Document 2DOk0NzoDJ241X7VgV4mZVma

3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 TNO-Report for the study entitled: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA TNO Metabolic Health Research Gaubius Laboratory Zernikedreef 9, 2333 CK Leiden The Netherlands General Phone +31 888 669 000 Specific Fax +31 888 660 603 Signature and date: E.J. Pieterman, B.Sc. Study coordinator Drafted by: Elsbet J. Pieterman Hans M.G. Princen In assignment of: 3M Company, Medical Department Project number: 031.20699 (3M ToxDocs reference 10-088) Date: 07-Apr-2016 Amended Final version Previous versions: Draft 24-03-2011 Draft 11-04-2011 Draft 20-04-2011 Draft 20-06-2011 Final 15-11-2012 Number of pages: 65 CONFIDENTIAL Dr. H.M.G. Princen Study director Dr. Ir. J.J.M. van de Sandt Research Manager All rights reserved. No part of this report may be reproduced and/or published in any form by print, photoprint, microfilm or any other means without the previous written permission from TNO. All information which is classified according to Dutch regulations shall be treated by the recipient in the same way as classified information of corresponding value in his own country. No part of this information will be disclosed to any third party. In case this report was drafted on instructions, the rights and obligations of contracting parties are subject to either the Standard Conditions for Research Instructions given to TNO, or the relevant agreement concluded between the contracting parties. Submitting the report for inspection to parties who have a direct interest is permitted. 2016 TNO 1 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Summary Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are exceptionally stable, amphiphilic, anionic compounds with strong surface-active properties and are essentially completely ionized in nature. Both PFOA and PFOS are widely distributed in nature and are present in the blood (serum/plasma) of the human general population at low ng/mL concentrations. Once present in the body of humans, both compounds clear slowly, with estimated serum elimination half-lives of several years. Although both PFOA and PFOS have been found to reduce serum cholesterol in experimental studies, epidemiological studies have found positive associations of both PFOA and PFOS with serum non-HDL cholesterol at serum PFOA and PFOS concentrations below approximately 50 ng/mL. At greater serum PFOA and PFOS concentrations, these associations are either much weaker or not present. The paradoxical associations found at low serum concentrations have prompted the investigation of potential non-causal factors that may account for positive associations between serum cholesterol and general population serum concentrations of PFOA and PFOS. One potential non-causal hypothesis for the observed associations is that dietary factors that may affect serum cholesterol may also affect serum PFOA and PFOS in the same direction. Based on dietary questionnaire data, serum concentrations of PFOA and PFOS among pregnant Danish women were found to be positively associated with higher consumption of dietary fat, among other factors. However, in another study based on the United States Centers for Disease Control National Health and Nutrition Examination Survey, questionnaire-based consumption of dietary saturated fat was not associated with serum PFOA and PFOS. Epidemiological and observational studies have noted that intake of saturated fat is associated with increased plasma lipid levels. The experiment reported herein was based on the hypothesis that, assuming equal oral intake of PFOS or PFOA on a mass/kg body-weight basis, absorption of PFOS or PFOA from the gastrointestinal tract will increase with increasing dietary fat in a similar manner to cholesterol. Based on increased absorption, plasma concentrations of cholesterol, PFOS, and PFOA will also increase with increasing dietary fat consumption. Aim: to test the hypothesis that increased consumption of saturated fat leads to both increased plasma lipid and increased plasma PFOS and PFOA concentrations, using male E3L.CETP mice on diets with increasing amounts of saturated fat with equal dietary doses of PFOS and PFOA. The PFOS and PFOA doses used do not affect plasma lipid levels and result in PFOS and PFOA plasma levels in the range of those found in the general population (determined from previous studies of the sponsor). Forty male APOE*3Leiden.CETP mice on a regular chow diet were weighed and blood samples were taken after a 4-hour fasting period to measure plasma cholesterol and triglycerides (t=-4 weeks). Thereafter, the mice were sub-divided into four groups of 10 mice each, matched for body weight, plasma total cholesterol and triglycerides, and the 4 weeks run-in period was started. Group 1 received a diet with 5% fat, group 2 received a diet with 15% fat, group 3 received a diet with 25% fat, and group 4 received a diet with 35% fat. At t=-3 and t=-2 weeks food intake was measured. At t=0 weeks body weight and food intake were measured and blood samples were taken after a 4-hour fasting period to measure plasma total cholesterol, HDL-cholesterol and triglycerides. The run-in period was prolonged with one week, hereafter the 4 weeks treatment period was started. Mice were treated with the different diets with PFOA/PFOS (nominal dose 0.03 ppm for the diet with 35% fat and equal oral amounts for the other diets based on food intake data of t=-3 weeks and -2 weeks). Food intake was measured weekly during treatment. At t=2 and 4 weeks, body weight was measured and blood samples were taken after a 4-hour fasting period to measure plasma cholesterol, HDL-cholesterol and triglycerides. At t=-4, 0, 2 and 4 weeks lipoprotein profiles were measured at group level. Extra plasma samples were collected at t=-4, 0, 1, 2 and 4 weeks for PFOA/PFOS analysis. Mice were sacrificed, and plasma was collected via heart puncture and liver was isolated and weighed. Markers of general safety and well-being Specific clinical signs were not observed during the study. At sacrifice, macroscopic aberrations were not observed. 2 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Body weight, liver weight and food intake The different fat percentages in the diets did not significantly influence body weights. Only at t=2 weeks, body weights were significantly higher in the 15% fat + PFOA/PFOS treatment when compared to the 25% fat + PFOA/PFOS treatment. Liver weights were decreased by the fat concentration in the different diets in a fatconcentration-dependent manner. When compared to the 5% fat diet + PFOA/PFOS group, the largest reduction was seen in the 35% fat + PFOA/PFOS group (by -24%, p=0.001). The different fat diets did not significantly influence food intake, except for t=-2 weeks and at t=2 weeks. At t=-2 weeks, the group on 35% fat diet showed a significantly lower food intake when compared to the group on 5% fat diet (by -18%, p=0.029); at t=2 weeks, the group on 25% fat diet + PFOA/PFOS showed a significantly decreased food intake when compared to the group on 15% fat diet + PFOA/PFOS (by -16%, p=0.032), consistent with the body-weight observations in these groups at t=2 weeks. PFOA/PFOS intake and plasma and liver levels The PFOA and PFOS doses in the regular chow diet (mice were fed this diet until the start of the run-in period at t=-4 weeks) and in the different high fat diets without PFOA/PFOS added (mice were fed these diets during the run-in period), were below the lower limit of quantitation (LLOQ, 0.5 ng/g). At t=1, 2, and 4 weeks, PFOA plasma levels were decreased in a dietary fat-concentration-dependent manner. PFOS plasma levels were decreased by the 15%, 25% and 35% fat diets at t=2 and 4 weeks, when compared to the 5% fat diet levels. The PFOA and the PFOS intakes were significantly different between most of the groups at the different time points of the treatment period. Therefore, the measured plasma PFOA/PFOS levels were corrected for PFOA and PFOS intake, by calculating the estimated PFOA/PFOS burden in the body as % of PFOA/PFOS intake. When corrected for the PFOA and PFOS intake, the estimated PFOA and PFOS burden in the body at the different time points were decreased by the different fat diets in a fatconcentration-dependent manner. When comparing the 5% fat diet group the group to the 35% fat diet, the 35% fat diet showed the highest reduction in estimated whole-body PFOA content, estimated based on plasma PFOA concentration and volume of distribution (200 mL/kg) at all time points (by -29% (p=0.002), -27% (p=0.001), and -27% (p=0.009) for 1, 2, and 4 weeks, respectively). When comparing to the 5% fat diet group to the 35% fat diet group, the 35% fat diet group also had significantly lowered estimated whole-body PFOS content at all time points (by -47% (p=0.001), -55% (p<0.001) and 50% (p<0.001) after 1, 2, and 4 weeks of treatment, respectively). Both total liver PFOS and PFOA levels (liver PFOS or PFOA concentration times liver weight) seemed to be decreased by the different fat diets in a fat-concentration dependent manner. Liver PFOA and PFOS levels as percentage of PFOA/PFOS intake, seemed to be decreased by the different fat diets in a fat-concentration dependent manner. When compared to the 5% fat diet group the group on 35% fat diet showed a significant reduction in liver PFOA and PFOS (by -26% (p=0.019) and by -61% (p=0.003); respectively). Plasma lipids Prior to the run-in period when mice were on a regular chow diet, plasma total cholesterol levels were similar in all groups (approximately 2.5 mM). The fat content in the semisynthetic diets had a major effect on plasma total cholesterol levels in a dietary fatconcentration-dependent manner. Plasma total cholesterol ranged from approximately 12 mM for the 5% fat diet to approximately 5 mM for the 35% fat diet. When comparing the lowest fat content diet (5% fat) with the highest (35% fat), the latter showed a robust reduction of total cholesterol levels (by -59% (p<0.001), -65% (p<0.001), and -61% (p<0.001) at t=0, 2, and 4 weeks, respectively). Prior to the run-in period when mice were on a regular chow diet, plasma triglycerides levels were similar in all groups. The fat content in the diets had a major effect on plasma triglycerides levels in a dietary fat-concentration-dependent manner and in a similar direction as plasma total cholesterol levels were affected. When comparing the lowest fat content diet (5% fat) with the highest (35% fat), the latter showed a robust reduction of triglycerides levels (by -51% (p=0.001), -61% (p<0.001), and -54% (p<0.001) at t=0, 2, and 4 weeks, respectively). The fat content in the diets had a major effect on plasma HDL-cholesterol levels in a dietary fat-concentration-dependent manner. When comparing the lowest fat content diet (5% fat) with the highest (35% fat), the latter showed a robust elevation of HDL- 3 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 cholesterol levels (by +147% (p=0.001), +171% (p<0.001) and +140% (p<0.001) at t=0, 2, and 4 weeks, respectively). The lipoprotein profiles were in line with the measured plasma lipid levels. At t=-4 weeks (mice were on a regular chow diet), profiles were similar between groups. Fat in the diets decreased the ApoB-containing lipoproteins (VLDL and LDL) and increased HDL in a fatconcentration dependent manner. Conclusion In male ApoE3L.CETP mice, the increased consumption of saturated fat led to decreased plasma total cholesterol and triglycerides levels and increased HDL-cholesterol levels. Because intake of PFOA and PFOS was not equal between mice on the different fat diets, uptake of PFOA and PFOS from the diets was estimated based on plasma PFOA and PFOS concentrations