9.46 20 8.58 20 8.76 20 22.50 20 2.60 20 18.64 20 39.93 20 8.43 20 7.11 20 9.21 20 22.71 20 9.46 20 7.35 20 15.80 20 9.43 20 11.22 20 15.31 20 3.42 20 12.32 20 40.53 20 13.66 20 6.35 20 6.82 20 4.74 20 7.03 20 3.52 20 14.71 20 29.06 13$ p 129 04-12-2006 Page A129of A172 Appendix to TNQ-report of 3M-02____________ Final version p. 130 04-12-2006 HPTLC measurements, liver lipids: mouse# 1 13 21 31 45 51 61 2 14 22 32 46 52 62 3 17 23 33 Peak# 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Name Width [mm] Pos[mm] Free cholesterol 4.57 6.73 Triqlycerides 8.13 21.84 Internal standard 3.94 Cholesterolester 4.19 27.3 35.31 Free cholesterol 4.57 6.22 Triqlvcerides 6.22 19.94 Internal standard 4.06 26.42 Cholesterolester 5.08 34.8 Free cholesterol 4.57 Triglycerides 7.87 5.97 19.68 Internal standard 3.94 Cholesterolester 4.06 26.16 34.42 Free cholesterol 3.81 6.35 Triglycerides 5.46 19.43 Internal standard 4.19 Cholesterolester 4.32 26.42 34.8 Free cholesterol 4.06 6.35 Triglycerides 6.99 20.32 Internal standard 4.19 Cholesterolester 4.83 26.42 34.67 Free cholesterol 4.57 Triglycerides 7.62 6.48 20.07 Internal standard 4.32 Cholesterolester 4.7 26.29 34.42 Free cholesterol 4.19 6.48 Triglycerides 7.75 20.07 Internal standard 4.06 26.16 Cholesterolester 4.32 34.16 Free cholesterol 4.57 6.99 Triglycerides 6.73 20.32 Internal standard 4.06 26.54 Cholesterolester 4.45 34.54 Free cholesterol 4.32 6.86 Triglycerides 7.11 20.83 Internal standard 4.06 26.16 Cholesterolester 4.32 33.66 Free cholesterol 4.06 6.86 Triglycerides 7.49 21.97 Internal standard 3.17 26.29 Cholesterolester 3.81 33.66 Free cholesterol 4.45 7.62 Triglycerides 5.71 23.24 Internal standard 3.81 27.56 Cholesterolester 4.19 34.16 Free cholesterol 4.45 6.48 Triglycerides 8.64 22.86 Internal standarc 3.17 27.56 Cholesterolester 4.57 35.31 Free cholesterol 4.83 6.48 Triglycerides 8 20.83 Internal standard 4.32 26.54 Cholesterolester 4.57 34.54 Free cholesterol 4.45 6.22 Triglycerides 6.6 19.56 Internal standard 3.94 Cholesterolester 4.83 25.65 34.16 Free cholesterol 4.95 5.97 Triglycerides 4.83 18.41 Internal standard 4.06 Cholesterolester 3.94 25.4 33.78 Free cholestero 4.45 6.22 Triglycerides 6.6 20.07 Internal standard 3.68 Cholesterolester 4.7 26.16 34.29 Free cholesterol 4.06 6.35 Triglycerides 6.86 21.34 Internal standard 3.81 Cholesterolester 5.08 26.67 34.54 Free cholesterol 4.57 6.86 Triglycerides 6.22 22.23 Internal standard 4.19 Cholesterolester 4.57 27.3 34.92 od-Bkg MSlipid/ 200 pg protein 62604 5.44 287988 50.09 22996 2.00 105220 9.15 52524 4.33 197880 32.62 24262 2.00 125607 10.35 43766 3.24 251729 37.32 26977 2.00 68880 5.11 34466 2.55 123478 18.26 27048 2.00 67378 4.98 52422 4.22 260322 41.92 24841 2.00 141768 11.41 50585 4.09 265253 42.87 24749 2.00 127056 10.27 48224 3.38 248371 34.87 28494 2.00 130184 9.14 44510 3.51 192428 30.37 25345 2.00 79085 6.24 54357 4.54 258846 43.24 23946 2.00 89672 7.49 57023 5.09 299353 53.39 22427 2.00 84768 7.56 39787 3.78 115041 21.84 21069 2.00 81470 7.73 51326 4.75 318787 59.01 21609 2.00 111253 10.30 41327 3.28 242241 38.43 25212 2.00 94035 7.46 46397 3.41 221527 32.53 27238 2.00 133113 9.77 35829 2.77 104710 16.20 25858 2.00 42916 3.32 44899 4.06 206251 37.26 22142 2.00 78487 7.09 51469 3.99 216844 33.62 25800 2.00 74388 5.77 46317 3.55 162768 24.94 26102 2.00 89651 6.87 3age A130 of A172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 mouse# 41 53 63 4 11 24 34 42 54 64 6 12 25 35 43 55 65 5 Peak# 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Name Width [mm] Pos [mm] Free cholesterol 5.08 7.37 Triglycerides 5.59 22.61 Internal standard 4.06 27.69 Cholesterolester 4.06 35.31 Free cholesterol 4.19 7.24 Trialvcerides 6.86 22.99 Internal standard 3.94 27.81 Cholesterolester 3.94 35.31 Free cholesterol 4.45 6.73 Triglycerides 7.49 23.11 Internal standard 3.3 27.43 Cholesterolester 4.45 35.05 Free cholesterol 4.32 7.37 Triglycerides 5.33 23.37 Internal standard 4.32 28.45 Cholesterolester 3.56 36.07 Free cholesterol 4.19 5.46 Triglycerides 8.51 21.21 Internal standard 3.56 26.16 Cholesterolester 5.33 34.04 Free cholesterol 3.94 5.46 Triglycerides 5.97 18.8 Internal standard 4.45 25.27 Cholesterolester 4.95 33.66 Free cholesterol 4.19 4.95 Triglycerides 6.48 17.02 Internal standard 4.06 24 Cholesterolester 5.33 33.02 Free cholesterol 4.45 4.57 Triglycerides 6.48 16 Internal standard 3.94 23.24 Cholesterolester 5.08 32.64 Free cholesterol 4.06 4.7 Triglycerides 6.6 15.75 Internal standard 3.94 23.11 Cholesterolester 5.59 32.64 Free cholesterol 4.06 5.21 Triglycerides 6.99 16 Internal standard 4.32 23.24 Cholesterolester 6.1 32.64 Free cholesterol 4.7 6.22 Triglycerides 5.97 19.68 Internal standard 3.56 26.29 Cholesterolester 4.06 34.29 Free cholesterol 3.94 4.45 Triglycerides 5.97 15.37 Internal standard 4.32 22.86 Cholesterolester 5.59 32.13 Free cholesterol 3.81 5.33 Triglycerides 5.46 17.14 Internal standard 3.94 Cholesterolester 4.45 24.13 33.02 Free cholesterol 4.32 5.59 Triglycerides 6.73 19.05 Internal standard 4.06 24.77 Cholesterolester 4.95 33.15 Free cholesterol 4.7 5.46 Triglycerides 7.24 20.57 Internal standard 3.94 25.53 Cholesterolester 4.95 33.4 Free cholesterol 3.68 3.94 Triglycerides 6.6 13.97 Internal standard 3.56 21.21 Cholesterolester 5.59 30.35 Free cholesterol 3.81 3.94 Triglycerides 6.6 13.84 Internal standard 3.81 20.96 Cholesterolester 5.84 30.35 Free cholesterol 3.56 4.19 Triglycerides Internal standard Cholesterolester 5.71 4.19 5.59 13.08 20.7 30.23 od-Bkg 39765 132487 27644 37392 49635 246286 23327 123642 60183 294481 22239 141459 38024 104943 29874 26558 35962 307860 24049 107934 40358 115325 25364 52857 42991 172245 24996 73646 56968 192630 26017 123337 56224 215795 27127 153105 55767 238689 31249 162067 53651 199383 25618 75588 38266 151763 32037 54371 49570 149995 26617 46177 61124 219127 28657 145012 56257 295337 28462 97524 48925 249297 28191 157664 65477 276227 27578 178277 49411 189703 29682 95600 pg lipid/ 200 pgprotein 2.88 19.17 2.00 2.71 4.26 42.23 2.00 10.60 5.41 52.97 2.00 12.72 2.55 14.05 2.00 1.78 2.99 51.20 2.00 8.98 3.18 18.19 2.00 4.17 3.44 27.56 2.00 5.89 4.38 29.62 2.00 9.48 4.15 31.82 2.00 11.29 3.57 30.55 2.00 10.37 4.19 31.13 2.00 5.90 2.39 18.95 2.00 3.39 3.72 22.54 2.00 3.47 4.27 30.59 2.00 10.12 3.95 41.51 2.00 6.85 3.47 35.37 2.00 11.19 4.75 40.07 2.00 12.93 3.33 25.57 2.00 6.44 Page A131 of A172 135 p. 132 Appendix to TNO-report of 3M-02____________ Final version 04-12-2006 mouse# 15 26 36 44 56 66 7 16 27 37 47 57 67 8 18 28 38 48 Peak# 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Name Width [mm] Pos [mm] Free cholesterol Trialvcerides 3.3 5.71 3.94 12.57 Internal standard 4.19 Cholesterolester 5.33 20.45 30.1 Free cholesterol 3.43 Trialvcerides 7.49 3.94 13.84 Internal standard 3.81 Cholesterolester 5.59 21.08 30.73 Free cholesterol 3.94 Trialvcerides 6.35 4.32 14.48 Internal standard 3.81 Cholesterolester 5.46 22.1 31.5 Free cholesterol 3.56 4.19 Trialvcerides 5.71 14.73 Internal standard 3.81 Cholesterolester 4.95 22.48 31.62 Free cholesterol Trialvcerides 3.94 6.6 4.57 16.13 Internal standard 3.68 Cholesterolester 5.33 23.37 32.26 Free cholesterol 4.19 5.33 Trialvcerides 7.37 18.16 Internal standard 3.68 Cholesterolester 5.46 24.51 33.02 Free cholesterol 4.45 Trialvcerides 5.97 5.33 18.92 Internal standard 4.19 Cholesterolester 4.7 25.27 33.27 Free cholesterol 4.7 5.59 Trialvcerides 6.35 20.32 Internal standard 4.19 Cholesterolester 3.68 25.91 33.27 Free cholesterol 4.83 5.84 Trialvcerides 6.99 21.21 Internal standard 4.7 Cholesterolester 4.57 27.69 35.69 Free cholesterol 4.32 5.71 Trialvcerides 6.22 20.45 Internal standard 4.06 26.92 Cholesterolester 4.45 34.92 Free cholesterol Trialvcerides 3.94 6.6 5.97 20.07 Internal standard 3.81 Cholesterolester 4.06 26.42 34.54 Free cholesterol 4.19 5.97 Trialvcerides 5.71 19.3 Internal standard 3.68 Cholesterolester 4.06 25.65 33.78 Free cholesterol 4.7 6.48 Trialvcerides 7.49 20.45 Internal standard 3.43 Cholesterolester 5.08 25.65 33.66 Free cholesterol 3.81 6.99 Trialvcerides 5.59 20.45 Internal standard 3.81 Cholesterolester 4.06 25.91 33.66 Free cholesterol 3.94 7.11 Trialvcerides 5.84 21.21 Internal standard 3.94 25.91 Cholesterolester 4.45 33.53 Free cholesterol 4.32 6.48 Trialvcerides 5.71 20.83 Internal standard 3.81 Cholesterolester 4.45 25.53 32.89 Free cholesterol 4.06 Trialvcerides 5.59 7.37 22.23 Internal standarc 3.81 Cholesterolester 4.45 26.8 33.91 Free cholesterol 4.32 7.75 Trialvcerides Internal standard Cholesterolester 6.6 3.17 4.7 23.88 27.43 34.16 od-Bkg |jg lipid/ 200 M9protein 51117 3.29 186121 23.97 31056 108021 2.00 6.96 51578 3.43 314298 41.81 30069 134094 2.00 8.92 49352 209014 3.64 30.84 27111 2.00 117556 8.67 43993 173724 24940 3.53 27.86 2.00 61245 4.91 53573 223574 4.32 36.04 24814 2.00 161398 13.01 55464 4.56 270083 24336 153330 44.39 2.00 12.60 51342 4.32 176085 29.66 23750 2.00 76943 6.48 47717 3.80 148024 23.58 25114 2.00 60069 4.78 45004 3.22 193690 27.70 27970 60245 2.00 4.31 43432 3.40 150439 23.55 25551 2.00 98765 7.73 42568 194874 3.64 33.29 23418 72154 2.00 6.16 40720 3.01 177809 26.24 27101 2.00 69768 5.15 52458 292884 3.50 39.10 29963 160643 2.00 10.72 50307 3.70 162127 23.85 27192 2.00 70970 5.22 56774 4.17 225869 33.16 27244 2.00 91446 6.71 49756 3.78 171023 25.99 26319 2.00 87109 6.62 41083 2.99 144165 20.96 27516 2.00 61184 4.45 55902 4.23 285090 43.14 26432 119215 2.00 9.02 Page A132 of A172 A3& Appendix to TNO-report of 3M-02 Final version mouse# 58 68 9 19 29 39 49 59 69 10 20 30 40 50 60 70 Peak# 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Name Width [mm] Pos [mm] Free cholesterol Triglycerides 3.81 6.73 7.11 23.24 Internal standard 3.43 Cholesterolester 4.57 27.43 34.16 Free cholesterol 3.56 4.57 Triglycerides 8.89 14.61 Internal standard 4.06 22.1 Cholesterolester 5.71 32.13 Free cholesterol 3.17 4.57 Triglycerides 5.59 12.19 Internal standard 3.81 Cholesterolester 4.95 20.83 31.24 Free cholesterol 3.3 4.32 Triglycerides 4.7 Internal standard 3.94 11.68 20.57 Cholesterolester 4.95 31.12 Free cholesterol 3.3 3.81 Triglycerides 6.99 12.57 Internal standard 3.68 Cholesterolester 5.33 20.57 31.12 Free cholesterol 3.56 3.81 Triglycerides 5.08 Internal standard 4.19 11.68 20.7 Cholesterolester 5.33 31.24 Free cholesterol 3.94 4.06 Triglycerides 4.83 11.81 Internal standard 4.32 21.34 Cholesterolester 4.95 32.13 Free cholesterol 3.94 3.81 Triglycerides 5.97 12.57 Internal standard 4.06 21.84 Cholesterolester 5.71 32.38 Free cholesterol 3.17 4.7 Triglycerides 8.13 14.86 Internal standard 4.32 22.99 Cholesterolester 5.46 33.4 Free cholesterol 3.43 4.57 Triglycerides 5.21 Internal standard 3.68 13.59 22.99 Cholesterolester 4.83 33.27 Free cholesterol 3.43 4.83 Triglycerides Internal standard Cholesterolester 6.22 3.68 4.7 14.99 23.75 33.91 Free cholesterol Triglycerides 3.17 5.08 5.08 15.49 Internal standard 3.68 Cholesterolester 4.32 24.38 34.16 Free cholesterol Triglycerides internal standard Cholesterolester 4.06 5.46 2.92 4.83 8.76 27.43 30.1 35.18 Free cholesterol Triglycerides Internal standard Cholesterolester 3.56 4.83 3.43 3.94 8.89 25.78 29.34 35.18 Free cholesterol 4.19 8 Triglycerides 6.35 Internal standard 2.79 24.77 28.57 Cholesterolester 4.83 35.05 Free cholesterol 4.7 7.49 Triglycerides Internal standard Cholesterolester 7.49 3.17 4.45 23.11 27.81 34.8 od-Bkg 50057 254148 23859 136982 59495 368098 29699 172395 47364 186870 29695 97139 44604 114977 29508 85494 55495 290613 29976 133502 54475 146150 30815 126727 63475 140542 33224 84388 64340 238585 30644 139703 56623 368599 30442 178078 37007 124055 26723 78711 47883 216520 26901 83366 34039 136553 27780 92480 54261 222500 24418 136191 36509 87808 28028 35295 58652 238639 21843 134627 58311 269150 31958 124736 pg lipid/ 200 pg protein 4.20 42.61 2.00 11.48 4.01 49.58 2.00 11.61 3.19 25.17 2.00 6.54 3.02 15.59 2.00 5.79 3.70 38.78 2.00 8.91 3.54 18.97 2.00 8.23 3.82 16.92 2.00 5.08 4.20 31.14 2.00 9.12 3.72 48.43 2.00 11.70 2.77 18.57 2.00 5.89 3.56 32.20 2.00 6.20 2.45 19.66 2.00 6.66 4.44 36.45 2.00 11.15 2.61 12.53 2.00 2.52 5.37 43.70 2.00 12.33 3.65 33.69 2.00 7.81 04-12-2006 151 Page A133ofA172 p. 134 Appendix to TNO-report of 3M-02____________ Final version 04-12-2006 G roup# M ouse# 1 2 3 4 5 6 7 8 9 10 ri* 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 b' N j 4 4 4 4 4 4 4 4 4 AVERAGE. 5 5 .b . 5' s& j & 5 'S E R a S T . *SD; 6 o ;; b 6 AVERAGE i____ r ____ 7 `r . -7 ... 7. 7' 7 ... 7 7 7 AVERAGE SO 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 Liver Kpids (pg/200 \ ig protein) Liver Hpkls {pg/m g protein) | Free cholesterol Triglycerides Chotesterofester Free cholesterol Triglycerides ChoJesteroiester 5.44 3.51 2.77 50.09 30.37 16.20 9.15 6.24 3.32 2722 17.56 13.86 250.5 151.8 81.0 45.76 31.20 16.60 2.55 3.33 4.19 14.05 25.57 31.13 1.78 6.44 5.90 12.73 16.65 20.94 70.3 127.8 155.7 8.89 3221 29.51 4.32 29.66 6.48 21.62 148.3 32.40 3.70 23.85 5.22 18.50 119.2 26.10 3.19 2.77 25.17 18.57 6.54 5.89 15.95 1385 125.9 92.8 32.71 29.45 17.89 132.3 28.48 4.41 51.0 9.92 2.99 51.20 8.98 14.95 256.0 4488 2.39 4.33 18.95 32.62 3.39 10.35 11.94 21.65 94.7 163.1 16.97 51.77 4.54 3.29 3.80 4.06 43.24 23.97 23.58 37.26 7.49 6.96 4.78 7.09 22.70 16.46 19.00 20.28 216.2 119.9 117.9 186.3 37.45 34.78 23.92 35.45 4.17 3.02 3.56 33.16 15.59 32.20 6.71 5.79 620 20.84 15.12 17.80 165.8 77.9 161.0 33.57 28.97 30.99 18.07 iS 5!9 33.87 3.44 55.1 9.66 3.24 37.32 5.11 5.09 53.39 7.56 3.99 33.62 5.77 16.22 25.43 19.95 1868 267.0 168.1 25.53 37.80 28.83 3.18 3.72 343 3.22 18.19 22.54 41.81 27.70 4.17 3.47 8.92 4.31 15.91 18.62 17.15 16.09 90.9 112.7 209.0 138.5 20.84 17.35 44.59 21.54 3.78 25.99 6.62 3.70 38.78 8.91 2.61 12.53 2.52 18.90 18.51 13.03 130.0 193.9 62.7 33.10 44.54 12.59 17.98 155.9 2887 328 61.0 11.14 2.55 3.78 3.55 3.44 4.27 3.64 3.40 18.26 21.84 24.94 27.56 30.59 30.84 23.55 4.98 7.73 687 589 10.12 8.67 7.73 12.74 18.86 17.74 17.20 21.33 18.20 17.00 91.3 109.2 124.7 137.8 152.9 1542 1178 24.91 3887 34.35 29.46 50.60 43.36 38.65 2.99 3.54 2.45 2Q.96 18.97 19.66 4.45 823 6 .6 6 14.93 17.68 1225 1048 94.9 98.3 22.24 41.13 33.29 1680 118.6 35.67 2.78 232 8.64 2.88 4.38 3.95 19.17 29.62 41.51 2.71 9.48 685 14.38 21.90 19.77 95.9 148.1 207.5 13.53 47.41 34.26 3.53 4.22 4.75 3.64 27.86 41.92 59.01 33.29 4.91 11.41 10.30 6.16 17.64 21.10 23.75 18.18 139.3 209.6 295.1 166.4 24.56 57.07 51.49 30.81 4.23 43.14 9.02 21.15 215.7 45.10 3.82 5.37 16.92 43.70 5.08 12.33 19.10 26.85 84.6 218.5 25.40 61.64 20.38 178.1 39.13 3.45 6 3 8 15.76 4.09 3.28 4.26 42.87 38.43 42.23 10.27 7.46 10.60 20.44 16.39 21.28 214.4 192.2 211.2 51.34 37.30 53.00 4.15 3.47 4.32 3.01 31.82 35.37 36.04 26.24 11.29 11.19 13.01 5.15 20.73 17.35 21.59 15.03 159.1 176.9 1802 131.2 56.44 55.93 65.04 25.74 450 4.20 4.44 42.61 31.14 36.45 11.48 9.12 11.15 20.98 21.00 22 22 213.0 155.7 182.2 57.41 45.59 55.77 19.70 181.6 50.36 2.49 27.5 11.40 3.38 3.41 5.41 3.57 34.87 32.53 52.97 30.55 9.14 9.77 12.72 10.37 16.92 17.03 27.06 17.85 174.3 162.7 2648 152.8 45.69 4887 6381 51.86 4.75 40.07 12.93 23.74 200.3 64.65 4.56 44.39 12.60 22.79 222.0 63.01 3.50 4.01 39.10 49.58 10.72 11.61 17.51 20.03 195.5 247.9 53.61 58.05 3.72 48.43 11.70 3 6 5 33.69 7.81 18.60 1825 242.2 168.4 58.50 39.03 19.98 3.43 203.1 39.4 54.69 8.45 Page A134of A172 /38 Appendix to TNO-report of 3M-02 Final version lipid contents liver, p values Mann-Whitney U-test p. 135 04-12-2006 3/ ? Page A135ofA172 P 136 Appendix to TNO-report of 3M-02____________ Final version_________________________ 04-12-2006 H. HMG-CoA reductase and cholesterol-7a-hydroxylase activity in liver microsomes and sterol27-hydroxylase activity in liver mitochondria H1. HMG-CoA reductase and cholesterol-7a-hydroxylase activity in liver microsomes Protein concentration isolated microsomes interesme for HM G C o A reductase determination mouse 1 13 21 31 45 51 61 2 14 22 32 46 52 62 3 17 23 33 41 53 63 4 11 24 34 42 54 64 6 12 25 35 43 55 65 5 15 26 36 44 56 66 7 16 27 37 47 57 67 8 18 28 38 48 58 68 9 19 29 39 49 59 69 10 20 40 60 30 50 70 measured prot cone fmo/mi) well 1 weB 2 0.828 0.866 0.725 0.739 1.052 1.102 1.42 1.525 0.927 0.977 0.659 0.654 0.654 0.688 0.763 0.801 1.019 1.073 0.788 0.818 1.667 1.803 1.031 1.056 0.64 0.647 0.691 0.709 0.789 0.828 0.669 0.688 0.718 0.739 0.876 0.888 0.664 0.677 1.86 1.983 1.733 1.851 0.583 0.604 0.7S2 0.773 0.718 0.729 1.124 1.161 0.734 0.752 1.555 1696 1.756 1.961 0.7 0.723 0.519 0.521 0.822 0.846 1223 1.308 1.136 1.176 1.649 1.791 0.651 0.643 0.622 0.628 0S8 1.041 0.98 0.991 0.763 0.796 0.881 0.91 1.149 1216 1.2 1.281 0.66 0.734 0.597 0.64 1.127 1.198 1254 1.315 0.67 0.699 1.697 1.712 1.641 1.823 0.637 0.668 0.794 0.826 0.832 0.859 1.63 1.553 0.796 0.817 1.747 1.899 0.636 0.632 1.097 1.149 0.736 0.766 0.92 0.906 1.503 1.47 0.98 1.034 0.621 0.612 1.154 1219 0.798 0.82 0.807 0.794 1.17 1203 1.685 1.612 0.629 0.657 1.092 1.048 1.529 1.598 dilution Protein cone imgAnl) 20 16,94 20 14.64 20 21.54 20 29.45 20 19.04 60 39.39 60 40.26 20 15.64 20 20.92 20 16.06 20 34.70 20 20.87 60 38.61 60 42.00 20 16.17 20 13.57 20 14.57 20 17.64 20 13.41 20 38.43 20 35.84 20 11.87 20 15.25 20 14.47 20 22.8S 20 14.86 20 32.51 20 37.17 20 14.23 20 10.40 20 16.68 20 25.31 20 23.12 20 34.40 60 38.82 20 12.50 20 20.21 20 19.71 20 15.59 20 17.91 20 23.65 20 24.81 20 13.94 20 12.37 20 23.25 20 25.69 20 13.69 20 34.09 20 34.64 20 13.05 20 16.20 20 16.91 20 31.83 20 16.13 20 36.46 60 38.04 20 22.46 20 15.02 20 18.26 20 29.73 20 20.14 60 36.99 20 23.73 20 18.18 20 16.01 20 23.73 20 32.97 20 12.86 20 21.40 20 31.27 M icrosom es for 7-aloha-hvdroxvtase determination mouse 1 13 21 31 45 51 61 2 14 22 32 46 52 62 3 17 23 33 41 53 63 4 11 24 34 42 54 64 6 12 25 35 43 55 65 5 15 26 36 44 56 66 7 16 27 37 47 57 67 8 18 28 38 48 58 68 9 19 29 39 49 59 69 10 20 40 60 30 50 70 Code 1.1 12 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 1.13 1.14 1.15 1.16 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 3.16 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 4.14 4.15 4.16 5.1 5.2 5.3 5.4 5.5 5.6 measured prot cone (mo/ml) wefl 1 we1 2 0.952 0.996 0.683 0.703 1.097 1.048 1.543 1.438 1.222 1218 0.652 0.644 0.612 0.694 1.103 1.113 1.366 1229 0.999 0.963 1.524 1.447 0.921 0.909 0.569 0.575 0.577 0.571 0.708 0.679 0.553 0.545 0.916 0.874 1.15 1.14 0.69 0.694 0.5 0.436 0.543 0.479 0.599 0.569 0.887 0.837 0.726 0.724 0.882 0.893 0.772 0.829 0.541 0.515 0.61 0.579 0.604 0.624 0.486 0.498 0.674 0.67 1263 1.329 1.023 1.055 0.507 0.472 0.505 0.492 0.595 0.581 0.808 0.79 0.994 0.988 0.673 0.646 1.164 1.183 0.472 0.488 0.385 0.376 0.792 0.772 0.561 0.592 0.942 0.933 1.404 1.527 0.933 0.948 0.462 0.479 0.35 0.366 0.74 0.699 0.818 0.821 0.837 0.859 1.604 1.463 0.829 0.824 0.45 0.46 0.468 0.455 0.821 0.665 0.832 0.826 0.916 0.954 1.164 1.132 0.893 0.951 0.455 0.468 0.432 0.41 0.821 0.84S 0.994 1.039 1.268 1.517 0.483 0225 0.779 0.785 0.919 0.87 0.553 0.511 dilution 20 20 20 20 20 60 60 20 20 20 20 20 60 60 20 20 20 20 20 60 60 20 20 20 20 20 60 60 20 20 20 20 20 60 60 20 20 20 20 20 60 60 20 20 20 20 20 60 60 20 20 20 20 20 60 60 20 20 20 20 20 60 60 20 20 20 60 20 20 60 Protein cone (mg/ml) 19.48 13.86 21.45 29.81 24.40 38.88 39.18 22.16 26.95 19.62 29.71 18,30 34.32 34.44 13.87 10.98 17.92 22.90 13.84 28.08 30.66 11.68 17.24 14.50 17.75 16.01 31.68 35.67 12.28 9.86 13.44 25.92 20,78 29.37 29.91 11.78 15.98 19.82 13.19 23.47 28.80 22.83 15.64 11.53 18.75 29.31 18.81 28.23 21.48 14.39 16.39 16.96 30.87 16.53 27.30 27.69 16.86 16.58 18.70 22.96 18.44 27.69 2526 16.66 20.33 27.85 3024 15.64 17.89 31.92 Page A136ofA172 Appendix to TNQ-rePort of 3M-02 Final version 137 04-12-2006 A m ount isolated liver m icrosom es m ouse# 1 2 3 4 5 6 7 8 9 10 I 11 12 I 13 I 14 15 I 16 17 I 18 19 20 21 22 1 23 1 24 25 1 26 1 27 1 28 1 29 I 30 1 31 t 32 | 33 V 34 1 35 1 36 1 37 1 38 | 39 1 40 5 v L1 aVLAia^F 41 42 43 44 45 46 47 48 49 50 51 52 1 53 5* 1 55 1 5$ 1 57 1f 1 59 I 60 / T tT 7 : T"7 *~ r 61 62 63 64 65 66 67 68 69 70 P ro te in c o n e (m o frn l) m ic ro s o m e s m ic ro s o m e s 7 a lp h a h y d ro x ^ a s e H M G C o A re d u e t 1 9 .4 8 2 2 .1 6 1 3 .8 7 1 1 .6 8 1 6 .9 4 1 5 .6 4 1 6 .1 7 1 1 .8 7 1 1 .7 8 1 2 .5 0 1128 1 5 .6 4 1 4 .2 3 