and volume of distribution compared against PFOA and PFOS intake based on diet consumption and dietary concentrations of PFOA and PFOS. The corrected estimated uptake of PFOA and PFOS levels were decreased with increasing consumption of saturated fat. Liver PFOA and PFOS levels, measured at the end of the study, confirmed the plasma results; decreased PFOA and PFOS levels with increasing consumption of saturated fat. The study indicated that in male ApoE3L.CETP mice, the increased consumption of saturated fat led to decreased PFOA and PFOS uptake and plasma concentrations while at the same time leading to decreased plasma total and non-HDL cholesterol. The results of the current study in mice do not support the hypothesis that increasing dietary fat increases the uptake and plasma concentrations of PFOA and PFOS while also increasing the plasma concentration of cholesterol. Although the original hypothesis was not demonstrated, an important finding emerges from the experiment. Increasing dietary fat consumption in the presence of low and constant dietary concentrations of PFOA and PFOS leads not only to lower serum non-HDL cholesterol, but also lower serum PFOA and PFOS plasma concentrations, resulting from lower uptake of PFOA and PFOS. Therefore, plasma PFOA and PFOS were associated with plasma total and non-HDL cholesterol, a finding similar to that observed in epidemiological studies. The basis of these associations is not known currently, and the extent to which these experimental findings may explain the epidemiological associations observed in human populations is also not known. However, this experiment does demonstrate that a dietary factor, increasing fat, can create associations in ApoE3L.CETP mice similar to those observed in human populations in the same range of serum PFOA and PFOS concentrations. 4 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Contents Summary .......................................................................................................................... 2 Contents ........................................................................................................................... 5 Sponsor of the study ........................................................................................................ 6 Testing facility.................................................................................................................. 6 Responsible personnel ..................................................................................................... 6 1. Introduction & aim .................................................................................................. 7 2. Materials and methods ............................................................................................ 8 2.1 Test substance and reference substance........................................................... 8 2.2 Mice .......................................................................................................... 8 2.3 Animal welfare .............................................................................................. 8 2.4 Diets .......................................................................................................... 8 2.5 Drug administration ....................................................................................... 8 2.6 Treatment groups.......................................................................................... 9 2.7 Study design................................................................................................. 9 2.8 Determinations (see also, Figure 1) ................................................................10 2.9 Methods ......................................................................................................10 2.10 Sacrifice ......................................................................................................10 2.11 Statistical analysis ........................................................................................10 3. Deviations from the protocol ................................................................................. 11 4. Results .................................................................................................................. 12 4.1 Markers of general well-being ........................................................................12 4.2 Body weight.................................................................................................12 4.3 Liver weight.................................................................................................13 4.4 Food and fat intake.......................................................................................15 4.5 Cumulative PFOA and PFOS intake..................................................................18 4.6 PFOA and PFOS plasma levels ........................................................................20 4.7 PFOA and PFOS dose in body as % of dietary intake .........................................22 4.8 Plasma total cholesterol ................................................................................24 4.9 Plasma triglycerides......................................................................................25 4.10 Plasma HDL-cholesterol.................................................................................26 4.11 Lipoprotein profiles .......................................................................................27 4.12 PFOA and PFOS liver levels ............................................................................28 5. Conclusions ........................................................................................................... 32 6. Comments ............................................................................................................. 32 7. References............................................................................................................. 36 8. Appendices ............................................................................................................ 37 Appendix I Body weight ........................................................................................37 Appendix II Liver weight ........................................................................................39 Appendix III Food and fat intake ..............................................................................41 Appendix IV Accumulated PFOA and PFOS intake.......................................................43 Appendix V Plasma PFOA and PFOS levels ...............................................................47 Appendix VI PFOA and PFOS dose as percentage of intake ..........................................50 Appendix VII Plasma total cholesterol........................................................................53 Appendix VIII Plasma triglycerides .............................................................................54 Appendix IX Plasma HDL-cholesterol ........................................................................55 Appendix X Lipoprotein profiles ..............................................................................56 Appendix XI Average plasma cholesterol levels and relative PFOA/PFOS doses ..............58 Appendix XII PFOA and PFOS liver levels ...................................................................60 Appendix XIII Report on statistical analysis of associations among effects resulting from feeding fat, PFOA, and PFOS in the diet of mice for four weeks by David W. Gaylor, Ph.D. ......................................................................................63 5 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Sponsor of the study 3M, Medical Department 3M Center, Building 0220-06-W-08 St.Paul, Minnesota 55144-1000 USA Study contact John L. Butenhoff, Ph.D., CIH, DABT Corporate Scientist 3M, Medical Department 3M Center, Building 0220-06-W-08 St.Paul, Minnesota 55144-1000, USA Phone: +1.651.733.1962 Fax: +1.651.733.9066 E-mail: jlbutenhoff@mmm.com Testing facility TNO Metabolic Health Research Mail address: P.O.Box 2215, 2301 CE Leiden Delivery address: Zernikedreef 9, 2333 CK Leiden The Netherlands Responsible personnel Study director Study coordinator Advisor Technical staff Dr. Hans Princen Phone: +31 888 666 124 Fax: +31 888 660 603 E-mail: hans.princen@tno.nl Elsbet Pieterman B.Sc. Phone: +31 888 666 028 Fax: +31 888 660 603 E-mail: elsbeth.pieterman@tno.nl Prof. Dr. Louis Havekes Phone: +31 888 666 116 Fax: +31 888 660 603 E-mail: louis.havekes@tno.nl Ing. Simone van der Drift-Droog Ing. Annemarie Maas Ing. Anita van Nieuwkoop-van Straalen Ing. Erik Offerman 6 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 1. Introduction & aim Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are exceptionally stable, amphiphilic, anionic compounds with strong surface-active properties and are essentially completely ionized in nature. Both PFOA and PFOS are widely distributed in nature and are present in the blood (serum/plasma) of the human general population at low ng/mL concentrations. Once present in the body of humans, both compounds clear slowly, with estimated serum elimination half-lives of several years. Although both PFOA and PFOS have been found to reduce serum cholesterol in experimental studies, epidemiological studies have found positive associations of both PFOA and PFOS with serum non-HDL cholesterol at serum PFOA and PFOS concentrations below approximately 50 ng/mL. At greater serum PFOA and PFOS concentrations, these associations are either much weaker or not present. The paradoxical associations found at low serum concentrations have prompted the investigation of potential non-causal factors that may account for positive associations between serum cholesterol and general population serum concentrations of PFOA and PFOS. One potential non-causal hypothesis for the observed associations is that dietary factors that may affect serum cholesterol may also affect serum PFOA and PFOS in the same direction. Based on dietary questionnaire data, serum concentrations of PFOA and PFOS among pregnant Danish women were found to be positively associated with higher consumption of dietary fat, among other factors. However, in another study based on the United States Centers for Disease Control National Health and Nutrition Examination Survey, questionnaire-based consumption of dietary saturated fat was not associated with serum PFOA and PFOS. Epidemiological and observational studies have noted that intake of saturated fat is associated with increased plasma lipid levels. The experiment reported herein was based on the hypothesis that, assuming equal oral intake of PFOS or PFOA on a mass/kg body-weight basis, absorption of PFOS or PFOA from the gastrointestinal tract will increase with increasing dietary fat in a similar manner to cholesterol. Based on increased absorption, plasma concentrations of cholesterol, PFOS, and PFOA will also increase with increasing dietary fat consumption. Aim: to test the hypothesis that increased consumption of saturated fat leads to both increased plasma lipid and increased plasma PFOS and PFOA concentrations, using male E3L.CETP mice on diets with increasing amounts of saturated fat with equal dietary doses of PFOS and PFOA. The PFOS and PFOA doses used do not affect plasma lipid levels and result in PFOS and PFOA plasma levels in the range of those found in the general population (determined from previous studies of the sponsor). 7 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 2. Materials and methods 2.1 Test substance and reference substance Test substances: Perfluorooctanoic acid, ammonium salt, NH4+PFOA, lot 15822 Perfluorooctanesulfonate, potassium salt, K+PFOS: FC-95, lot 217 Both substances were supplied by the 3M company (PFOS was sent to TNO on 10-Jun-04, PFOA was sent to TNO on 19-Aug-09). Upon receipt, the substances were stored at 4C. 2.2 Mice 40 Male APOE*3Leiden.CETP transgenic mice, (10-11 weeks of age at the start of the run-in period), from the SPF breeding stock at TNO-Biosciences (Leiden) were used, and were housed during the experiment in macrolon cages (1-4 mice per cage), in clean-conventional animal rooms at TNO-Leiden (relative humidity 50-60%, temperature ~21C, light cycle 7 am to 7 pm). Individual animals were marked by ear punch-holes. Mice were supplied with food and acidified tap water ad libitum. 2.3 Animal welfare Experiments were performed in conformance with the rules and regulations set forward by the Netherlands Law on Animal Experiments. Experiments had been approved by the Animal Experiment Committee of TNO under registration number 2956. The study director was entitled to terminate experiments in case of serious unexpected animal discomfort. 2.4 Diets Semi-synthetic diets were used that contained different amounts of cacao butter and sucrose and equal amounts of corn oil, cholesterol and casein based on an isocaloric intake. Sucrose in the diets was replaced by cacao butter to a fat concentration in the diet of 5%, 15%, 25 % and 35%. All diets were prepared by ABdiets (Woerden, Netherlands). Description st. vit. premix st. spor. premix CaHPO4*2H2O CaCO3 KH2PO4 KCl NaCl MgSO4*7H2O MgO Methionine DL Choline CL 50% acid casein cellulose/diacel corn starch sucrose Cocoa butter corn oil Cholesterol kCal per gram 4 kCal/g - 4 kCal/g - 3.61 kCal/g - 3.664 kCal/g 4 kCal/g 9 kCal/g 9 kCal/g - 5% fat diet gram kCal 0.25 1 0.25 0 1.3 0 1 0 0.7 0 0.7 0 0.3 0 0.4 0 0.2 0 0.2 0.8 2 0 20 72.2 6.2 0 10 36.6 51.5 206 4 36 1 9 0.223 0 total % fat kcal/gram Theoretical food intake at isocaloric intake: gram kcal 100 362 5% 3.61 3.4 g/m/d 2.5 Drug administration 15% fat diet gram kCal 0.25 1 0.25 0 1.3 0 1 0 0.7 0 0.7 0 0.3 0 0.4 0 0.2 0 0.2 0.8 2 0 20 72.2 6.2 0 6.9 25.3 35.5 142 12.4 111.6 1 9 0.223 0 gram kcal 90 362 15% 4.04 3.0 g/m/d 25% fat diet gram kCal 0.25 1 0.25 0 1.3 0 1 0 0.7 0 0.7 0 0.3 0 0.4 0 0.2 0 0.2 0.8 2 0 20 72.2 6.2 0 4.4 16.1 22.6 90.4 19.2 172.8 1 9 0.223 0 gram kcal 81 362 25% 4.48 2.7 g/m/d 35% fat diet gram kCal 0.25 1 0.25 0 1.3 0 1 0 0.7 0 0.7 0 0.3 0 0.4 0 0.2 0 0.2 0.8 2 0 20 72.2 6.2 0 2.3 8.4 12 48 24.7 222.3 1 9 0.223 0 gram kcal 74 362 35% 4.91 2.5 g/m/d Equal amounts of PFOA and PFOS were administered orally as admix to the 4 different diets for 4 weeks (0.03 ppm for the diet with 35% fat and equal oral amounts for the other diets based on food intake data of t=-3 weeks and -2 weeks). Blood collections at the start of the study (t=-4 weeks) and following the run-in period (t=0 weeks) were added to be able to measure the background levels of PFOS and PFOA in plasma of mice on chow diet and on the different diets without added PFOS and PFOA. To be able to measure the accumulation of PFOS and PFOA in 8 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 plasma during treatment, blood was collected after 1, 2 and 4 weeks of treatment. Every week diets were changed and left over diet was collected per cage. 2.6 Treatment groups 1) Diet with 5% fat + PFOS/PFOA 2) Diet with 15% fat + PFOS/PFOA 3) Diet with 25% fat + PFOS/PFOA 4) Diet with 35% fat + PFOS/PFOA 2.7 Study design The study was performed according to the scheme as outlined in figure 1. Figure 1. Flow chart of the study week of treatment: start different fat diets without PFOA/PFOS -4 -3 -2 -1 0 1: Diet with 5% fat + PFOA/PFOS x 2: Diet with 15% fat + PFOA/PFOS x 3: Diet with 25% fat + PFOA/PFOS x 4: Diet with 35% fat + PFOA/PFOS x Randomization x Body weight x x Food intake xx x Plasma cholesterol + triglycerides + lipoprotein profile (group) x x HDL x Collection plasma for compound analysis x x *: run-period prolonged for 1 week start treatment diets with PFOA/PFOS 0* 1 2 3 4 x x x x x x x x x x xxxx x x x x xx x Forty male APOE*3Leiden.CETP mice on a regular chow diet were weighed and blood samples were taken after a 4-hour fasting period to measure plasma cholesterol and triglycerides (t=-4 weeks). Thereafter, the mice were sub-divided into four groups of 10 mice each, matched for body weight, plasma total cholesterol and triglycerides, and the 4 weeks run-in period was started. Group 1 received a diet with 5% fat, group 2 received a diet with 15% fat, group 3 received a diet with 25% fat, and group 4 received a diet with 35% fat. At t=-3 and t=-2 weeks food intake was measured. At t=0 weeks body weight and food intake were measured and blood samples were taken after a 4-hour fasting period to measure plasma total cholesterol, HDL-cholesterol and triglycerides. The run-in period was prolonged with one week, hereafter the 4 weeks treatment period was started. Mice were treated with the different diets with PFOA/PFOS (nominal dose 0.03 ppm for the diet with 35% fat and equal oral amounts for the other diets based on food intake data of t=-3 weeks and -2 weeks). Food intake was measured weekly during treatment. At t=2 and 4 weeks, body weight was measured and blood samples were taken after a 4-hour fasting period to measure plasma cholesterol, HDL-cholesterol and triglycerides. At t=-4, 0, 2 and 4 weeks lipoprotein profiles were measured at group level. Extra plasma samples were collected at t=-4, 0, 1, 2 and 4 weeks for PFOA/PFOS analysis. Mice were sacrificed, and plasma was collected via heart puncture and liver was isolated and weighed. One day after the last blood sampling, mice (non fasted) were sacrificed and plasma was collected via heart puncture and liver was isolated and weighed. Two pieces of liver tissue were snap-frozen in liquid nitrogen for optional measurements, one piece of liver was fixed in 10% formalin for optional histology analysis. The in-life mouse work started on July 1st 2010 and ended on September 3rd 2010. 9 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 2.8 Determinations (see also, Figure 1) Body weight at week -4, 0, 2, 4. Food intake per cage at week -3, -2, 0, 1, 2, 3, 4. Plasma cholesterol and triglycerides after 4h fasting at week -4, 0, 2 and 4. Lipoprotein profiles (cholesterol and phospholipids) at group level at week 0 and 4. HDL-cholesterol measurement at week 0 ,2 and 4. Extra plasma collection for PFOA/PFOS analysis at week -4, 0, 1, 2, 4 Sacrifice: heart puncture with EDTA-plasma collection, liver weight, collection of 4 liver parts (N2) and 1 liver part (formalin). 2.9 Methods Total plasma cholesterol and triglycerides were determined using kit "Cholesterol CHODPAP" and kit "Triglycerides GPO-PAP" both from Roche (Mannheim, Germany). Measurement of pooled lipoprotein profiles by FPLC analysis using an AKTA apparatus. Analyses were performed in freshly obtained samples or samples stored at 4C for one day. Cholesterol and phospholipid profiles were measured in the fractions using kit "Cholesterol CHOD-PAP" from Roche (Mannheim, Germany) and kit "Phospholipids" from Instruchemie (Delfzijl, the Netherlands), respectively. Plasma HDL-cholesterol levels were determined using kit "Cholesterol CHOD-PAP" from Roche (Mannheim, Germany) after the precipitation of ApoB-containing lipids by MnCl2 (0.2 mol/L) and heparin (500 IU/mL). 2.10 Sacrifice Mice were sacrificed via CO2 suffocation. After sacrifice, blood was collected via heart puncture in CB 300 K2E microvettes (Sarstedt, Nrnbrecht, Germany) containing EDTA-dipotassium salt. Microvettes containing blood were placed on ice immediately. EDTA-plasma was obtained after centrifugation (10 min at 6000 rpm) of the samples in a lab centrifuge at 4C and samples were then stored at -80C. Livers were isolated and weighed and two parts were snap-frozen in liquid N2 and stored individually at -80C, one part was fixed in 10% buffered formalin solution. 2.11 Statistical analysis Significance of differences between the groups were calculated non-parametrically, using the computer program SPSS. For non-parametric calculations a Kruskall-Wallis test for several independent samples was used, followed by a Mann-Whitney U-test for independent samples. 10 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 3. Deviations from the protocol The following deviations from the original protocol were observed. Since at t=0, the results of the plasma lipid measurements of the different groups were unexpected, start of treatment was postponed for one week to discuss the results of the t=0 weeks measurements with the Sponsor (email correspondence of 02 Aug 2010). It was decided not to exclude 2 mice per group during the treatment period (so the number of mice per group during the treatment period remained 10). Extra food intake measurements were performed at t=1 and t=3 weeks to be able to calculate more precisely the PFOA/PFOS intake. After discussion with the sponsor, it was decided to also perform HDL-cholesterol measurements at t=2 weeks (see email correspondence 13-14 April 2011). At sacrifice, EDTA-plasma was collected via heart puncture and liver was isolated and weighed. Four pieces of liver tissue were snap-frozen in liquid nitrogen for optional measurements; one piece of liver was fixed in 10% formalin for optional histology analysis. The sponsor used one of the liver pieces for PFOA and PFOS liver level measurements. 11 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 4. Results 4.1 Markers of general well-being No adverse clinical signs were observed during the study. At sacrifice, no abnormal macroscopic aberrations were observed. 4.2 Body weight Values are absolute values (grams) and are means S.D. of 10 mice per group. Individual body weight per mouse and number of mice per group are given in appendix I. Group 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS Body weight (g) Time (weeks) t=-4 AVG 25.7 SD 2.4 AVG 25.6 SD 2.4 AVG 25.1 SD 1.7 AVG 25.1 SD 1.7 t=0 28.1 2.6 28.3 2.7 26.8 1.5 27.2 1.7 t=2 28.8 2.4 29.1 3.1 27.1 1.5 28.1 1.9 t=4 29.1 2.1 29.9 3.3 27.9 1.8 28.6 2.3 Body weight, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 35% fat + PFOA/PFOS t=-4 0.602 0.739 0.315 0.353 0.436 0.353 0.853 Time (weeks) t=0 t=2 0.183 0.154 0.353 0.353 0.280 0.105 0.393 0.436 0.052 0.043 0.105 0.280 0.971 0.353 t=4 0.234 0.353 0.247 0.579 0.052 0.247 0.529 The different fat percentages in the diets did not significantly influence body weights. Only at t=2 weeks, body weights were significantly higher in the 15% fat + PFOA/PFOS treatment when compared to the 25% fat + PFOA/PFOS treatment. Since there are no significant differences between the body weights of mice fed the more extreme diets (5% and 35% fat diets), the significant difference in body weight between 15% fat + PFOA/PFOS treatment and 25% fat + PFOA/PFOS treatment is likely due to chance. 