1 3 .9 4 1 4 .3 9 1 6 .8 6 1 3 .0 5 2 2 .4 6 1 6 .6 6 1 6 .1 8 e s tim a te d p i m ic ro s o m e s 600 600 600 600 600 600 600 600 600 600 m g m ic ro s o m e s 7 a lp h a h y d ro x y la s e 1 1 .6 8 8 1 3 .2 9 6 8 .3 2 2 7 .0 0 8 7 .0 5 6 7 .3 6 8 9 .3 8 4 8 .6 3 4 1 0 .1 1 6 9 .9 9 6 1 7 .2 4 9 .8 6 1 3 .8 6 2 6 .9 5 1 5 .9 6 1 1 .5 3 1 0 .9 8 1 6 .3 9 1 6 .5 8 2 0 .3 3 1 5 .2 5 1 0 .4 0 1 4 .6 4 2 0 .9 2 2 0 .2 1 1 2 .3 7 1 3 .5 7 1 6 .2 0 1 5 .0 2 1 6 .0 1 600 600 600 600 600 600 600 600 600 600 1 0 .3 4 4 5 .9 1 6 8 .3 1 6 1 6 .1 7 9 .5 6 8 6 .9 1 8 6 .5 8 8 9 .8 3 4 9 .9 4 8 1 2 .1 9 8 2 1 .4 5 1 9 .6 2 1 7 .9 2 1 4 .5 1 3 .4 4 1 9 .8 2 1 8 .7 5 1 6 .9 6 1 8 .7 1 5 .6 4 2 1 .5 4 1 6 .0 6 1 4 .5 7 1 4 .4 7 1 6 .6 8 19.7 1 2 3 .2 5 1 6 .9 1 1 8 .2 6 1 2 .8 6 600 600 600 600 600 600 600 600 600 600 1 2 .8 7 1 1 .7 7 2 1 0 .7 5 2 8 .7 8 .0 6 4 1 1 .8 9 2 1 1 .2 5 1 0 .1 7 6 1 1 .2 2 9 .3 8 4 2 9 .8 1 2 9 .7 1 2 2 .9 1 7 ,7 5 2 5 .9 2 1 3 .1 9 2 9 .3 1 3 0 .8 7 2 2 .9 6 2 7 .8 5 2 9 .4 5 3 4 .7 0 1 7 .6 4 2 2 .8 5 2 5 .3 1 1 5 .5 9 2 5 .6 9 3 1 .8 3 2 9 .7 3 2 3 .7 3 700 700 700 700 700 600 700 700 700 700 2 0 .8 6 7 2 0 .7 9 7 1 6 .0 3 1 2 .4 2 5 1 8 .1 4 4 7 .9 1 4 2 0 .5 1 7 2 1 .6 0 9 1 6 .0 7 2 1 9 .4 9 5 1 3 .8 4 16.0 1 2 0 .7 8 2 3 .4 7 2 4 .4 1 8 .3 18.8 1 1 6 .5 3 1 8 .4 4 1 7 .8 9 1 3 .4 1 1 4 .8 6 2 3 .1 2 1 7 .9 1 1 9 .0 4 2 0 .8 7 1 3 .6 9 1 6 .1 3 2 0 .1 4 2 1 .4 0 600 600 600 600 600 600 600 600 600 600 8 .3 0 4 9 .6 0 6 1 2 .4 6 8 1 4 .0 8 2 1 4 .6 4 1 0 .9 8 1 1 .2 8 6 9918 1 1 .0 6 4 1 0 .7 3 4 3 8 .8 8 3 4 .3 2 2 8 .0 8 3 1 .6 8 2 9 .3 7 2 8 .8 2 8 .2 3 2 7 .3 2 7 .6 9 3 0 .2 4 3 9 .3 9 3 8 .6 1 3 8 .4 3 3 2 .5 1 3 4 .4 0 2 3 .6 5 3 4 .0 9 3 6 .4 6 3 6 .9 9 3 2 .9 7 700 700 700 700 700 700 700 700 700 700 2 7 .2 1 6 2 4 .0 2 4 1 9 .6 5 6 2 2 .1 7 6 2 0 .5 5 9 2 0 .1 6 1 9 .7 6 1 1 9 .1 1 1 9 .3 8 3 2 1 .1 6 8 3 9 .1 8 3 4 .4 4 3 0 .6 6 3 5 .6 7 2 9 .9 1 2 2 .8 3 2 1 .4 8 2 7 .6 9 2 5 .2 6 3 1 .9 2 4 0 .2 6 4 2 .0 0 3 5 .8 4 3 7 .1 7 3 8 .8 2 2 4 .8 1 3 4 .6 4 3 8 .0 4 2 3 .7 3 3 1 .2 7 700 700 700 700 700 700 700 700 700 700 2 7 .4 2 6 2 4 .1 0 8 2 1 .4 6 2 2 4 .9 6 9 2 0 .9 3 7 1 5 .9 8 1 1 5 .0 3 6 1 9 .3 8 3 1 7 .6 8 2 2 2 .3 4 4 m g m ic ro s o m e s H M G C o A re d u c t. 1 0 .1 6 4 9 .3 8 4 9 .7 0 2 7 .1 2 2 7 .5 8 .5 3 8 6 .3 6 4 7 .8 3 1 3 .4 7 6 9 .7 0 8 9 .1 5 614 6 .7 8 4 1 2 .5 5 2 1 2 .1 2 6 7 .4 2 2 8 .1 4 2 9 .7 2 9 .0 1 2 9 .6 0 6 1 2 .9 2 4 9 .6 3 6 8 .7 4 2 8 .6 6 2 1 0 .0 0 8 1 1 .8 2 6 1 3 .9 5 1 0 .1 4 6 1 0 .9 5 6 7 .7 1 6 2 0 .6 1 5 2419 1 2 .3 4 8 1 5 .9 9 $ 1 7 .7 1 7 9 .3 5 4 1 7 .9 8 3 22181 2 0 .8 1 1 16511 8 .0 4 6 8 .9 1 6 13572 1 0 .7 4 6 1 1 .4 2 4 1 2 .5 2 2 8 .2 1 4 9 .6 7 8 1 2 .0 8 4 1 2 .8 4 2 7 .5 7 3 2 7 .0 2 7 2 6 .9 0 1 2 2 .7 5 7 2 4 .0 8 1 6 .5 5 5 2 3 .8 6 3 2 5 .5 2 2 2 5 .8 9 3 2 3 .0 7 9 2 8 .1 8 2 2 9 .4 2 5 .0 8 8 2 6 .0 1 9 2 7 .1 7 4 1 7 .3 6 7 24148 2 6 .6 2 8 1 6 .6 1 1 2 1 .8 8 9 (mg)is o la te d m ic ro s o m e s 2 1 .8 5 2 2 .6 8 1 8 .0 2 1 4 .1 3 1 4 .5 6 1 5 .9 1 1 7 .7 5 1 6 .4 6 2 3 .5 9 1 9 .7 0 1 8 .4 7 3 .3 7 1 9 .4 9 1 2 .1 6 1 7 .1 0 2 8 .7 2 2 1 .7 1 1 4 .3 4 1 4 .7 3 1 9 .5 5 1 8 .9 6 2 1 .8 0 1 8 .8 6 4 .7 2 2 1 .4 1 1 9 .4 9 1 7 .3 8 1 8 .0 7 2 3 .7 2 2510 2 0 .3 2 2 2 .1 8 1 7 .1 0 2 t3 ? 3 .1 5 4 1 .4 8 4 5 .0 9 2818 2 8 .4 2 3 5 .8 6 1 7 .2 7 3 8 .5 0 4 3 .8 9 3 6 .8 8 3 5 936.11 8 .4 4 1 6 .3 5 1 8 .5 2 2614 2 4 .8 3 2 6 .0 6 2 3 .5 0 1 9 .5 0 1 9 .6 0 2 3 .1 5 2 3 .5 7 2 2 .1 4 3 .4 2 5 4 .7 9 5 1 .0 5 4 6 .5 8 4 4 .9 3 4 4 .6 4 3 6 .7 2 4 3 .8 2 4 4 .6 3 4518 43451553 4 .7 4 5 5 .6 1 5 3 .5 1 4 6 .5 5 5 0 .9 9 4 8 .1 1 3 3 .3 5 3918 4 6 .0 1 3419 4413 4 5 .1 9 7 .5 6 used ver m g m ic ro s o m e s fo r is o la tio n {g ) p e r g Rver 1 .9 2 1 .7 6 1 .4 5 1 .1 9 1 .4 9 148 1 .4 2 1 .8 1 .5 2 1 .3 1 2 .5 2 1 .0 18 213 1 .4 8 1 .0 5 1 .1 6 1 .6 7 1 .3 4 1 .7 9 1 .7 B 1 .9 8 117 1 .5 119 1 .7 8 167 115 2 .1 8 1 .1 4 3 .1 313 313 3 .5 3 3 .8 9 1 .7 2 311 3 .4 3 3 .6 4 3 .1 9 1 .7 1 151 212 1 .4 7 2 .1 4 2 .1 7 1 .3 2 1 .6 5 162 186 3 .4 9 416 4 .1 4 .1 7 4 .1 3 3 .1 3 2 .7 8 317 4 .1 7 3 .8 5 315 3 .8 6 3 .2 7 3 .9 2 3 .7 3 .4 8 317 3 .3 6 2 .9 2 3 .4 8 1 1 .3 6 1Z89 1 2 .4 3 1 1 .8 7 9 .7 7 1 0 .7 5 1 2 .5 0 1 0 .2 9 1 5 .5 2 1 5 .0 4 1214 189 7 .7 4 1169 9 .5 0 1 2 .8 8 1 4 .6 7 1 3 .6 6 1 2 .7 0 1 1 .7 1 1 4 .1 5 112..1t8 2 .1 2 1 4 .6 6 1 0 .8 1 9 .9 0 1119 1 3 .0 0 1 3 .3 2 1 5 .0 9 1 5 .0 5 1 0 .1 7 1 5 .0 0 1 2 .8 6 2 .1 0 1 3 .3 8 1 3 .5 4 8 .5 2 8 .0 5 9 .2 2 1 0 .0 4 1199 1 2 .8 0 1 0 .1 3 1112 1010 2 .0 0 9 .5 6 1217 1115 1619 1 2 .1 8 1013 1 4 .7 7 1 1 .8 8 1419 1 2 .6 7 1 2 .6 7 2 .1 3 1 5 .7 0 1171 1 1 .3 6 1 0 .7 8 1011 1173 1519 1 2 .5 0 1 0 .8 6 1 1 .4 9 1216 168 1 4 .4 4 1 3 .8 6 1414 1 3 .0 1 1 3 .0 0 9 .5 8 1 2 .0 1 1 3 .6 9 1 1 .7 4 1 2 .7 1 1 2 .8 3 145 AVERAGE SD g ro u p 1 -7 1216 117 Page A137 of A172 /? / Appendix to TNO-report of 3M-02____________ Final version p. 138 04-12-2006 Cholesterol-7-a-hydroxylase activity measurements in microsomes used per assay 420 pg protein 20 min incubation internal standard 20 OH: 0.5 pg per determination Mouse 1 13 21 31 45 51 61 2 14 22 32 46 52 62 3 17 23 33 energy generating mix added + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix area 20 OH 7.489 7.764 8.344 7.459 7.921 8.048 7.859 8.072 8.614 8.284 8.279 7.708 7.956 8.246 8.569 8.034 8.103 8.551 8.177 7.462 8.615 8.176 7.997 8.678 7.153 8.014 7.760 7.119 8.211 8.341 7.684 7.654 8.062 8.155 8.529 8.412 8.674 8.281 9.215 7.851 8.260 8.138 9.023 9.148 8.783 7.856 7.946 7.525 7.842 7.885 8.094 7.994 7.866 7.808 area 7aOH 1.417 1.459 0.055 2.470 2.542 0.046 2.287 2.372 0.036 0.709 0.725 0.012 2.363 2.406 0.054 0.580 0.602 0.017 0.525 0.452 n.d. 2.048 2.039 0.036 0.912 0.988 0.018 1.032 1.302 0.026 0.389 0.369 n.d. 1.034 0.991 0.027 0.344 0.332 n.d. 0.414 0.408 n.d. 2.248 2.242 n.d. 2.554 2.519 0.055 2.020 2.034 0.031 0.878 0.863 n.d. pg 7aOH formed 0.095 0.094 0.003 0.166 0.160 0.003 0.146 0.147 0.002 0.043 0.044 0.001 0.149 0.146 0.003 0.036 0.037 0.001 0.032 0.030 0.000 0.125 0.127 0.002 0.064 0.062 0.001 0.072 0.079 0.002 0.025 0.024 0.000 0.063 0.058 0.002 0.020 0.020 0.000 0.026 0.025 0.000 0.125 0.123 0.000 0.163 0.159 0.004 0.129 0.129 0.002 0.055 0.055 0.000 7 alpha hydroxylase activity (nmol/h/mg protein) 1.681 2.959 2.662 0.785 2.661 0.658 0.576 2.296 1.137 1.373 0.456 1.093 0.368 0.472 2.282 2.898 2.346 1.014 Page A138ofA172 WX Appendix to TNO-report of 3M-02____________ Final version p. 139 04-12-2006 Mouse 41 53 63 4 11 24 34 42 54 64 6 12 25 35 43 55 65 5 15 26 energy generating mix added + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix -N A D P H mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix +NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix area 20 OH 8.173 7.392 7.547 7.601 8.694 8.113 8.815 8.171 8.490 8.332 8.091 8.957 8.472 7.915 8.828 8.352 8.133 8.018 7.148 8.432 6.831 8.519 8.106 8.352 8.875 7.996 8.907 8.561 9.027 9.222 8.466 8.414 8.950 7.978 8.371 9.549 8.359 7.939 7.754 7.600 8.216 7.859 8.187 7.860 7.683 9.259 8.480 8.305 7.459 8.254 8.066 8.428 8.346 7.805 8.127 8.333 8.348 7.974 7.537 8.761 area 7aOH 3.471 2.911 0.024 0.537 0.542 n.d. 0.556 0.583 n.d. 2.127 2.288 n.d. 1.862 1.780 0.029 1.753 1.984 n.d. 0.368 0.396 n.d. 2.052 1.990 n.d. 0.496 0.455 n.d. 0.678 0.653 n.d. 1.697 1.674 0.029 6.870 6.821 0.075 1.183 1.091 0.011 0.546 0.559 n.d. 2.021 1.895 0.020 0.619 0.595 0.024 1.066 1.116 n.d. 2.606 2.667 0.010 3.369 3.371 n.d. 2.884 2.681 0.043 pg 7aOH formed 0.212 0.197 0.002 0.035 0.031 0.000 0.032 0.036 0.000 0.128 0.141 0.000 0.110 0.112 0.002 0.105 0.122 0.000 0.026 0.023 0.000 0.120 0.123 0.000 0.028 0.028 0.000 0.040 0.036 0.000 0.100 0.099 0.002 0.431 0.407 0.004 0.071 0.069 0.001 0.036 0.034 0.000 0.123 0.121 0.001 0.033 0.035 0 .0 0 0 0.071 0.068 0 .0 0 0 0.155 0.160 0.001 0.207 0.202 0 .0 0 0 0.181 0.178 0.002 7 alpha hydroxylase activity (nmol/h/mg protein) 3.751 0.614 0.621 2.485 2.023 2.096 0.455 2.246 0.521 0.700 1.815 7.668 1.275 0.646 2.229 0.633 1.284 2.892 3.783 3.268 Page A139ofA172 Appendix to TNO-report of 3M-02____________ Final version p. 140 04-12-2006 Mouse 36 44 56 66 7 16 27 37 47 57 67 8 18 28 38 48 58 68 9 19 29 energy generating mix added + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix area 20 OH 7.543 7.412 8.080 8.523 8.450 7.586 8.189 9.455 7.871 9.543 8.293 8.583 6.707 8.792 8.695 7.664 8.129 9.510 9.979 9.559 9.372 8.482 9.335 7.948 8.375 8.607 9.266 7.959 10.692 8.987 8.115 9.262 8.761 4.538 4.178 4.434 3.873 4.184 4.643 8.343 9.026 9.171 3.989 3.669 4.843 4.001 4.331 5.012 3.910 4.155 5.027 4.100 4.005 4.765 4.192 4.391 4.288 4.084 4.075 4.527 4.149 3.944 4.428 area 7aOH 4.629 4.514 0.158 4.801 4.542 0.494 0.921 0.949 0.014 1.148 0.974 0.026 3.465 4.504 0.064 6.853 7.362 n.d. 2.836 2.618 n.d. 0.848 0.932 n.d. 4.765 4.853 0.083 0.895 1.034 0.006 0.890 0.924 0.028 2.079 2.064 0.049 1.823 1.740 0.032 4.317 4.463 0.031 0.463 0.409 n.d. 1.789 1.905 0.014 0.450 0.453 n.d. 0.821 0.866 0.037 2.296 2.352 0.023 2.755 2.820 n.d. 1.052 1.025 0.036 |jg 7aOH formed 7 alpha hydroxylase activity (nmol/h/mg protein) 0.307 0.305 0.010 5.466 0.282 0.269 0.033 4.482 0.056 0.050 0.001 0.967 0.060 0.059 0.000 1.098 0.258 0.256 0.004 4.684 0.447 0.453 8.312 0.000 0.142 0.137 2.577 0.000 0.050 0.050 0.000 0.923 0.284 0.282 0.004 5.149 0.056 0.048 0.000 0.960 0.055 0.050 0.000 0.967 0.229 0.247 0.006 4.295 0.235 0.208 0.003 4.031 0.259 0.247 0.002 4.642 0.058 0.056 1.051 0 .0 0 0 0.224 0.220 0.001 4.071 0.058 0.055 1.035 0 .0 0 0 0.100 0.108 0.004 1.852 0.274 0.268 0.003 4.954 0.337 0.346 6.311 0 .0 0 0 0.127 0.130 0.004 2.296 Page A140 of A172 Appendix to TNO-report of 3M-02____________ Final version p. 141 04-12-2006 Mouse 39 49 59 69 10 20 40 60 30 50 70 energy generating mix added + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix -N A D P H mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix + NADPH mix + NADPH mix - NADPH mix area 20 OH 4.145 4.132 3.371 4.812 3.305 4.276 3.994 3.969 4.551 3.328 3.792 10.237 9.887 11.010 8.101 9.894 12.152 11.089 10.748 10.280 11.854 9.866 10.231 11.422 10.852 10.771 11.044 10.087 10.560 10.687 10.035 10.174 10.971 n.d.: not detectable area 7aOH 0.417 0.483 n.d. 1.356 0.979 n.d. 0.219 0.210 n.d. 0.710 0.735 n.d. 3.986 4.342 0.026 3.083 3.063 0.073 0.839 0.883 n.d. 0.951 0.893 0.040 2.471 2.346 n.d. 4.003 3.663 n.d. 1.536 1.606 n.d. pg 7aOH formed 0.050 0.058 0.000 0.141 0.148 0.000 0.027 0.026 0.000 0.107 0.097 0.000 0.202 0.197 0.002 0.156 0.126 0.003 0.039 0.043 0.000 0.048 0.044 0.002 0.114 0.109 0.000 0.198 0.173 0.000 0.077 0.079 0.000 7 alpha hydroxylase activity (nmol/h/mg protein) 1.004 2.669 0.498 1.880 3.654 2.542 0.757 0.816 2.057 3.435 1.436 jif6 Page A141 of A172 Appendix to TNO-report of 3M-02____________ Final version P- 142 04-12-2006 Group mouse# 10 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 5. S* S' * 5- . AVi_KK5f SD 41 42 43 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 -m - AVERAGE > SD 61 62 63 64 65 67 68 70 liver weight (9) 2.19 2.00 1.66 1.41 1.68 1.70 1.61 1.77 1.68 1.49 2.77 1.17 1.88 2.53 1.23 1.35 1.84 1.53 1.98 2.00 2.23 2.27 1.68 1.56 2.06 1.85 1.51 2.42 2.14 3.46 3.68 3.69 3.93 4.23 1.94 3.62 3.90 4.03 3.39 1.90 1.71 2.48 1.69 2.25 2,46 1.47 2.14 3.90 4.87 4.10 4.13 3.48 3.13 4,74 4.33 4.29 4.33 4.28 4.15 3.85 3.91 3.83 3.33 3.92 average mg microsomes/g liver 1226 12.26 12.26 12.26 1226 12.26 12.26 1226 1226 1226 12.26 1226 1226 1226 12.26 12.26 1226 12.26 12.26 12.26 12.26 1226 1226 1226 1226 1226 1226 1226 1226 12.26 12.26 1226 1226 1226 1226 1226 1226 12.26 12.26 12.26 1226 1226 12.26 12.26 12.26 12.26 12.26 12.26 1226 1226 1226 12.26 1226 12.26 12.26 1226 12.26 12.26 12.26 1226 cholesterol 7 alpha hydroxylase activity (nmol/h/mg proteg) per liver (nmol/h) 1.681 45.14 2.296 56.32 2.485 2.892 1.815 4.684 4.295 4.954 3.654 3.104 1.206 2.023 7.668 2.959 1.137 3.783 8.312 2.898 4.031 6.311 4.166 2.442 2.662 1.373 2.346 2.096 1275 3.268 2.577 4.642 2.296 2.459 0.965 0,785 1.014 0.455 0.923 1.051 1.004 0.757 1.256 1.495 3.751 2.229 4.482 2.661 4.071 3.435 1221 0.658 0.521 0.967 0.960 0.498 0.816 0.226 0.576 0.472 0.621 0.700 1284 0.967 1.852 1.880 1.436 1.089 0.516 42.97 59.59 37.83 92.49 9324 102.07 66.76 64,29 23.55 66.74 110.02 68.22 35.27 77.94 125.39 47.98 90.96 116.43 61.74 80.47 30.16 37.55 65.30 43.19 24.40 82.56 85.97 68.15 54.00 58.49 19.37 33.32 20.60 21.91 33.51 130.06 40.97 5026 49.65 31.48 45.77 87.40 47.11 67.81 92.91 73.43 32.96 92.83 93.86 59.59 90.15 73.80 21.60 22.00 30.88 26.64 32.05 36.84 52.17 28.93 43.33 34.56 30.32 25.05 36.74 65.37 51.84 46.38 86.98 76.80 69.03 21.79 Page A142ofA172 Appendix to TNO-report of 3M-02 Final version 04-12-2006 HMG CoA reductase activity measurements internal standard mix 1 t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i 1 1 1 1 1 1 t t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 t 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 mouse# 1 13 21 31 45 51 61 2 14 22 32 46 52 3 17 23 33 53 4 11 24 34 42 54 64 6 12 25 35 blanc blanc 3Hdpm (internal standard) 14802 15138 14416 16500 16182 18200 16197 16086 15302 17401 19233 18151 18408 16484 14705 17314 no sample 19044 17259 17962 16815 18678 17627 16776 18636 13567 12055 12101 13870 12322 13846 15599 13354 14885 17744 17935 15926 15147 17598 18276 17701 13443 16084 13092 12500 12547 12475 15313 15836 11436 13617 15941 12877 13025 17786 15984 16769 15482 14676 12984 14Cdpm 222 207 199 294 310 278 437 321 187 224 296 360 192 169 132 159 172 124 137 205 265 140 158 226 146 118 92 143 138 116 144 197 241 231 266 190 172 221 316 187 156 260 209 127 116 114 145 119 86 104 110 196 254 405 279 378 214 162 118 14C dpm 14C mevaionate HMG CoA activity -bianc formed (dpm) pmol/min/mg protein 82 169 67 135 5.111 4.083 59 125 154 284 3.776 8.611 170 320 138 231 9.692 6.995 297 558 16.917 181 342 10.381 47 93 84 147 2.834 4.454 156 247 220 369 7.483 11.182 52 86 29 54 2.606 1.623 -8 -17 19 33 -0.502 1.012 32 51 -16 -28 -3 -5 65 118 125 204 00 18 33 86 140 6 13 -22 -56 -48 -121 37 -2 -5 -24 -53 48 57 130 101 207 91 156 126 214 50 96 32 64 81 140 176 293 47 81 16 36 120 227 69 160 -13 -32 -24 -58 -26 -63 5 10 -21 -40 -54 -144 -36 -80 -30 -57 56 132 114 266 265 453 139 247 238 403 74 136 1.550 -0.855 -0.154 3.566 6.174 0.000 0.990 4.257 0.408 -1.684 -3.659 0.200 -0.150 -1.599 0.237 3.938 6.260 4.731 6.481 2.896 1.949 4.246 8.885 2.450 1.098 6.883 4.862 -0.959 -1.765 -1.923 0.301 -1.223 -4.356 -2.439 -1.736 4.012 8.075 13.746 7.482 12.211 4.112 mean 4.60 6.19 8.34 13.65 3.64 9.33 2.11 0.26 1.55 0.00 4.87 0.49 2.33 0.00 0.02 0.00 5.10 5.61 2.42 6.57 1.77 5.87 0.00 0.00 0.00 0.00 6.04 10.61 8.16 Input internal standard (dpm): average ^ mix 1 30176 29765 31362 30434 | mix 2 26810 28307 28031 28383 ,4C dpm HMG CoA mix: I average 58465 57242 55833 57180 11436 HMG CoA mix: 2.6 Specific activity 14C HMGCoA; 4398.46 incubation time: 30.0 liver microsomes: 0.250 mmol/l HMG CoA dpm/nmot min mg per 5 pi mix per pi mix Page A143ofA172 Appendix to TNO-report of 3M-02____________ Final version 59575 p. 144 04-12-2006 Input internal standard (dpm): average 27932 27694 28968 28198 C HMGCoAmix: average 58407 55714 65147 59756 11951.2 per 5 pi mix per pi mix HMGCoAmix: 2.6 Specific activity 14C HMGCoA: 4596.62 incubation time: 30.0 liver microsomes: 0.250 mmol/l HMG CoA dpm/nmol min mg mouse# 67 8 9 19 29 39 49 59 50 70 blanc Wane 3Hdpm (internal standard) 16112 16092 14295 17696 17286 16982 14651 19083 16755 16504 17225 17750 18651 15076 15831 16644 16807 14453 16679 15823 16121 16049 Input internal standard (dpm): average 27956 27723 28126 27935 14C dpm 284 180 129 164 379 317 145 113 142 115 247 218 209 152 159 154 195 321 191 287 95 72 14C dpm 14C mevalonate -blanc formed (dpm) 201 348 97 168 46 89 81 127 296 478 234 384 62 117 30 43 59 98 32 53 164 265 135 212 126 188 69 127 76 133 71 118 112 185 238 459 108 180 204 359 HMG CoA act. pmol/min.mg eiwit 9.646 4.648 2.467 3.526 13.251 10.658 3.254 1.198 2.706 1.479 7.358 5.874 5.216 3.522 3.697 3.283 5.142 12.738 4.996 9.969 mean 7.15 3.00 11.95 2.23 2.09 6.62 4.37 3.49 8.94 7.48 ,4C HMG CoA mix: average 63731 60065 63603 62466 12493.27 per 5 pi mix per pi mix HMG CoA mix: Specific activity 14C HMGCoA: incubation time: liver microsomes: 2.6 4805.1 30.0 0.250 mmol/l HMG CoA dpm/nmol min mg Page A144 of A172 Appendix to TNO-report of 3M-02____________ Final version p. 145 04-12-2006 mouse# 18 28 38 48 58 68 69 10 20 40 30 62 41 63 56 47 Wane 3Hdpm (internal standard) 18621 18163 16885 17274 15495 18485 15821 17041 19175 17310 16607 14530 16586 14581 15189 15445 16021 16747 15999 14205 20405 19844 16228 14978 15343 13316 15166 16339 17431 14614 15456 15823 16766 Input internal standard (dpm): average 32959 31221 31297 31826 14C dpm 513 361 265 260 682 691 341 312 412 318 241 348 352 360 275 241 224 235 404 355 591 403 328 218 233 317 214 302 407 514 650 287 164 14Cdpm 14C mevalonate HMG CoA act. -blanc formed (dpm) pmol/min.mg eiwit 349 596 17.513 197 345 10.135 101 190 96 177 5.589 5.193 518 1064 31.238 527 907 26.640 177 356 10.454 148 276 8.115 248 412 12.085 154 283 8.313 77 148 4.333 184 403 11.833 188 361 10.592 196 428 12.561 111 233 6.829 77 159 4.658 60 119 3.499 71 135 3.962 240 477 191 428 14.017 12.564 427 6 6 6 239 383 19.554 11.254 164 322 54 115 9.443 3.369 69 143 4.202 153 366 10.736 50 105 3.081 138 269 7.892 243 444 13.026 350 762 22.379 486 1001 123 247 29.382 7.264 mean 13.82 5.39 28.94 9.28 10.20 8.08 11.58 5.74 3.73 13.29 15.40 6.41 7.47 5.49 17.70 18.32 MC HMG CoA mix: average 61119 60406 55583 59036 11807.2 per5 pi mix per pi mix HMG CoA mix: 2.6 mmol/l HMG CoA Specific activity 14C HMGCoA: 4541.23 dpm/nmol incubationtime: 30.0 min liver microsomes: 0.250 mg 49 Page A145 of A172 Appendix to TNO-report of 3M-02____________ Final version Group mouse# 1 2 3 4 5 6 7 8 9 10 v h . -P-'- 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 4 4 4 4 -1 4 4 4 4 AVERAGE / so ; 5 5 5 5 & S 5- 5` : 5 ' AVERAGE SD r; 0 ti b AVERAGE 5D r 7 t 7 7 7 r., .7 7 7 r7 Ia v f r a iv e I so 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 liver weight average mg (9) mkrosomes/g Hver 2.19 12.26 2.00 12.26 1.66 1226 1.41 1226 1.68 12.26 1.70 12.26 1.61 12.26 1.77 12.26 1.68 12.26 1.49 12.26 2.77 12.26 1.17 1226 1.88 12.26 2.53 1226 1.68 1226 1.23 1226 1.35 1226 1.84 1226 1.53 1226 1.98 12.26 2.00 1226 2.23 1226 2.27 1226 1.68 12.26 1.56 1226 2.06 1226 1.85 1226 1.51 1226 2.42 1226 2.14 12.26 3.46 1226 3.68 1226 3.69 1226 3.93 12.26 4.23 1226 1.94 1226 3.62 1226 3.90 1226 4.03 1226 3.39 12.26 1.90 12.26 1.71 12.26 2.48 1226 1.69 12.26 2.25 1226 2.46 1226 1.47 1226 1.88 1226 1.82 1226 2.14 12.26 3.90 1226 4.87 1226 4.10 1226 4.17 1226 4.13 1226 3.48 1226 3.13 1226 4.11 1226 4.74 1226 4.33 12.26 4.29 4.33 3.88 4.28 4.15 3.85 3.91 3.83 I 3.33 3.92 1226 12.26 1226 12.26 1226 1226 1226 1226 1226 12.26 HMG CoA reductase activity I (pmot/min/ing protein) per Sver (pmol/mtn) 4.60 123.43 026 6.26 0.00 0.00 2.42 41.88 0.00 0.00 0.00 0.00 4.84 95.49 3.00 65.03 11.95 5.74 246.23 104.92 3.28 68.32 3.77 78.41 6.57 222.96 6.04 86.69 6.19 142.75 1.55 48.08 0.00 0.00 0.00 0.00 0.02 0.41 13.82 311.85 223 41.76 3.73 90.56 4.02 94.51 4.33 104.24 8.34 204.59 0.00 0.00 0.00 0.00 1.77 36.54 10.61 203.00 0.00 0.00 0.00 0.00 5.39 99.81 2.09 62.10 15.40 404.15 4.36 101.02 5.44 133.13 13.65 4.87 578.98 219.73 5.10 230.67 5.87 282.96 8.16 423.27 0.00 0.00 1.13 50.24 28.94 1383.68 6.62 1329 326.87 552.38 8.76 404.88 8.36 392.39 7.47 173.99 0.00 0.00 2.77 1022 3.64 84.30 211.77 100.51 0.49 18.32 14.93 33022 928 214.00 4.37 97.48 8.94 234.56 6.55 5.51 9.33 2.33 5.61 0.00 3.51 17.70 6.15 10.20 3.49 6.69 146.18 104.48 446 23 139.28 281.81 0.00 177.86 755.28 236.05 513.93 202.82 355.05 6.50 310.83 5.01 216.26 2.11 111.22 6.41 340.07 5.49 260.98 0.00 0.00 6.52 331.58 0.00 0.00 7.15 342.61 8.08 379.53 11.58 472.60 7.48 359.61 5.48 259.82 3.71 165.03 0 p 146 04-12-2006 Page A146 of A172 Appendix to TNO-report of 3M-02____________ Final version micfomes; mg per g liver. 