12 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Figure 4.2 Body weight 40 30 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS * 35% Fat diet + PFOA/PFOS Body weight (g) 20 10 0 t=-4 t=0 t=2 t=4 Time (weeks) *: p<0.05, Mann-Whitney U test 4.3 Liver weight Values are absolute values (grams) or relative values (% of body weight) and are means S.D. of 10 mice per group. Individual liver weight per mouse and number of mice per group are given in appendix II. Group Liver weight at t=4 weeks g 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS AVG 1.8 SD 0.2 AVG 1.6 SD 0.2 AVG 1.4 SD 0.1 AVG 1.3 SD 0.1 % of body weight 6.1% 0.9% 5.4% 0.4% 5.0% 0.3% 4.7% 0.2% Liver weight, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 35% fat + PFOA/PFOS g <0.001 0.089 0.001 0.001 0.015 0.005 0.393 % of body weight <0.001 0.005 0.002 0.002 0.063 <0.001 0.015 Liver weights were dose dependent decreased by the fat concentration in the different diets. When compared to the 5% fat diet + PFOA/PFOS group, the largest reduction was seen in the 35% fat + PFOA/PFOS group (by -24%, p=0.001). Relative liver weights (corrected for body weight) were 13 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 also dose dependent decreased by the fat concentration in the different diets. When compared to the 5% fat diet + PFOA/PFOS group, the largest reduction in relative liver weight was seen in the 35% fat + PFOA/PFOS group (by -23%, p=0.001). Figure 4.3a 2.5 Absolute liver weight * 2.0 * * * 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS 1.5 liver weight (g) 1.0 0.5 0.0 t=4 weeks liver weight (% of body weight) *: p<0.05, Mann-Whitney U test Figure 4.3b Liver weight as % of body weight 10% 8% * * * * 6% * 4% 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS 2% 0% *: p<0.05, Mann-Whitney U test t=4 weeks 14 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 4.4 Food and fat intake Values are absolute values (gram/mouse/day) and are means S.D. of 2-5 cages per group. Individual food and fat intake data per cage and number of cages per group are given in appendix III. Group 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS Food intake (g/day/mouse) Time (weeks) t=-3 t=-2 t=0 AVG 3.6 3.6 3.5 SD 0.5 0.3 0.4 AVG 3.4 3.5 3.5 SD 0.1 0.3 0.3 AVG 3.1 3.3 3.1 SD 0.2 0.5 0.3 AVG 3.1 SD 0.1 3.0 3.1 0.4 0.3 t=1 3.5 0.6 3.6 0.4 3.1 0.3 3.3 0.3 Food intake, P value t=-3 t=-2 Between groups 0.120 0.074 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS no analysis* no analysis* 25% fat + PFOA/PFOS no analysis* 0.343 35% fat + PFOA/PFOS 0.057 0.029 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS no analysis* no analysis* 35% fat + PFOA/PFOS no analysis* no analysis* 25% fat + PFOA/PFOS vs 35% fat + PFOA/PFOS no analysis* 0.343 *: no statistical analysis could be performed; not enough cages per group. Time (weeks) t=0 t=1 0.152 0.277 1.000 0.886 0.413 0.286 0.286 0.730 0.063 0.111 0.063 0.190 0.548 0.548 t=2 3.5 0.6 3.7 0.3 3.1 0.2 3.4 0.3 t=2 0.191 0.486 0.286 1.000 0.032 0.190 0.421 t=3 3.5 0.7 3.5 0.5 3.2 0.3 3.4 0.5 t=3 0.811 1.000 0.556 0.730 0.413 0.905 0.841 t=4 3.4 0.5 3.6 0.5 3.2 0.3 3.3 0.4 t=4 0.628 0.686 0.730 0.905 0.190 0.413 0.841 Group 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS Fat intake (g/day/mouse) Time (weeks) t=-3 t=-2 AVG 0.18 0.18 SD 0.02 0.01 AVG 0.51 0.53 SD 0.02 0.05 AVG 0.78 0.83 SD 0.05 0.12 AVG 1.08 1.04 SD 0.03 0.13 t=0 0.18 0.02 0.53 0.04 0.79 0.07 1.10 0.09 t=1 0.18 0.03 0.54 0.05 0.78 0.07 1.16 0.10 t=2 0.18 0.03 0.55 0.05 0.78 0.05 1.18 0.12 t=3 0.18 0.04 0.53 0.07 0.79 0.07 1.18 0.18 t=4 0.17 0.03 0.54 0.08 0.80 0.07 1.17 0.15 Fat intake, P value t=-3 t=-2 Between groups 0.017 0.005 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS no analysis* no analysis* 25% fat + PFOA/PFOS no analysis* 0.029 35% fat + PFOA/PFOS 0.029 0.029 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS no analysis* no analysis* 35% fat + PFOA/PFOS no analysis* no analysis* 25% fat + PFOA/PFOS vs 35% fat + PFOA/PFOS no analysis* 0.057 *: no statistical analysis could be performed; not enough cages per group. Time (weeks) t=0 t=1 0.001 0.001 0.029 0.029 0.016 0.016 0.016 0.016 0.016 0.016 0.016 0.016 0.008 0.008 t=2 0.001 0.029 0.016 0.016 0.016 0.016 0.008 t=3 0.001 0.029 0.016 0.016 0.016 0.016 0.008 Although food intake seemed to be somewhat decreased by a higher fat content in the diet, the different fat diets did not significantly influence food intake, except for t=-2 weeks and at t=2 weeks. At t=-2 weeks, the group on 35% fat diet showed a significantly lower food intake when compared to the group on 5% fat diet (by -18%, p=0.029); at t=2 weeks, the group on 25% fat diet + PFOA/PFOS showed a significantly decreased food intake when compared to the group on 15% fat diet + PFOA/PFOS (by -16%, p=0.032). t=4 0.001 0.029 0.016 0.016 0.016 0.016 0.008 Fat intake was dose dependent increased and significantly different between all groups, except at t=-3 weeks and t=-2 weeks. At these time points not enough food intake data were available for the 15%, 25% and 35% fat groups to perform statistical analysis. 15 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Figure 4.4a Food intake Food intake (g/mouse/day) 6 5 * 4 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS * 3 2 1 0 t=-3 t=-2 t=0 t=1 t=2 t=3 t=4 Time (weeks) *: p<0.05, Mann-Whitney U test Figure 4.4b Dietary fat intake 2.0 1.5 * 1.0 0.5 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS * ** ** ** * * * * * * * * * * * * * * * * * * * * * * * * * Fat intake (g/mouse/day) 0.0 t=-3 t=-2 t=0 t=1 t=2 t=3 t=4 Time (weeks) *: p<0.05, Mann-Whitney U test 16 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Based on the t=-3 and t=2 weeks food intake measurements the PFOA/PFOS doses in the different high fat diets were calculated. The target PFOA/PFOS doses in the different diets were 0.03 ppm PFOA/PFOS for the diet with 35% fat and equal oral intake amounts for the other diets based on food intake data of t=-3 weeks and -2 weeks. In appendix IV the calculation and measured PFOA/PFOS doses in the diets are given. The measured PFOA and PFOS doses deviated from the target PFOA/PFOS doses in all diets. Group Group 1: 5% fat diet Group 2: 15% fat diet Group 3: 25% fat diet Group 4: 35% fat diet Target concentration PFOA/PFOS ng/g diet 36/36 34/34 32/32 30/30 Average measured concentration PFOA/PFOS in diet ng/g diet 28.4/17.8 27.4/12.1 25.3/15.5 26.2/28.8 Based on the measured PFOA/PFOS doses in the diet and measured food intake the cumulative PFOA and PFOS dietary intake was calculated (see paragraph 4.5). 17 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 4.5 Cumulative PFOA and PFOS intake Values are absolute values (g/kg mouse) and are means S.D. of 10 mice per group. Individual PFOA and PFOS intake per mouse and number of mice per group are given in appendix IV. Accumulated PFOA intake per kg mouse (g/kg) Time (weeks) Group t=1 t=2 5% fat + PFOA/PFOS AVG 21.4 42.2 SD 2.6 5.2 15% fat + PFOA/PFOS AVG 21.1 42.1 SD 2.4 4.4 25% fat + PFOA/PFOS AVG 17.6 35.0 SD 1.0 1.8 35% fat + PFOA/PFOS AVG 18.8 37.4 SD 1.7 3.8 t=4 89.8 11.1 88.2 11.2 74.3 3.4 79.9 9.6 Accumulated PFOA intake per kg mouse, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS vs 35% fat + PFOA/PFOS Time (weeks) t=1 t=2 t=4 0.001 0.001 0.005 0.853 0.912 0.579 <0.001 <0.001 <0.001 0.035 0.023 0.043 0.002 <0.001 0.019 0.035 0.035 0.123 0.105 0.280 0.247 Accumulated PFOS intake per kg mouse (g/kg) Time (weeks) Group t=1 t=2 5% fat + PFOA/PFOS AVG 13.4 26.5 SD 1.6 3.3 15% fat + PFOA/PFOS AVG 9.3 18.6 SD 1.1 1.9 25% fat + PFOA/PFOS AVG 10.8 21.4 SD 0.6 1.1 35% fat + PFOA/PFOS AVG 20.7 41.2 SD 1.9 4.2 t=4 56.3 7.0 39.0 4.9 45.4 2.1 88.0 10.6 Accumulated PFOS intake per kg mouse, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS vs 35% fat + PFOA/PFOS Time (weeks) t=1 t=2 t=4 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.002 0.002 0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 The PFOA and PFOS doses in the regular chow diet (mice were fed this diet until the start of the run-in period at t=-4 weeks) and in the different high fat diets without PFOA/PFOS added (mice were fed these diets during the run-in period), were below detection limit (<0.5 ng/mL). The PFOA and the PFOS intake were significantly different between most of the groups at the different time points of the treatment period. Therefore the measured plasma PFOA/PFOS levels (see paragraph 4.6) were corrected for PFOA and PFOS intake, by calculating the estimated PFOA/ PFOS dose in the body as % of PFOA/FPFOS intake (see paragraph 4.7). 18 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Figure 4.5a 140 Accumulated PFOA intake 120 Accumulated PFOA intake (g/kg mouse) 100 80 60 * 40 * * * 20 * * * * 0 t=1 t=2 Time (weeks) *: p<0.05, Mann-Whitney U test Figure 4.5b Accumulated PFOS intake Accumulated PFOS intake (g/kg mouse) 140 120 100 80 60 40 * * * * 20 * * 0 t=1 *: p<0.05, Mann-Whitney U test * * * * * * t=2 Time (weeks) * * * 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS t=4 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS * 35% Fat diet + PFOA/PFOS * * * * * t=4 19 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 4.6 PFOA and PFOS plasma levels Values are absolute values (ng/mL) and are means S.D. of 10 mice per group. Individual PFOA and PFOS levels per mouse and number of mice per group are given in appendix V. Group 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS Plasma PFOA levels (ng/mL) Time (weeks) t=-4 t=0 AVG <0.5 <0.6 SD - - AVG <0.5 <0.7 SD - - AVG <0.5 <0.7 SD - - AVG SD <0.5 - <1.0 - t=1 46.0 9.7 35.5 5.3 29.9 2.3 28.6 4.5 Plasma PFOA levels, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 35% fat + PFOA/PFOS Time (weeks) t=1 t=2 t=4 <0.001 <0.001 <0.001 0.005 0.001 0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.009 <0.001 0.063 0.015 0.003 <0.001 0.684 0.796 0.023 Group 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS Plasma PFOS levels (ng/mL) Time (weeks) t=-4 t=0 AVG <0.5 <0.5 SD - - AVG <0.5 <0.5 SD - - AVG SD <0.5 - <0.5 - AVG <0.5 <0.5 SD - - t=1 20.5 10.8 17.3 7.0 15.7 6.0 16.5 5.8 Plasma PFOS levels, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 35% fat + PFOA/PFOS t=1 0.564 0.280 0.190 0.529 0.579 0.912 0.739 Time (weeks) t=2 <0.001 0.005 <0.001 0.143 0.063 0.089 0.003 t=4 0.010 0.089 0.002 0.105 0.043 0.739 0.043 t=2 104.0 13.4 81.9 12.4 65.6 5.4 66.9 9.4 t=2 56.9 37.1 33.0 5.9 27.6 6.1 41.1 13.8 t=4 194.7 26.2 157.4 13.5 143.2 13.0 128.3 14.5 t=4 97.5 31.8 80.9 23.0 69.6 27.0 78.1 12.6 At the different time points, PFOA plasma levels seemed to be dose dependent decreased by the fat concentration in the different diets. Since the PFOA dietary intake was different in the different dietary fat groups, the measured plasma PFOA levels were corrected for accumulated PFOA intake, by calculating the estimated PFOA dose in the body as percentage of PFOA dietary intake (see paragraph 4.7). When compared to the 5% fat diet, PFOS plasma levels seemed to be decreased by the 15%, 25% and 35% fat diets at t=2 and 4 weeks. Since the PFOS dietary intake was different in the different 20 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 dietary fat groups, the measured plasma PFOS levels were corrected for accumulated PFOS intake, by calculating the estimated PFOS dose in the body as percentage of PFOS intake (see paragraph 4.7). Figure 4.6a 350 PFOA plasma levels 300 250 * * * * 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS Plasma PFOA (ng/mL) 200 * * * 150 * * * * * 100 * * * 50 0 t=1 t=2 t=4 Time (weeks) *: p<0.05, Mann-Whitney U test Figure 4.6b 200 PFOS plasma levels 150 * 100 * 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS * * * * 50 Plasma PFOS (ng/mL) 0 t=1 t=2 t=4 Time (weeks) *: p<0.05, Mann-Whitney U test 21 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 4.7 PFOA and PFOS dose in body as % of dietary intake Values are relative values (%) and are means S.D. of 10 mice per group. Individual values per mouse and number of mice per group are given in appendix VI. Estimated PFOA dose in body as percentage of PFOA intake (%) Time (weeks) Group t=1 t=2 t=4 5% fat + PFOA/PFOS AVG 43% 50% 44% SD 8% 7% 10% 15% fat + PFOA/PFOS AVG 34% 40% 36% SD 8% 9% 7% 25% fat + PFOA/PFOS AVG SD 34% 3% 38% 3% 39% 4% 35% fat + PFOA/PFOS AVG 31% 36% 32% SD 5% 7% 4% Estimated PFOA dose in body as percentage of PFOA intake, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS vs 35% fat + PFOA/PFOS t=1 0.008 0.052 0.009 0.002 0.631 0.353 0.063 Time (weeks) t=2 0.001 0.007 <0.001 0.001 1.000 0.315 0.247 t=4 0.018 0.123 0.315 0.009 0.393 0.280 0.004 Estimated PFOS dose in body as percentage of PFOS intake (%) Time (weeks) Group t=1 t=2 t=4 5% fat + PFOA/PFOS AVG 31% 44% 36% SD 16% 33% 15% 15% fat + PFOA/PFOS AVG 37% 36% 41% SD 14% 8% 10% 25% fat + PFOA/PFOS AVG 29% 26% 31% SD 10% 5% 12% 35% fat + PFOA/PFOS AVG 16% 20% 18% SD 6% 6% 3% Estimated PFOS dose in body as percentage of PFOS intake, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS vs 35% fat + PFOA/PFOS Time (weeks) t=1 t=2 t=4 <0.001 <0.001 <0.001 0.105 1.000 0.063 0.796 0.015 0.218 0.001 <0.001 <0.001 0.218 0.002 0.001 <0.001 <0.001 <0.001 0.002 0.035 <0.001 When corrected for the PFOA and PFOS dietary intake, the estimated PFOA and PFOS doses in the body at the different time points seemed to be decreased by the different fat diets in a fatconcentration dependent manner. When compared to the 5% fat diet group the group on 35% fat diet showed the highest reduction in whole body PFOA concentration at all time points (by -29% (p=0.002), -27% (p=0.001) and 27% (p=0.009); respectively). 