7-alpha-hydroxylase activity and HMG CoA reductase aclivilY. p. 147 04-12-2006 Page A147 of A172 Appendix to TNO-report of 3M-02____________ Final version H2. Cholesteroi-27-hydroxylase activity in mitochondria Protein concentration isolated mitochondria Mitochondria mouse 1 13 21 31 45 51 61 2 14 22 32 46 52 62 3 17 23 33 41 53 63 4 11 24 34 42 54 64 6 12 25 35 43 55 65 5 15 26 36 44 56 66 7 16 27 37 47 57 67 8 18 28 38 48 58 68 9 19 29 39 49 59 69 10 20 40 60 30 50 70 measured prat cone (mg/ml) sveli 1 well 2 0.772 0.8 0.699 0.699 0.848 1.508 0.908 1.499 0.925 0.973 0.784 0.865 0.813 0.881 1.143 1.162 0.918 0.931 0.945 1.022 1.192 1.104 1.043 1.062 1.26 1.236 1.201 1.204 0.81 0.81 0.57 0.54 0.915 0.949 1.036 0.699 1.092 0.655 1.033 0.942 0.898 0.576 0.839 0.587 0.726 0.672 0.969 0.959 1.532 1.43 0.784 0.852 0.98 0.997 1.201 1.28 0.699 0.658 0.383 0.359 0.931 1.37 0.861 1.235 0.842 0.845 0.945 0.976 0.949 0.901 0,649 0.637 0.648 0.635 0.775 0.799 0.678 0.757 0.705 0.757 0.84 1.031 0.754 0.467 0.843 0.998 0.714 0.443 0.876 0.886 1.171 0.554 1.1 0.554 0.847 0.809 0.578 0.596 0.616 0.596 0.697 0.7 0.728 0.745 1.808 1.903 1.086 0.85 1.261 1.147 0.734 0.766 0.616 0.611 0.691 0.659 0.847 0.847 1.255 1.251 0.76 0.751 0.85 0.796 0.751 0.728 0.837 0.821 0.84 0.809 1.344 1.34 0.769 0.827 0.642 0.598 0.987 0.965 1.039 0.926 dilution Protein cone (ms/ml) 50 39.30 50 34.95 50 43.90 50 75.18 50 47.45 100 79.85 100 87.30 50 57.63 50 46.23 50 49.18 100 114.80 50 52.63 100 124.80 100 120.25 50 40.50 50 27.75 50 46.60 100 106.40 50 33.85 100 98.75 100 86.85 50 29.08 50 34.95 50 48.20 100 148.10 50 40.90 100 98.85 100 124.05 50 33.93 50 18.55 50 44.80 100 130.25 50 42.18 100 96.05 100 92.50 50 32.15 50 32.08 50 39.35 50 34.58 50 37.85 100 84.15 100 101.45 50 36.70 50 22.75 50 44.05 100 113.55 50 27.70 100 82.80 100 58.70 50 30.30 50 34.93 50 36.83 100 185.55 50 48.40 100 120.40 100 75.00 50 30.68 50 33.75 50 42.35 100 125.30 50 37.78 100 82.30 100 73.95 50 41.45 50 41.23 100 134.20 100 79.80 50 31.00 50 48.80 100 98.25 p. 148 04-12-2006 Page A148ofA172 Appendix to TNO-report of 3M-02____________ Final version A m ount isolated m itochondria g ro u p # m ouse# P ro te in c o n o estimated pt m ito c h o n d ria (m g frn l) 600 600 600 600 isolated mitochondria used liver mg mitochondria for isolation (g) per g ver 3 4 .5 8 2 4 .3 0 11 0 .9 7 12 13 14 15 16 17 18 19 20 21 1.78 22 23 24 25 26 27 28 29 3 0 18.3 2 15 61 p 149 04-12-2006 3 5-AVERAGE ' 20 .3 1 "W 15.74 14.2 3 ~rrr so 1 2 0 .2 5 6 8 .7 6 6 4 .2 8 1 8 .2 0 AVERAGE SO 1 AVERAGE L so AVERAGE SO g ro u p 1 -3 .5 1428 2 .5 5 g ro u p 4 2 4 .4 5 7 .0 3 g ro u p s ,? 17.9 7 2 .9 5 ISI Page A149of A172 Appendix to TNQ-report of 3M-02____________ Final version p. 150 04-12-2006 Cholesterol-27-hydroxylase activity measurements used per assay 420 pg protein 20 min incubation internal standard 20 OH: 0.5 pg per determination Mouse energy generating area 20 OH area 2 7 0 H mix added 1 + NADPH mix 10.798 13.936 + NADPH mix -N A D P H mix 10.39 7.98 13.174 0.136 13 + NADPH mix 11.806 20.962 + NADPH mix 12.439 19.566 - NADPH mix 8.742 0.125 21 + NADPH mix 11.5 19.885 + NADPH mix 11.782 16.14 - NADPH mix 8.867 0.126 31 + NADPH mix 9.271 8.456 + NADPH mix 10.042 9.881 - NADPH mix 8.477 0.038 45 + NADPH mix 12.431 18.514 + NADPH mix - NADPH mix 12.184 8.816 17.151 0.07 51 + NADPH mix 10.388 10.217 + NADPH mix - NADPH mix 10.595 8.259 11.997 0.113 61 + NADPH mix 9.893 11.615 + NADPH mix 10.305 11.839 - NADPH mix 8.75 0.068 2 + NADPH mix 9.624 11.398 + NADPH mix - NADPH mix 10.36 8.95 13.32 n.a. 14 + NADPH mix + NADPH mix - NADPH mix 11.022 11.6 9.308 14.253 14.61 0.023 22 + NADPH mix 10.919 15.555 + NADPH mix - NADPH mix 10.512 9.52 16.346 0.074 32 + NADPH mix 8.987 8.043 + NADPH mix - NADPH mix 9.298 8.538 8.372 0.023 46 + NADPH mix 10.614 15.727 + NADPH mix - NADPH mix 10.223 9.715 15.178 0.048 52 + NADPH mix 9.353 + NADPH mix 10.539 - NADPH mix 8.851 9.827 9.649 0.071 62 + NADPH mix 10.024 + NADPH mix 9.352 - NADPH mix 8.92 6.711 6.677 n.a. 3 + NADPH mix 12.459 19.765 + NADPH mix 11.384 15.393 - NADPH mix 9.006 n.a. 17 + NADPH mix 10.383 12.005 + NADPH mix - NADPH mix 14.276 9.026 18.144 n.a. 23 + NADPH mix 9.646 14.544 + NADPH mix - NADPH mix 10.293 7.233 14.524 n.a. 33 + NADPH mix + NADPH mix - NADPH mix 8.216 8.156 7.185 7.206 6.744 0.249 pg 27 OH formed 0.645 0.634 0.009 0.888 0.786 0.007 0.865 0.685 0.007 0.456 0.492 0.002 0.745 0.704 0.004 0.492 0.566 0.007 0.587 0.574 0.004 0.592 0.643 0.000 0.647 0.630 0.001 0.712 0.777 0.004 0.447 0.450 0.001 0.741 0.742 0.002 0.525 0.458 0.004 0.335 0.357 0.000 0.793 0.676 0.000 0.578 0.635 0.000 0.754 0.706 0.000 0.439 0.413 0.017 27- hydroxylase activity (nmot/h/mg protein) 11.659 15.332 14.181 8.715 13.306 9.645 10.656 11.407 11.766 13.689 8.267 13.654 9.007 6.389 13.571 11.209 13.480 7.549 Page A150ofA172 Appendix to TNO-report of 3M-02____________ Final version p. 151 04-12-2006 Mouse energy generating area 20 OH area 2 7 0 H mix added 41 + NADPH mix 10.176 + NADPH mix 9.541 4.943 4.889 - NADPH mix 7.88 n.a. 53 + NADPH mix 7.856 4.981 + NADPH mix - NADPH mix 7.979 7.279 4.873 n.a. 63 + NADPH mix 10.998 + NADPH mix 8.797 - NADPH mix 7.705 7.897 6.181 0.086 4 + NADPH mix 9.576 11.39 + NADPH mix 10.192 12.068 - NADPH mix 7.719 n.a. 11 + NADPH mix 11.137 + NADPH mix 9.797 8.145 7.601 - NADPH mix 8.556 0.038 24 + NADPH mix 10.407 11.002 + NADPH mix - NADPH mix 9.642 6.778 11.512 n.a. 34 + NADPH mix 6.577 3.674 + NADPH mix 9.528 5.095 - NADPH mix 7.252 n.a. 42 + NADPH mix 9.538 11.731 + NADPH mix 9.074 10.387 - NADPH mix 7.471 0.027 54 + NADPH mix 8.887 6.229 + NADPH mix 8.267 5.477 - NADPH mix 7.436 n.a. 64 + NADPH mix 8.585 6.457 + NADPH mix - NADPH mix 8.945 6.454 6.783 n.a. 6 + NADPH mix 11.823 13.325 + NADPH mix 8.846 9.816 - NADPH mix 8.775 0.057 12 + NADPH mix 9.224 1.784 + NADPH mix 9.098 1.721 - NADPH mix 9.234 n.a. 25 + NADPH mix 10.72 7.132 + NADPH mix 9.542 7.327 - NADPH mix 8.864 0.031 35 + NADPH mix + NADPH mix - NADPH mix 9.856 10.84 8.053 5.695 4.8 n.a. 43 + NADPH mix 10.178 12.222 + NADPH mix - NADPH mix 10.114 7.765 10.741 n.a. 55 + NADPH mix 8.696 5.552 + NADPH mix - NADPH mix 8.713 8.251 5.349 0.032 65 +NADPH mix + NADPH mix 9.579 9.473 6.842 7.217 - NADPH mix 8.811 0.056 5 + NADPH mix 9.569 + NADPH mix 9.56 - NADPH mix 8.442 7.618 7.922 0.004 15 + NADPH mix + NADPH mix - NADPH mix 10.061 10.559 9.105 8.488 7.934 n.a. 26 + NADPH mix + NADPH mix - NADPH mix 9.863 9.469 8.926 7.929 8.269 n.a. pg 27 OH formed 0.243 0.256 0.000 0.317 0.305 0.000 0.359 0.351 0.006 0.595 0.592 0.000 0.366 0.388 0.002 0.529 0.597 0.000 0.279 0.267 0.000 0.615 0.572 0.002 0.350 0.331 0.000 0.376 0.379 0.000 0.564 0.555 0.003 0.097 0.095 0.000 0.333 0.384 0.000 0.289 0.221 0.000 0.600 0.531 0.000 0.319 0.307 0.002 0.357 0.381 0.003 0.398 0.414 0.000 0.422 0.376 0.000 0.402 0.437 0.000 27- hydroxylase activity (nmol/h/mg protein) 4.610 5.749 6.458 10.962 6.920 10.396 5.049 10.933 6.297 6.976 10.270 1.767 6.619 4.714 10.450 5.748 6.758 7.499 7.366 7.746 Page A151 of A172 /55 Appendix to TNO-report of 3M-02____________ Final version p. 152 04-12-2006 Mouse energy generating area 20 OH area 270H mix added 36 + NADPH mix + NADPH mix - NADPH mix 9.147 9.469 7.56 4.853 4.856 0.022 44 + NADPH mix 9.972 + NADPH mix 11.897 - NADPH mix 8.394 8.19 8.239 n.a. 56 + NADPH mix + NADPH mix - NADPH mix 9.607 9.685 8.852 5.319 4.876 0.035 66 + NADPH mix 9.829 7.898 + NADPH mix - NADPH mix 10.203 9.701 7.893 0.042 7 + NADPH mix 10.39 + NADPH mix 9.963 - NADPH mix 9.004 6.813 7.322 n.a. 16 + NADPH mix + NADPH mix 9.887 9.808 6.158 5.884 - NADPH mix 8.749 n.a. 27 + NADPH mix 10.948 + NADPH mix 11.307 - NADPH mix 9.47 8.509 8.456 0.018 37 + NADPH mix 10.004 6.283 + NADPH mix 9.859 6.029 - NADPH mix 8.538 n.a. 47 + NADPH mix 9.054 6.579 + NADPH mix - NADPH mix 10.166 9.566 8.773 n.a. 57 + NADPH mix 10.482 + NADPH mix 9.237 - NADPH mix 9.439 5.812 5.663 n.a. 67 -t- NADPH mix 9.185 8.637 + NADPH mix - NADPH mix 9.321 8.555 10.313 0.041 8 + NADPH mix 10.123 14.048 + NADPH mix - NADPH mix 10.395 8.227 11.26 n.a. 18 + NADPH mix + NADPH mix - NADPH mix 9.534 9.641 7.802 9.63 8.353 n.a. 28 + NADPH mix 10.422 10.021 + NADPH mix - NADPH mix 10.894 7.384 12.138 n.a. 38 + NADPH mix + NADPH mix - NADPH mix 9.189 8.998 8.56 3.667 3.925 n.a. 48 + NADPH mix 10.497 11.218 + NADPH mix - NADPH mix 10.009 8.178 9.378 0.01 58 + NADPH mix + NADPH mix - NADPH mix 8.418 9.913 8.293 4.05 4.887 n.a. 68 + NADPH mix 9.401 + NADPH mix 10.176 - NADPH mix 8.704 6.674 6.558 n.a. 9 + NADPH mix 8.909 7.488 + NADPH mix 9.904 10.247 - NADPH mix 7.036 n.a. 19 + NADPH mix + NADPH mix - NADPH mix 6.336 8.809 6.461 5.573 7.57 n.a. 29 + NADPH mix + NADPH mix - NADPH mix 6.725 9.786 4.961 5.613 7.53 0.002 pg 27 OH formed 0.265 0.256 0.001 0.411 0.346 0.000 0.277 0.252 0.002 0.402 0.387 0.002 0.328 0.367 0.000 0.311 0.300 0.000 0.389 0.374 0.001 0.314 0.306 0.000 0.363 0.431 0.000 0.277 0.307 0.000 0.470 0.553 0.002 0.694 0.542 0.000 0.505 0.433 0.000 0.481 0.557 0.000 0.200 0.218 0 .0 0 0 0.534 0.468 0.001 0.241 0.246 0 .000 0.355 0.322 0 .0 0 0 0.420 0.517 0 .0 0 0 0.440 0.430 0 .0 0 0 0.417 0.385 0 .000 27- hydroxylase activity (nmol/h/mg protein) 4.792 6.991 4.846 7.244 6.422 5.647 7.026 5.725 7.341 5.392 9.408 11.412 8.666 9.586 3.858 9.251 4.499 6.255 8.660 8.031 7.405 Page A152ofA172 Appendix to TNO-report of 3M-02____________ Final version p. 153 04-12-2006 Mouse energy generating area 20 OH area 2 7 0 H mix added 39 + NADPH mix + NADPH mix - NA DPH mix 8.832 9.279 8.084 4.203 4.385 n.a. 49 + NA DPH mix 9.633 + NA DPH mix 11.234 6.413 7.141 - NA DPH mix 7.927 n.a. 59 + N A DPH mix + NADPH mix 9.799 9.597 4.515 4.761 - NADPH mix 9.087 n.a. 69 + NADPH mix 9.836 6.674 + NADPH mix 9.96 6.877 - NADPH mix 1.235 n.a. 10 + NADPH mix 9.549 8.229 + NADPH mix - NADPH mix 10.004 7.526 9.15 n.a. 20 + NADPH mix 9.543 7.346 + NADPH mix - NA DPH mix 9.019 8.008 7.444 0.031 40 + NADPH mix 7.696 2.057 + NADPH mix 8.096 2.046 - NADPH mix 7.695 0.01 60 + NADPH mix 8.639 3.465 + NA DPH mix 8.551 3.757 - NADPH mix 8.104 0.047 30 + NA DPH mix 8.271 5.462 + NA DPH mix - NADPH mix 8.406 7.768 5.704 n.a. 50 + NADPH mix 9.353 8.537 + NA DPH mix 9.445 7.619 - NADPH mix 7.913 n.a. 70 + NA DPH mix 9.568 4.626 + NA DPH mix - NADPH mix 8.325 7.941 4.716 n.a. pg 27 OH formed 0.238 0.236 0.000 0.333 0.318 0.000 0.230 0.248 0.000 0.339 0.345 0.000 0.431 0.457 0.000 0.385 0.413 0.002 0.134 0.126 0.001 0.201 0.220 0.003 0.330 0.339 0.000 0.456 0.403 0.000 0.242 0.283 0.000 27- hydroxylase activity (nmol/h/mg protein) 4.380 6.010 4.419 6.322 8.204 7.331 2.390 3.828 6.184 7.941 4.849 Page A153ofA172 Appendix to TNO-reoort of 3M-02____________ Final version Group Mouse# mouse# liver weight average mg M mitochondria/g irver 2.19 14.28 2.00 14.28 1.66 14.28 1.41 14.28 1.68 14.28 1.70 14.28 1.61 14.28 1.77 14.28 1428 10 1.49 1428 2.77 12 1.17 13 1.88 14 2.53 15 1.68 16 16 1.23 17 1.35 18 1.84 19 19 20 20 1.98 1428 1428 14.28 14.28 1428 1428 14.28 1428 14.28 14.28 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 30 2.00 2.23 2.27 1.68 1.56 2.06 1.85 1.51 2.42 2.14 1428 1428 14.28 14.28 14.28 14.26 1428 14.28 1428 14.28 32 32 33 33 34 34 35 35 36 36 37 38 39 39 40 40 3.46 3.68 3.69 4.23 3.62 3.90 4.03 3.39 24.45 24.45 24.45 24.45 24.45 24.45 24.45 24.45 5 I ' x : +`5`" ' 5 5* 5 AVERAGE 43 44 45 46 47 48 49 42 43 44 45 46 47 48 49 50 52 52 53 54 54 55 55 56 56 57 57 58 58 59 59 60 190 1.69 2.25 1.47 1.82 3.90 4.87 4.17 4.13 3.13 4.11 4.33 1428 14.28 14.28 14.28 1428 14.28 14.28 14.28 14.28 14.28 17.97 17.97 17.97 17.97 17.97 17.97 17.97 17.97 17.97 17.97 77 :> ;:| *7- i ; AVERAGE 61 62 63 64 65' 66 67 68 70 62 63 64 65 66 67 66 69 70 4.29 4.33 3.88 4.28 4.15 3.85 3.91 3.83 3.33 17.97 17.97 17.97 17.97 17.97 17.97 17.97 17.97 17.97 17.97 cholesterol 27 hydroxylase activity (nmol/h/mg protein) per Hver(nmotfh) 11.66 364.7 325.9 13.57 321.8 10.96 220.8 7.50 1027 6.42 147.7 11,41 288.5 8.66 207.8 8.20 174.6 10.01 222 248.1 73.9 6.92 273.8 1.77 29.5 15.33 411.7 11.77 425.2 7.37 176.8 5.65 1121 8.67 99.2 216.1 227.8 8.03 175.5 7.33 207.3 8.40 224.3 3.70 123.2 14.18 405.1 13.69 13.48 10.40 249.5 6.62 7.75 227.9 7.03 185.6 9.59 206.8 7.40 255.9 6.18 189.0 9.63 274.0 3.14 109.9 8.72 737.3 8.27 743.8 7.55 681.1 5.05 485.2 4.71 4.79 227.3 5.72 3.86 367.9 4.38 431.6 2.39 198.1 5.54 486.7 2.03 192.9 4.61 10.93 10,45 370.2 6.99 168.8 13.31 427.6 13.65 479.8 7.34 154.1 248.4 9.05 3.02 9.01 6.30 5.39 4.50 5.94 193 10.66 6.39 6.46 6.98 6.76 724 9.41 6.25 6.32 4.85 7.13 123.5 676.0 788.2 423.5 426.6 303.3 376.4 297.8 439.9 167.2 8215 497.1 450.3 536.5 504.0 501.1 661.0 430.5 378.3 341.6 512.2 140.0 P- 154 04-12-2006 Page A154ofA172 Appendix to TNO-report of 3M-02____________ Final version mitochondria; mg per 9 liver and 27-hydroxylase activity, p values Mann-WhHnev U-tesI p. 155 04-12-2006 /s f Page A155ofA172 Appendix to TNO-rePort of 3M-02____________ Final version p. 156 04-12-2006 I. Measurements in vitro experiment (rat hepatocytes) Experiment 1 Protein concentration determination sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Measured dilution average protein cone pg protein protein cone (mg/ml) (mg/ml) per well 97.5 4 376.2 125.4 90.6 119.5 4 490.0 163.3 125.5 172.9 4 626.8 208.9 140.5 155.8 4 672.2 224.1 180.3 167.7 4 689.6 229.9 177.1 131.6 4 505.6 168.5 121.2 139.7 4 508.4 169.5 114.5 257.5 4 944.4 314.8 214.7 176 4 702.0 234.0 175 155.8 4 594.8 198.3 141.6 155.8 4 555.8 185.3 122.1 167.7 4 645.0 215.0 154.8 129 4 512.4 170.8 127.2 136.1 4 555.2 185.1 141.5 170.8 4 695.8 231.9 177.1 172.9 4 717.2 239.1 185.7 168.7 4 648.6 216.2 155.6 177.1 4 685.6 228.5 165.7 151.9 4 605.6 201.9 150.9 188.9 4 744.8 248.3 183.5 190.1 4 840.0 280.0 229.9 168.7 4 662.8 220.9 162.7 167.7 4 650.8 216.9 157.7 229.9 4 896.6 298.9 218.4 150.9 4 594.2 198.1 146.2 127.2 4 521.2 173.7 133.4 172.9 4 649.6 216.5 151.9 170.8 4 713.0 237.7 185.7 170.8 4 697.8 232.6 178.1 163.7 4 669.0 223.0 170.8 sample 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 Measured dilution average protetn corte protein cone (mg/ml) (mg/ml) 134.3 4 523.0 127.2 149.9 4 563.0 131.6 202.7 4 796.8 195.7 194.1 4 821.8 216.8 188.7 4 784.8 203.7 173.8 4 670.2 161.3 194.1 4 821.8 216.8 188.7 4 785.0 203.8 173.9 4 670.4 161.3 168.6 4 685.0 173.9 140.8 4 569.2 143.8 139.7 4 569.0 144.8 155.1 4 606.0 147.9 162.4 4 653.6 164.4 164.4 4 649.4 160.3 179.2 4 697.8 169.7 198.4 4 732.0 167.6 209.1 195.1 207 216.8 239.9 255.6 241.1 249.9 182.33 132.7 4 4 4 4 4 808.4 847.6 991.0 982.0 630.1 201.6 4 793.4 195.1 212.4 4 867.2 221.2 210.4 4 806.8 193 211.4 4 909.4 243.3 208.1 4 828.0 205.9 239.9 4 926.6 223.4 213.5 4 849.8 211.4 198.4 4 759.4 181.3 129.7 4 526.8 133.7 156.1 4 674.8 181.3 pg protein per well 174.3 187.7 265.6 273.9 261.6 223.4 273.9 261.7 223.5 228.3 189.7 189.7 202.0 217.9 216.5 232.6 244.0 269.5 282.5 330.3 327.3 210.0 264.5 289.1 268.9 303.1 276.0 308.9 283.3 253.1 175.6 224.9 /LO Page A156of A172 Appendix to TNO-report of 3M-02____________ Final version sample Measured protein cone (mg/ml) 63 171.8 193 64 146.9 140.8 63 166.5 177 64 147.9 132.7 63 130.7 127.7 64 135.7 145.9 69 131.7 139.7 70 77.6 72.1 71 153.1 160.2 72 132.7 126.7 73 96.4 90.2 74 118.4 125.3 75 1622 140.6 76 \ 152.9 177.5 77 165.9 175.4 78 129.7 119.4 79 138.7 113.3 80 281.1 217.1 81 176.4 175.4 82 153.9 138.7 83 154.8 120.1 84 167.9 154.9 85 126.1 125.3 86 133.2 140.6 87 169 176.1 88 174.3 186.3 89 167.9 155.9 90 176.4 164.9 91 150 149 92 188.5 184.1 93 189.6 221.4 94 166.9 161.9 95 166.9 156.9 96 230.1 218.4 97 149 145.2 98 125.3 132.3 99 172.2 TSTj 100 170.1 185.2 101 170.1 176.4 102 163.9 170.1 103 132.3 126.1 104 148.1 130.6 105 203.6 196.5 106 212.1 195.4 107 146.2 213.4 dilution 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 average protein cone (moAnl) 729.6 575.4 687.0 5^12 516.8 563.2 542.8 299.4 626.6 518.8 373.2 487.4 605.6 660.8 682.6 488.2 504.0 956.4 703.6 585.2 549.8 645.6 502.8 547.6 690.2 721.2 647.6 682.6 598.0 745.2 822.0 657.6 647.6 897.0 588.4 515.2 648.2 710.6 693.0 668.0 516.8 557.4 800.2 815.0 719.2 pg protein per well 243.2 191.8 229.0 187.1 172.3 187.7 180.9 99.8 208.9 172.9 124.4 162.5 201.9 220.3 227.5 166.1 168.0 318.8 234.5 195.1 183.3 215.2 167.6 182.5 230.1 240.4 215.9 227.5 199.3 248.4 274.0 219.2 215.9 299.0 196.1 171.7 216.1 236.9 231.0 222.7 172.3 185.8 286.7 271.7 239.7 p. 157 04-12-2006 Page A157ofA172 sample compound concentration 1 control 2 control 3 control 4 control 5 control 6 control 7 control 8 control 9 control 10 control 11 control 12 control 13 PFBS 1 pM 14 PFBS 1 pM * 15 P F B S 10 pM 16 PFBS 10 pM 17 PFBS 100 uM 18 PFBS 100 uM 19 PFHS 1 pM 20 PFHS 1 pM 21 PFHS 10 pM 22 PFHS 10 pM 23 PFHS 100 pM 24 PFHS 100 pM 25 PFOS 1 uM 26 PFOS 1 pM 27 PFOS 10 uM 28 PFOS 10 uM 29 PFOS 1 0 0 pM 30 PFOS 1 0 0 pM 31 Rosuvastatln 0.003 pM 32 Rosuvastatin 0.003 uM 33 Rosuvastatln 0.03 pM 34 Rosuvastatin 0.03 pM 35 Rosuvastatln 0.3 uM 36 Rosuvastatin 0.3 pM 37 PFBS 1 pM u 38 Q) 39 co 40 CD 41 > 42 PFBS 1 gM PFBS 10 uM PFBS 10 pM PFBS 100 pM PFBS 100 pM O l 43 00 44 2 45 , 46 PFHS 1 PM PFHS 1 pM P F H S 1 0 pM P F H S 10 pM > 47 PFHS 100 pM 48 PFHS 100 pM IO 49 50 PFO S1 pM PFOS 1 uM 51 PFOS 10 pM 52 PFOS 10 pM 53 PFOS 100 pM 54 PFOS 100 pM " C Label "*C acetic acid ,4C acetic acid '*C acetic acid i4C butyric acid ,4C butyric acid " C butyric acid U C lauric add 14C lauric add UC lauric add l4C palmitic add ,4C palmitic add '*C palmitic add " C acetic add U C acetic add J4C acetic add 14C acetic add <4C acetic acid l4C acetic add 14C acetic add " C acetic acid ,4C acetic acid ,4C acetic Bdd ,4C acetic acid ,4C acetic add 14C acetic add " C acetic add i4C acetic add 14C acetic acid ,4C acetic acid l4C acetic acid ,4C acetic acid l4C acetic acid ,4C acetic acid ,4C acetic acid 14C acetic acid l4C acetic add ,4C butyric acid 14C butyric add ,4C butyric acid WC butyric acid " C butyric add ,4C butyric add ,4C butyric add ,4C butyric add " C butyric add " C butyric acid " C butyric acid I4C butyric add "*C butyric acid ,4C butyric add MC butyric acid WC butyric acid " C butyric add `"C butyric add ^H'Cholestero (dpm) 12705 12827 11820 14124 12699 15542 12531 11864 11644 14154 12949 14391 15327 14468 14439 18148 12484 13353 15401 14081 14983 13978 14523 16419 13313 13999 13096 14417 12438 12519 13661 14096 12455 12791 14394 13181 13006 13189 13331 12774 13454 16489 12397 12966 13703 13864 15672 15719 12626 13507 15475 13164 15165 15228 " C-choleserol (dpm) 7957 10612 12414 8971 11979 14954 17327 16561 15565 7968 7804 6763 11500 11220 13928 17531 10821 10981 13298 16955 15087 14244 14505 18682 9191 14418 11960 13268 8472 9940 6222 5074 1670 2268 624 465 7667 10370 17500 14299 8093 14468 13939 11630 10974 15549 16480 17411 9783 16521 19586 12135 9834 10776 14C'Chotesterol corrected for recovery (dpm) 16067 21225 26944 16295 24200 24684 35474 35812 34294 14442 15462 12056 19249 19896 24747 24783 22237 21098 22152 30891 25833 26143 25623 29191 17712 26423 23430 23610 17475 20370 11685 9235 3440 4549 1112 905 15124 20172 33678 28718 15432 22511 28846 23012 20546 28773 26978 28417 19876 31380 32470 23650 16636 18155 pg protein 125.4 183.3 208.9 224.1 229.9 168,5 169.5 314.8 234 198.3 185.3 215 170.8 185.1 231.9 239.1 216.2 228.5 201.9 248.3 280 220.9 216.9 298.9 198.1 173.7 216.5 237.7 232.6 223.0 174.3 187.7 265.6 273.9 261.6 223.4 273.9 261.7 223.5 228.3 189.7 189.7 202.0 217.9 216.5 232.6 244.0 269.5 282.5 330.3 327.3 210.0 264.5 269.1 14C-choiesterol per mg protein (dpm/mg) 128130 129974 128982 72713 105265 146495 209286 113761 146556 72832 83441 56077 112700 107486 106714 103650 102856 92331 109717 124411 92261 11B349 118134 97661 89408 152118 108220 99328 75127 91345 67038 49200 12951 16608 4251 4051 55216 77079 150685 125790 81351 118664 142802 105606 94899 123702 110564 105442 70366 95004 99207 112617 62898 62797 A v e ra g e ,4C-choestero per mg protein (dpm/mg) 129029 108158 156534 70783 110093 105182 97594 117064 105305 107898 120763 103774 83236 58119 14780 4151 66147 138237 100007 124204 109301 108003 82685 105912 62847 % o f control Average % o f control 99 100 1Q1 100 67 100 97 135 134 100 73 94 103 100 118 79 87 85 83 83 82 80 80 76 72 85 91 96 72 82 92 92 64 76 69 94 118 84 80 77 58 65 71 52 45 38 10 11 13 33 3 51 81 71 139 128 116 75 92 110 132 115 98 68 101 114 102 100 97 65 76 68 92 98 104 58 58 58 SO 0.7 34.2 31.0 19.5 2.9 1.7 5.8 8.1 14.3 11.2 34.4 4.9 8.9 9.8 2.0 0.1 14.3 16.3 24.4 24.3 18.8 3.3 16.1 8.8 0.1 O o CD &CD 3 W >< 3 r - - =T <D W C/T 21 rr CD o DJ o o v> P> o a CD 3 a. 9 s o o O co 5 6 ho V) o 3 a Ul 00 Page A159 of A172 sample 55 56 57 58 59 60 61 62 63 64 65 66 67 66 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 86 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 compound concentration "C Label Rosuvastalin 0.003 uM Rosuvastatin 0.003 uM Rosuvastalin 0.03 pM Rosuvastatin 0.03 uM Rosuvastatin 0.3 uM Rosuvastatin 0.3 pM PFBS 1 uM PFBS 1 uM PFBS 10 uM PFSS10 uM PFBS 100 uM PFBS 100 uM PFHS 1 uM PFHS 1 uM PFHS 10 mM PFHS 10 uM PFHS 100 uM PFHS 100 uM PFOS 1 uM PFOS 1 uM PFOS 10 uM PFOS10 uM PFOS 100 uM PFOS 100 uM Rosuvastatin 0.003 uM Rosuvastatin 0.003 uM Rosuvastatin 0.03 uM Rosuvastatin 0.03 uM Rosuvastatin 0.3 gM Rosuvastatin 0.3 pM PFBS 1 jjM PFBS 1 uM PFBS 10 uM PFBS 10 uM PFBS 100 uM PFBS 100 pM PFHS 1 uM PFHS 1 pM PFH S 1 0 uM PFH S 1 0 uM PFHS 100 pM PFHS 100 gM PFOS 1 uM PFOS 1 pM PFOS 10 uM PFOS 10 mM PFOS 100 uM PFOS 100 uM Rosuvastatin 0.003 pM Rosuvastatin 0.