22 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 When compared to the 5% fat diet group the group on 35% fat diet significantly lowered the whole body PFOS concentration at all time points (by -47% (p=0.001), -55% (p<0.001) and -50% (p<0.001) after 1, 2 and 4 weeks of treatment; respectively). Figure 4.7a Estimated PFOA dose in body as % of intake 80% 60% * * * * * 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS * * 40% Estimated PFOA dose in body as % of PFOA intake (%) 20% 0% t=1 t=2 Time (weeks) *: p<0.05, Mann-Whitney U test Figure 4.7b Estimated PFOS dose in body as % of intake 120% 100% t=4 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS Estimated PFOS dose in body as % of PFOS intake (%) 80% 60% 40% 20% * * * * * * * * * * * * 0% t=1 t=2 t=4 Time (weeks) *: p<0.05, Mann-Whitney U test 23 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 4.8 Plasma total cholesterol Values are absolute values (mM) and are means S.D. of 10 mice per group. Individual cholesterol levels per mouse and number of mice per group are given in appendix VII. Group 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS Plasma total cholesterol(mmol/L) Time (weeks) t=-4 t=0 AVG 2.5 12.0 SD 0.4 3.5 AVG 2.5 7.5 SD 0.5 2.2 AVG 2.4 5.5 SD 0.5 1.0 AVG 2.6 4.9 SD 0.5 1.0 t=2 14.0 4.9 8.6 3.0 5.4 1.1 4.9 0.6 t=4 12.4 3.1 6.8 2.3 5.6 0.6 4.8 0.8 Plasma total cholesterol, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 35% fat + PFOA/PFOS t=-4 0.736 0.853 0.190 0.912 0.971 0.739 0.353 Time (weeks) t=0 t=2 <0.001 <0.001 0.005 0.011 <0.001 0.001 <0.001 <0.001 0.015 0.005 0.007 0.002 0.247 0.190 t=4 <0.001 0.001 0.001 <0.001 0.393 0.011 0.023 On a regular chow diet (at t=-4 weeks, before the start of the run-in period with different fat diets), plasma total cholesterol levels were similar in all groups (approximately 2.5 mM). The fat content in the diets majorly affected plasma total cholesterol levels in a fat-concentration dependent manner (ranging from approximately 12 mM for the 5% fat diet to approximately 5 mM for the 35% fat diet) . When comparing the lowest fat content diet (5% fat) with the highest (35% fat), the latter showed a robust reduction of total cholesterol levels (by -59% (p<0.001), -65% (p<0.001), and -61% (p<0.001) at t=0, 2 and 4 weeks; respectively). Cholesterol (mM) Figure 4.8 25 Plasma total cholesterol 20 * * * 15 * * 10 * * * * * 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS * * * * * 5 0 t=-4 t=0 t=2 t=4 *: p<0.05, Mann-Whitney U test Time (weeks) 24 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 4.9 Plasma triglycerides Values are absolute values (mM) and are means S.D. of 10 mice per group. Individual triglycerides levels per mouse and number of mice per group are given in appendix VIII. Group 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS Plasma triglycerides (mmol/L) Time (weeks) t=-4 AVG 3.5 SD 1.0 AVG 3.6 SD 1.2 AVG 3.7 SD 0.9 AVG 3.6 SD 0.7 t=0 3.9 1.1 3.7 0.9 2.5 1.1 1.9 0.4 t=2 3.6 0.9 3.0 1.3 1.5 0.5 1.4 0.4 t=4 3.0 0.7 3.1 0.9 1.9 0.5 1.4 0.3 Plasma triglycerides, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 35% fat + PFOA/PFOS t=-4 0.951 0.739 0.481 0.481 1.000 0.912 0.971 Time (weeks) t=0 t=2 0.001 <0.001 0.796 0.190 0.011 <0.001 0.001 <0.001 0.023 0.007 <0.001 0.003 0.143 0.436 t=4 <0.001 0.684 0.001 <0.001 0.002 <0.001 0.043 On a regular chow diet (at t=-4 weeks, before the start of the run-in period with different fat diets), plasma triglycerides levels were similar in all groups. The fat content in the diets majorly affected plasma triglycerides levels in a fat-concentration dependent manner and in a similar way as plasma total cholesterol levels were affected. When comparing the lowest fat content diet (5% fat) with the highest (35% fat), the latter showed a robust reduction of triglycerides levels (by 51% (p=0.001), -61% (p<0.001), and -54% (p<0.001) at t=0, 2 and 4 weeks; respectively). Figure 4.9 Plasma triglycerides 8 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 6 * 25% Fat diet + PFOA/PFOS * * 35% Fat diet + PFOA/PFOS * * * * * * * * * 4 Triglycerides (mM) * 2 0 t=-4 t=0 t=2 t=4 *: p<0.05, Mann-Whitney U test Time (weeks) 25 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 4.10 Plasma HDL-cholesterol Values are absolute values (mM) and are means S.D. of 10 mice per group. Individual data per mouse and number of mice per group are given in appendix IX. Group 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS Plasma HDL-cholesterol(mmol/L) Time (weeks) t=0 AVG 0.43 SD 0.19 AVG 0.63 SD 0.22 AVG 0.91 SD 0.16 AVG 1.06 SD 0.44 t=2 0.43 0.16 0.66 0.28 1.12 0.42 1.16 0.36 t=4 0.54 0.22 0.67 0.18 1.26 0.27 1.29 0.29 Plasma HDL-cholesterol, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 35% fat + PFOA/PFOS Time (weeks) t=0 t=2 t=4 <0.001 <0.001 <0.001 0.043 0.075 0.165 <0.001 <0.001 <0.001 0.001 <0.001 <0.001 0.004 0.011 <0.001 0.023 0.005 <0.001 0.529 0.579 0.631 The fat content in the diets majorly affected plasma HDL-cholesterol levels in a fat-concentration dependent manner. When comparing the lowest fat content diet (5% fat) with the highest (35% fat), the latter showed a robust elevation of HDL-cholesterol levels (by +147% (p=0.001), +171% (p<0.001) and +140% (p<0.001) at t=0, 2 and 4 weeks; respectively). Figure 4.10 2.5 Plasma HDL-cholesterol 2.0 * * 1.5 * * 1.0 * * * * * * * * * 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS HDL-Cholesterol (mM) 0.5 0.0 t=0 t=2 t=4 Time (weeks) *: p<0.05, Mann-Whitney U test 26 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 4.11 Lipoprotein profiles Lipoproteins were separated on a Superose column. Values are absolute values (mM) from cholesterol (left graphs) and phospholipids (right graphs) measurements in pooled plasma samples per group (with 10 mice per group) at t=0 and 4 weeks. Fractions 3-8 represent VLDL, fractions 916 represent LDL, and fractions 17-24 represent HDL. The cholesterol and phospholipids amounts per fraction are given in appendix X. The lipoprotein profiles were in line with the measured plasma lipid levels. At t=-4 weeks (mice were on a regular chow diet), profiles were similar between groups. The fat concentration in the diets decreased the ApoB containing lipoproteins (VLDL and LDL) and increased HDL in a fatconcentration dependent manner. Figure 4.11 lipoprotein profiles 1.0 Lipoprotein profiles t=-4 weeks 20 Lipoprotein profiles t=-4 weeks Cholesterol (mmol/L) 0.8 0.6 0.4 0.2 0.0 0 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS VLDL LDL HDL 5 10 15 20 25 Fraction Lipoprotein profiles t=0 weeks VLDL 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS LDL HDL 5 10 15 20 25 Fraction Phospholipids (mg/dL) Phospholipids (mg/dL) 5% Fat diet + PFOA/PFOS 15 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS 10 VLDL LDL HDL 5 0 0 50 40 30 20 10 0 0 5 10 15 20 25 Fraction Lipoprotein profiles t=0 weeks VLDL 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS HDL LDL 5 10 15 20 25 Fraction Cholesterol (mmol/L) 27 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Cholesterol (mmol/L) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 Lipoprotein profiles t=2 weeks VLDL 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS LDL HDL 5 10 15 20 25 Fraction Lipoprotein profiles t=4 weeks VLDL 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS LDL HDL 5 10 15 20 25 Fraction Phospholipids (mg/dL) Phospholipids (mg/dL) 50 40 30 20 10 0 0 50 40 30 20 10 0 0 Lipoprotein profiles t=2 weeks VLDL 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS HDL LDL 5 10 15 20 25 Fraction Lipoprotein profiles t=4 weeks VLDL 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS HDL LDL 5 10 15 20 25 Fraction Cholesterol (mmol/L) 4.12 PFOA and PFOS liver levels Values are absolute values (ng/g liver or ng per liver) or relative values (% of food intake) and are means S.D. of 10 mice per group. Individual PFOA and PFOS liver levels per mouse and number of mice per group are given in appendix XII. Group 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS PFOA and PFOS liver levels PFOA ng/g liver ng per liver AVG 400 714 SD 107 218 AVG 382 611 SD 73 134 AVG 351 488 SD 41 95 AVG 353 471 SD 40 61 PFOS ng/g liver ng per liver 553 999 234 507 478 760 202 336 362 500 97 134 455 608 85 124 28 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 PFOA and PFOS liver levels, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 35% fat + PFOA/PFOS PFOA ng/g liver ng per liver 0.061 0.002 0.218 0.063 0.043 0.005 0.035 0.004 0.143 0.029 0.165 0.015 0.684 0.579 PFOS ng/g liver ng per liver 0.067 0.011 0.247 0.218 0.063 0.011 0.165 0.043 0.105 0.015 0.912 0.165 0.019 0.089 Since liver weights were significantly different between groups, PFOA and PFOS levels were also expressed as ng per liver to correct for the different liver weights. When corrected for liver weight, PFOA levels seemed to be decreased by the different fat diets in a fat-concentration dependent manner. PFOS levels, corrected for liver weight, seemed to be decreased by the different fat diets in a fat-concentration dependent manner to some extent. Figure 4.12a Liver PFOA levels 1000 800 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS * 600 * * * * * 400 liver PFOA 200 0 ng/g liver ng per liver *: p<0.05, Mann-Whitney U test 29 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Figure 4.12b 2000 Liver PFOS levels 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS 25% Fat diet + PFOA/PFOS 35% Fat diet + PFOA/PFOS 1500 1000 * * * liver PFOS * 500 0 ng/g liver ng per liver *: p<0.05, Mann-Whitney U test Since the PFOA and PFOS intake was different in the different dietary fat groups, the measured liver PFOA and PFOS levels were corrected for accumulated PFOA and PFOS intake, by calculating the liver PFOA/PFOS as percentage of PFOA/PFOS intake. Group Liver PFOA and PFOS (% of PFOA and PFOS intake) PFOA PFOS 5% fat + PFOA/PFOS 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS AVG SD AVG SD AVG SD AVG SD 28% 10% 23% 4% 24% 4% 21% 4% 63% 34% 65% 25% 40% 12% 24% 5% Liver PFOA and PFOS as % of food intake, P value Between groups 5% fat + PFOA/PFOS vs 15% fat + PFOA/PFOS 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 15% fat + PFOA/PFOS vs 25% fat + PFOA/PFOS 35% fat + PFOA/PFOS 25% fat + PFOA/PFOS vs 35% fat + PFOA/PFOS PFOA 0.045 0.052 0.063 0.019 0.739 0.315 0.190 PFOS <0.001 0.971 0.043 0.003 0.007 <0.001 0.001 30 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Liver PFOA and PFOS levels as percentage of PFOA/PFOS intake, seemed to be decreased by the different fat diets in a fat-concentration dependent manner. When compared to the 5% fat diet group the group on 35% fat diet showed