(X)3 pM Rosuvastatin 0.03 pM Rosuvastalin 0.03 uM Rosuvastatin 0.3 uM UC butyric acid <4C butyric acid 14C butyric acid i4C butyric add butyric add "C butyric acid ,4C Iauric add t4C laurlc add WC lauric acki lauric add U C lauric add "C lauric add UC lauric add lauric add ,4C lauric add 14C lauric add '"C lauric add ,4C lauric add " C lauric add " C lauric add ,4C lauric acid )4C lauric add lauric add " C lauric add 14C lauric add 14C lauric add ,4C lauric add ,4C lauric acid ,4C lauric acid MC lauric add '4C palmitic acid UC palmitic acid UC palmitic acid ,4C palmitic acid palmitic acid " C palmitic acid ,4C palmitic add ,4C palmitic acid *4C palmitic add NC palmitic add UC palmitic add palmitic acid UC palmitic acid 14C palmitic acid ,4C palmitic add l4C palmitic add palmitic acid UC palmitic add "C palmitic add 14C palmitic acid ,4C palmitic acid ,4C palmitic acid ,4C palmitic acid input *H cholesterol input JH cholesterol input JH cholesterol average input VH cholesterol ' H-chotestero (dpm) 15609 15687 14378 13730 15870 12832 13019 16614 16141 15873 17296 16841 10210 15012 15875 16131 16397 14452 15496 17004 13663 13951 14919 16126 15126 15117 15845 15353 16583 15158 14410 15961 16275 14927 14899 2959 14864 15988 14366 15716 13749 16009 14012 15284 14474 12042 14571 13686 13478 13285 12506 13476 14963 26014 25923 25028 25655 14C*cholesterol (dpm) 4123 4793 901 984 311 512 11827 19187 21002 19906 21050 16813 11716 18465 17804 20213 12748 11356 14299 17756 13984 16405 11557 12122 4914 5462 1201 1756 465 505 5450 5718 5596 5781 5367 1505 6675 4773 5602 8024 5977 5872 4048 9125 5125 5104 4478 4542 3476 2874 1940 1859 1145 14C-cholesterol corrected for recovery (dpm) 6777 7839 1608 1839 503 1024 23306 29628 33381 32173 31225 25612 29439 31556 28772 32147 19946 20159 23673 26790 26258 30166 19874 19285 8335 9270 1945 2934 719 855 9703 9191 8821 9936 9242 13049 11521 7659 10004 13098 11153 9410 7412 15317 9084 10874 7884 8514 6616 5550 3980 3539 1963 pg protein 268.9 303.1 276.0 308.9 283.3 253.1 175.6 224.9 243.2 191.8 229.0 187.1 172.3 187.7 180.9 99.8 208.9 172.9 124.4 162.5 201.9 220.3 227.5 166.1 168.0 318.8 234.5 195.1 183.3 215.2 167.6 182.5 230.1 240.4 215.9 228.5 199.3 248.4 274.0 219.2 215.9 299.0 196.1 171.7 216.1 236.9 231.0 222.7 172.3 185.8 266.7 271.7 239.7 MC-cholesterot per mg protein (dpm/mg) 25201 25861 5825 5952 1775 4044 132722 131739 137256 167745 136354 136891 170860 168120 159051 322115 95480 116594 190300 164859 130053 136939 87357 116105 49611 29076 8292 15040 3925 3972 57894 50361 38336 41330 42605 57105 57807 30833 36511 59756 51657 31472 37795 89207 42036 45901 34131 38232 38401 29871 14922 13026 8190 Average,4C-chalesterol per mg protein (dpm/mg) 25531 5889 2910 132231 152501 136623 168120 240583 106037 177579 133496 101731 39343 1166B 3948 54127 39833 42805 44320 48134 41565 63501 43968 36182 34136 13974 8190 % of control Average % of control 23 24 24 55 6 23 4 85 84 84 88 97 107 87 87 87 109 108 107 102 154 206 61 68 74 122 113 105 83 85 87 56 65 74 32 25 19 57 10 33 3 82 76 71 54 56 58 60 71 81 82 63 44 52 68 84 73 59 44 53 90 126 59 62 65 48 51 54 54 48 42 21 20 18 12 12 _____________________ 0 4 -12 -200 6 Appendix to TNO -report of 3 M -0 2_______________Final version Appendix to TNO-report of 3M-02____________ Final version p. 160 04-12-2006 Experiment 2 Protein concentration determination sample Measured dilution average protein cone pg protein protein cone (mg/ml) (mg/ml) per well 1 191.5 4 792.2 264.1 204.6 2 218.1 4 896.8 298.9 230.3 3 234.8 4 911.6 303.9 221 4 267.6 4 965.6 321.9 215.2 5 187.7 4 803.8 267.9 214.2 6 222.9 4 913.4 304.5 233.8 7 235.8 4 1004.8 334.9 266.6 8 250 4 1 0 2 0 .6 340.2 260.3 9 232.8 4 949.4 316.5 241.9 10 246.9 4 1033.2 344.4 269.7 13 211.4 4 813.2 271.1 195.2 14 155.9 4 620.0 206.7 154.1 15 123.5 4 591.2 197.1 172.1 16 156.8 4 777.2 259.1 231.8 17 139.1 4 508.0 169.3 114.9 18 147 4 682.6 227.5 194.3 19 256.2 4 952.6 317.5 220.1 20 126.6 4 540.2 180.1 143.5 21 221 4 876.2 292.1 217.1 22 158.6 4 676.0 225.3 179.4 23 235.8 4 921.4 307.1 224.9 24 175.8 4 835.2 278.4 241.8 25 196.3 4 801.8 267.3 204.6 26 206.6 4 814.8 271.6 2 0 0 .8 27 240.8 4 967.4 322.5 242.9 28 190.5 4 764.0 254.7 191.5 29 150.6 4 609.4 203.1 154.1 30 142.6 4 567.0 189.0 140.9 sample Measured dilution average protein cone pg protein protein cone (mg/mi) (mg/ml) per well 31 231.9 4 945.6 315.2 240.9 32 211.3 4 839.6 279.9 208.5 33 216.2 4 912.0 304.0 239.8 34 203.7 4 879.0 293.0 235.8 35 292.4 340.6 4 1266.0 422.0 36 167.6 4 729.4 243.1 197.1 37 177.5 4 777.6 259.2 211.3 38 237.4 4 939.6 313.2 232.4 39 217.3 4 924.4 308.1 244.9 40 202.1 206.2 41 240 266.7 4 816.6 272.2 4 1013.4 337.8 42 181.8 4 769.4 256.5 202.9 43 217.3 4 880.8 293.6 223.1 44 188.5 4 725.2 241.7 174.1 45 238.2 4 976.4 325.5 250 46 173.3 4 755.8 251.9 204.6 47 235.8 4 934.8 311.6 231.6 48 229.9 4 939.8 313.3 240 49 183.5 4 744.0 248.0 188.5 50 163.1 4 630.2 210.1 152 51 202.9 4 862.0 287.3 228.1 52 213.8 4 917.4 305.8 244.9 53 211.4 4 859.0 286.3 218.1 54 168.2 4 635.2 211.7 149.4 55 211.4 4 848.8 282.9 213 56 232.4 4 973.2 324.4 254.2 57 245.8 4 939.6 313.2 224 58 156.2 4 585.4 195.1 136.5 59 215.6 4 904.4 301.5 236.6 60 133.9 4 585.2 195.1 158.7 61 210.5 4 840.4 280.1 209.7 62 156.2 4 681.0 227.0 184.3 Page A160of A172 Appendix to TNQ-reoort of 3M-02____________ Final version sample 63 64 63 64 63 64 69 70 71 72 73 74 75 76 77 76 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 Measured dilution average protein cone pg protein protein cone (mg/ml) (nxmJ) per weft 252.5 4 252 84.0 225.7 153.6 4 256 85.3 142.5 228.2 4 252 84.0 210.5 228.1 4 Ss 85.3 204.6 220.6 4 252 84.0 244.2 133.9 4 256 85.3 150.2 220.6 4 276 92.0 224.8 234.9 4 280 93.3 236.6 247.5 4 284 94.7 230.7 137.3 4 288 96.0 156.4 206.2 4 292 97.3 194.7 150 4 296 98.7 175 152.7 4 300 100.0 142.8 141.9 4 304 101.3 140.1 230.3 233.8 4 308 102.7 237.3 211.6 4 312 104.0 184 146.9 4 316 105.3 118.4 4 320 106.7 200.1 180.4 4 324 108.0 235.6 162.6 4 328 108.3 132 211.6 4 332 110.7 252.4 168.8 4 336 112.0 170.6 222.3 4 340 113.3 242.6 257.7 4 344 114.7 247.1 223.1 208.9 4 348 116.0 178.7 4 352 117.3 215.1 179.6 4 356 118.7 194.7 166.8 142.9 4 360 120.0 188.5 4 364 121.3 222.3 196.5 4 366 122.7 180.4 226.7 4 372 124.0 240.9 4 376 125.3 182.2 228.4 4 380 126.7 240 165.2 4 384 128 0 142.8 272.7 4 388 129.3 243.5 201.8 4 392 130.7 167.1 213.4 4 396 132.0 236.5 254.2 219.6 4 400 133.3 153.6 4 404 134.7 122 232 281.5 4 408 136.0 207.2 227,6 4 412 1373 208.1 4 416 138.7 207.2 192.9 161.7 4 420 140.0 281.6 218.7 4 424 141.3 145.5 4 428 142.7 151.8 162.6 137.4 4 432 144.0 / 5 P- 161 04-12-2006 Page A161 of A172 sampie compound concentration 1 contrai 2 control 3 control 4 control 5 control 6 control 7 control 8 control 9 control 13 PFBS 1 pM 14 PFBS 1 pM 15 PFBS 10uM 16 PFBS10pM 17 PFBS 100 pM 18 PFBS 100 pM 19 PFHS1 pM 20 PFHS 1 pM 21 PFHS 10 pM 22 PFHS 10 uM 23 PFHS 100 UM 24 PFHS 100 pM 25 PFOS 1 pM 26 PFOS 1 pM 27 PFOS 10 pM 28 PFOS 10 pM 29 PFOS 100 pM 30 PFOS 100 pM 31 Rosuvastatin 0.003 pM 32 Rosuvastatin 0.003 pM 33 Rosuvastatin 0.03 uM 34 Rosuvastatin 0.03 pM 35 Rosuvastatin 0.3 pM 36 Rosuvastatin 0.3 pM 37 PFBS 1 pM 38 PFBS 1 uM 39 PFBS 10 pM 40 PFBS 10 pM 41 PFBS 100 PM 42 PFBS100pM 43 PFHS 1 pM 44 PFHS 1 pM 45 PFHS10pM 46 PFHS10pM 47 PFHS 100 pM 48 PFHS 100 pM 49 PFOS 1 pM 50 PFOS 1 uM 51 PFOS10uM 52 PFOS10pM 53 PFOS 100 pM 54 PFOS 100 pM "C Label ,4C acetic acid ,4C acetic acid "*C acetic acid ,4C butyric acki ,4C butyric acid `"C butyric acb 14C (auric add 14C fauric acid ,4C lauric add 14C acetic acid ,4C acetic acid 14C acetic acid 14C acetic acid ,4C acetic acid 14C acetic acid 14C acetic K id ,4C acetic acid ,4C acetic acid " C acetic add i4C acetic add ,4C acetic acid ,4C acetic acid ,4C acetic acid ,4C acetic acid <4C acetic acid 14C acetic acid 14C acetic acid ,4C acetic acid ,4C acetic acid ,4C acetic acid 14C acetic acid ,4C acetic acid '"C acetic acid ,4C butyric acid 14C butyric acid WC butyric acid *4C butyric acid "C butyric acid ,4C butyric add ,4C butyric acid ,4C butyric add ,4C butyric acid ,4C butyric acid U C butyric acid ,4C butyric acid ,4C butyric add UC butyric acid ,4C butyric acid ,4C butyric acid i4C butyric acid ,4C butyric acid ' H-choiestero (dpm) 10176 15067 8013 7102 13239 16182 11285 9341 14585 10415 11106 13209 17775 11729 16244 15747 13331 15569 8199 15486 14084 15835 16929 15411 9454 15895 17054 18199 17403 16235 14014 15647 9439 17104 16962 16583 15691 14615 16380 16700 15447 15347 15658 16797 16476 14120 13621 16279 16347 15609 16385 ' 4C-cholesterol dpm) 13013 29921 20565 3628 7313 13946 19393 14887 19635 15531 22304 31195 41007 24081 42002 34491 30717 28923 21682 25911 36299 30330 46461 37008 20098 26782 29629 16110 11966 3127 1836 499 439 8190 23749 11608 16193 17212 17173 12743 20907 18404 22910 19524 25363 11804 21880 28289 17820 18990 28115 ,4C-dho(esterol corrected for recovery (dpm) 34518 53601 69272 13788 14910 23262 46384 43017 36337 40250 54206 63744 62269 55416 69791 59120 62193 50143 71378 45162 69565 51699 74077 64817 57380 45479 46894 23893 18559 5199 3536 861 1255 12924 37791 18894 27855 31788 28298 20596 36532 32368 39492 31373 41550 24616 43357 46904 29423 32838 46314 pg protein 264.1 298.9 303.9 321.9 267.9 304.5 334.9 340.2 316.5 271.1 206.7 197.1 259.1 169.3 227.5 317.5 180.1 292.1 225.3 307.1 270.4 267.3 271.6 322.5 254.7 203.1 189.0 315.2 279.9 304.0 293.0 422.0 243.1 259.2 313.2 308.1 272.2 337.8 256.5 293.6 241.7 325.5 251.9 311.6 313.3 248.0 210.1 287.3 305.8 286.3 211.7 ,4C-cholesterd per mg protein (dpm/mg) 130711 179328 227969 42839 55647 76402 138487 126446 114821 148487 262280 323464 240359 327262 306730 186184 345324 171682 316785 147042 249876 193434 272742 201004 225315 223922 248115 75803 66313 17101 12069 2040 5163 49863 120662 61317 102332 94102 110338 70149 151125 99450 156757 100685 132635 99258 206396 163241 96218 114684 218739 Average,4C-cholesterol per mg protein (dpm/mg) 179336 58296 126585 205388 261912 316996 265754 244224 198459 233088 213159 236019 71058 14585 3601 85262 81825 102220 110637 128104 116660 152828 129729 166712 % of control Average % of control 73 100 100 127 73 100 95 131 83 115 146 180 157 134 182 177 171 104 148 193 96 136 177 82 111 139 108 130 152 112 119 128 125 132 138 42 40 37 10 8 7 12 3 86 146 207 105 140 176 161 175 189 120 190 259 171 220 269 173 200 228 170 262 354 280 223 165 197 286 375 SD 27.1 29.1 44.9 32.8 8.1 62.7 57.2 40.5 31.3 9.6 9.5 3.7 20 1.2 85.9 49.8 19.7 98.2 69.5 38.8 130.0 81.3 126.2 [14C]-Cholesterol synthesis in rat hpatocytes 0 41* o> IO sample compound concentration " C Label 55 Rosuvastatin 0.003 uM 56 Rosuvastatln 0.003 uM WC butyric wiki " C butyric acid 57 Rosuvastatin 0.03 uM 58 Rosuvastatin 0.03 uM 14C butyric acid "C butyric acid 59 Rosuvastatin 0.3 uM 60 Rosuvastatin 0.3 uM 14C butyric add "C butyric add 61 PFBS1 uM 14C 1auric add 62 PFBS1 uM 63 PFBS 10 uM 64 PFBS 10 uM 65 PFBS 100 uM 66 PFBS 100 uM 67 PFHS 1 uM "C laurtc acid "C lauric add " C lauric add lauric acid ,4C lauric acid "C lauric add 68 PFHS1 uM 69 PFHS 10 uM 14C lauric add 14C lauric add 70 PFHS 10 uM 71 PFHS 100 uM 72 PFHS 100 uM 73 PFOS 1 uM 74 PFOS 1 gM 14C lauric add "C lauric add 14C lauric add " C lauric add ,4C lauric add 75 PFOS 10 uM 76 PFOS 10 uM 77 PFOS 100 gM " C lauric add 14C lauric add 14C lauric add 76 PFOS 100 uM 79 Rosuvastatin 0.003 uM ,4C lauric add 14C lauric add 80 Rosuvastatin 0.003 uM ,4C lauric add 81 Rosuvastatin 0.03 uM ,4C lauric add 82 Rosuvastatin 0.03 pM " C lauric add 83 Rosuvastatin 0.3 uM 14C lauric acid 84 Rosuvastatin 0.3 uM 14C lauric add input 3H cholesterol input aH cholesterol input ->H cholesterol average input JH didesterol JH-choJesteroi "C-choesterol (dpm) (dpm) 16224 4328 16103 5533 16808 975 17593 1781 14906 452 15758 284 14491 19019 17266 28110 16567 21487 17868 19412 17520 21648 19364 28972 16638 26282 17741 30159 17115 25762 16449 23842 15800 21356 16793 27449 16050 18811 17035 24894 16660 16687 15663 19295 16046 27259 13213 24544 13776 6081 12989 3529 15098 1767 11184 2114 13447 566 14569 329 26701 27378 26895 26991.33333 ' "C-cholesterol corrected for recovery (dpm) 7200 9274 1566 2732 818 486 38645 43943 35007 29324 33351 40384 42637 45884 40628 39123 36483 44119 31635 39444 27035 33250 45853 50138 11915 7333 3159 5102 1136 610 pg protein 282.9 324.4 313.2 195.1 301.5 195.1 280.1 227.0 318.8 197.4 292.5 288.5 309.9 189.4 296.9 314.3 318.8 195.8 267.3 216.7 197.0 188.0 309.4 299.3 220.6 212.3 277.3 196.4 309.3 226.3 14C-cholesterol per mg protein (dpm/mg) 25449 28589 4999 14003 2715 2494 137952 193583 109809 148550 114033 139995 137582 242261 136826 124462 114438 225325 118383 182048 137234 176863 148200 167537 54010 34537 11390 25977 3673 2694 Average^C-cholesterol per mg protein (dpm/mg) 27019 9501 2604 165768 129179 127014 168120 130644 169881 150205 157048 157668 44273 18684 3183 % of control Average % of control 44 46 49 9 16 24 54 4 109 131 153 87 102 117 90 100 111 109 150 191 108 103 98 90 134 178 94 119 144 108 124 140 117 125 132 43 35 27 9 15 21 33 2 so 3.8 10.9 0.3 31.1 21.6 14.5 58.5 6.9 61.9 35.6 22.1 10.8 10.9 81 0.5 T3 03 CO CD O) W a > ro Appendix to TNO-report of 3M-02____________Final version________________________ 04-12-2006 C7> w Appendix to TNO-report of 3M-02____________ Final version Experiment 3 Protein concentration determination sample Measured protein cone (mg/ml) 1 246.35 3 298.46 4 229.35 5 266.32 6 232.70 7 299.74 8 186.54 9 154.56 10 143.00 11 169.33 12 153.58 13 301.02 14 220.50 15 370.42 16 222.70 17 224.91 18 171.32 19 272.34 20 180.40 21 240.62 22 227.12 23 209.66 24 295.91 25 248.65 26 295.91 27 331.42 28 268.72 29 222.71 30 282.13 31 236.08 32 172.33 33 263.93 34 175.34 35 171.32 36 247.50 37 249.81 38 393.62 39 348.03 40 289.60 41 153.58 42 227.12 43 209.66 44 317.98 45 272.34 46 211.81 47 258.00 48 195.88 49 304.88 50 389.03 51 352.27 52 258.00 53 279.67 54 363.75 55 295.91 dilution 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 protein cone pg protein (mg/ml) per well 985.4 328.5 1193.8 397.9 917.4 305.8 1065.3 355.1 930.8 310.3 1199.0 399.7 746.2 248.7 618.2 206.1 572.0 190.7 677.3 614.3 1204.1 225.8 204.8 401.4 882.0 1481.7 890.8 294.0 493.9 296.9 899.6 685.3 299.9 228.4 1089.4 363.1 721.6 240.5 962.5 320.8 908.5 302.8 838.6 279.5 1183.6 394.5 994.6 331.5 1183.6 1325.7 394.5 441.9 1074.9 890.8 358.3 296.9 1128.5 376.2 944.3 314.8 689.3 1055.7 229.8 351.9 701.4 685.3 233.8 228.4 990.0 999.2 330.0 333.1 1574.5 524.8 1392.1 1158.4 464.0 386.1 614.3 908.5 838.6 204.8 302.8 279.5 1271.9 424.0 1089.4 847.2 1032.0 363.1 282.4 344.0 783.5 1219.5 1556.1 261.2 406.5 518.7 1409.1 469.7 1032.0 344.0 1118.7 1455.0 1183.6 372.9 485.0 394.5 /f p. 164 04-12-2006 Page A164 of A172 Appendix to TNO-rePort of 3M-02____________ Final version sample Measured dilution protein cone (mg/ml) 56 356.51 57 319.45 58 356.51 59 319.45 60 226.01 61 2 4 1.05 4 4 4 4 4 4 62 333.17 63 251.23 64 297.09 65 232.80 66 268.15 67 294.15 68 222.82 69 295.13 70 244.74 71 2 3 7 .3 8 72 207.61 73 324.00 74 253.09 75 241.05 76 283.46 77 267.20 78 275.77 79 347.64 80 216.53 81 3 6 8 .7 5 82 219.00 83 329.09 84 221,92 85 285.39 86 348.30 87 312.93 88 212.95 89 288.30 90 281.53 91 30 5.97 92 245.66 93 274.81 94 289.28 95 254.03 96 230.98 97 288.30 98 332.15 99 294.15 100 219.22 101 3 3 5.22 102 252.16 103 297.09 104 247.52 105 280.57 106 250.30 107 296.11 108 217.43 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 protein cone pg protein (mg/ml) per well 1426.0 1277.8 1426.0 1277.8 904.0 964.2 475.3 425.9 475.3 425.9 301.3 321.4 1332.7 1004.9 1188.4 931.2 1072.6 1176.6 891.3 1180.5 979.0 949.5 830.4 1296.0 1012.4 964.2 1133.8 1068.8 1103.1 1390.6 866.1 1475.0 876.0 1316.4 887.7 1141.6 1393.2 1251.7 851.8 1153.2 1126.1 1223.9 982.6 1099.2 1157.1 1016.1 923.9 1153.2 1328.6 1176.6 876.9 1340.9 1008.6 1188.4 990.1 1122.3 1001.2 1184.4 869.7 444.2 335.0 396.1 310.4 357.5 392.2 297.1 393.5 326.3 316.5 276.8 432.0 337.5 321.4 377.9 356.3 367.7 463.5 288.7 491.7 292.0 438.8 295.9 380.5 464.4 417.2 283.9 384.4 375.4 408.0 327.5 366.4 385.7 338.7 308.0 384.4 442.9 392.2 292.3 447.0 336.2 396.1 330.0 374.1 333.7 394.8 289.9 p. 165 04-12-2006 Page A165 of A172 [14C]-Cholesterol synthesis in rat hepatocytes Page A 166of A172 3H cholesterol sample mix 11 13 14 15 16 17 18 1 11 1 12 1 13 1 14 1 15 t 16 1 17 1 16 1 19 1 20 1 21 1 22 1 23 1 24 1 25 1 26 1 27 1 28 1 29 1 30 1 31 1 32 1 33 1 34 1 35 1 36 1 37 1 38 1 39 1 41 1 42 1 43 1 44 1 45 1 46 1 47 1 48 1 49 1 50 1 51 1 52 1 53 1 54 1 55 1 56 1 57 1 58 1 59 1 60 compound concentration control control control control control control control control control PF8S 1 yM PFBS 1 uM PFBS 10 uM PFBS 10 uM PFBS 100 uM PFBS 100 yM PFHS 1 yM PFHS 1 UM PFHS 10 yM PFHS 10 UM PFHS 100 yM PFHS 100 yM PFOS 1 yM PFOS1 yM PFOS 10 yM PFOS 10 uM PFOS 100 yM PFOS 100 yM Rosuvastatin 0.003 yM Rosuvastatin 0.003 yM Rosuvastatin 0.03 uM Rosuvastatin 0.03 yM Rosuvastatin 0.3 yM Rosuvastaiin 0.3 yM PFBS 1 yM PFBS 1 yM PFBS 10 yM PFBS 100 yM PFBS 100 uM PFHS 1 uM PFHS 1 yM PFHS 10 yM PFHS 10 yM PFHS 100 uM PFHS 100 uM PFOS 1 uM PFOS 1 yM PFOS 10 yM PFOS 10 yM PFOS 100 yM PFOS 100 UM Rosuvastatin 0.003 yM Rosuvastaiin 0.003 uM Rosuvastatin 0.03 yM Rosuvastatin 0.03 uM Rosuvastatin 0.3 yM Rosuvastatin 0.3 yM " C Label ,4C acetic acid " C acetic acid 14C butyric acid 14C butyric acid ,4C butyric acid 14C (auric add ,4C tauric add UC palmitic add ,4C palmitic add ,4C acetic add ,4C acetic add 14C acetic add acetic add ,4C acetic add ,4C acetic add ,4C aceticadd ,4C acetic add " C acetic add acetic add ,4C acetic acid ,4C acetic acid ,4C acetic acid ,4C acetic add " C acetic add l4C acetic acid ,4C acetic add l4C acetic add 14C acetic add ,4C acetic acid 14C acetic add ,4C acetic acid ,4C acetic acid ,4C acetic acid MC butyric acid MC butyric acid MC butyric acid ,4C butyric acid MC butyric acid ,4C butyric acid ,4C butyric acid " C butyric add ,4C butyric acid ,4C butyric acid MC butyric acid ,4C butyric acid MC butyric acid ,4C butyric add MC butyric add >4C butyric add ,4C butyric add ,4C butyric add ,4C butyric add ,4C butyric add ,4C butyric add ,4C butyric acid " C butyric acid '` H-cbo(estero! ,4C-cholesterol (dpm) (dpm) 12507 44451 19023 99078 16324 18935 15491 15714 14301 17937 15647 40817 14301 32511 16834 5000 17733 4789 17724 22246 15643 21849 13898 22496 17481 26287 17149 23557 18303 17772 22881 35195 16907 39844 17592 23683 16152 24871 18689 27769 17098 22118 17841 27624 19413 43559 17762 25533 14828 24114 16243 22967 15814 26323 15332 2953 14969 3029 14770 574 14215 626 16761 257 16727 248 15968 5934 14142 11778 16118 13512 16293 9931 19457 7789 18922 6439 17479 14976 17443 7993 16118 9922 18498 6585 17002 17226 10436 7974 16924 8198 15074 12993 17062 18014 15031 12106 18507 9530 14681 930 15247 687 15905 276 17593 212 13507 94 18268 120 ,4C-cholesterol corrected yg protein l4C-cholesteroi for recovery (dpm) per mg protein (dpm/mg) 91111 133519 328.5 397.9 277384 335520 29736 305.8 97240 26005 32153 68874 58279 355.1 310.3 399.7 248.7 73233 103632 167329 234314 7614 6923 32176 35806 225.6 204.8 401.4 294.0 33725 33809 80166 121789 41495 38550 35215 32048 50768 493.9 296.9 299.9 228.4 363.1 84018 129626 117430 140296 139610 60414 34512 240.5 320.8 251169 107571 39474 38091 302.8 279.5 130352 136259 33162 39693 57521 36651 41690 394.5 331.5 394.5 441.9 358.3 84052 119725 145791 83394 116357 36248 42672 296.9 376.2 122069 113436 4938 5187 996 1129 393 380 9527 314.8 229.6 351.9 233.8 228.4 330.0 333.1 15686 22576 2631 4829 1721 1152 28602 21350 21491 15626 10236 524.6 464.0 204.6 302.8 40681 46312 76307 33802 8724 21965 11747 15781 11898 279.5 424.0 363.1 282.4 344.0 31206 51607 32351 55878 34586 15738 11867 12416 22097 27068 261.2 406.5 518.7 469.7 344.0 60261 29192 23940 47045 78660 20647 13201 1624 1155 445 372.9 485.0 394.5 475.3 425.9 55370 27218 4116 2430 1044 309 475.3 650 178 425.9 419 168 301.3 559 Average ,4C-chdesterol per mg protein (dpm/mg) 306452 91366 200821 33767 100979 106922 128864 195490 118962 110155 132758 99670 117752 19131 3830 1436 34641 46312 55054 41506 44115 47423 26566 62663 41294 3273 847 489 % of control Average SD % of control 90.5 100.0 13.4 109.5 106.4 100.0 17.5 80.2 113.4 83.3 100.0 23.6 116.7 99.9 100.0 0.18 100.1 26.2 33.0 9.6 39.7 27.4 34.9 10.6 42.4 38.3 42.1 5.3 45.8 45.6 63.B 25.7 62.0 35.1 38.8 5.3 42.5 44.5 35.9 12.0 27.4 39.1 43.3 6.0 47.6 27.2 32.6 7.6 38.0 39.8 38.4 2 0 37.0 5.1 6.2 1.6 7.4 0.9 1.2 0.5 1.6 0.6 0.5 0.1 0.4 31.3 379 9.3 44.5 50.7 50.7 63.5 60.3 32.9 37.0 34.2 45.4 15.9 56.7 35.4 48.3 18.2 61.2 37.9 51.9 19.9 66.0 32.0 29.1 4.1 26.2 51.5 68.8 24.5 86.1 60.6 45.2 21.6 29.8 4.5 3.6 1.3 2.7 1.1 0.9 0.3 0.7 0.5 0.5 0.1 0.6 9> 9> Appendix to TNO -report of 3M -0 2______________ Final version______________________________ 0 4 -12 -200 6 Page A 16 7 o fA 1 7 2 3H cholesterol sample mix 1 61 1 62 1 63 1 64 1 65 1 66 1 67 1 68 1 69 1 70 1 71 1 72 2 73 2 74 2 75 2 76 2 77 2 78 2 79 2 60 2 81 2 62 2 83 2 85 2 86 2 67 2 88 2 89 2 90 2 91 2 92 2 93 2 94 2 95 2 96 2 97 2 98 2 99 2 100 2 101 2 102 2 103 2 104 2 105 2 106 2 107 2 108 compound concentration "C Label PFBS 1 UM PFBS 1 uM PFBS10 PM PFBS 10 uM PFBS 100uM PFBS100 pM PFHS 1 pM PFHS 1 uM PFHS 10 pM PFHS10 PM PFHS 100pM PFHS 100pM PFOS 1 uM PFOS 1 pM PFOS10 pM PFOS 10 uM PFOS 100pM PFOS 100 PM Rosuvastatin 0.003 uM Rosuvastatin 0.003 uM Rosuvastatin 0.03 pM Rosuvastatin 0.03 uM Rosuvastatin 0.3 pM PFBS 1 pM PFBS 1 uM PFBS 10 pM PFBS 10 pM PFBS 100 pM PFBS 100 pM PFHS 1 pM PFHS 1 uM PFHS 10 uM PFHS10pM PFHS 100 pM PFHS 100uM PFOS 1 pM PFOS 1 pM PFOS 10pM PFOS 10 pM PFOS 100 pM PFOS 100 pM Rosuvastatin 0.003 pM Rosuvastatin 0.003 pM Rosuvastatin 0.03 pM Rosuvastatin 0.03 uM Rosuvastatin 0.3 pM Rosuvastatin 0 3 uM "C lauric acid ,4C lauric add UC lauric acid "C lauric add ,4C lauric acid 14C lauric acid lauric acid ,4C lauric add UC lauric add "*C lauric add 14C lauric add WC lauric add MC lauric add 14C lauric add WC lauric add 14C lauric add WC lauric add " C lauric add ,4C lauric add ,4C lauric add 14C lauric add nC lauric add 14C lauric add UC palmitic acid 14C palmitic acid 14C palmitic acid >4C palmitic add ,4C palmitic acid 14C palmitic acid l4C palmitic acid WC palmitic add UC palmitic add 14C palmitic add UC palmitic acid 14C palmitic acid ,4C palmitic acid UC palmitic add " C palmitic acid ,4C palmitic acid "C palmitic acid WC palmitic add UC palmitic acid ,JIC palmitic add 14C palmitic add palmitic acid palmitic add MC palmitic add input JH cholesterol input JH cholesterol input JH cholesterol average input "H cholesterol JH-cholestero (dpm) 18064 16312 16632 16430 17140 16929 17947 17680 17511 18652 17842 17390 12194 17125 16554 16019 15880 17209 15981 14109 17448 16191 15558 16555 17040 15827 16659 15285 17337 14572 14708 15562 14849 16477 15761 16923 19055 16210 17729 16090 17458 12554 17205 16635 17350 13361 15847 mix 1 25860 25671 25376 25635.7 ,4C-cholesteroi {dpm) 35197 33843 31024 42519 24245 22983 34462 35488 31561 34500 20913 30612 39952 49945 29563 42312 22742 31390 3218 3171 754 686 261 4312 3219 3366 4602 2953 3284 2399 3322 3238 3857 2952 3697 3859 4608 4074 5568 2981 3072 711 986 534 544 313 359 mix 2 24418 25195 24950 24854.3 MC-cholesterol corrected pg protein for recovery (dpm) 49950 321.4 53187 444.2 47819 335.0 66342 396.1 36262 310.4 34803 357.5 49226 392.2 51457 297.1 46205 393.5 47417 326.3 30048 316.5 45127 276.8 81432 432.0 72488 337.5 44386 321.4 65649 377.9 35594 356.3 45335 367.7 5005 463.5 5586 288.7 1074 491.7 1053 292.0 417 438.8 6474 380.5 4695 464.4 5286 417.2 6866 283.9 4802 384.4 4708 375.4 4092 408.0 5614 327.5 5171 366.4 6456 385.7 4453 338.7 5830 308.0 5668 384.4 6010 442.9 6247 392.2 7806 292.3 4605 447.0 4373 336.2 1408 1424 396.1 330.0 798 374.1 779 333.7 582 394.8 563 289.9 "C-cholesterol 155414 119730 142754 167480 116825 97343 125512 173201 117417 145310 94937 163023 188500 214808 138103 173700 99909 123297 10797 19348 2185 3606 950 17013 10110 12669 24182 12492 12542 10030 17139 14114 16738 13147 18930 14744 13572 15927 26705 10302 13008 3554 4316 2133 2335 1475 1942 Average'4C-chofe8terol % of contro! Average SD per mg protein (dpm/mg) % of control 137572 77.4 68.5 12.6 59.6 155117 71.1 77.2 8.7 83.4 107064 58.2 53.3 6.9 48 5 168120 62.5 74.4 16.8 86.2 131364 58,5 65.4 9.8 72.4 128980 47.3 64.2 24.0 81.2 201654 93.9 100.4 9.3 107.0 155901 68.8 77.6 12.5 86.5 111603 49.8 55.6 8.2 61.4 15073 5.4 7.5 3.0 9.6 2695 1.1 1.4 0.5 1.8 1050 0.5 0.5 0.1 13562 50.4 50.4 18425 42805 13584 15426 16038 14158 21316 11655 3935 2234 8190 37.5 54.6 24.1 71.6 37.0 37.1 0.1 37.1 29.7 40.2 14.9 50.8 41.8 45.7 5.5 49.6 38.9 47.5 12.1 56.1 43.7 41.9 2.5 40.2 47.2 63.1 22.6 79.1 30.5 34.5 5.7 38.5 10.5 11.7 1.6 12.8 6.3 6.6 0.4 6.9 4.4 5.1 1.0 5.8 9> vj Appendix to TNO-report of 3M-02____________ Final version p. 168 04-12-2006 Experiment 4 Protein concentration determination sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Measured protein cone (mq/ml) well 1 well 2 207.81 223.77 214.31 224.72 189.73 202.31 205.97 234.4 230.5 252.34 2 2 1 .8 6 218.07 205.97 224.72 225.68 225.68 214.31 223.77 210.58 214.31 210.58 220.91 199.58 205.97 212.44 214.31 241.29 245.28 230.5 226.54 234.4 239.31 272.05 262.61 292.63 272.24 254.37 242.28 231.47 237.34 269.94 274.18 272.05 253.35 253.35 250.31 242.28 2 2 1 .8 6 202.31 210.58 241.29 242.28 245.28 239.31 230.5 235.37 245.28 233.42 218.07 209.66 250.31 240.3 253.35 242.28 244.28 249.3 233.42 237.34 215.24 204.14 227.6 249.52 210.58 228.83 256.56 277.1 246.52 284.44 248.52 238.6 251.52 254.54 266.75 258.59 238.6 246.52 265.73 254.54 251.52 253.53 242.55 245.53 237.62 229.8 212.56 208.8 211.62 203.19 206.92 194.87 209.73 192.13 206.92 2 1 0 .6 8 222.08 177.7 234.67 186.68 214.45 220.17 228.83 214.45 dilution 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 protein cone (mg/ml) 1079.0 1097.6 980.1 1091.2 1216.9 1069.6 1107.0 1128.4 1095.2 1062.2 1078.7 1013.9 1066.9 1216.4 1142.6 1184.3 1336.7 1412.2 1241.6 1172.0 1360.3 1313.5 1259.2 1160.4 1032.2 1208.9 1211.5 1164.7 1196.8 1069.3 1226.5 1239.1 1234.0 1176.9 1048.5 1095.5 1195.9 1257.7 1403.9 1217.8 1265.2 1313.4 1 2 1 2 .8 1300.7 1262.6 1 2 2 0 .2 1168.6 1053.4 1037.0 1041.6 967.5 1044.0 1141.9 911.0 1108.2 1086.6 pg protein per well 359.7 365.9 326.7 363.7 405.6 356.5 369.0 376.1 365.1 354.1 359.6 338.0 355.6 405.5 380.9 394.8 445.6 470.7 413.9 390.7 453.4 437.8 419.7 386.8 344.1 403.0 403.8 388.2 398.9 356.4 408.8 413.0 411.3 392.3 349.5 365.2 398.6 419.2 468.0 405.9 421.7 437.8 404.3 433.6 420.9 406.7 389.5 351.1 345.7 347.2 322.5 348.0 380.6 303.7 369.4 362.2 Page A168ofA172 Appendix to TNO-report of 3M-02____________ Final version p. 169 04-12-2006 sample 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 91 92 94 95 96 97 98 100 101 103 104 105 106 107 109 110 112 113 114 115 116 118 119 Measured protein cone (mq/ml) well 1 well 2 224.96 223.04 219.21 213.51 199.48 210.68 218.26 204.12 205.99 204.12 186.68 160.17 193.04 161.9 209.73 203.19 205.99 217.3 229.8 237.62 225.93 246.52 222.08 234.67 261.64 263.68 259.6 235.65 239.59 278.14 255.55 285.49 212.61 232.92 215.22 229.35 225.79 231.13 233.82 225.79 241.03 240.13 233.82 241.03 239.22 238.32 239.22 232.03 217.85 221.37 224.02 234.72 220.49 225.79 283 208.27 287.81 190.32 204.81 203.09 218.73 216.10 205.67 224.02 218.73 233.82 226.68 242.85 214.35 217.85 214.35 212.61 203.95 217.85 211.74 224.02 218.73 235.62 252.91 239.22 242.85 218.73 255.68 242.85 245.58 209.13 256.60 245.58 248.32 229.35 251.99 245.58 231.13 251.07 237.42 242.85 243.76 259.39 217.85 255.68 272.51 291.69 219.61 243.74 255.52 267.47 234.05 216.10 246.67 269.48 271.50 235.01 dilution 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 protein cone (mg/ml) 1120.0 1093.7 1044.1 1013.7 1037.0 949.3 805.2 1032.3 1058.2 1139.3 1210.4 1141.9 1313.3 1238.1 1237.9 1409.1 1069.6 1155.7 1164.8 1162.4 1187.1 1162.5 1198.4 1178.1 1098.1 1146.9 1115.7 1427.0 996.5 1047.9 1052.4 1082.7 1071.8 1024.1 1104.7 1076.2 1144.6 1113.5 1196.2 1230.3 1221.1 1069.7 1280.7 1221.1 1214.4 1180.5 1239.4 1262.4 1122.5 1266.9 1410.5 1089.3 1226.0 1312.5 1347.4 1172.7 pg protein per well 373.3 364.6 348.0 337.9 345.7 316.4 268.4 344.1 352.7 379.8 403.5 380.6 437.8 412.7 412.6 469.7 356.5 385.2 388.3 387.5 395.7 387.5 399.5 392.7 366.0 382.3 371.9 475.7 332.2 349.3 350.8 360.9 357.3 341.4 368.2 358.7 381.5 371.2 398.7 410.1 407.0 356.6 426.9 407.0 404.8 393.5 413.1 420.8 374.2 422.3 470.2 363.1 408.7 437.5 449.1 390.9 Page A169ofA172 [14C]-Cholesterol synthesis in rat hepatocytes Page A170of A172 3H cholestero sample mix 11 12 13 14 15 17 1B 19 1 10 1 11 1 12 1 13 1 15 1 17 1 18 1 19 1 20 22 1 23 1 24 1 25 1 26 1 27 1 28 1 30 1 31 1 32 1 33 1 34 1 35 1 36 1 38 1 39 2 40 2 41 2 42 2 43 2 44 2 45 2 46 2 47 2 48 2 49 2 50 2 51 2 52 2 53 2 54 2 56 2 57 2 58 compound concentration control control control control control control control control control control control PFBS 1 gM PFBS 1 gM PFBS 10 gM PFBS 10 mM PFBS 100 uM PFBS 100 gM PFHS 1 uM PFHS 1 gM PFHS 1 gM PFHS 10 uM PFHS 10 gM PFHS10 gM PFHS 100 gM PFHS 100 uM PFOS 1 gM PFOS 1 gM PFOS 1 gM PFOS 10 gM PFOS10 gM PFOS 10 gM PFOS 100 gM PFOS 100 gM Rosuvastatln 0.003 gM Rosuvastatin 0.003 gM Rosuvastatln 0.003 uM Rosuvastatin 0.03 uM Rosuvastatin 0.03 gM Rosuvastatin 0.03 gM Rosuvastatin 0.3 uM Rosuvastatin 0.3 gM Rosuvastatin 0.3 gM PFBS1 gM PFBS 1 gM PFBS 1 uM PFBS 10 gM PFBS 10 gM PFBS 10 gM PFBS 100 uM PFBS 100 gM PFHS 1 gM | "C Label ,4C acetic acid ,4C acetic acid 14C acetic acid "C acetic acid ,4C butyric acid ,4C butyric acid ,4C butyric acid 14C palmitic acid "C palmitic acid 14C palmitic acid 14C palmitic acid 14C acetic acid ,4C acetic add 14C acetic add 14C acetic add 14C acetic acid ,4C acetic add " C acetic add ,4C acetic add 14C acetic add ,4C acetic add ,4C acetic add ,4C acetic add ,4C acetic add ,4C acetic add 14C acetic acid ,4C acetic acid 14C acetic acid ,4C acetic acid UC acetic acid MC acetic acid MC acetic add "C acetic add ,4C acetic acid ,4C acetic add ,4C acetic add "C acetic add <4C acetic add "C acetic add "C acetic add ,4C acetic add '"C acetic acid ,4C butyric acid ,4C butyric add l4C butyric add *4C butyric acid ,4C butyric acid butyric acid ,4C butyric acid WC butyric acid " C butyric acid aH-cholesterol (dpm) 11557 13921 12789 14330 14143 12409 13395 12206 13316 12616 13307 14019 14018 13123 12859 12350 12583 12002 12074 10469 12178 13070 11021 12494 11696 12079 12015 12687 12477 12560 10462 10416 9390 9534 11731 12187 11494 12998 10852 9480 8849 11450 9541 11879 12114 11350 11456 9935 11036 9818 12175 l4C-chotesterol (dpm) 1815 1877 2094 2430 2257 2888 4007 1142 1694 1574 1703 2569 1506 1714 1837 1427 2709 1811 1822 2089 2935 3091 3398 2059 1924 3650 2929 2772 2383 2407 1828 1647 1807 81 95 61 36 42 18 7 10 12 1216 2158 3192 998 1499 1125 4223 1517 1686 ",C-choiesterol corrected for recovery (dpm) 3220 2765 3357 3477 3272 4772 6134 1918 2608 2558 2624 3757 2203 2678 2929 2369 4414 3094 3094 4091 4942 4849 6322 3379 3373 6196 4998 4480 3916 3929 3583 3242 3946 171 163 100 63 65 33 15 23 21 2559 3647 5290 1765 2627 2273 7682 3102 2780 pg protein '"C-cholesterol per mg protein (dpm/mg) 359.7 8953 365.9 7556 326.7 10276 363.7 9559 405.6 369.0 8067 12932 376.1 385.1 16307 5255 354.1 7367 359.6 7114 338.0 7764 355.6 380.9 10566 5784 445.6 6011 470.7 6223 413.9 5724 390.7 11299 437.8 7066 419.7 7372 386.8 10578 344.1 14362 403.0 12033 403.8 15655 388.2 8704 356.4 9463 408.8 15155 413.0 12102 411.3 10692 392.3 9982 349.5 11243 365.2 9811 419.2 7733 468.0 8432 405.9 420 421.7 386 437.8 230 404.3 156 433.6 150 420.9 79 406.7 36 389.5 351.1 58 60 345.7 7402 347.2 10504 322.5 16403 348.0 5073 380.6 6902 303.7 7487 362.2 21211 373.3 6309 364.8 7626 % of control Average % of control 98.5 100.0 83.2 113.1 105.2 64.9 100.0 104.0 131.1 76.4 100.0 107.2 103.5 112.9 116.3 90.0 63.7 66.2 67.3 68.5 63.0 93.7 124.4 77.8 91.8 81.1 116.4 158.1 154.3 132.4 172.3 95.8 100.0 104.1 166.8 139.9 133.2 119.9 109.9 113.9 123.7 108.0 85.1 89.0 92.8 4.6 3.8 4.2 2.5 1.7 1.4 1.6 0.9 0.4 0.6 0.6 0.7 59.5 92.0 84.5 131.9 40.8 52.2 55.5 60.2 170.6 118.7 66.8 61.3 124.5 SD 12.7 33.3 16.2 37.2 1.6 43.4 21.4 20.2 5.9 24.2 8.6 5.4 1.1 0.5 0.1 36.8 10.1 73.4 62.2 vj O Appendix to TNO -report of 3 M -0 2_______________ Final version___________________________ 04-12 -200 6 Page A171 of A172 3H cholesterol sample mix 2 59 2 60 2 61 2 62 2 64 2 66 2 67 2 66 2 69 2 70 2 71 2 72 2 73 2 74 2 75 2 76 2 77 2 78 2 79 2 80 2 81 2 82 2 83 2 85 2 86 2 87 2 88 2 89 2 91 2 92 2 94 2 95 2 96 2 97 2 98 2 100 2 101 2 103 2 104 2 105 2 106 2 107 2 109 2 110 2 112 2 113 2 114 2 115 2 116 2 118 2 119 compound concentration PFHS 1uM PFHS 1 uM PFHS 10 liM PFHS 10 tiM PFHS 100 uM PFHS 100 mM PFOS 1uM PFOS 1 mM PFOS 1 uM PFOS 10(jM PFOS10uM PFOS 10 uM PFOS 100 uM PFOS 100 uM PFOS 100 pM Rosuvastatin 0.003 uM Rosuvastatin 0.003 uM Rosuvastatin 0.003 uM Rosuvastatin 0.03 uM Rosuvastatin 0.03 uM Rosuvastatin 0.03 uM Rosuvastatin 0.3 uM Rosuvastatin 0.3 uM PFBS 1 uM PFBS 1 uM PFBS 1 uM PFBS 10uM PFBS 10uM PFBS 100 uM PFBS 100 UM PFHS 1pM PFHS 1 tiM PFHS 1 uM PFHS10uM PFHS 10 uM PFHS 100 uM PFHS 100 uM PFOS 1 uM PFOS 1 pM PFOS 1 pM PFOS 10 pM PFOS 10 pM PFOS 100 pM PFOS 100 uM Rosuvastatin 0.003 uM Rosuvastatin 0.003 uM Rosuvastatin 0.003 uM Rosuvastatin 0 03 uM Rosuvastatin 0.03 uM Rosuvastatin 0.3 pM Rosuvastatin 0.3 uM "C Label UC butyric acid 14C butyric acid ,4C butyric acid i4C butyric acid ,4C butyric acid "C butyric acid ,4C butyric acid "C butyric acid ,4C butyric add '**0butyric acid MC butyric acid '*C butyric acid "C butyric add " C butyric add '*C butyric add ,4C butyric add ,4C butyric add 14C butyric add `*C butyric add MC butyric acid " C butyric acid ,4C butyric add l4C butyric add ,4C palmitic add ,4C palmitic add "C palmitic add ,4C palmitic acid "C palmitic add ,4C palmitic acid ,4C palmitic acid " C palmitic acid 14Cpalmitic acid 14C palmitic add 14C palmitic acid 14C paimitic acid "C palmitic add <4C palmitic add ,4C palmitic add 14C palmitic add 14C palmitic acid ,4C palmitic add ,4C palmitic add ,4C palmitic acid ,4C palmitic add 14C palmitic add ,4C palmitic acid palmitic acid ,4C palmitic add ,4C palmitic add ,4C palmitic add palmitic acid input JHchdeaterol input JHcholesterol input *H cholesterol average input *H cholesterol ^H-chofestero (dpm) 11671 11805 11641 11848 11906 12251 12599 10509 12593 12245 11423 12013 11885 11791 11061 12385 12579 12107 12774 11196 12355 13007 12240 14199 12961 13307 13107 12361 14351 12742 13653 12703 12038 13137 11960 11398 14337 11689 13496 13069 13862 14202 9771 8624 6247 8729 10667 6797 4697 9054 4674 mix 1 21387 20596 19529 20504 "C-cholesterol (dpm) 3180 4589 3479 4837 2793 3204 1926 2215 2141 2079 2076 2892 2655 2641 2162 137 127 162 35 33 43 30 43 978 745 963 863 429 1098 954 1536 1392 1184 1031 991 1551 1171 851 1062 1193 1377 1377 742 980 152 122 128 127 107 141 125 mix 2 21723 17423 21082 20076 ''C-choiesterol corrected for recovery (dpm) 5470 7804 6000 8337 4710 5250 3069 4231 34,13 3409 3649 4833 4485 4497 3924 222 203 289 55 59 70 46 71 1383 1154 1453 1322 697 1536 1503 2259 2200 1975 1576 1663 2732 1640 1462 1580 1833 1994 1947 1525 2281 488 281 241 375 457 313 537 M9 protoin ''C-cholesterol per mg protein (dpnVmg) 348.0 15718 337.9 23097 345.7 17357 316.4 26346 344.1 13687 379.8 13825 403.5 7607 380.6 11117 437.8 7797 412.7 6259 412.6 8843 469.7 10290 356.5 12579 385.2 11673 388.3 10107 387.5 573 395.7 512 387.5 693 399.5 136 392.7 151 368.0 191 382.3 121 371.9 190 332.2 4163 349.3 3304 350.8 4142 360.9 3663 357.3 1950 341.4 4500 368.2 4082 358.7 6296 381.5 5766 371.2 5320 398.7 3952 410.1 4056 407.0 6712 358.6 4599 426.9 3424 407.0 3881 404.8 4527 393.5 5068 413.1 4712 420.8 3623 374.2 6097 422.3 1157 470.2 597 363.1 663 408.7 918 437.5 1045 449.1 696 390.9 1374 % of control Average % of control 126.4 185.7 139.8 175.7 211.9 110.1 110.6 111.2 61.2 71.1 89.4 62.7 66.4 73.4 71.1 82,7 101.2 92.1 93.9 81.3 4.6 4.B 4.1 5.6 1.1 1.3 1.2 1.5 1.0 1.2 1.5 60.6 56.3 48.1 60.2 53.3 40.8 28.4 65.5 62.4 594 91.6 84.3 83.9 77.4 57.5 58.2 59.0 97.6 82.3 66.9 49.8 57.4 56.5 65.9 73.7 71.1 68.5 52.7 70.7 88.7 16.8 11.7 8.7 9.7 13.4 14.3 15.2 10.1 15.1 20.0 SO 64.8 0.8 15.9 8.4 10.1 0.7 0.2 0.4 7.1 17.6 4.3 7.1 1.1 21.7 8.1 3.7 25.5 4.4 1.3 7.0 Appendix to TNO-report of 3M-02 Final version 04-12-2006 Average [14C]-Cholesterol synthesis in rat hepatocytes (% of control) Concentration: 1 uM I 10 PM I 100 pM | 0.003 uM 0.03 MM experiment MC acetate incorporation. n=4, PFBS 1 850 82 76 2 115 157 1Y r 3 33 34.9 42.1 4 90 67.3 93.7 AVERAGE 80.8 863 972 $D 34.4 51.7 57.3 experiment 1 2 3 4 AVERAGE SO MC acetate incorporation. n=4. PFHS 91 82 84 148 136 111 63.8 38.8 35.9 91.8 154.3 100 98.7 'i& U 82.7 35.4 52.5 33.1 experiment MC acetate incorporation, n=4, PFOS 1 76 84 65 2 3 4 AVERAGE SD 130 43.3 139.9 97.3 45.7 119 32.6 113.9 87.4 39.6 132 38.4 E 81.1 39.7 experiment 1 2 3 4 AVERAGE SO '*C acetate incorporation. n*4, Rosuvastatin 45 40.0 8.2 3.8 23.8 21.8 11 8 1 1 5.4 4.9 0.3 uM 3 2 0.5 0.6 16 12 Concentration: 1 uM I 10 uM I tOO uM | 0 003 uM 0.03 uM experiment 1 2 3 4 "C butyrate incorporation. n=4, PFBS 61.0 12B 92 24 146 140 175 376 50.7 60.3 92 52.2 116.7 5 AVERAGE 842 92> SD 46.8 47.9 1113 48.6 experiment MC butyrate incorporation, rr*4, PFHS 1 115 101 100 2 190 220 200 3 45,4 48.3 51.9 4 124.5 175.7 110.6 AVERAGE SO 118.7 136.3 02 76.5 115.6 61.8 experiment WC butyrate incorporation. n=4, PFOS 1 76 98 58 2 262 223 286 3 29.1 68.6 45.5 4 71.1 73.4 92.1 AVERAGE 109 6 115.8 120.4 SD 103.8 72.6 112.1 experiment " C butyrate incorportion. n=4. rosuvastatin 1 24 S 2 46.0 16 3 3.7 1 4 4.8 1 AVERAGE 5 19.6 5.8 19.9 7.0 0.3 uM 3.0 3 4 0.5 12 2.2 1.6 Concentration: 1 uM I 10 uM 1 100 uM i 0.003 uM 0.03 uM experiment " C (aurate incorporation, n=3, PFBS 1 84.0 97 87 2 131 102 100 3 68.5 Y.2 53.3 AVERAGE 94.5 92.1 SD 32.5 13.1 80.1 24.1 **C (aurate incorporation, n=3, PFHS 1 108 102 68 2 150 103 134 3 74.4 65.4 84.2 AVERAGE SD 110.8 37.9 90.1 21.4 88.7 392 " C (aurate incorporation. n=3, PFOS 1 113 65 65 2 119 124 125 3 100.4 77.6 55.6 AVERAGE 110.8 95.5 SD 9.5 24.9 81.9 37.6 "C (aurate incorporation. n=3. rosuvastatin t 25 7 2 35.0 15 3 7.5 AVERAGE SD 22.5 7.8 13.9 6.8 0.3 uM 3 3 0.5 2.2 1.4 Concentration: 1|iM | 10 pM 100 uM I 0.003 uM 0.03 pM experiment 1 3 4 "C patiniate neonration. n3, PFBS 76.0 56 71 50.4 54.6 37.1 56.3 40 8 62.4 AVERAGE 60.9 50.5 SO 13.4 8.4 17.6 "C patmitate neonration, r>=3. PFHS 1 63 68 59 3 40.2 45.7 47.5 4 84.3 582 82.3 AVERAGE 62.5 57.3 SD 22.1 112 .9 17.7 MC palmitate incorporation. n=3. PFOS 1 90 62 51 3 41.6 63.1 34.5 4 57.4 71.1 70.7 AVERAGE 63.0 6&.4 SO 24.7 5.0 52.1 18.1 MC patiniate incorporation, n=3. rosuvastatin 1 48 20 3 11.7 7 4 11,7 14 AVERAGE SD 23.8 13.8 1.0 6-5 0.3 uM 12 5 15.1 10.7 5.2 Page A 172ofA172 Appendix to TNO-rePort of 3M-02____________ Final version p. 173 04-12-2006 area a-murichoate deoxycholate cholate lithocholate B-muricholate w-muricholate hyodeo/urso 216 631 176 110 543 386 response area corrected % Mgbile add ng bile add/ nmol bHeadd/ factor for response factor mg feces mg feces 1.38 298.08 12.4 0.542 98.6 0.241 1 631 26.3 1.148 208.7 0.532 1 206 8.6 0.375 68.1 0.167 1 110 4.6 0.200 36.4 0.097 1.08 586.44 24.4 1.067 194.0 0.475 1 