a significant reduction in liver PFOA and PFOS (by -26% (p=0.019) and by -61% (p=0.003); respectively). Figure 4.12C Liver PFOA/PFOS as % of PFOA/PFOS intake * 100% 5% Fat diet + PFOA/PFOS 15% Fat diet + PFOA/PFOS * * * 25% Fat diet + PFOA/PFOS 80% 35% Fat diet + PFOA/PFOS liver PFOA or PFOS (% of PFOA or PFOS intake) 60% * * 40% 20% 0% PFOA PFOS *: p<0.05, Mann-Whitney U test 31 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 5. Conclusions In male ApoE3L.CETP mice, the increased consumption of saturated fat led to decreased plasma total cholesterol and triglycerides levels and increased HDL-cholesterol levels. Because intake of PFOA and PFOS was not equal between mice on the different fat diets, uptake of PFOA and PFOS from the diets was estimated based on plasma PFOA and PFOS concentrations and volume of distribution compared against PFOA and PFOS intake based on diet consumption and dietary concentrations of PFOA and PFOS. The corrected estimated uptake of PFOA and PFOS levels were decreased with increasing consumption of saturated fat. Liver PFOA and PFOS levels, measured at the end of the study, confirmed the plasma results; decreased PFOA and PFOS levels with increasing consumption of saturated fat. The study indicated that in male ApoE3L.CETP mice, the increased consumption of saturated fat led to decreased PFOA and PFOS uptake and plasma concentrations while at the same time leading to decreased plasma total and non-HDL cholesterol. The results of the current study in mice do not support the hypothesis that increasing dietary fat increases the uptake and plasma concentrations of PFOA and PFOS while also increasing the plasma concentration of cholesterol. Although the original hypothesis was not demonstrated, an important finding emerges from the experiment. Increasing dietary fat consumption in the presence of low and constant dietary concentrations of PFOA and PFOS leads not only to lower serum non-HDL cholesterol, but also lower serum PFOA and PFOS plasma concentrations, resulting from lower uptake of PFOA and PFOS. Therefore, plasma PFOA and PFOS were associated with plasma total and non-HDL cholesterol, a finding similar to that observed in epidemiological studies. The basis of these associations is not known currently, and the extent to which these experimental findings may explain the epidemiological associations observed in human populations is also not known. However, this experiment does demonstrate that a dietary factor, increasing fat, can create associations in ApoE3L.CETP mice similar to those observed in human populations in the same range of serum PFOA and PFOS concentrations. 6. Comments In this study, male ApoE3L.CETP mice were fed four different semi-synthetic diets containing different amounts of cacao butter and sucrose. The diets contained 5%, 15%, 25% and 35% saturated fat, respectively. In previous studies, a semi-synthetic diet with 15% cacao butter and 1% corn oil (diet T) with the addition of different amounts of cholesterol has frequently been used at TNO. High-fat diets containing 24% and 35% fat have also been used previously by TNO in studies with male ApoE3L.CETP mice; however, the source of fat was lard. TNO had no prior experience in ApoE3L.CETP mice with the 5%, 25% and 35% cacao butter saturated fat diets. As mentioned in the protocol, TNO had no prior experience with the effect of large variation of saturated fat intake on plasma lipids. The effect on plasma cholesterol of feeding mice the 5% fat diet gave especially surprising results. Plasma cholesterol levels in male ApoE3L.CETP mice fed the 5% fat diet were increased approximately 60% as compared to mice fed the 15% fat diet. TNO suspects that the amount of fast carbohydrates and saturated fat in the diet is linked to VLDL secretion. By decreasing the amount of fast carbohydrates and increasing the fat content in the diet, the VLDL secretion is reduced, and thereby, the levels of the apoB-containing lipoproteins and triglycerides are also decreased in the mice. Treatment with the 15% fat diet (+0.25% cholesterol) resulted in plasma cholesterol levels of approximately 8 mM and triglycerides levels of approximately 3 mM, which were in line with what TNO has previously observed in male ApoE3L.CETP mice on diet T + 0.25% cholesterol (cholesterol levels of approximately 7-9 mM, triglycerides levels of approximately 2-3 mM). The measured PFOA and PFOS concentrations in the diet deviated from the target PFOA/PFOS doses in all diets. Since the target dietary concentration of PFOA and PFOS were very low (30 ppb), we prepared the PFOA/PFOS-containing diets in 4 dilution steps. We think that these multiple dilution steps in the preparation of the PFOA/PFOS diets could have caused the deviations from the target PFOA/PFOS doses. In addition, it is possible that the distribution of PFOS and PFOA in the diets were not completely homogenous. However, all diets contained PFOA/PFOS levels in the ppb range, which was not expected to affect plasma lipid levels based on prior toxicological studies. Because the dietary concentrations of PFOA and PFOS in the different diets were somewhat variable, and in order to correct for potential differences in diet consumption, we used the dietary 32 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 concentrations of PFOA and PFOS along with the individual diet consumption as a measure of dietary intake of PFOA and PFOS and compared the dietary intake of PFOA and PFOS to the estimated uptake to the body, the latter being estimated by multiplying the plasma PFOA and PFOS concentrations with volume of distribution. This allowed us to estimate the proportions of the dietary intake of PFOA and PFOS that were absorbed. It should be noted that: - Food intake could not be precisely measured per mouse at all time points and with all diets. - There was substantial variation in the levels of PFOA and PFOS measured in the diet, possibly due to non-homogeneity. - There are some potential outliers in the measurement of plasma and liver PFOA and PFOS levels. Based on Chauvenet's criterion, 3 of 120 PFOA plasma determinations, 9 of 120 PFOS plasma determinations, 2 of 40 PFOA liver determinations and 3 of 40 PFOS liver determinations may have been outliers, which may be indicative of "hotspots" in the diet. The current study indicated that in male ApoE3L.CETP mice, after correcting for PFOA and PFOS intake, estimated uptakes of PFOA and PFOS from the diet were decreased with increasing consumption of saturated fat. Although the question remains whether the results in ApoE3L.CETP mice can be extrapolated to the human situation, the current study in mice does not support the hypothesis that increased consumption of saturated fats lead to increased serum/plasma concentrations of PFOA and PFOS as well as increased serum/plasma cholesterol concentration. The finding in the current study that the uptakes of PFOA and PFOS were decreased with increasing consumption of saturated fat may be related to a decreased intestinal absorption of dietary PFOA and PFOS with increasing consumption of saturated fat. Perhaps the increased fat content in the diet caused a less efficient intestinal fat absorption that coincided with decreased absorption of PFOA and PFOS. The observations that the mice consumed more of the higher fat diets than what we would expect based on an isocaloric intake and that the mice fed the higher fat diets did not gain more weight than the low fat group are consistent with less efficient intestinal fat absorption when fed the higher fat diets. It is possible that at higher fat intakes the potentially reduced fat uptake and consequently increased fecal excretion carried away PFOA and PFOS. Unfortunately, we did not collect fecal samples in the current study to examine the fecal excretion of fatty acids and PFOA/PFOS. Increasing consumption of saturated fat could also affect PFOA and PFOS storage in various tissues, leading to decreased plasma PFOA and PFOS levels. In the current study, liver weights were decreased significantly by the increasing fat diets. PFOA and PFOS are not known to distribute into fat, and fat tissues were not evaluated at the end of the study. In order to evaluate the effect of saturated fat consumption on PFOA and PFOS storage in liver, PFOA and PFOS liver levels were measured. The liver levels confirmed the plasma results; decreased PFOA and PFOS levels with increasing consumption of saturated fat. Another possible explanation for the observed decreased PFOA and PFOS levels with increasing consumption of saturated fat could lie in the results of the different fat diets on plasma lipid levels. In male ApoE3L.CETP mice, the increased consumption of saturated fat and decreased consumption of fast carbohydrates led to decreased plasma total cholesterol levels. The estimated PFOA and PFOS intake levels and plasma concentrations were also decreased with increased dietary fat and decreased fast carbohydrates. Because positive associations of serum PFOA or PFOS with serum non-HLD cholesterol have been observed in epidemiological studies, we have tested whether there is a significant correlation between PFOA/PFOS levels and plasma total cholesterol levels. Per mouse the average estimated PFOA and PFOS levels during treatment (t=1-4 weeks) and the average cholesterol levels (t=0-4 weeks) were calculated (see appendix XI for individual values). Significance of correlation between parameters were calculated non-parametrically, using the computer program SPSS. The Spearman's rho non-parametric correlations algorithm was used for the calculation of the correlation coefficient and the significance. 33 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Correlation relative PFOA/PFOS dose and total cholesterol levels 80% 70% PFOA PFOS Relative PFOA/PFOS dose 60% 50% 40% 30% 20% 10% 2 4 6 8 10 12 14 16 18 20 Plasma total cholesterol (mM) Correlation PFOA dose-cholesterol PFOS dose-cholesterol Spearman's rho correlation coefficient 0.445 0.417 P value 0.004 0.008 In the current study, there is a significant positive correlation between both the PFOA levels and plasma cholesterol and the PFOS levels and plasma cholesterol. The increase in PFOA and PFOS levels could be caused by the increase in plasma cholesterol by a yet unknown mechanism. To exclude that the increase in PFOA/PFOS levels and the increase in plasma cholesterol levels are two independent effects, both caused by the dietary fat content, ApoE3L.CETP mice could be fed diets with the same dietary fat and PFOA/PFOS content but with different dietary cholesterol concentrations. To investigate what the effect of a diet containing fibers and plant sterols next to a low fat is on PFOA and PFOS plasma levels, a group fed a regular chow diet could also be added in the same experiment. Taking into account that the current study has been performed in mice, the results do not support the hypothesis that the observed association between serum PFOA or PFOS levels and serum total cholesterol in humans is the result of an increased consumption of saturated fat. Perhaps by only changing the fat and sucrose content in the diets, we have not taken into account that in humans the increased consumption of saturated fat could coincide with an increased consumption of dietary cholesterol and could also coincide with a decreased consumption of dietary fibers and plant sterols (for example by eating a meat-rich and vegetable-poor diet). In studies performed at TNO, it has been observed that the intestinal absorption of lipophilic compounds was higher in a diet with a higher cholesterol concentration and higher in a diet with lower dietary fiber and plant sterol content. It is quite possible that feeding mice higher dietary cholesterol leads to a high bile acid production and a higher biliary pool, causing a higher intestinal absorption of the amphiphilic PFOA and PFOS. The addition of fibers and plant sterols leads to a lower intestinal cholesterol absorption and perhaps also to a lower intestinal absorption of PFOA and PFOS. In the proposed study, the effect of dietary cholesterol on plasma PFOA and PFOS levels (and PFOA and PFOS fecal excretion) and the effect of a chow diet (high on dietary fiber and plant sterol 34 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 content) versus a semi synthetic fat diet on plasma PFOA and PFOS levels (and PFOA and PFOS fecal excretion) could be examined. A further limitation of the present study was that a fat composition was used which does not represent the composition of dietary fat in humans. The present fat is very rich in saturated fat with lower amounts of monounsaturated fat and a low amount of polyunsaturated fat. In man much more mono- and polyunsaturated fat is consumed. Moreover, the fat intake lies in general between 25 and 45 kcal%. In the present study, the range was 12 - 64 kcal%. Therefore, a suggestion for the proposed study is to use a westernized diet instead of a diet in which the main fat source is cacoa butter. In westernized diet the dietary fat composition has been humanized (with 45% saturated fat, 35% mono unsaturated fat and 20% poly unsaturated fat and an energy of 37kcal% fat), but the fat sources are not from animal source (the humanized fat contains soy bean oil, olive oil, cacoa butter, coconut oil and palm oil). 35 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 7. References 1. Van den Maagdenberg AMJM, Hofker MH, Krimpenfort PJA, de Bruijn IG, van Vlijmen B, van der Boom H, Havekes LM, Frants RR. Transgenic mice carrying the apolipoprotein E3Leiden gene exhibit hyperlipoproteinemia. J Biol Chem 1993; 268: 10540-10545. 2. Van Vlijmen B, van den Maagdenberg AMJM, Gijbels MJJ, van der Boom H, HogenEsch H, Frants RR, Hofker MH, Havekes LM. Diet-induced hyperlipoproteinemia and atherosclerosis in apolipoprotein E3-Leiden transgenic mice. J Clin Invest 1994; 93: 1403-1410. 3. Groot PHE, van Vlijmen BJM, Benson GM, Hofker MH, Schiffelers R, Vidgeon-Hart M, Havekes LM. Quantitative assessment of aortic atherosclerosis in apoE3Leiden transgenic mice and its relationship to serum cholesterol exposure. Arterioscler Thromb Vasc Biol 1996; 16: 926-933. 4. Zadelaar S, Kleemann R, Verschuren L, de Vries-van der Weij J, van der Hoorn J, Princen HM, Kooistra T. Mouse models for athereosclerosis and pharmaceutical modifiers. Arterioscler Thromb Vasc Biol 2007: 27; 1706-1721. 5. Westerterp M, Van der Hoogt CC, de Haan W, Offerman EH, Dallinga-Thie GM, Jukema JW, Havekes LM, Rensen PCN. Cholesteryl ester transfer protein decreases HDL and severely aggravates atherosclerosis in APOE*3Leiden mice. Arterioscler Thromb Vasc Biol 2006: 26; 2552-2559. 6. Van der Hoogt CC, de Haan W, Westerterp M, Hoekstra M, Dallinga-Thie GM, Romijn JA, Princen HMG, Jukema JW, Havekes LM, Rensen PCN. Fenofibrate increases HDL cholesterol by reducing cholesteryl ester transfer protein expression. J Lipid Res 2007; 48: 17631771. 7. De Haan W, van der Hoogt CC, Westerterp M, Hoekstra M, Dallinga-Thie GM, Princen HMG, Romijn JA, Jukema JW, Havekes LM, Rensen PCN. Atorvastatin increases HDL cholesterol by reducing CETP expression in cholesterol-fed APOE*3-Leiden.CETP mice. Atherosclerosis 2008; 197: 57-63. 8. Van der Hoorn JW, de Haan W, Berbee JF, Havekes LM, Jukema JW, Rensen PC, Princen HM Niacin Increases HDL by Reducing Hepatic Expression and Plasma Levels of Cholesteryl Ester Transfer Protein in APOE*3Leiden.CETP Mice. Arterioscler Thromb Vasc Biol 2008; 28: 2016-2022. 9. De Haan W., de Vries-Van der Weij J, van der Hoorn JWA, Gautier T, van der Hoogt CC, Westerterp M, Romijn JA, Jukema JW, Havekes LM, Princen HMG, Rensen PCN. Torcetrapib does not reduce atherosclerosis beyond atorvastatin and induces more proinflammatory lesions than atorvastatin. Circulation 2008; 117: 2515-2522. 36 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 8. Appendices Appendix I Body weight Group 1: 5% fat diet + PFOA/PFOS Mouse# Cage# Body weight (g) in time t=-4 t=0 t=2 t=4 1 1 24.4 27.1 27.8 28.5 2 1 27.1 28.7 28.6 28.7 3 1 25.7 28.7 29.4 30.1 4 2 25.3 27.0 27.3 28.5 5 2 20.9 23.8 25.1 25.7 6 3 24.6 26.4 27.8 27.2 7 3 25.4 26.6 27.4 27.8 8 4 26.3 29.3 29.8 30.3 9 4 26.6 29.7 30.8 31.1 10 4 30.4 33.4 33.8 33.0 AVG 25.7 28.1 28.8 29.1 SD 2.4 2.6 2.4 2.1 n 10 10 10 10 Group 2: 15% fat diet + PFOA/PFOS Mouse# Cage# Body weight (g) in time t=-4 t=0 t=2 t=4 11 5 23.1 25.7 25.6 26.8 12 5 25.2 27.2 27.9 28.2 13 5 27.3 29.8 30.9 31.9 14 6 20.2 22.5 22.4 22.9 15 6 24.4 27.4 28.8 30.2 16 7 27.8 31.5 33.2 34.2 17 7 26.0 29.9 30.8 33.1 18 8 26.4 29.8 29.6 30.2 19 8 27.8 30.3 31.4 30.9 20 8 27.5 29.0 30.0 30.3 AVG 25.6 28.3 29.1 29.9 SD 2.4 2.7 3.1 3.3 n 10 10 10 10 Group 3: 25% fat diet + PFOA/PFOS Mouse# Cage# Body weight (g) in time t=-4 t=0 t=2 t=4 21 9 25.2 26.4 26.4 27.4 22 9 24.4 27.3 28.9 30.4 23 9 25.3 27.1 27.8 28.0 24 9 25.2 27.2 27.4 28.0 25 10 25.6 26.9 26.4 26.6 26 10 23.3 24.4 24.1 24.3 27 11 28.7 29.2 28.8 30.2 28 11 26.7 28.0 28.7 29.2 29 12 23.3 26.5 26.8 28.1 30 12 23.1 24.5 25.7 26.3 AVG 25.1 26.8 27.1 27.9 SD 1.7 1.5 1.5 1.8 n 10 10 10 10 37 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 4: 35% fat diet + PFOA/PFOS Mouse# Cage# Body weight (g) in time t=-4 t=0 t=2 t=4 31 13 24.2 26.4 26.8 27.4 32 13 24.3 27.0 27.7 28.2 33 14 22.6 24.1 25.5 25.3 34 14 25.0 26.9 28.9 29.5 35 15 28.3 29.6 30.1 30.6 36 15 26.6 29.1 30.2 31.1 37 16 25.1 29.3 31.0 32.6 38 16 23.2 26.0 26.2 26.5 39 17 25.5 26.8 27.3 27.3 40 17 26.0 26.8 27.0 27.8 AVG 25.1 27.2 28.1 28.6 SD 1.7 1.7 1.9 2.3 n 10 10 10 10 38 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix II Liver weight Group 1: 5% fat diet + PFOA/PFOS Mouse# Cage# Liver weight (g) t=4 weeks 1 1 1.71 2 1 2.08 3 1 2.12 4 2 1.80 5 2 1.76 6 3 1.65 7 3 1.70 8 4 1.72 9 4 1.23 10 4 1.87 AVG 1.8 SD 0.2 n 10 Group 2: 15% fat diet + PFOA/PFOS Mouse# Cage# Liver weight (g) t=4 weeks 11 5 1.35 12 5 1.68 13 5 1.90 14 6 1.17 15 6 1.71 16 7 1.65 17 7 1.87 18 8 1.58 19 8 1.68 20 8 1.47 AVG 1.6 SD 0.2 n 10 Group 3: 25% fat diet + PFOA/PFOS Mouse# Cage# Liver weight (g) t=4 weeks 21 9 1.34 22 9 1.52 23 9 1.44 24 9 1.38 25 10 1.37 26 10 1.29 27 11 1.52 28 11 1.50 29 12 1.27 30 12 1.16 AVG 1.4 SD 0.1 n 10 39 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 4: 35% fat diet + PFOA/PFOS Mouse# Cage# Liver weight (g) t=4 weeks 31 13 1.35 32 13 1.32 33 14 1.17 34 14 1.28 35 15 1.45 36 15 1.53 37 16 1.36 38 16 1.26 39 17 1.29 40 17 1.35 AVG 1.3 SD 0.1 n 10 40 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix III Food and fat intake Group 1: 5% fat diet + PFOA/PFOS Cage# Number Food intake (g/day/mouse) in time of mice t=-3 t=-2 t=0 t=1 t=2 t=3 1 3 3.42 3.48 3.39 3.26 3.21 3.30 2 2 3.51 3.73 3.58 3.65 3.48 3.75 3 2 3.16 3.35 3.02 2.94 2.94 2.74 4 3 4.28 3.95 4.06 4.23 4.42 4.40 AVG 3.6 3.6 3.5 3.5 3.5 3.5 SD 0.5 0.3 0.4 0.6 0.6 0.7 n 4 4 4 4 4 4 Group 2: 15% fat diet + PFOA/PFOS Cage# Number of mice t=-3 t=-2 5 3 3.54 3.32 6 2 3.28 3.38 7 2 * 3.89 8 3 * * AVG 3.4 3.5 SD/Range 0.1 0.3 n 2 3 Food intake (g/day/mouse) in time t=0 t=1 t=2 3.71** 3.86** 3.83 ** 3.25 3.20 3.21 3.37** 3.38** 3.85** 3.82** 3.94** 3.90** 3.5 3.6 3.7 0.3 0.4 0.3 4 4 4 t=3 3.94** 3.19 3.06** 3.86** 3.5 0.5 4 t=4 3.10 3.56 2.91 4.06 3.4 0.5 4 t=4 3.96** 2.91 3.62** 4.01** 3.6 0.5 4 Group 3: 25% fat diet + PFOA/PFOS Mouse# Cage# Food intake (g/day/mouse) in time t=-3 t=-2 t=0 t=1 t=2 9 4 3.33 2.95 3.09** 3.25** 3.24** 10 2 2.91 2.96 2.87 2.71 2.78 11B 1 11A 1 * 3.93 3.63 3.50 3.12 3.44 3.03 3.12 3.18 12 2 * * 3.08 3.08 3.21 AVG 3.1 3.3 3.1 3.1 3.1 SD/Range 0.2 0.5 0.3 0.3 0.2 n 2 4 5 5 5 Mouse from cage 11 were split in week -3, because of fighting wounds t=3 3.14** 2.76 3.39 3.47 3.11 3.2 0.3 5 t=4 3.38** 2.73 3.47 3.19 3.16 ** 3.2 0.3 5 Group 4: 35% fat diet + PFOA/PFOS Mouse# Cage# t=-3 t=-2 13 2 3.04 3.30 14 2 3.00 2.50 15 2 3.13 2.93 16 2 * 3.21 17 2 3.18 * AVG 3.1 3.0 SD 0.1 0.4 n 4 4 Food intake (g/day/mouse) in time t=0 t=1 t=2 3.13 3.15 3.09 2.73 3.03 3.01 3.23 3.19 3.33 3.42** 3.73** 3.52** 3.13** 3.43** 3.84** 3.1 3.3 3.4 0.3 0.3 0.3 5 5 5 t=3 3.10 2.73 3.37 4.12** 3.57** 3.4 0.5 5 t=4 2.89 3.12** 3.16 4.01** 3.52** 3.3 0.4 5 *: a lot of small crums on bottom of cage, food intake was not measured accurately **: mice crumbled food, crums on bottom of cage were also collected for accurate food intake measurements 41 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 1: 5% fat diet + PFOA/PFOS Cage# Number of mice t=-3 t=-2 1 3 0.17 0.17 2 2 0.18 0.19 3 2 0.16 0.17 4 3 0.21 0.20 AVG 0.18 0.18 SD 0.02 0.01 n 4 4 Group 2: 15% fat diet + PFOA/PFOS Cage# Number of mice t=-3 t=-2 5 3 0.53 0.50 6 2 0.49 0.51 7 2 * 0.58 8 3 * * AVG 0.51 0.53 SD/Range 0.02 0.05 n 2 3 Fat intake (g/day/mouse) in time t=0 t=1 t=2 0.17 0.16 0.16 0.18 0.18 0.17 0.15 0.15 0.15 0.20 0.21 0.22 0.18 0.18 0.18 0.02 0.03 0.03 4 4 4 Fat intake (g/day/mouse) in time t=0 t=1 t=2 0.56** 0.58** 0.58 ** 0.49 0.48 0.48 0.51** 0.51** 0.58** 0.57** 0.59** 0.59** 0.53 0.54 0.55 0.04 0.05 0.05 4 4 4 t=3 0.16 0.19 0.14 0.22 0.18 0.04 4 t=3 0.59** 0.48 0.46** 0.58** 0.53 0.07 4 t=4 0.15 0.18 0.15 0.20 0.17 0.03 4 t=4 0.59** 0.44 0.54** 0.60** 0.54 0.08 4 Group 3: 25% fat diet + PFOA/PFOS Mouse# Cage# Fat intake (g/day/mouse) in time t=-3 t=-2 t=0 t=1 t=2 9 4 0.83 0.74 0.77** 0.81** 0.81** 10 2 0.73 0.74 0.72 0.68 0.69 11B 1 11A 1 * 0.98 0.91 0.88 0.78 0.86 0.76 0.78 0.80 12 2 * * 0.77 0.77 0.80 AVG 0.78 0.83 0.79 0.78 0.78 SD/Range 0.05 0.12 0.07 0.07 0.05 n 2 4 5 5 5 Mouse from cage 11 were split in week -3, because of fighting wounds t=3 0.79** 0.69 0.85 0.87 0.78 0.79 0.07 5 t=4 0.85** 0.68 0.87 0.80 0.79 ** 0.80 0.07 5 Group 4: 35% fat diet + PFOA/PFOS Mouse# Cage# t=-3 t=-2 13 2 1.06 1.16 14 2 1.05 0.88 15 2 1.09 1.02 16 2 * 1.12 17 2 1.11 * AVG 1.08 1.04 SD 0.03 0.13 n 4 4 Fat intake (g/day/mouse) in time t=0 t=1 t=2 1.10 1.10 1.08 0.96 1.06 1.05 1.13 1.12 1.17 1.20** 1.30** 1.23** 1.10** 1.20** 1.34** 1.10 1.16 1.18 0.09 0.10 0.12 5 5 5 t=3 1.09 0.96 1.18 1.44** 1.25** 1.18 0.18 5 t=4 1.01 1.09** 1.11 1.41** 1.23** 1.17 0.15 5 *: a lot of small crums on bottom of cage, food intake was not measured accurately **: mice crumbled food, crums on bottom of cage were also collected for accurate food intake measurements 42 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix IV Accumulated PFOA and PFOS intake Aimed and measured PFOA/PFOS doses in diets Measured PFOA/PFOS doses in regular chow diet and different fat diets without PFOA/PFOS added, were below detection limit (<0.5 ppb). PFOA/PFOS in diet preparation Weighed: 0.1249 g PFOS 0.1250 g PFOA weighed PFOS mixed with weighed PFOA 3 parts per thousand PFOA/PFOS: 0.3 parts per thousand PFOA/PFOS: 3 ppm PFOA/PFOS: 0.125 g PFOA/PFOS in 10 gram 3 parts per thousand PFOA/PFOS + 10 gram 0.3 parts per thousand PFOA/PFOS + 41.7 g 5% fat diet 90 g 5% fat diet 990 g 5% fat diet 35% fat diet: 30 ppb PFOA/PFOS 60 g 3 ppm PFOA/PFOS mixed in 6 kg 35% fat diet PFOA/FPOS amount in other diets based on food intake t=-3/t=-2 (equal dosing as 35% fat diet) Food intake t=-3/t=-2: Group Group 1: 5% fat diet Group 2: 15% fat diet Group 3: 25% fat diet Group 4: 35% fat diet Food intake (g/d/mouse) 3.61 3.47 3.22 3.03 60.0 gram 3 ppm PFOA+ PFOS 56.5 gram 3 ppm PFOA+ PFOS 52.5 gram 3 ppm PFOA+ PFOS 50.4 gram 3 ppm PFOA+ PFOS mixed in 6 kg 35% fat diet 25% fat diet 15% fat diet 5% fat diet Aimed PFOA/PFOS in diet (ppb) 30 32 34 36 Measured PFOA/PFOS in diet Group Group 1: 5% fat diet Group 2: 15% fat diet Group 3: 25% fat diet Group 4: 35% fat diet