571 23.8 1.039 189.0 0.463 1 0 0.0 0.000 0.0 0.000 total I 2062 | | 2403 | 100 | 4 I 795 I 2.0 nor-hyodeoxy | 5496 | 1 | 5496 | 5496 weightfeces 5.9 mg a-muricholate deoxycholate cholate lithocholate S-muricholate w-muricholate hyodeo/urso area 872 3015 1451 382 2004 3004 653 response area corrected factor for response factor % Mgbile add ng bile acid/ nmol bile add/ mg feces mgfeces 1.38 1203.36 8.9 0.443 75.1 0.184 1 3015 22.2 1.110 188.2 0.479 1 1698 12.5 0.625 106.0 0.259 1 382 2.8 0.141 23.8 0.063 1.08 2164.32 16.0 0.797 135.1 0.331 1 4446 32.8 1.637 277.5 0.679 1 653 4.8 0.240 40.8 0.104 total | 11381 | I 13561 t 100 | 5 | 846 | 2.1 | nor-hyodeoxyI 27158 | 1 | 27158 | 27158 [ 2.7-21 weight feces 5.3 mg v- | a-muricholate deoxycholate cholate lithocholate B-muricholate w-muricholate hyodeo/urso area 665 3474 1201 397 1600 2024 155 response area corrected % pg bile acid ng bile acid/ nmol bile acid/ factor for response factor mg feces mg feces 1.38 917.7 8.3 0.585 110.4 0.270 1 3474 31.4 2.215 418.0 1.065 1 1405 12.7 0.896 169.1 0.414 1 397 3.6 0.253 47.8 0.127 1.08 1728 15.6 1.102 207.9 0.509 1 2996 27.1 1.910 360.4 0.882 1 155 1.4 0.099 18.6 0.047 tota! I 9516 | | 11Q72 | 100 | 7 | 1332 | 3.3 | nor-hyodeoxy | 15683 | 1 | 15683 1 15683 a-muri deoxy cholate lithochot b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 m Page A79 of A172 Appendix to TIMO-report of 3M-02 Final version 04-12-2006 weight feces 6.7 mg area response area corrected % pgbile acid ngbileacid/ nmol bile acid/ factor forresponse factor mgfeces mgfeces a-muricholate 535 deoxycholate 850 1.38 1 738.3 850 12.0 0.631 94.1 13.8 0.726 108.4 0.230 0.276 cholate 877 1 1026 16.7 0.877 130.8 0.320 lithocholate 325 1 325 5.3 0.278 41.4 0.110 a-muricholate 1150 1.08 1242 20.2 1.061 158.4 0.388 w-muricholate 921 1 1363 22.2 1.164 173.8 0.425 hyodeo/urso 605 1 605 9.8 0.517 77.1 0.196 total | 5263 I | 6149 | 100 | 5 | 784 | 1.9 nor-hyodeoxy| 11706 | 1 | 11706 | 11706 weightfeces 6.5 mg oppervlakte response factor a-muricholate deoxycholate cholate lithocholate 290 571 462 258 1.38 1 1 1 B-muricholate w-muricholate hyodeo/urso 300 422 1.08 1 1 oppervlakte 400.2 571 541 258 324 625 0 % pg bile acid ng bile acid/ nmol bile acid/ mgfaeces mgfaeces 14.7 0.950 21.0 1.355 146.1 208.5 0.358 0.531 19.9 1.283 197.3 0.483 9.5 0.612 94.2 0.250 11.9 0.769 118.3 0.289 23.0 1.482 228.0 0.558 0.0 0.000 0.0 0.000 total I 2303 I I 2718' | 100 | 6 | 992 ~"f....... 7.5 nor-hyodeoxy| 4214 | 1 | 4214 | 4214 weight feces 7.2 mg area response area corrected % pg bile acid ngbile acid/ nmol bile acid/ factor forresponse factor mgfeces mgfeces a-muricholate deoxycholate cholate 1184 3208 2501 1.38 1 1 1633.92 3208 2926 9.1 0.503 17.8 0.987 16.2 0.900 69.8 137.1 125.1 0.171 0.349 0.306 lithocholate B-muricholate w-muricholate hyodeo/urso 1182 3049 3105 1201 1 1.08 1 1 1182 3292.92 4595 1201 6.6 0.364 18.3 1.013 25.5 1.414 6.7 0.370 50.5 140.7 196.4 51.3 0.134 0.344 0.481 0.131 total I 15430 I I 18039 I 100 | 6 [ 771 1 1.9 nor-hyodeoxy| 32495 | 1 | 32495 | 32495 a-muri deoxy cholate lithochol b-muri w-muri hyodeoxy/urso 408.6 392.6 408.6 376.6 408.6 408.6 392.6 Page A80 of A172 Appendix to TNO-report of 3M-02 Final version p. 175 04-12-2006 1 1 ap 2 I a 5 s ss s o 8 pi 2 R M V 8 d oN 1 CO e o ao CM h; O8 O 2 8 CM 8 CPMS0 s d d PSCM CM CM A d AA 8 0 K o A A AAA s 8 o h* CaMn 8 dA s 8 3 8 O0 d 00 o 8 d 3 o 3 s 8 3 3 3 3 8 2 8& 3 5 2 8 8 2 2 0 2s fi ? 5 CM? s s s CM jp PUSStCoD s h* 3 A r PS 0 A s r i 0 A s s CM2 n 6 A 2 i u> C sr w>S B d s 0 h> s i s & MS * s 0s s 00 H B2 g CM n 0 CM S S S CM CAM S A 3 3 2 2 3 2 2 8ds s S b A s hi a> d d 3s s s 9 Mr R S to dCM 0 s a CM o CM | A d CM A H 10 0 0 0d A sr. s d 0 0 tr A0 CM 2S N 0 AA S>3 n 3 9A A N e 0 28 0 ai h CO g SS rdA h. 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CM S K d c o o o d d d 03us CM n * 00 ED CM CP i Qodd dodd 5 9 PS us h. n CM 23 d d add dd a CM 0 o CO us oCM CM CO s 2 s PS o d ddd 3 3 5 8CM rs 9 o 3 A dd d d d d Q d iB CM 3 o a d d dddddd CM o 33 dd A CM PM A r i A <0 3 ce a s ' CM a3 i so 0h. 0 O3 sO 3 O a ddo 3 1sh* PS a I PoS 0 3 d d d d dd 1 5 0a PS a 0 s ? s 00 ddd ddd aPSt PS 8 CM d d o o O d O d CM 8s 0I h N CM 0 0 ddddddd 6 80CM CM CPMS h. us $0 dd dddd i o 3 h> 3 CM Cm i i d ddd dd d 0PS A uS S A A A A A A AA AA 3 8 0 80c^ CM d NA CM A s' s As CM CM 1 1 1 1PM a 3% ddd a0 di o 8 0 0 0h. 8 PS s a ddC d s n 3h P0PMS dO 9o o d A o d o 1 s0 68 O CM 2 d PM 1 i d 3 d dd d CM 0 CM o o CM CM 3 3 dd dddd d 6 1 3 30 ddd ddd 3 d S h 3 h> O o PM PM noo dd dd dd 0 w IM 0A ( A A A A A A A AAm A 0 0 dCM CM s CM CM 2 PM 0 2 0 8" CM o S o O 83 3 o dddddd d 1i a 0 hw i 0CwM t B d d dd d 1d i2 CgM d 00 0 i gl dd ddd d to d R S h8 ddO w s d M d o d P0M d S 2 8CM h- <* 0 3 0a 0 B 30 ddddd dd 2 2g CM 3 S 0 dOoddi ddO 1 3i 3 ohCM. s i ddd dO 1 O g CM g cl ci AAAAA A 0000 3 s 2 08 8 3A 00 n1 3 A0 ps 0 w ro i Il M . W .17 Page A81 ofA172 Appendix to TNO-report of 3M-02____________ Final version p. 176 04-12-2006 /W Page A82 of A172 Appendix to TNO-report of 3M-02____________ Final version E3. Fecal sterol measurements weightfeces 6.2 mg area response area corrected % pgsterol ng sterol/ nmol sterol/ factor for responsefactor mgfeces mgfeces coprostanol 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol 284344 cholestanol 9225 lathosterol 10102 0.98 0.91 1 278657 8395 10102 85.2 60.3 9718 25.13 2.6 1.8 293 0.75 3.1 2.2 352 0.91 campestero! 4671 1.56 7287 2.2 1.6 254 0.63 stigmasterol 1682 1.49 2506 0.8 0.5 87 0.21 b-sitosterol 13713 b-sitostanol 1.47 1 20158 0 6.2 4.4 703 1.70 0.0 0.0 0 0.00 total I 323737 | | 327105 | 100 | 71 | 11407 | 29.3 I cholestane | 111178 | 1,04 | 115625 ~H weightfeces 6.5 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 313994 9355 9015 4796 1808 13562 response area corrected factor for responsefactor 10 10 0.98 307714 0.91 8513 1 9015 1.56 7482 1.49 2694 1.47 19936 10 % 0.0 0.0 86.6 2.4 2.5 2.1 0.8 5.6 0.0 pgsterol 0.0 0.0 76.7 2.1 2.2 1.9 0.7 5.0 0.0 ngsterol/ mgfeces 0 0 11803 327 346 287 103 765 0 nmol sterol/ mg feces 0.00 0.00 30.52 0.84 0.89 0.72 0.25 1.84 0.00 total I 352530 | I 355354 | 100 | 89 ""| '1331" | 35.1 | cholestane I 96413 | 1.04 | 100270 ~1 100270 weightfeces 7.1 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitostero! b-sitostanol area 298829 8558 10265 4356 1589 12044 response area corrected factor for responsefactor 10 10 0.98 292852 0.91 7788 1 10265 1.56 6795 1.49 2368 1.47 17705 10 % 0.0 0.0 86.7 2.3 3.0 2.0 0.7 5.2 0.0 total 335641 337773 100 cholestane 95828 1.04 99661 99661 pgsterol 0.0 0.0 73.5 2.0 2.6 1.7 0.6 4.4 0.0 ngsterol/ nmol sterol/ mgfeces mgfeces 0 0.00 0 0.00 10347 26.76 275 0.71 363 0.94 240 0.60 84 0.20 626 1.51 0 0.00 85 11934 30.7 coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 177 04-12-2006 Page A83of A172 Appendix to TNQ-report of 3M-02 Final version weightfeces 7 mg coprostanoi coprostanon cholesterol cholestanol lathosterol campesterei stigmasterol b-sitosterol b-sitostanol area 319680 11436 10467 5023 1268 13626 response areacorrected factor forresponsefactor 10 10 0.98 313286 0.91 10407 1 10467 1.56 7836 1.49 1889 1.47 20030 10 % 0.0 0.0 86.1 2.9 2.9 2.2 0.5 5.5 0.0 pgsterol ngsterol/ nmol sterol/ mgfeces mgfeces 0.0 0 0.00 0.0 0 0.00 87.7 12532 32.41 2.9 416 2.9 419 1.07 1.08 2.2 313 0.78 0.5 76 0.18 5.6 801 1.93 0.0 0 0.00 total I 361500 | j 363916 | 100 | 102 | 14557 | 37.5 | cholestane | 85850 I 1.04 | 89284 ~| 83284 weightfeces 10.4 mg coprostanoi coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 508256 15011 13004 7404 2781 19988 response areacorrected lector for responsefactor 10 10 0.98 498091 0.91 13660 1 13004 1.56 11550 1.49 4144 1.47 29382 10 % 0.0 0.0 87.4 2.4 2.3 2.0 0.7 5.2 0.0 pgsterol 0.0 0.0 131.0 3.6 3.4 3.0 1.1 7.7 0.0 ngsterol/ nmol sterol/ mgfeces mgfeces 0 0.00 0 0.00 12591 345 329 32.56 0.89 0.85 292 0.73 105 0.25 743 1.79 0 0.00 total I 566444 I I S69831 I 100 | 150 I 14405 | 37.1 | cholestane | 91434 | 1.04 | 95091 | weightfeces 6.8 mg area response area corrected % pgsterol ngsterol/ nmol sterol/ factor forresponsefactor mqfeces mgfeces coprostanoi 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol 292666 0.98 286813 86.8 89.9 13215 34.17 cholestanol 8651 0.91 7872 2.4 2.5 363 0.93 lathosterol 9600 1 9600 2.9 3.0 442 1.14 campesterol 4193 1.56 6541 2.0 2.0 301 0.75 stigmasterol 1436 1.49 2140 0.6 0.7 99 0.24 b-stosterol 11876 1.47 17458 5.3 5.5 804 1.94 b-sitostanol 1 0 0.0 0.0 0 0.00 tota! I 328422 | | 330424 | 100 | 104 | 15225 | 39,2 | cholestane [ 76721 II 1 79790 1 79790 coprostanoi coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 178 04-12-2006 /n Page A84 of A172 Appendix to TNO-report of 3M-02____________ Final version weight feces 6.1 mg area response area corrected % M9sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol coprostanon 1 0 0.0 0.0 0 0.00 1 0 0.0 0,0 0 0.00 cholesterol 440429 0.98 431620 87.6 73.9 12121 31.34 cholestanoi lathosterol 13442 11803 0.91 1 12232 11803 25 2.1 344 0.88 2.4 z o 331 0.86 campesterol 6241 1.56 9736 2.0 1.7 273 0.68 stigmasterol 2114 1.49 3150 0.6 0.5 88 0.21 b-sitosterol b-sitostanol 16460 1.47 1 24196 0 4.9 4.1 679 1.64 0.0 0.0 0 0.00 total I 490489 I | 492738 I 100 | 84 | 13837 | 35.6 | cholestane | 140330 I 1.04 | 145943 145943 1 coprostanol coprostanon cholesterol cholestanoi lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 237491 7441 6444 3777 962 10254 response area corrected factor for response factor 10 10 0.98 232741 0.91 6771 1 6444 1.56 5892 1.49 1463 1.47 15073 10 % 0.0 0.0 86.7 2.5 2.4 2.2 0.5 5.6 0.0 pg sterol 0.0 0.0 55.6 1.6 1.5 1.4 0.3 3.6 0.0 ng sterol/ mg feces 0 0 10481 305 290 265 66 679 0 nmol sterol/ mg feces 0.00 0.00 27.10 0.78 0.75 0.66 0.16 1.64 0.00 total | 266389 | | 268385 | 100 " f .........64 | '"12086.....|" 31."j.........| cholestane | 100714 | 1,04 | 104743 104743 | coprostanol coprostanon cholesterol cholestanoi lathosterol campesterol stigmasterol b-srtosteroi b-sitostanol area 404372 11102 11373 5436 2003 14146 response area corrected factor for response factor 10 10 0.98 396285 0.91 10103 1 11373 1.56 8480 1.49 2984 1.47 20795 10 % 0.0 0.0 88.1 2.2 2.5 1.9 0.7 4.6 0.0 total 448432 450020 100 cholestane 131190 1.04 136438 136438 pg sterol 0.0 0.0 72.6 1.9 2.1 1.6 0.5 3.8 0.0 ng sterol/ mq feces 0 0 10838 276 311 232 82 569 0 nmol sterol/ mg feces 0.00 0.00 28.03 0.71 0.80 0.58 0.20 1.37 0.00 82 12307 3T7 ! coprostanol coprostanon cholesterol cholestanoi lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 179 04-12-2006 Page A85of A172 m Appendix to TNO-report of 3M-02____________ Final version weight feces 9.1 mg area response areacorrected % pgsterol ngsterol/ nmol sterol/ factor for responsefactor mgfeces mgfeces coprostanol 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol 473290 0.98 463824 88.5 123.6 13579 35.12 cholestanol 9709 0.91 8835 1.7 2.4 259 0.67 lathosterol 12754 1 12754 2.4 3.4 373 0.97 campesterol 6031 1.56 9408 1.8 2.5 275 0.69 stigmasterol 2406 1.49 3585 0.7 1.0 105 0.25 b-sitosterol 17298 1.47 25428 4.9 6.8 744 1.80 b-sitostanol 1 0 0.0 0.0 0 0.00 total | 521488 | | 523835 | 100 | 140 | 15336 | 39.5 | cholestane | 90227 | 1.04 | 93836 1 :2-5 weight feces 7.2 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 446804 6124 14209 4560 1811 12767 response area corrected factor for responsefactor 10 10 0.98 437868 0.91 5573 1 14209 1.56 7114 1.49 2698 1.47 18767 10 % 0.0 0.0 90.1 1.1 2.9 1.5 0.6 3.9 0.0 total | 486275 | [ 486229 | 100 cholestane I 92255 | 1.04 j 95945 95945 | pg sterol 0.0 0.0 114.1 1.5 3.7 1.9 0.7 4.9 0.0 ngsterol/ nmol sterol/ mgfeces mgfeces 0 0.00 0 0.00 15846 40.98 202 0.52 514 1.33 257 0.64 98 0.24 679 1.64 0 0.00 127 | 17596 | 45.3 | weight feces 6.2 mg area response area corrected % pgsterol ngsterol/ nmol sterol/ factor forresponsefactor mgfeces mgfeces coprostanol 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol 298782 0.98 292806 84.8 65.1 10502 27.16 cholestanol 11596 0.91 10552 3.1 2.3 378 0.97 lathosterol 8473 1 8473 2.5 1.9 304 0.79 campesterol 5240 1.56 8174 2.4 1.8 293 0.73 stigmasterol 1956 b-sitosterol 15108 1.49 1.47 2914 22209 0.8 0.6 105 0.25 6.4 4.9 797 1.92 b-sitostanol 1 0 0.0 0.0 0 0.00 total | 341155 | | 345129 | 100 | 77 '[...12379 | 31.8 | cholestane | 108095 | 1.04 | 112419 112419 | coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanot 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 180 04-12-2006 Page A86of A172 Appendix to TNO-report of 3M-02____________ Final version weight feces 8.6 mg coprostanol coprostanon cholesterol choiestanol lathosterol campesterol stigmasterol b-sitosterol b-sltostanol area 481673 11693 12978 6203 1175 17089 response area corrected factor for response factor 10 10 0.98 472040 0.91 10641 1 12978 1.56 9677 1.49 1751 1.47 25121 10 % 0.0 0.0 88.7 2.0 2.4 1.8 0.3 4.7 0.0 pg sterol 0.0 0.0 118.2 2.7 3.3 2.4 0.4 6.3 0.0 ng sterol/ mg feces 0 0 13750 310 378 282 51 732 0 nmol sterol/ mg feces 0.00 0.00 35.56 0.80 0.98 0.70 0.12 1.76 0.00 total I 530811 I I 532206 | 100 | 133 | 15502 | 39.9 | cholestane | 9S962 | 1.04*~T 99800 99800 I weight feces 9.9 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol coprostanon cholesterol choiestanol lathosterol 434117 8812 13243 1 1 0.98 0.91 1 0 0 425435 8019 13243 0.0 0.0 0 0.0 0.0 0 88.8 152.2 15373 1.7 2.9 290 2.8 4.7 479 0.00 0.00 39.75 0.75 1.24 campesterol stigmasterol 5232 2068 1.56 1.49 8162 3081 1.7 2.9 295 0.74 0.6 1.1 111 0.27 b-sitosterol b-sitostanoi 14440 1.47 1 21227 0 4.4 7.6 767 1.85 0.0 0.0 0 0.00 tota! I 477912 | | 479167 | 100 | 171 | 17315 | 44.6 | cholestane | 6796 | 1.04 | 69884 69884 | coprostanol coprostanon cholesterol choiestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 268071 10152 6784 4476 1045 12736 response area corrected factor for response factor 10 10 0.98 262710 0.91 9238 1 6784 1.56 6983 1.49 1557 1.47 18722 10 % 0.0 0.0 85.9 3.0 2.2 2.3 0.5 6.1 0.0 total 303264 305993 100 cholestane [ 87400 II 1-0 I 90896 90896 1 pg sterol 0.0 0.0 72.3 2.5 1.9 1.9 0.4 5.1 0.0 84 ng sterol/ nmol sterol/ mg feces mg feces 0 0.00 0 0.00 12043 31.14 423 1.09 311 0.80 320 0.80 71 0.17 zzm858 0 14027 2.07 0.00 coprostanol coprostanon cholesterol choiestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 181 04-12-2006 ns Page A87 of A172 Appendix to TNO-report of 3M-02____________ Final version weight feces 5.6 mg coprostanol coprastanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 269271 7132 7058 3976 1401 10601 response area corrected factor for response factor 10 10 0.98 263886 0.91 6490 1 7058 1.56 6203 1.49 2087 1.47 15583 10 % 0.0 0.0 87.6 2.2 2.3 2.1 0.7 5.2 0.0 pg sterol 0.0 0.0 66.6 1.6 1.8 1.6 0.5 3.9 0.0 ng sterol/ mg feces 0 0 11896 293 318 280 94 702 0 nmol sterol/ mg feces 0.00 0.00 30.76 0.75 0.82 0.70 0.23 1.69 0.00 total I 299439 | j 301307 | 100 | 76 | 13582 I 35.0 | cholestane | 95225 | 1.04 | :2-5 weight feces 6.9 mg 99034 99034 1 coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 398275 7905 11474 4821 1507 11865 response area corrected factor for response factor 10 10 0.98 390310 0.91 7194 1 11474 1.56 7521 1.49 2245 1.47 17442 10 % 0.0 0.0 89.5 1.6 2.6 1.7 0.5 4.0 0.0 pg sterol 0.0 0.0 79.9 1.5 2.3 1.5 0.5 3.6 0.0 ng sterol/ mg feces 0 0 11581 213 340 223 67 518 0 nmol sterol/ mg feces 0.00 0.00 29.95 0.55 0.88 0.56 0.16 1.25 0.00 total I 435647 | I cholestane | 117414 | 1.04 ~T 436185 112222111111 I 100 | 89 j 12942 I 33~3 I weight feces area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol 336997 0.98 330257 86.2 80.7 11206 26.98 cholestanol 12422 0.91 11304 2.9 2.8 384 0.99 lathosterol 7963 1 7963 2.1 1.9 270 0.70 campesterol 5484 1.56 8555 2.2 2.1 290 0.72 stigmasterol 2009 1.49 2993 0.8 0.7 102 0.25 b-sitoslerol 15075 1.47 22160 5.8 5.4 752 1.81 b-sitostanol 1 0 0.0 0.0 0 0.00 total | 379950 | | 383233 | 100 | 94 | 13003 | 33.4 ~~1 cholestane [ 98396 |I ^ I 102332 102332 | coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 182 04-12-2006 Page A88 of A172 Appendix to TNO-report of 3M-02____________ Final version weight teces 6 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanoi coprostanon cholesterol cholestanol lathosterol 240788 7267 9283 1 1 0.98 0.91 1 0 0 235972 6613 9283 0.0 0.0 0 0.00 0.0 0.0 0 0.00 85.5 67.4 11229 29.04 2.4 1.9 315 0.81 3.4 2.7 442 1.14 campesterol 3808 1.56 5940 2.2 1.7 283 0.71 stigmasterol 1488 1.49 2217 0.8 0.6 106 0.26 b-sitostefol b-sitostanol 10778 1.47 1 15844 0 5.7 4.5 754 1.82 0.0 0.0 0 0.00 total | 273412 | | 275869 | 100 | 79 | 13128 | 33.8 | chotestane II----8-4-1-9-1---- 1I----1-.0--4---I1--------8-7-5-5-9--------I weight feces 7 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanoi 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol 283543 0.98 277872 85.5 71.7 10245 26.49 cholestanol 10969 0.91 9982 3.1 2.6 368 0.95 lathosterol 8285 1 8285 2.5 2.1 305 0.79 campesterol 4663 1.56 7274 2.2 1.9 268 0.67 stigmasterol b-s&osterol b-sitostanol 1774 12953 1.49 1.47 1 2643 19041 0 0.8 0.7 97 0.24 5.9 4.9 702 1.69 0.0 0.0 0 0.00 total | 322187 | | 325097 | 100 | 84 I 11986 | 30.8 | cholestane | 93139 | 1.04 | 96865 96865 1 area response area corrected factor for response factor % pg sterol ng sterol/ nmol sterol/ mg feces mg feces coprostanoi 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol 529012 0.98 518432 89.0 153.4 14205 36.73 cholestanol 14038 0.91 12775 2.2 3.8 350 0.90 lathosterol 11702 1 11702 2.0 3.5 321 0.83 campesterol 6647 1.56 10369 1.8 3.1 284 0.71 stigmasterol 1317 1.49 1962 0.3 0.6 54 0.13 b-sitosterol b-sitostanol 18688 1.47 1 27471 0 4.7 8.1 753 0.0 0.0 0 1.82 0.00 total | 581404 | | 582711 | 100 | 172 | 15966 I 41.1 | chotestane f 81233 |I V M 1 84482 84482 | coprostanoi coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 386.7 386.7 400.7 412.7 414.7 416.7 p. 183 04-12-2006 Page A89 of A172 m Appendix to TNO-report of 3M-02____________ Final version weight feces 8 mg coprostanol coprostanon cholesterol cholestartol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 330311 9369 11675 5157 1936 14114 response area corrected factor for response factor 10 10 0.98 323705 0.91 8526 1 11675 1.56 8045 1.49 2885 1.47 20748 10 % 0.0 0.0 86.2 2.3 3.1 2.1 0.8 5.5 0.0 yg sterol 0.0 0.0 95.0 2.5 3.4 2.4 0.8 6.1 0.0 ng sterol/ mg feces 0 0 11875 313 428 295 106 761 0 nmol sterol/ mg feces 0.00 0.00 30.71 0.80 1.11 0.74 0.26 1.84 0.00 total I 372562 | | 375583 | 100 j 110 I 1377B | 35.4 | cholestane I 81910 | 1.04 | 85186 ~1 weight feces 7.3 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol coprostanon 1 0 0.0 0.0 0 0.00 1 0 0.0 0.0 0 0.00 cholesterol cholestanol lathosterol 361988 10955 10492 0.98 0.91 1 354748 9969 10492 87.7 89.9 12322 31.86 2.5 2.5 346 0.89 2.6 2.7 364 0.94 campesterol stigmasterol b-sitosterol b-sltostanol 4873 1595 13274 1.56 1.49 1.47 1 7602 2377 19513 0 1.9 1.9 264 0.66 0.6 0.6 83 0.20 4.8 4.9 678 1.63 0.0 0.0 0 0.00 tota! I 403177 | I 404701 | 100 | 103 | 14057 | 36.2 | chiestane | 94804 | 1.04 | 98596 | weight feces 7.7 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 409014 11289 9106 5174 1042 14716 response area corrected factor for response factor 10 10 0.98 400834 0.91 10273 1 9106 1.56 8071 1.49 1553 1.47 21633 10 % 0.0 0.0 86.8 2.3 2.0 1.8 0.3 4.8 0.0 total 450341 451469 100 cholestane | 88054 1.04 91576 91576 pg sterol 0.0 0.0 109.4 2.8 2.5 2.2 0.4 5.9 0.0 ng sterol/ mg feces 0 0 14211 364 323 286 55 767 0 nmol sterol/ mg feces 0.00 0.00 36.75 0.94 0.83 0.71 0.13 1.85 0.00 123 16006 41*2 i coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 184 04-12-2006 PageA90 of A172 Appendix to TNO-report of 3M-02____________ Final version weightfeces 8.2 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol coprostanon 1 0 0.0 0.0 0 0.00 1 0 0.0 0.0 0 0.00 cholesterol 428635 cholestanol 12166 0.98 0.91 420062 11071 87.0 95.1 11598 29.99 2.3 2.5 306 0.79 lathosteral 13406 1 13406 2.8 3.0 370 0.96 campesterol 6496 1.56 10134 2.1 2.3 280 0.70 stigmasterol 2441 1.49 3637 0.8 0.8 100 0.24 b-sitosterol b-sitostanol 16860 1.47 1 24784 0 5.1 5.6 684 0.0 0.0 0 1.65 0.00 total I 480004 | | 483094 | 100 | 109 | 13338 | 34.3 | cholestane I 106174 | 1.04 | 110421 110421 I weight feces 7.6 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol coprostanon 1 0 0.0 0.0 0 0.00 1 0 0.0 0.0 0 0.00 cholesterol 370433 0.98 363024 86.3 74.7 9828 25.41 cholestanol 13556 0.91 12336 2.9 2.5 334 0.86 lathosterol 9317 1 9317 2.2 1.9 252 0.65 campesterol 6050 1.56 9438 2.2 1.9 256 0.64 stigmasterol 2043 1.49 3044 0.7 0.6 82 0.20 b-sitosterol b-sitostanol 15825 1.47 1 23263 0 5.5 4.8 630 1.52 0.0 0.0 0 0.00 total I 417224 j I 420422 | 100 | 87 | ..'38 2 | 29,3 | cholestane I 116833 I 1.04 j 121506 ~ | coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 327522 9565 7411 4458 1015 11869 response area corrected factor for response factor 10 10 0.98 320972 0.91 8704 1 7411 1.56 6954 1.49 1512 1.47 17447 10 % 0.0 0.0 88.4 2.4 2.0 1.9 0.4 4.8 0.0 total 361840 363001 100 cholestane 108272 1.04 T2&3 112603 pg sterol 0.0 0.0 71.3 1.9 1.6 1.5 0.3 3.9 0.0 ng sterol/ mg feces 0 0 11877 322 274 257 56 646 0 nmol sterol/ mg feces 0.00 0.00 30.71 0.83 0.71 0.64 0.14 1.56 0.00 81 13432 34.6 coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 P- 185 04-12-2006 PageA91 ofA172 m Appendix to TNO-report of 3M-02____________ Final version o w eight fe ce s 5.9 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 362101 9381 7899 4622 1882 12850 response area corrected factor for response factor 10 10 0.98 354859 0.91 8537 1 7899 1.56 7210 1.49 2804 1.47 18890 10 % 0.0 0.0 88.7 2.1 2.0 1.8 0.7 4.7 0.0 pg sterol 0.0 0.0 81.6 2.0 1.8 1.7 0.6 4.3 0.0 ng sterol/ mg feces 0 0 13829 333 308 281 109 736 0 nmol sterol/ mg feces 0.00 0.00 35.76 0.86 0.80 0.70 0.26 1.78 0.00 total I 398735 I I 400199 I 100 | 92 I 15595 | 40.2 | cholestane I 104552 I 1.04 I 108734 108734 weight feces 8 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sltosterol b-sitostanol area 489702 15062 10888 4869 748 13552 response area corrected factor for response factor 10 10 0.98 479908 0.91 13706 1 10888 1.56 7596 1.49 1115 1.47 19921 10 % 0.0 0.0 90.0 2.6 2.0 1.4 0.2 3.7 0.0 pg sterol 0.0 0.0 107.0 3.1 2.4 1.7 0.2 4.4 0.0 ng sterol/ mg feces 0 0 13380 382 304 212 31 555 0 nmol sterol/ mg feces 0.00 0.00 34.60 0.98 0.78 0.53 0.08 1.34 0.00 total I 534821 | | 533134 | 100 | 119 | 14864 | 38.3 I cholestane | __________ 2 weight feces 107776 | 1.04 | _________ 6.1 mg 112087 112087 | coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 459088 15039 11182 4797 686 13085 response area corrected factor for response factor 10 10 0.98 449906 0.91 13685 1 11182 1.56 7483 1.49 1022 1.47 19235 10 % 0.0 0.0 89.5 2.7 2.2 1.5 0.2 3.8 0.0 total 503877 502514 100 (M estane [ 111810 1 1 116282 I 116282 pg sterol 0.0 0.0 96.7 2.9 2.4 1.6 0.2 4.1 0.0 ng sterol/ mg feces 0 0 15857 482 394 264 36 678 0 nmol sterol/ mg feces 0.00 0.00 41.01 1.24 1.02 0.66 0.09 1.63 000 108 17711 456 coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 186 04-12-2006 Page A92of A172 Appendix to TNO-report of 3M-02 Final version weight feces 7.7 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanoi 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol 521217 0.98 510793 90.7 142.2 18473 47.77 cbolestanoi 14915 0.91 13573 2.4 3.8 491 1.26 iathosterol 11055 1 11055 2.0 3.1 400 1.03 campesterei 4779 1.56 7455 1.3 2.1 270 0.67 starnasterol 743 1.49 1107 0.2 0.3 40 0.10 b-sitosterol 13109 1.47 19270 3.4 5.4 697 1.68 b-sitostanol 1 0 0.0 0.0 0 0.00 total I 565818 | | 563253 | 100 | 157 | 20370 | S2.S | choiestane | 86322 | 1.04 | 8977S | weight feces 6.4 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanoi coprostanon cholesterol cholestanol Iathosterol cam pesterei stigmasterol b-sitosterol b-sitostanol 497491 13351 10137 4786 727 12456 1 1 0.98 0.91 1 1.56 1.49 1.47 1 0 0 487541 12149 10137 7466 1083 18310 0 0.0 0.0 0 0.00 0.0 0.0 0 0.00 90.8 118.8 18567 48.01 2.3 3.0 463 1.19 1.9 2.5 386 1.00 1.4 1.8 284 0.71 0.2 0.3 41 0.10 3.4 4.5 697 1.68 0.0 0.0 0 0.00 total I 538948 | | 536687 | 100 | 131 | 20439 | 52~ | choiestane I 98627 | 1.04 __________________________ 2:4-12 weight feces 8.9 mg | 102572 102572 I coprostanoi coprostanon cholesterol cholestanol Iathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 572090 15045 12013 5734 676 15286 response area corrected factor for response factor 10 10 0.98 560648 0.91 13691 1 12013 1.56 8945 1.49 1007 1.47 22470 10 % 0.0 0.0 90.6 2.2 1.9 1.4 0.2 3.6 0.0 pg sterol 0.0 0.0 171.6 4.2 3.7 2.7 0.3 6.9 0.0 ng sterol/ mg feces 0 0 19286 471 413 308 35 773 0 nmol sterol/ mg feces 0.00 0.00 49.87 1.21 1.07 0.77 0.08 1.86 0.00 total 620844 1 1 618775 | I T9 r"2 1 2 8 5 I 549 ! choiestane 78519 I 1.04 1 81660 81660 ! coprostanoi coprostanon cholesterol cholestanol Iathosterol campesterol stigmasterof b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 04-12-2006 Page A93 of A172 Appendix to TNO-report of 3M-02 Final version 0 weight feces 6.8 mg coprostanol coprostanon cholesterol choiestanol lathosterol campesterol stigmasterol b-sitosterol b-sKostanol area 434209 12431 9196 4448 651 11421 response area corrected factor for response factor 10 10 0.98 425525 0.91 11312 1 9196 1.56 6939 1.49 970 1.47 16789 10 % 0.0 0.0 90.4 2.4 2.0 1.5 0.2 3.6 0.0 pg sterol 0.0 0.0 89.6 2.4 1.9 1.5 0.2 3.5 0.0 ng sterol/ mg feces 0 0 13179 350 285 215 30 520 0 nmol sterol/ mg feces 0.00 0.00 34.08 0.90 0.74 0.54 0.07 1.25 0.00 total I 472356 | | 470731 | 100 | 99 | 14579 | 37.6 | cholestane | 114142 | 1.04 | 118708 118708 | weight feces 6.8 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol coprostanon cholesterol choiestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 690765 15497 14645 6317 805 16234 1 1 0.98 0.91 1 1.56 1.49 1.47 1 0 0 676950 14102 14645 9855 1199 23864 0 0.0 0.0 0 0.00 0.0 0.0 0 0.00 91.4 122.7 18042 46.66 1.9 2.6 376 0.97 2.0 2.7 390 1.01 1.3 1.8 263 0.66 0.2 0.2 32 0.08 3.2 4.3 636 1.53 0.0 0.0 0 0.00 total | 744263 | | 740615 | 100 I 134 I 19739 (... 50.9 | cholestane | 13263 | 1.04 | 137944 137944 | w ig h t feces 7.7 mg coprostanol coprostanon cholesterol choiestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 657017 16315 13781 6401 812 16291 response area corrected factor for response factor 10 10 0.98 643877 0.91 14847 1 13781 1.56 9986 1.49 1210 1.47 23948 10 % 0.0 0.0 91.0 2.1 1.9 1.4 0.2 3.4 0.0 total 710617 707648 100 cholestane [ 127109 I 1.04 | 132193 132193 | pg sterol 0.0 0.0 121.8 2.8 2.6 1.9 0.2 4.5 0.0 ng sterol/ mg feces 0 0 15814 365 338 245 30 588 0 nmol sterol/ mg feces 0.00 0.00 40.89 0.94 0.88 0.61 0.07 1.42 0.00 134 17380 44.8 coprostanol coprostanon cholesterol choiestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 04-12-2006 /? Page A94 of A172 Appendix to TNO-report of 3M-02____________ Final version weight feces 7.8 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor fo r response factor mg feces mg feces coprostanol coprostanon 1 0 0.0 0.0 0 0.00 1 0 0.0 0.0 0 0.00 cholesterol 482166 cholestanol 15148 0.98 0.91 472523 13785 89.1 129.2 16561 42.83 2.6 3.8 483 1.24 lathosterol 13001 1 13001 2.5 3.6 456 1.18 campesterol 5131 1.56 8004 1.5 2.2 281 0.70 stigmasterol 1060 1.49 1579 0.3 0.4 55 0.13 b-sitosterol b-sitostanol 14512 1.47 1 21333 0 4.0 5.8 748 0.0 0.0 0 1.80 0.00 1total I 531018 c 530225 I 100 I 145 1 18583 | 47.9 I chotestane I 87934 1.04 91451 91451 weight feces 13.4 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 634279 12599 16167 6696 2927 18799 response area corrected factor for response factor 10 10 0.98 621593 0.91 11465 1 16167 1.56 10446 1.49 4361 1.47 27635 10 % 0.0 0.0 89.9 1.7 2.3 1.5 0.6 4.0 0.0 pg sterol 0.0 0.0 187.2 3.5 4.9 3.1 1.3 8.3 0.0 ng sterol/ mg feces 0 0 13972 258 363 235 98 621 0 nmol sterol/ mg feces 0.00 0.00 36.13 0.66 0.94 0.59 0.24 1.50 0.00 tota! I 691467 | | 691667 | 100 | 208 | 15547 | 40.1 | chotestane | 79811 | 1.04 | | 13-15 weight feces ........ j 6.2 mg 83003 83003 I area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol 1 0 0.0 0.0 0 0.00 coprostanon cholesterol cholestanol 255399 13187 1 0.98 0.91 0 250291 12000 0.0 0.0 0 0.00 85.5 66.1 10669 27.59 4.1 3.2 512 1.32 lathosterol campesterai stigmasterol b-sitosterol b-sitostanol 6307 3843 729 11657 1 1.56 1.49 1.47 1 6307 5995 1086 17136 0 2.2 1.7 269 0.70 2.0 1.6 256 0.64 0.4 0.3 46 0.11 5.9 4.5 730 1.76 0.0 0.0 0 0.00 tota! I 291122 I I cholestane [ 90959 II 104 1 292815 94597 94597 | 100 | 77 I 12481 | 32.----- 1 1 coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 189 04-12-2006 Page A95of A172 Appendix to TNO-report of 3M-02 Final version weight feces 7.9 mg area response area corrected factor for response factor % pg sterol ng sterol/ nmol sterol/ mg feces mQfeces coprostanol 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol 455921 0.98 446803 89.0 124.6 15774 40.79 cholestanoi 13655 0.91 12426 2.5 3.5 439 1,13 lathosterol 12928 1 12928 2.6 3.6 456 1.18 campester! 5203 1.56 8117 1.6 2.3 287 0.72 stigmasterol 898 1.49 1338 0.3 0.4 47 0.11 b-sitosterol 13941 1.47 20493 4.1 5.7 723 1.74 b-sitostanol 1 0 0.0 0.0 0 0.00 total I 502546 I I 502105 | 100 | 140 | 17726 | 45.7 | choiestane | 86190 | 1.04 | 89638 | weight feces 6.9 mg area response area corrected % Mgsterol ng sterol/ nmol sterol/ factor for response factor mg feces mq feces coprostanol coprostanon 1 0 0.0 0.0 0 0.00 1 0 0.0 0.0 0 0.00 cholesterol cholestanoi lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 302926 6271 8314 3149 541 8475 0.98 0.91 1 1.56 1.49 1.47 1 296867 5707 8314 4912 806 12458 0 90.2 101.4 14697 38.01 1.7 1.9 283 0.73 2.5 2.8 412 1.06 1.5 1.7 243 0.61 0.2 0.3 40 0.10 3.8 4.3 617 1.49 0.0 0.0 0 0.00 total I 329676 | | 329065 | 100 T " " " '2 | 16291 I 42. | choiestane 70369 2:5-15weight feces 4.8 1.04 mg 73184 73184 coprostanol coprostanon cholesterol cholestanoi lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 210645 13493 5461 3832 733 10573 response area corrected factor for response factor 10 10 0.98 206432 0.91 12279 1 5461 1.56 5978 1.49 1092 1.47 15542 10 % 0.0 0.0 83.6 5.0 2.2 2.4 0.4 6.3 0.0 pg sterol 0.0 0.0 52.2 3-1 1.4 1.5 0.3 3.9 0.0 ng sterol/ mq feces 0 0 10870 647 288 315 58 818 0 nmol sterol/ mq feces 0.00 0.00 28.11 1.66 0.74 0.79 0.14 1.97 0.00 tota! I 244737 | | 246784 I 100 | 62 | 12996 | 33.4 | choiestane [ 95106 II 1 98910 98910 I coprostanol coprostanon cholesterol cholestanoi lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 04-12-2006 Page A96 of A172 Appendix to TNO-report of 3M-02 Final version weight feces 11.8 mg coprostand coprostanon cholesterol cholestanol lathosterol campesteroi stigmasterol b-sitosterol b-sitostanol area 760655 21051 19855 9056 3585 24867 response area corrected factor for response factor 10 10 0.98 745442 0.91 19156 1 19855 1.56 14127 1.49 5342 1.47 36554 10 % 0.0 0.0 88.7 2.3 2.4 1.7 0.6 4.3 0.0 pg sterol 0.0 0.0 146.6 3.8 3.9 2.8 1.1 7.2 0.0 ng sterol/ mg feces 0 0 12425 319 331 235 89 609 0 nmoi sterol/ mg feces 0.00 0.00 32.13 0.82 0.86 0.59 0.22 1.47 0.00 total | 839069 | I 840477 | 100 | 165 I 14009 | 36.1 | cholestane | 122218 | 1.04 | 127107 weight feces 7.2 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesteroi stigmasterol b-sitosterol b-sitostanol area 475539 8894 11319 4936 2133 13417 response area corrected factor fo r response factor 10 10 0.98 466028 0.91 8094 1 11319 1.56 7700 1.49 3178 1.47 19723 10 % 0.0 0.0 90.3 1.6 2.2 1.5 0.6 3.8 0.0 pg sterol 0.0 0.0 88.3 1.5 2.1 1.5 0.6 3.7 0.0 ng sterol/ mg feces 0 0 12257 213 298 203 84 519 0 nmol sterol/ mg feces 0.00 0.00 31.70 0.55 0.77 0.51 0.20 1.25 0.00 total I 516238 | | 516042 | 100 I 98 i 13573 | 35.0 | cholestane | 126938 1 1.04 | 132016 | 3:5-15 weight feces 7.1 rW & fr ; mg coprostanol coprostanon cholesterol cholestanol lathosterol campesteroi stigmasterol b-sitosterol b-sitostanol area 369324 21096 8285 6116 1212 15819 response area corrected factor for response factor 10 10 0.98 361938 0.91 1 1.56 1.49 1.47 1 19197 8285 9541 1806 23254 0 % 0.0 0.0 85.4 4.5 2.0 2.3 0.4 5.5 0.0 total 421852 424021 | 100 cholestane [ 139834 | i 145427 | 145427 pg stenol 0.0 0.0 62.2 3.3 1.4 1.6 0.3 4.0 0.0 ng sterol/ mg feces 0 0 8763 465 201 231 44 563 0 nmol sterol/ mg feces 0.00 0.00 22.66 1.20 0.52 0.58 0.11 1.36 0.00 73 10267 26.4 coprostanol coprostanon cholesterol cholestanol lathosterol campesteroi stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 04-12-2006 Page A97 of A172 Appendix to TNO-report of 3M-02 Final version weight feces 5.5 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 316760 10679 7883 3863 573 10951 response area corrected factor for response factor 10 10 0.98 310425 0.91 9718 1 7883 1.56 6026 1.49 854 1.47 16098 10 % 0.0 0.0 88.4 2.8 2.2 1.7 0.2 4.6 0.0 pg sterol 0.0 0.0 75.3 2.4 1.9 1.5 0.2 3.9 0.0 ng sterol/ mg feces 0 0 13682 428 347 266 38 710 0 nmol sterol/ mg feces 0.00 0.00 35.38 1.10 0.90 0.66 0.09 1.71 0.00 total j 350709 | | 351004 | 100 | 85 | " 15471 | 39.8 | choiestane | 99161 | 1.04 | 103127 | 1 :6-1T weight feces 5.1 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol coprostanon cholesterol cholestanol lathosterol 283717 12638 7544 1 1 0.98 0.91 1 0 0 278043 11501 7544 0.0 0.0 0 0.00 0.0 0.0 0 0.00 86.7 61.2 12006 31.05 3.6 2.5 497 1.28 2.4 1.7 326 0.84 campesterol stigmasterol b-sitosterol b-sitostanol 4033 593 11162 1.56 1.49 1.47 1 6291 884 16408 0 2.0 1.4 272 0.68 0.3 0.2 38 0.09 5.1 3.6 709 1.71 0.0 0.0 0 0.00 tot I 319687 j 320670 100 71 I 13847 I 35.6" ~ 1 choiestane | 109156 | 1.04 113522 113522 coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 432750 15694 11216 5531 828 15866 response area corrected factor for response factor 10 10 0.98 424095 0.91 14282 1 11216 1.56 8628 1.49 1234 1.47 23323 10 % 0.0 0.0 87.8 3.0 2.3 1,8 0.3 4.8 0.0 total 481885 482778 100 choiestane | 64015 Il 1.04 I 87376 87376 I pg sterol 0.0 0.0 121.3 4.1 3.2 2.5 0.4 6.7 0.0 ng sterol/ mg feces 0 0 13048 439 345 265 38 718 0 nmol sterol/ mg feces 0.00 0.00 33.74 1.13 0.89 0.66 0.09 1.73 0.00 138 14853 38.2 coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 04-12-2006 Page A98 of A172 Appendix to TNO-report of 3M-02 Final version weight feces 5.2 mg coprostanol coprostanon cholesterol cholestanol lathosterol cam pesterol stigm asterol b-sitosterol t>-sitostanol area 306320 10414 7025 3400 583 9578 response factor 1 1 0.98 0.91 1 1.56 1.49 1.47 1 area corrected for response factor 0 0 300194 9477 7025 5304 869 14080 0 % 0.0 0.0 89.1 2.8 2.1 1.6 0.3 4.2 0.0 pg sterol 0.0 0.0 71.3 2.2 1.7 1.3 0.2 3.3 0.0 ng sterol/ mg feces 0 0 13705 433 321 242 40 643 0 nmol sterol/ mg feces 0.00 0.00 35.44 1.11 0.83 0.60 0.10 1.55 0.00 total | 337320 | | 336948 | 100 | 80 | 15383 | 39.6 | cholestane I 101256 I 1.04 I 105306 weight feces 4.6 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor fo r response factor mg feces mg feces coprostanol coprostanon 1 0 0.0 0.0 0 0.00 1 0 0.0 0.0 0 0.00 cholesterol cholestanol lathosterol cam pesterol stigm asterol 264813 13978 6351 4076 392 0.98 0.91 1 1.56 1.49 259517 12720 6351 6359 584 86.0 58.0 12612 32.61 4.2 2.8 618 1.59 2.1 1.4 309 0.80 2.1 1.4 309 0.77 0.2 0.1 28 0.07 b-sitosterol b-sitostanol 11081 1.47 1 16289 0 5.4 3.6 792 1.91 0.0 0.0 0 0.00 total I 300691 | | 301819 | HX) I 67 | 14667 I 37.8 I cholestane I 107534 I 1.04 | 111835 111835 | weight feces 7.1 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor fo r response factor mg feces mg feces coprostanol coprostanon cholesterol 415098 1 1 0.98 0 0 406796 0.0 0.0 0 0.00 0.0 0.0 0 0.00 88.0 89.1 12553 32.46 cholestanol lathosterol cam pesterol stigm asterol b-sitosterol b-sltostanol 15248 11257 5140 790 14501 0.91 1 1.56 1.49 1.47 1 13876 11257 8018 1177 21316 0 3.0 3.0 428 2.4 2.5 347 1.7 1.8 247 0.3 0.3 36 4.6 4.7 658 0.0 0.0 0 1.10 0.90 0.62 0.09 1.59 0.00 total | 462034 | | 462441 | 100 | 101 | 14270 | 36.8 | cholestane [ 109718 | 1.04 | 114107 114107 | coprostanol coprostanon cholesterol cholestanol lathosterol cam pesterol stigm asterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 193 04-12-2006 Page A99of A172 Appendix to TNO-report of 3M-02 Final version weight feces 9.4 mg coprostanoi coproslanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 528030 15892 10925 5503 761 14410 response area corrected factor fo r response factor 10 10 0.98 517469 0.91 14462 1 10925 1.56 8585 1.49 1134 1.47 21183 10 % 0.0 0.0 90.2 2.5 1.9 1.5 0.2 3.7 0.0 pg sterol 0.0 0.0 120.9 3.4 2.6 2.0 0.3 4.9 0.0 ng sterol/ mg feces 0 0 12861 359 272 213 28 526 0 nmol sterol/ mo feces 0.00 0.00 33.26 0.92 0.70 0.53 0.07 1.27 0.00 total | 575521 I | 573757 | 100 | 134 | 14260 | 36.8 | cholestane I 102895 I 1.04 I 107011 107011 weight feces 5.2 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanoi 1 0 0.0 0.0 0 0.00 copiostanon 1 0 0.0 0.0 0 0.00 cholesterol 325132 0.98 318629 87.3 55.5 10671 27.59 cholestanol 15757 0.91 14339 3.9 2.5 480 1.24 lathosterol 7236 1 7236 2.0 1.3 242 0.63 campesterol stigmasterol b-sitosterol b-sitostanol 4495 611 11479 1.56 1.49 1.47 1 7012 910 16874 0 1.9 1.2 235 0.59 0.2 0.2 30 0.07 4.6 2.9 565 1.36 0.0 0.0 0 0.00 total I 364710 I | 365001 | 100 | 64 | 12224 | 3".'s l cholestane | 138038 | 1.04 | 143560 143560 I coprostanoi coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 716301 24577 18733 8508 1102 22755 response area corrected factor for response factor 10 10 0.98 701975 0.91 22365 1 18733 1,56 13272 1.49 1642 1.47 33450 10 % 0.0 0.0 88.7 2.8 2.4 1.7 0.2 4.2 0.0 total 791976 791437 100 cholestane | 174978 | 1.04 | 181977 181977 | pg sterol 0.0 0.0 96.4 3.1 2.6 1.8 0.2 4.6 0.0 ng sterol/ mg feces 0 0 13976 445 373 264 33 666 0 nmol sterol/ mg feces 0.00 0.00 36.14 1.15 0.96 0.66 0.08 1.61 0.00 109 15758 40.6 coprostanoi coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 04-12-2006 Page A100 of A172 Appendix to TNO-report of 3M-02____________ Final version weight feces 7.5 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol cholestanol lathosterol 449806 25289 7667 0.98 0.91 1 440810 23013 7667 86.5 98.4 13115 33.92 4.5 5.1 685 1.76 1.5 1.7 228 0.59 campesterol 6477 1.56 10104 2.0 2.3 301 0.75 stigmasterol 917 1.49 1366 0.3 0.3 41 0.10 b-sitosterol b-sitostanol 18151 1.47 1 26682 0 5.2 6.0 794 1.91 0.0 0.0 0 0.00 total j 508307 I ( 509642 | 100 | 114 | 15163 I 39.0 I cholestane | 107726 | 1.04 | 112035 | weight feces 10.8 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol coprostanon 1 0 0.0 0.0 0 0.00 1 0 0.0 0.0 0 0.00 cholesterol 497481 0.98 487531 85.5 119.2 11041 28.55 cholestanol lathosterol 30777 9362 0.91 1 28007 9362 4.9 6.9 634 1.63 1.6 2.3 212 0.55 campesterol stigmasterol b-sitosterol b-sitostanol 7714 1041 21441 1.56 1.49 1.47 1 12034 1551 31518 0 2.1 2.9 273 0.68 0.3 0.4 35 0.09 5.5 7.7 714 1.72 0.0 0.0 0 0.00 total I 567816 | | 570004 | 100 | 139 j 12909 | 33.2 1 cholestane 1 98281 | 1.04 | 102212 102212 | weight feces 8.5 mg area response area corrected % pg sterol ng sterol/ nmol sterol/ factor for response factor mg feces mg feces coprostanol 1 0 0.0 0.0 0 0.00 coprostanon 1 0 0.0 0.0 0 0.00 cholesterol 328489 0.98 321919 86.3 100.2 11792 30.49 cholestanol 18232 0.91 16591 4.4 5.2 608 1.56 lathosterol 5881 1 5881 1.6 1.8 215 0.56 campesterol 5028 1.56 7844 2.1 2.4 287 0.72 stigmasterol b-sitosterol b-sitostanol 654 13624 1.49 1.47 1 974 20027 0 0.3 0.3 36 5.4 6.2 734 0.0 0.0 0 0.09 1.77 0.00 total 371908 | | 373237 | 100 | m | 13671 35.2 cholestane 77207 | 1.04 | 80295 80295 | coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanot 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 195 04-12-2006 /f7 Page A101 OA172 Appendix to TNO-report of 3M-02____________ Final version weight feces 7.3 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 393028 19805 6961 5235 2749 13255 response area corrected factor for response factor 10 10 0.98 385167 0.91 18023 1 6961 1.56 8167 1.49 4096 1.47 19485 10 % 0.0 0.0 87.2 4.1 1.6 1.8 0.9 4.4 0.0 pg sterol 0.0 0.0 87.9 4.1 1.6 1.9 0.9 4.4 0.0 ng sterol/ mg feces 0 0 12046 564 218 255 128 609 0 nmol sterol/ mg feces 0.00 0.00 31.15 1.45 0.56 0.64 0.31 1.47 0.00 total j 441033 | | 441898 | 100 | 101 | 13820 | 35.6 | chotestane j 105292 | 1,04 | 109504 109504 | coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sltosterol b-sitostanol area 363884 22163 6943 5485 752 14149 response area corrected factor for response factor 10 10 0.98 356606 0.91 20168 1 6943 1.56 8557 1.49 1120 1.47 20799 10 % 0.0 0.0 86.1 4.9 1.7 2.1 0.3 5.0 0.0 pg sterol 0.0 0.0 91.7 5.2 1.8 2.2 0.3 5.4 0.0 ng sterol/ mg feces 0 0 13105 741 255 314 41 764 0 nmol sterol/ mg feces 0.00 0.00 33.89 1.91 0.66 0.78 0.10 1.84 0.00 total | 413376 | I 414194 | 100 | 107 | 15222 | 39.2 | cholestane | 93444 || 1.04 T 97182 | 97182 _____________________________________ t 2-7-21 ................................................ - - > :,n weight feces 6.9 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 399924 19224 7234 5395 2024 14143 response area corrected factor for response factor 10 10 0.98 391926 0.91 17494 1 7234 1.56 8416 1.49 3016 1.47 20790 10 % 0.0 0.0 87.3 3,9 1.6 1.9 0.7 4.6 0.0 pg sterol 0.0 0.0 88.6 4.0 1.6 1.9 0.7 4.7 0.0 ng sterol/ mg feces 0 0 12845 573 237 276 99 681 0 nmot sterol/ mg feces 0.00 0.00 33.22 1.48 0.61 0.69 0.24 1.64 0.00 total l 447944 | | 448876 | 100 | 102 | 14711 | 37.9 1 cholestane [ 106300 | 1.04 | 110552 110552 | coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 196 04-12-2006 Page A102of A172 Appendix to TNO-report of 3M-02____________ Final version weight feces 10 mg coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sltosterol b-sitostand area 582689 23901 10390 7529 942 19466 response area corrected factor for response factor 10 10 0.98 571035 0.91 21750 1 10390 1.55 11745 1.49 1404 1.47 28615 10 % 0.0 0.0 88.5 3.4 1.6 1.8 0.2 4.4 0.0 jig sterol 0.0 0.0 114.6 4.4 2.1 2.4 0.3 5.7 0.0 ng sterol/ mg feces 0 0 11457 436 208 236 28 574 0 nmol sterol/ mg feces 0.00 0.00 29.63 1.12 0.54 0.59 0.07 1.38 0.00 total I 644917 | I 644939 I i I i 129 1 12940 I 33.3 I T 124603 I 124603 weight feces coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol area 282329 16221 5485 3642 437 9568 response area corrected factor for response factor 10 10 0.98 276682 0.91 14761 1 5485 1.56 5682 1.49 651 1.47 14065 10 % 0.0 0.0 87.2 4.7 1.7 1.8 0.2 4.4 0.0 pg sterol 0.0 0.0 56.9 3.0 1.1 1.2 0.1 2.9 0.0 ng sterol/ mg feces 0 0 11609 619 230 238 27 590 0 nmol sterol/ mg feces 0.00 0.00 30.02 1.59 0.60 0.59 0.07 1.42 0.00 total I 317682 I 1 317326 | 100 | 65 I 13314 | 34.3 | chotestane | 116924 | 1.04 | 121601 | __________________________________ 121601____________________ jte .gr.: re c o s j .p rig coprostand coprostanon cholesterol cholestanol lathosterol campesterol stigmasterd b-sitosterol b-sitostanol area 486322 21163 7823 6293 622 16081 response area corrected factor for response factor 10 10 0.98 476596 0.91 19258 1 7823 1.56 9817 1.49 927 1.47 23639 10 % 0.0 0.0 88.6 3.6 1.5 1.8 0.2 4.4 0.0 total 538304 538060 100 chdestane | 103492 | I 107633 1 107632 pg sterol 0.0 0.0 110.7 4.5 1.8 2.3 0.2 5.5 0.0 ng sterol/ mg feces 0 0 11653 471 191 240 23 578 0 nmol sterol/ mg feces 0.00 0.00 30.13 1.21 0.49 0.60 0.05 1.39 0.00 125 13155 339 I coprostanol coprostanon cholesterol cholestanol lathosterol campesterol stigmasterol b-sitosterol b-sitostanol 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 388.7 386.7 386.7 388.7 386.7 400.7 412.7 414.7 416.7 p. 197 04-12-2006 Page A103ofA172 Appendix to TNO-report of 3M-02 ss s s Final version ! S3 ; S 3 ! SS 8S II i6 g II f s S 38 S S3 88 8 88 a Iff | li SS ;r 88 ss 8 88 38 SS 198 04-12-2006 3 SS S 8 8 ss s 3 Si. 8 8 83 S 88 Page A104 of A172 Appendix to TNO-report of 3M-02 Final version P s I 5 S3 38 S 83 3 33 3 8 8 883 88 "R a Si & il ii 8 p. 199 04-12-2006 88 8 83 S3 p> n 3 88 3 8 - t i3 Page A105 of A172 Appendix to TNO-report of 3M-02 Final version p. 200 04-12-2006 cholestanol lathosterol total phvtosterols campesterol stiqmasterol b-sitosterol b-sitostanol M H MF <0.001 <0.001 H M lS h <0.001 H B fS f 0.008 W H I-4 . 0.040 H H -.9 0 190 HUHeSh 1.000 Page A106ofA172 Appendix to TNO-report of 3M-02____________ Final version p. 201 04-12-2006 E4. Fecal fatty acid measurements weight faces 8.6 mg area % C14:0 C16:0 C16:1 018:0 C18:1 C18.2 C18;3 13257 256605 2203 231285 22933 1279 2731 2.5 48.4 0.4 43.6 4.3 0.2 0.5 total 530293 100 C15:0 I 76166 | yg fatty add 13.9 269.5 2.3 242.9 24.1 1.3 2.9 ng fatty add nmol fatty acid/ mg feces mg feces 1619 7.09 31340 122.23 269 1.06 28247 99.29 2801 9.91 156 0.56 334 1.20 557.0 64766 241.33 weight feces 7.6 mg area % C14:0 016:0 C16:1 018:0 018:1 C18:2 018:3 8193 210520 1596 214647 24494 1701 2635 1.8 45.4 0.3 46.3 5.3 0.4 0.6 total 463786 100 015:0 | 86874 | yg fatty add 7.5 193.9 1.5 197.7 22.6 1.6 2.4 ngfatty add nmol fatty add/ mg feces mg feces 993 4.35 25508 99.49 193 0.76 26008 91.42 2968 10.51 206 0.73 319 1.15 427.1 56196 m I weight feces 10.1 mg 014:0 016:0 016:1 C18:0 018:1 0182 C18:3 area % Mg fatty ng fatty add nmolfatty add/ add mg feces mg feces 16324 1.9 14.8 1465 6.42 404265 46.1 366.5 36288 141.53 3337 0.4 3.0 300 1.18 411875 47.0 373.4 36971 129.95 37807 4.3 34.3 3394 12.01 2492 0.3 2.3 224 0.80 1091 0.1 1.0 98 0.35 total | 877191 | 100 | 795.3 | >739 | 292.24 | C15:0 I 88241 I weight feces 5.7 mg 014:0 C16:0 C16:1 018:0 C18:1 C18:2 018:3 area % yg fatty ng fatty add nmol fatty add/ add mg feces mg feces 7613 1.8 6.7 1172 5.13 201403 46.8 176.8 31014 120.96 1437 0.3 1.3 221 0.87 196561 45.7 172.5 30268 106.39 19865 4.6 17.4 3059 10.83 1352 0.3 1.2 208 0.74 2147 0.5 1.9 331 1.19 total I ~430378 I 100 I 377.8....| ~66274 "| 248.11 | C15:0 I 91143 | weight feces 8.8 mg area % yg fatty ng fatty add nmolfattyadd/ add mg feces mg feces C14:Q 18959 1.7 14.2 1618 7.09 C16:0 482473 44.2 362.4 41183 160.62 C16:1 4337 0.4 3.3 370 1.46 018:0 520971 47.7 391.3 44469 156.30 C18:1 55946 5.1 42.0 4775 16.90 C18:2 3057 0.3 2.3 261 0.93 018:3 6151 0.6 4.6 525 1.89 total | 1091894 | 100 | 820.2 | 93201 | C15:0 I 106504 | 345.18 | weight feces 7.2 mg C14:0 C16:0 C16:1 C18:0 018:1 C18:2 C18:3 area % M9 fatty ng fatty acid nmol fatty add/ add mg feces mg feces 9801 1.4 9.1 1262 5.52 284195 41.3 263.4 36587 142.70 2725 0.4 2.5 351 1.38 348778 50.7 323.3 44901 157.83 35118 5.1 32.6 4521 16.00 2252 0.3 2.1 290 1.03 4534 0.7 4.2 584 2.10 tota| | 687403 | 100 | 637.2 I 88496 ...| 32666 I C15:0 I 86307 I mofw C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 228.4 256.4 254.4 284.5 282.5 280.5 278.5 MYSTRIC ACID PALMITIC ACID PALMITOLEIC ACID STEARIC ACID OLEIC ACID LINOLEIC ACID LINOLENIC ACID Page A107of A172 m Appendix to TNO-report of 3M-02____________ Final version p. 202 04-12-2006 weight feces 7.3 mg C14:0 C16:0 C16:1 C18:0 018:1 C18:2 C18:3 area % M9 fatty ngfettyacid nmolfatty add/ acid mgfeces mgfeces 5463 1.2 7.5 1023 4.48 156085 35.1 213.4 29233 114.01 1332 0.3 1.8 249 0.98 248855 56.0 340.2 46607 163.82 26880 6.0 36.8 5034 17.82 1566 0.4 2.1 293 1.05 4158 0.9 5.7 779 2.80 tota! I 444339 I 100 I 607.5 I 83219 I 304.96 | C15:0 I 58514 | weight feces 5.7 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % M9 fatty ng fatty add nmol fatty add/ add mg feces mg feces 7866 1.5 7.3 1287 5.63 223765 43.6 208.6 36598 142.74 1702 0.3 1.6 278 1.09 248550 48.4 231.7 40651 142.89 26968 S3 25.1 4411 15.61 1646 0.3 1.5 269 0.96 2844 0.6 2.7 465 1.67 total I 513341 I 100 | 478.6 | 83959 I 10.59 | C15;0 I 85813 I weight feces $.3 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % M9 fatty ngfettyacid nmol fatty add/ acid mgfeces mgfeces 11108 1.5 9.5 1150 5.03 319405 42.1 274.4 33063 128.95 2509 380522 0.3 50.1 2.2 326.9 260 39390 1.02 138.45 37736 5.0 32.4 3906 13.83 2443 0.3 2.1 253 5057 0.7 4.3 523 0.90 1.88 tola! | 758780 | 100 | 651.9 I 78546 | 290.07 I C1S:0 I 93112 I 1:2-4 weight feces 13.4 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 ares % pg fatty ng fatty add nmol fatty add/ add mgfeces mg feces 27893 1.2 20.7 1545 6.76 881387 39.5 654.1 48810 190.37 8053 0.4 6.0 446 1.75 1181866 53.0 877.0 65450 230.05 109775 4.9 81.5 6079 21.52 5394 0.2 4.0 299 1.06 17094 0.8 12.7 947 3.40 total | 2231462 I 100 I 1665.9 || 123575 | 454.92 | C15:0 I 107806 I 1: 2*5 weight feces 9.2 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C l 8:3 area % pg fatty ng fatty add reno!fatty add/ add mgfeces mg feces 31737 1.4 19.1 2081 9.11 923618 40.8 557.2 60560 236.19 93 0.0 0.1 6 0.02 1135022 50.2 684.7 74421 261.59 147397 6.5 88.9 9665 34.21 7383 0.3 4.5 484 1.73 16098 0.7 9.7 1056 3.79 total | 2261348 I 100 | 1364.1 | 148272 | 546.64 | C15:0 | 132620 | 1:2-6 weight feces 9 mg C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % pg fatty ngfettyacid nmol fatty add/ add mgfeces mg feces 10087 1.7 6.6 728 3.19 262021 45.2 170.1 18905 73.73 2302 0.4 1.5 166 0.65 272616 47.0 177.0 19669 69.14 27573 4.8 17.9 1989 7.04 1825 0.3 1.2 132 0.47 3059 0.5 2.0 221 0.79 total I 579483 I 100 | 376.3 | 41810 | 155.".... | C15:0 | 123199 | Page A108of A172 Appendix to TNO-report of 3M-02____________ Final version p. 203 04-12-2006 weight feces 9.7 mg C14:0 C16:0 C16:1 C18:0 C18:1 C182 C18:3 area % tig fatty ng fatty add nmol fatty acid/ acid mg feces mg feces 18615 1.6 15.3 1579 6.91 488224 42.0 401.8 41421 161.55 4357 0.4 3.6 370 1.45 581814 50.0 478.8 49361 173.50 60179 5.2 49.5 5106 18.07 3098 0.3 2.5 263 0.94 7073 0.6 5.8 600 2.15 tota! 1 1163360 | 100 | 957.4 I 96699 | 364,56 I 015:0 I 97212 1 3: 2-4 weight feces 8.6 mg C14:0 C16;0 C16:1 018:0 C18:1 C18:2 C18;3 area % pg fatty ng fatty add nmolfatty add/ acid mg feces mg feces 18311 1.7 15.1 1754 7.68 420511 39.9 346.4 40284 157.11 3649 0.3 3.0 350 1.37 545499 51.7 449.4 52257 183.68 56501 5.4 46.5 5413 19.16 3001 0.3 2.5 287 1.02 7025 0.7 5.8 673 2.42 tota! I 1054497 I 100 | 868.7 | 101017 I 372.45 | C15:0 | 97105 I weight feces 9.3 mg C14:0 C16:0 C16:1 C18:0 018:1 018:2 C18:3 area % pg fatty ng fatty add nmol fatty add/ add mgfeces mgfeces 18128 1.3 14.7 1575 6.90 549910 39.1 444.5 47793 186.40 5378 0.4 4.3 467 1.84 733331 52.1 592.7 63734 224.02 85796 6.1 69.3 7457 26.39 4289 0.3 3.5 373 1.33 10396 0.7 8.4 904 3.24 total I 1407228 | 100 | 1137.4 I 122302 I 450.12 I c15:0 | 98978 | weight teces 5.9 mg CH-.0 016:0 016:1 018:0 018:1 018:2 018:3 area % pg fatty ng fatty add nmol fatty add/ add mgfaces mgfeces 5981 2.1 4.9 824 3.61 103637 47.0 84.2 14272 55.66 790 0.4 0.6 109 0.43 97059 44.0 78.9 13366 46.98 10984 5.0 8.9 1513 5.35 884 0.4 0.7 122 0.43 1019 0.5 0.8 140 0.50 tota! I 220354 I 100 I 179,0 | 30345 | 112.97 | 015:0 I 98463 | weight feces 7.7 mg 014:0 C16:0 C16:1 018:0 C18:1 C182 C18:3 area % pg fatty ng fatty add nmol fatty add/ add mg feces mg feces 14837 1.3 11.7 1515 6.63 453886 39.8 356.9 46352 180.78 4567 0.4 3.6 466 1.83 591094 51.9 464.8 60364 212.18 63874 5.6 50.2 6523 23.09 3296 0.3 2.6 337 1.20 7991 0.7 63 816 2.93 tota) | 1139545 I 100 I 896.1 l 42.S ~ l C15X) | 101736 1 !-6 weight feces 8.4 area C14:0 C16:0 016:1 018:0 C18:1 018:2 018:3 5585 169177 1364 196952 20806 1530 2302 total I 397716 | 015:0 | 90961 1 mg % pg fatty ng fatty add nmol fatty add/ add mg feces mg feces 1.4 4.9 585 2.56 42.5 148.8 17709 69.07 0.3 1.2 143 0.56 49.5 173.2 20617 72.47 53 18.3 2178 7.71 0.4 1.3 160 0.57 0.6 2.0 241 0.87 100 | 349,7 | 41633 I 153.80 ~1 Page A109of A172 Appendix to TNO-report of 3M-Q2____________ Final version P 204 04-12-2006 weight feces 11 mg 0 4 :0 C16:0 C16:1 C18:0 0 8 :1 0 8 :2 C18:3 area 14517 354931 3055 369554 36484 2417 4404 % pg fatty ng fatty add nmol fatty acid/ add mg feces mg feces 1.8 10.1 914 4.00 45.2 245.8 22349 87.16 0.4 2.1 192 0.76 47.1 256.0 23269 81.79 4.6 25.3 2297 8.13 0.3 1.7 152 0.54 0.6 3.1 277 1.00 total 1 785362 I 100 I 544.0 | 49451 | 183.38 | C15:0 I 115502 | weight feces 8.3 mg C14:0 C16:0 C16:1 0 8 :0 C18:1 C18:2 0 8 :3 area 7895 202071 1616 212839 21090 1476 2397 % M9 fatty ng fatty add nmd fatty add/ add mg feces mg feces 1.8 6.9 833 3.65 45.0 177.0 21327 83.18 0.4 1.4 171 0.67 47.4 186.4 22464 78.96 4.7 18.5 2226 7.88 0.3 1.3 156 0.56 0.5 2.1 253 0.91 total | 449384 | 100 | 393.7 | 47429 I 175.80 I C15:0 | 91324 | weight feces 11.1 mg C14:0 0 6 :0 016:1 C18:0 C18:1 C18:2 0 8 :3 area % pg fatty ng fatty add nmd fatty add/ add mg feces mg feces 20655 1.4 11.8 1062 4.65 613614 40.9 350.2 31548 123.04 5769 0.4 3.3 297 1.17 774403 51.6 441.9 39815 139.95 72889 4.9 41.6 3747 13.27 4402 0.3 2.5 226 0.81 10243 0.7 5.8 527 1.89 tota) I 1501975 I 100 I 687.2 I 77221 I 284.77 I 0 5 :0 I 140182 | weight feces 7.6 mg C14 C16:0 016:1 C18:0 08 :1 0 8 :2 C18:3 area 13707 374445 3351 433392 46463 2770 5202 % pg fatty ng fatty add nmd fatty add/ add mg feces mg feces 1.6 9.9 1300 5.69 42.6 269.9 35519 138.53 0.4 2.4 318 125 49.3 312.4 41111 144.50 5.3 33.5 4409 15.61 0.3 2.0 263 0.94 0.6 3.8 493 1.77 total I 879350 | 100 | 633.9 | 83414 | 308.29 ~ l 0 5 :0 I 110969 I weight feces 13.4 area 014:0 C16:0 016:1 0 8 :0 018:1 0 8 :2 018:3 26223 851207 7949 1186581 108707 7434 16281 total 2204382 mg % 1.2 38.6 0.4 53.8 4.9 0.3 0-7 100 C15:0 I 1-44789 I tig fatty add 14.5 470.3 4.4 655.6 60.1 4.1 9.0 ng fatty add nmol fatty add/ mg feces mg feces 1081 4.73 35098 136.89 328 48927 1.29 171.97 4462 15.87 307 1.09 671 2.41 1218.0 90894 33426 weight feces 7.4 mg area % C14:0 0 6 :0 C16:1 C18:0 C18:1 0 8 :2 C18:3 9500 267057 2386 302932 29071 2020 3014 1.5 43.4 0.4 49.2 4.7 0.3 0.5 total 615980 100 0 5 :0 | 101306 | pg fatty acid 7.5 210.9 1.9 239.2 23.0 1.6 2.4 ng fatty add nmd fatty acid/ mg feces mg feces 1014 4.44 28499 111.15 255 1.00 32327 113.63 3102 10.98 216 0.77 322 1.15 486.4 65734 243.12 Page A110 of A172 Appendix to TNIO-rePort of 3M-02____________ Final version p. 205 04-12-2006 weight feces 8,5 mg C14:0 016:0 C16:t C18:0 C18:1 C18:2 C18:3 area 7543 193432 1421 205498 21652 1375 2581 % pg fatty ng fatty add nmol fatty acid/ add mg feces mg feces 1.7 6.4 753 44.6 164.2 19312 3.30 75.32 0.3 1.2 142 47,4 174.4 20517 0.56 72.12 5.0 18.4 2162 7.65 0.3 1.2 137 0.6 2.2 258 0.49 0.93 total I 433502 I 100 I 367.9 | 43281 | 160.36 | C15:0 I 94269 | weight feces 6.5 mg I C14:0 C16:0 016:1 018:0 C18:1 C182 C18:3 area % pg fatty ng fatty acid nmol fatty add/ acid mg feces mg feces 6822 183086 1.6 44.2 6.4 172.2 987 26486 4.32 103.30 1710 0.4 1.6 247 0.97 197892 47.8 186.1 28628 100.63 21000 5.1 19.7 3038 10.75 1164 0.3 1.1 168 0.60 2282 0.6 2.1 330 1.19 total I 413956 I 100 I 389.3 | 59885 | 221,76 I C15:0 1.....85077.... | 1:4-10 weight feces 82 mg C14:0 C16:0 C16.-1 018:0 C18:1 C18:2 C18:3 area % pg fatty ng fatty add nmol fatty add/ add mg feces mg feces 16048 1.4 11.1 1350 5.91 495415 41.9 341.8 41686 162.58 3727 0.3 2.6 314 1.23 599026 50.7 413.3 50404 177.17 56734 4.8 39.1 4774 16.90 3322 0.3 2.3 280 1.00 7505 0.6 52 631 2.27 total I 1181777 | 100 I 815.4 | 99439 | 367.06 | C15:0 I 115946 I :d -tf weight feces 8.8 mg C14:0 C16:0 C16:1 018:0 C18:1 C182 C18:3 area % pg fatty ng fatty add nmol fatty add/ add mg feces mg feces 13693 1.5 8.5 966 4.23 392497 42.8 243.6 27681 107.96 3202 0.3 2.0 226 0.89 454998 49.7 282.4 32089 112.79 43482 4.7 27.0 3067 10.88 2667 0.3 1.7 188 0.67 5468 0.6 3.4 386 1.38 total | 916007 | 100 | 568.5 | 64603 | 238.78 | C15:0 I 128901 I weigh! feces 14 mg area % M9 fatty ng fatty acid nmol fatty add/ add mg feces mg feces C14:0 13974 1.4 12.4 883 3.87 C16:0 420919 41.7 372.5 26609 103.78 C16:1 4038 0.4 3.6 255 1.00 C18:0 511096 50.6 452.3 32310 113.57 C18:1 49923 4.9 44.2 3156 11.17 C18:2 3410 0.3 3.0 216 0.77 C18:3 6144 0.6 5.4 388 1.39 total I 1009504 | 100 | 893.5 I 63818 | 235.56 | C15:0 I 90391 | 1:4-12 weight feces 9 .8 mg C14:0 C16:0 C16:1 C18:0 C18:1 C182 C18:3 area % pg fatty ng fatty add nmol fatty add/ add mg feces mg feces 11857 1.3 12.4 1261 5.52 361984 40.3 377.2 38489 150.11 2570 0.3 2.7 273 1.07 460388 51.3 479.7 48952 172.06 51833 5.8 54.0 5511 19.51 3265 0.4 3.4 347 1.24 5863 0.7 6.1 623 2.24 total | 897760 | 100 | 935,5 | 95457 | 351.76 | 015:0 I 76774 I Page A111 ofA172 Appendix to TNIO-report of 3M-02____________ Final version p. 206 04-12-2006 2:< weight feces 7.1 mg 14:0 016:0 C16:1 C18;0 018:1 C18:2 C18:3 area % M9 fatty ng fatty add nmol fatty acid/ acid mg feces mg feces 10522 344618 12 40.8 8.5 278.0 1196 39156 5.23 152.71 2591 0.3 2.1 294 1.16 438053 51.9 353.4 49772 174.94 40213 4.8 32.4 4569 16.17 2602 0.3 2.1 296 1.05 5292 0.6 4.3 601 2.16 total I 843891 I 100 I 680.8 [ 95883 | 353.44 | C15:0 I 99169 | 2:4-11 weight feces 8.7 mg C14:0 C16:0 016:1 010:0 C18:1 C18:2 C18:3 area % pg fatty ng fatty acid nmol fatty add/ add mg feces mg feces 13021 1.5 10.2 1170 5.12 381006 42.6 297.9 34238 133.53 3180 0.4 2.5 286 1.12 444595 49.7 347.6 39952 140.43 44296 5.0 34.6 3981 14.09 2954 0.3 2.3 265 0.95 4928 0.6 3.9 443 1.59 total I 893980 I 100 I 696.9 I 80335 I 296.84 I C15.0 I 102328 1 weight feces 72 mg C14:0 016:0 016:1 C18:0 018:1 C18:2 018:3 area % pg fatty ng fatty acid nmolfatty add/ acid mg feces mg feces 12246 1.6 9.2 1273 5.58 332981 44.3 249.3 34627 135.05 2528 0.3 1.9 263 1.03 357246 47.5 267.5 37150 130.58 40701 5.4 30.5 4232 14.98 2826 0.4 2.1 294 1.05 3646 0.5 2.7 379 1.36 O*31 I 752174 I 100 I 563.2 | 78218 I 289.63 | 015:0 I 106848 | 3:4-10 weight feces 8.5 area 014:0 016:0 C16:1 C18-.0 C18:1 C18'2 C18:3 10172 310431 2449 394129 34766 2260 4580 total I 758787 | C15:0 I 86070 | mg % pg fatty ng fatty acid nmol fatty add/ add mg feces mg feces 1.3 9.5 1112 4.87 40.9 288.5 33946 132.39 0.3 2 2 268 1.05 51.9 366.3 43098 151.49 4.6 32.3 3802 13.46 03 2.1 247 0.88 0.6 4.3 501 1.80 100 | 705.3 | 82973 | 30S.94 3:4-11 weight feces 9.8 mg C14:0 016:0 C16:1 018:0 C18:1 018:2 018:3 area % pg fatty ng fatty add nmol fatty add/ add mgfeces mg feces 20169 422311 1.9 39.3 17.5 367.1 1789 37462 7.83 146.11 3653 565589 0.3 52.6 32 491.7 324 50171 1.27 176.35 52273 4.9 45.4 4637 16.41 2913 0.3 2.5 258 0.92 7488 0.7 6.5 664 2.39 total I 1074396 I 100 I 934.0 I 95305 I 351.28 ~| 015:0 I 32026 I 3 :4-12 weight feces 10.7 mg C14:0 C16:0 016:1 C18:0 018:1 018:2 018:3 area % pg fatty ng fatty acid nmol fatty add/ add mg feces mg feces 15056 1.1 13.7 1283 5.62 501243 38.2 456.9 42703 166.55 4206 0.3 3.8 358 1.41 708410 54.0 645.8 60353 212.14 68397 5.2 62.3 5827 20.63 4333 0.3 3.9 369 1.32 9322 0.7 8.5 794 2.85 total I 1310967 | 100 | 1195.1 I 111668 | 410.51 ~| 015:0 I 87759 | Page A112of A172 A p p e n d ix to T N O -re p o rt o f 3 M -0 2 ________________ Final version p. 207 0 4 -1 2 -2 0 0 6 weight feces 8 ? ^ mg area % C14:0 016:0 C16:1 018:0 018:1 018:2 018:3 13526 393457 3030 473949 44962 3228 5563 1.4 42.0 0.3 50.5 4.8 0.3 0.6 total | 937715 T 100 C15:0 | 114357 | pg fatty add 9.5 275.2 2.1 331.6 31.5 2.3 3.9 ng fatty add nmol fatty add/ mg feces mg feces 1140 4.99 33162 129.34 255 1.00 39947 140.41 3790 13.41 272 0.97 469 1.68 656.0 79035 291.81 014:0 016:0 016:1 018:0 018:1 018:2 018:3 tota! area % pg fatty ng fatty add nmol fatty add/ add mg feces mg feces 13428 1.5 10.8 1331 5.83 386269 43.2 310.0 38276 149.28 3253 0.4 2.6 322 1.27 437237 48.9 350.9 43327 152.29 45693 5.1 36.7 4528 16.03 2448 0.3 2.0 243 0.86 5034 0.6 4.0 499 1.79 | 893362 I 100 I 717,1 | 88S2S | 327.35 | C15:0 I 99670 | weight feces C14:0 C16:0 016:1 018:0 C18:1 018:2 018:3 area 33483 1006010 9704 1300166 160875 7669 18973 % 1.3 39.7 0.4 51.3 6.3 0.3 0.7 Mg fatty add 19.7 592.1 5.7 765.3 94.7 4.5 112 ng fatty add nmoi fatty add/ mg feces mg feces 1792 7.84 53832 209.95 519 2.04 69572 244.54 8608 30.47 410 1.46 1015 3.65 total | 2536880 I 100 I 1493.2 | 135749 j 499.96 | C15:0 | 135913 | weight feces 9.9 mg 014:0 C16:0 016:1 018.-0 018:1 018*2 C18:3 area 23143 686470 6696 830835 105595 4699 10267 % pg fatty ng fatty add nmol fatty add/ add mg feces mg feces 1.4 17.1 1731 7.58 41.2 508.5 51359 200.31 0.4 5.0 501 1.97 49.8 615.4 62160 218.49 6.3 78.2 7900 27.97 0.3 3.6 367 1.31 0.6 7.6 768 2.76 total | 1667905 I 100 | 1235.4 | I2477 I 460.38 I C150 | 106008 | I 1:5-15 weigni reces ...j--: 8.3 mg 1 >.- t | 014:0 C16:0 016:1 C18:0 C18:1 018:2 C18:3 area 11073 294462 2464 315545 28429 1608 3367 % pg fatty ng fatty add nmolfatty acid/ add mg feces mg feces 1.7 7.3 884 3.87 44.8 195.1 23500 91.65 0.4 1.6 197 0.77 48.0 209.0 25183 88.52 4.3 18.8 2269 6.03 0.2 1.1 128 0.46 0.5 22 269 0.96 tota! | 656948 | 100 | 435.2 | 52429 | 194.27 | 015:0 | 120773 I weight feces 8.3 mg C14:0 016:0 C16:1 C18:0 C18:1 C18:2 C18:3 area % pg fatty ng fatty add nmolfatty add/ add mg feces mg feces 7030 128252 2.7 50.0 6.9 126,1 833 15190 3.65 59.24 1016 0.4 1.0 120 0.47 106592 41.5 104.8 12624 44.37 11903 4.6 11.7 1410 4.99 760 0.3 0.7 90 0.32 1127 0.4 1.1 133 0.48 total | 256680 | 100 | 252.3 I 30401 | 113.53 I 015:0 I 81381 I Page A113ofA172