Measured PFOA in diet ppb 24.3 32.5 26.1 28.6 27.0 23.7 20.7 31.7 Average PFOA in diet ppb 28.4 27.4 25.3 26.2 Measured PFOS in diet ppb 20.6 15.0 10.8 13.4 17.5 13.4 37.4 20.3 Average PFOS in diet ppb 17.8 12.1 15.5 28.8 43 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 1: 5% fat diet Mouse# Cage# 1 1 2 1 3 1 4 2 5 2 6 3 7 3 8 4 9 4 10 4 AVG SD n Accumulated PFOA intake (ng) in time t=1 t=2 t=3 t=4 555 1102 1757 2373 555 1102 1757 2373 555 1102 1757 2373 622 1215 1961 2669 622 1215 1961 2669 502 1003 1547 2127 502 1003 1547 2127 722 1475 2349 3156 722 1475 2349 3156 722 1475 2349 3156 608 1217 1934 2618 88 192 318 413 10 10 10 10 Group 2: 15% fat diet Mouse# Cage# 11 5 12 5 13 5 14 6 15 6 16 7 17 7 18 8 19 8 20 8 AVG SD n Accumulated PFOA intake (ng) in time t=1 t=2 t=3 t=4 633 1263 2017 2776 633 1263 2017 2776 633 1263 2017 2776 525 1052 1663 2220 525 1052 1663 2220 556 1188 1774 2467 556 1188 1774 2467 648 1288 2028 2797 648 1288 2028 2797 648 1288 2028 2797 600 1213 1901 2609 53 93 162 243 10 10 10 10 Group 3: 25% fat diet Mouse# Cage# 21 9 22 9 23 9 24 9 25 10 26 10 27 11B 28 11A 29 12 30 12 AVG SD n Accumulated PFOA intake (ng) in time t=1 t=2 t=3 t=4 495 987 1544 2144 495 987 1544 2144 495 987 1544 2144 495 987 1544 2144 412 833 1323 1807 412 833 1323 1807 532 1006 1606 2221 474 957 1573 2138 469 956 1508 2068 469 956 1508 2068 474 949 1502 2069 38 63 98 144 10 10 10 10 Group 1: 5% fat diet Mouse# Cage# Accumulated PFOA intake in time per kg mouse (g/kg) t=1 t=2 t=4 1 1 20.2 39.6 83.3 2 1 19.4 38.5 82.7 3 1 19.1 37.5 78.8 4 2 22.9 44.5 93.6 5 2 25.5 48.4 103.8 6 3 18.5 36.1 78.2 7 3 18.6 36.6 76.5 8 4 24.4 49.5 104.2 9 4 23.9 47.9 101.5 10 4 21.5 43.6 95.6 AVG 21 42 90 SD 3 5 11 n 10 10 10 Group 2: 15% fat diet Mouse# Cage# Accumulated PFOA intake in time per kg mouse (g/kg) t=1 t=2 t=4 11 5 24.7 49.3 103.6 12 5 23.0 45.3 98.4 13 5 20.9 40.9 87.0 14 6 23.4 47.0 96.9 15 6 18.7 36.5 73.5 16 7 17.2 35.8 72.1 17 7 18.3 38.6 74.5 18 8 21.8 43.5 92.6 19 8 21.0 41.0 90.5 20 8 22.0 42.9 92.3 AVG 21 42 88 SD 2 4 11 n 10 10 10 Group 3: 25% fat diet Mouse# Cage# Accumulated PFOA intake in time per kg mouse (g/kg) t=1 t=2 t=4 21 9 18.7 37.4 78.3 22 9 17.6 34.2 70.5 23 9 18.0 35.5 76.6 24 9 18.1 36.0 76.6 25 10 15.4 31.6 67.9 26 10 17.0 34.6 74.4 27 11B 18.3 34.9 73.6 28 11A 16.7 33.4 73.2 29 12 17.6 35.7 73.6 30 12 18.7 37.2 78.6 AVG 18 35 74 SD 1 2 3 n 10 10 10 44 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 4: 35% fat diet Mouse# Cage# 31 13 32 13 33 14 34 14 35 15 36 15 37 16 38 16 39 17 40 17 AVG SD n Accumulated PFOA intake (ng) in time t=1 t=2 t=3 t=4 495 980 1548 2077 495 980 1548 2077 476 949 1449 2021 476 949 1449 2021 501 1025 1643 2222 501 1025 1643 2222 585 1137 1893 2628 585 1137 1893 2628 538 1141 1796 2441 538 1141 1796 2441 519 1047 1666 2278 41 84 170 240 10 10 10 10 Group 1: 5% fat diet Mouse# Cage# 1 1 2 1 3 1 4 2 5 2 6 3 7 3 8 4 9 4 10 4 AVG SD n Accumulated PFOS intake (ng) in time t=1 t=2 t=3 t=4 347.9 690.5 1101.4 1487.3 347.9 690.5 1101.4 1487.3 347.9 690.5 1101.4 1487.3 390.1 761.5 1229.1 1672.6 390.1 761.5 1229.1 1672.6 314.4 628.8 969.9 1333.3 314.4 628.8 969.9 1333.3 452.4 924.5 1472.5 1978.4 452.4 924.5 1472.5 1978.4 452.4 924.5 1472.5 1978.4 381 763 1212 1641 55 120 199 259 10 10 10 10 Group 2: 15% fat diet Mouse# Cage# Accumulated PFOS intake (ng) in time t=1 t=2 t=3 t=4 11 5 279.9 558.2 891.7 1227.3 12 5 279.9 558.2 891.7 1227.3 13 5 279.9 558.2 891.7 1227.3 14 6 232.3 465.2 735.0 981.2 15 6 232.3 465.2 735.0 981.2 16 7 245.6 525.1 784.0 1090.7 17 7 245.6 525.1 784.0 1090.7 18 8 286.3 569.5 896.5 1236.5 19 8 286.3 569.5 896.5 1236.5 20 8 286.3 569.5 896.5 1236.5 AVG 265 536 840 1154 SD 23 41 71 108 n 10 10 10 10 Group 4: 35% fat diet Mouse# Cage# Accumulated PFOA intake in time per kg mouse (g/kg) t=1 t=2 t=4 31 13 18.6 36.6 75.8 32 13 18.1 35.4 73.7 33 14 19.2 37.2 79.9 34 14 17.1 32.8 68.5 35 15 16.8 34.0 72.6 36 15 16.9 33.9 71.5 37 16 19.4 36.7 80.6 38 16 22.4 43.4 99.2 39 17 19.9 41.8 89.4 40 17 20.0 42.3 87.8 AVG 19 37 80 SD 2 4 10 n 10 10 10 Group 1: 5% fat diet Mouse# Cage# Accumulated PFOS intake in time per kg mouse (g/kg) t=1 t=2 t=4 1 1 12.7 24.8 52.2 2 1 12.1 24.1 51.8 3 1 12.0 23.5 49.4 4 2 14.4 27.9 58.7 5 2 16.0 30.3 65.1 6 3 11.6 22.6 49.0 7 3 11.6 22.9 48.0 8 4 15.3 31.0 65.3 9 4 15.0 30.0 63.6 10 4 13.5 27.4 60.0 AVG 13 26 56 SD 2 3 7 n 10 10 10 Group 2: 15% fat diet Mouse# Cage# Accumulated PFOS intake in time per kg mouse (g/kg) t=1 t=2 t=4 11 5 10.9 21.8 45.8 12 5 10.2 20.0 43.5 13 5 9.2 18.1 38.5 14 6 10.3 20.8 42.8 15 6 8.3 16.2 32.5 16 7 7.6 15.8 31.9 17 7 8.1 17.0 33.0 18 8 9.6 19.2 40.9 19 8 9.3 18.1 40.0 20 8 9.7 19.0 40.8 AVG 9 19 39 SD 1 2 5 n 10 10 10 45 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 3: 25% fat diet Mouse# Cage# Accumulated PFOS intake (ng) in time t=1 t=2 t=3 t=4 21 9 301.9 602.5 942.7 1308.7 22 9 301.9 602.5 942.7 1308.7 23 9 301.9 602.5 942.7 1308.7 24 9 301.9 602.5 942.7 1308.7 25 10 251.2 508.6 807.8 1103.1 26 10 251.2 508.6 807.8 1103.1 27 11B 324.7 613.8 980.1 1355.8 28 11A 289.1 584.4 960.0 1304.8 29 12 286.0 583.6 920.6 1262.3 30 12 286.0 583.6 920.6 1262.3 AVG 290 579 917 1263 SD 23 39 60 88 n 10 10 10 10 Group 4: 35% fat diet Mouse# Cage# Accumulated PFOS intake (ng) in time t=1 t=2 t=3 t=4 31 13 544.8 1079.5 1705.1 2287.3 32 13 544.8 1079.5 1705.1 2287.3 33 14 524.6 1044.9 1595.5 2225.4 34 14 524.6 1044.9 1595.5 2225.4 35 15 552.0 1128.5 1808.8 2447.3 36 15 552.0 1128.5 1808.8 2447.3 37 16 644.3 1252.5 2084.1 2894.1 38 16 644.3 1252.5 2084.1 2894.1 39 17 592.4 1256.8 1977.5 2688.0 40 17 592.4 1256.8 1977.5 2688.0 AVG 572 1152 1834 2508 SD 45 92 187 264 n 10 10 10 10 Group 3: 25% fat diet Mouse# Cage# Accumulated PFOS intake in time per kg mouse (g/kg) t=1 t=2 t=4 21 9 11.4 22.8 47.8 22 9 10.7 20.8 43.0 23 9 11.0 21.7 46.7 24 9 11.1 22.0 46.7 25 10 9.4 19.3 41.5 26 10 10.4 21.1 45.4 27 11B 11.2 21.3 44.9 28 11A 10.2 20.4 44.7 29 12 10.7 21.8 44.9 30 12 11.4 22.7 48.0 AVG 11 21 45 SD 1 1 2 n 10 10 10 Group 4: 35% fat diet Mouse# Cage# Accumulated PFOS intake in time per kg mouse (g/kg) t=1 t=2 t=4 31 13 20.5 40.3 83.5 32 13 19.9 39.0 81.1 33 14 21.2 41.0 88.0 34 14 18.8 36.2 75.4 35 15 18.5 37.5 80.0 36 15 18.6 37.4 78.7 37 16 21.4 40.4 88.8 38 16 24.7 47.8 109.2 39 17 21.9 46.0 98.5 40 17 22.0 46.5 96.7 AVG 21 41 88 SD 2 4 11 n 10 10 10 46 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix V Plasma PFOA and PFOS levels Group 1: 5% fat diet Mouse# Cage# t=-4 1 1 <0.5 2 1 <0.5 3 1 <0.5 4 2 <0.5 5 2 <0.5 6 3 <0.5 7 3 <0.5 8 4 <0.5 9 4 <0.5 10 4 <0.5 AVG <0.5 SD - n 10 Group 2: 15% fat diet Mouse# Cage# t=-4 11 5 <0.5 12 5 <0.5 13 5 <0.5 14 6 <0.5 15 6 <0.5 16 7 <0.5 17 7 <0.5 18 8 <0.5 19 8 <0.5 20 8 <0.5 AVG <0.5 SD - n 10 PFOA conc in plasma (ng/mL) t=0 t=1 t=2 <0.5 37.3 96.3 <0.5 45.0 93.3 <0.5 43.5 102.3 <0.5 51.2 95.9 <0.5 56.7 96.8 <0.5 39.5 108.5 0.6 50.7 101.0 <0.5 35.3 101.2 0.5 64.7 139.8 <0.5 36.5 104.7 <0.6 46.0 104.0 - 9.7 13.4 10 10 10 t=4 173.0 226.0 197.5 178.0 161.5 206.5 222.0 160.0 192.5 229.8 194.7 26.2 10 PFOA conc in plasma (ng/mL) t=0 t=1 t=2 <0.5 28.6 75.3 0.7 31.3 81.8 <0.5 31.0 77.9 <0.5 36.3 85.6 <0.5 44.8 86.1 <0.5 42.4 109.5 <0.5 32.0 68.9 <0.5 39.0 92.7 <0.5 33.0 71.7 0.6 36.4 70.1 <0.7 35.5 81.9 - 5.3 12.4 10 10 10 t=4 141.5 154.0 143.0 165.5 159.0 177.5 152.5 155.5 145.0 180.0 157.4 13.5 10 Group 3: 25% fat diet Mouse# Cage# t=-4 21 9 <0.5 22 9 <0.5 23 9 <0.5 24 9 <0.5 25 10 <0.5 26 10 <0.5 27 11B <0.5 28 11A <0.5 29 12 <0.5 30 12 <0.5 AVG <0.5 SD - n 10 PFOA conc in plasma (ng/mL) t=0 t=1 t=2 <0.5 26.5 61.7 0.5 29.6 60.1 <0.5 31.4 70.3 <0.5 28.1 74.5 0.5 27.3 63.4 <0.5 34.0 67.2 0.5 29.2 56.1 0.7 32.1 69.2 0.5 30.7 67.7 0.6 30.8 66.0 <0.7 29.9 65.6 - 2.3 5.4 10 10 10 t=4 124.0 136.8 157.0 154.0 144.5 143.0 122.5 154.3 138.0 158.0 143.2 13.0 10 47 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 4: 35% fat diet Mouse# Cage# t=-4 31 13 <0.5 32 13 <0.5 33 14 <0.5 34 14 <0.5 35 15 <0.5 36 15 <0.5 37 16 <0.5 38 16 <0.5 39 17 <0.5 40 17 <0.5 AVG <0.5 SD - n 10 Group 1: 5% fat diet Mouse# Cage# t=-4 1 1 <0.5 2 1 <0.5 3 1 <0.5 4 2 <0.5 5 2 <0.5 6 3 <0.5 7 3 <0.5 8 4 <0.5 9 4 <0.5 10 4 <0.5 AVG <0.5 SD - n 10 Group 2: 15% fat diet Mouse# Cage# t=-4 11 5 <0.5 12 5 <0.5 13 5 <0.5 14 6 <0.5 15 6 <0.5 16 7 <0.5 17 7 <0.5 18 8 <0.5 19 8 <0.5 20 8 <0.5 AVG <0.5 SD - n 10 PFOA conc in plasma (ng/mL) t=0 t=1 t=2 0.7 28.5 65.8 <0.5 27.6 65.9 0.8 21.2 60.1 1 32.3 89.0 0.6 24.7 56.4 0.6 23.6 60.8 0.7 33.5 75.9 0.8 28.7 61.8 0.8 35.1 68.7 0.7 31.6 65.3 <1.0 28.6 66.9 - 4.5 9.4 10 10 10 t=4 119.5 129.0 133.0 134.5 105.5 117.0 145.5 132.0 114.5 152.8 128.3 14.5 10 PFOS conc in plasma (ng/mL) t=0 t=1 t=2 <0.5 16.0 36.4 <0.5 12.1 27.9 <0.5 13.3 157.5 <0.5 21.3 46.8 <0.5 21.2 41.7 <0.5 16.4 46.6 <0.5 18.4 42.0 <0.5 18.0 42.9 <0.5 18.1 58.7 <0.5 50.1 68.4 <0.5 20.5 56.9 - 10.8 37.1 10 10 10 t=4 68.7 84.4 178.0 87.1 72.1 88.5 109.0 75.7 102.1 110.0 97.5 31.8 10 PFOS conc in plasma (ng/mL) t=0 t=1 t=2 <0.5 16.4 29.8 <0.5 23.3 32.1 <0.5 12.9 28.6 <0.5 11.5 35.6 <0.5 11.6 30.3 <0.5 16.0 40.3 <0.5 15.1 26.7 <0.5 20.2 45.6 <0.5 12.5 29.4 <0.5 34.2 32.1 <0.5 17.3 33.0 - 7.0 5.9 10 10 10 t=4 96.2 74.8 66.8 82.3 63.5 72.0 55.5 82.7 138.0 77.2 80.9 23.0 10 48 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 3: 25% fat diet Mouse# Cage# t=-4 21 9 <0.5 22 9 <0.5 23 9 <0.5 24 9 <0.5 25 10 <0.5 26 10 <0.5 27 11B <0.5 28 11A <0.5 29 12 <0.5 30 12 <0.5 AVG <0.5 SD - n 10 Group 4: 35% fat diet Mouse# Cage# t=-4 31 13 <0.5 32 13 <0.5 33 14 <0.5 34 14 <0.5 35 15 <0.5 36 15 <0.5 37 16 <0.5 38 16 <0.5 39 17 <0.5 40 17 <0.5 AVG <0.5 SD - n 10 PFOS conc in plasma (ng/mL) t=0 t=1 t=2 <0.5 18.2 39.8 <0.5 11.2 23.0 <0.5 19.7 28.9 <0.5 29.2 32.2 <0.5 10.0 21.0 <0.5 16.6 29.4 <0.5 16.9 28.5 <0.5 15.2 23.6 <0.5 9.9 30.7 <0.5 10.6 19.2 <0.5 15.7 27.6 - 6.0 6.1 10 10 10 t=4 69.9 48.1 64.0 71.1 65.4 143.3 61.1 53.3 66.1 53.5 69.6 27.0 10 PFOS conc in plasma (ng/mL) t=0 t=1 t=2 t=4 <0.5 12.9 30.8 58.2 <0.5 12.0 43.4 84.7 <0.5 8.2 24.8 70.8 <0.5 22.0 41.2 69.2 <0.5 25.1 32.3 64.0 <0.5 10.5 34.8 83.5 <0.5 19.7 37.8 87.2 <0.5 15.0 36.2 75.7 <0.5 23.2 70.5 89.9 <0.5 16.6 58.9 98.0 <0.5 16.5 41.1 78.1 - 5.8 13.8 12.6 10 10 10 10 49 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix VI PFOA and PFOS dose as percentage of intake Group 1: 5% fat diet Mouse# Cage# Estimated PFOA dose in body (g/kg) t=1 t=2 t=4 1 1 7.5 19.3 34.6 2 1 9.0 18.7 45.2 3 1 8.7 20.5 39.5 4 2 10.2 19.2 35.6 5 2 11.3 19.4 32.3 6 3 7.9 21.7 41.3 7 3 10.1 20.2 44.4 8 4 7.1 20.2 32.0 9 4 12.9 28.0 38.5 10 4 7.3 20.9 46.0 AVG 9.2 20.8 38.9 SD 1.9 2.7 5.2 n 10 10 10 Group 2: 15% fat diet Mouse# Cage# Estimated PFOA dose in body (g/kg) t=1 t=2 t=4 11 5 5.7 15.1 28.3 12 5 6.3 16.4 30.8 13 5 6.2 15.6 28.6 14 6 7.3 17.1 33.1 15 6 9.0 17.2 31.8 16 7 8.5 21.9 35.5 17 7 6.4 13.8 30.5 18 8 7.8 18.5 31.1 19 8 6.6 14.3 29.0 20 8 7.3 14.0 36.0 AVG 7.1 16.4 31.5 SD 1.1 2.5 2.7 n 10 10 10 Group 1: 5% fat diet Mouse# Cage# 1 1 2 1 3 1 4 2 5 2 6 3 7 3 8 4 9 4 10 4 AVG SD n PFOA dose in body (% of PFOA intake) t=1 t=2 t=4 37% 49% 42% 46% 48% 55% 45% 55% 50% 45% 43% 38% 45% 40% 31% 43% 60% 53% 55% 55% 58% 29% 41% 31% 54% 58% 38% 34% 48% 48% 43% 50% 44% 8% 7% 10% 10 10 10 Group 2: 15% fat diet Mouse# Cage# PFOA dose in body (% of PFOA intake) t=1 t=2 t=4 11 5 23% 31% 27% 12 5 27% 36% 31% 13 5 30% 38% 33% 14 6 31% 36% 34% 15 6 48% 47% 43% 16 7 49% 61% 49% 17 7 35% 36% 41% 18 8 36% 43% 34% 19 8 31% 35% 32% 20 8 33% 33% 39% AVG 34% 40% 36% SD 8% 9% 7% n 10 10 10 Group 3: 25% fat diet Mouse# Cage# Estimated PFOA dose in body (g/kg) t=1 t=2 t=4 21 9 5.3 12.3 24.8 22 9 5.9 12.0 27.4 23 9 6.3 14.1 31.4 24 9 5.6 14.9 30.8 25 10 5.5 12.7 28.9 26 10 6.8 13.4 28.6 27 11B 5.8 11.2 24.5 28 11A 6.4 13.8 30.9 29 12 6.1 13.5 27.6 30 12 6.2 13.2 31.6 AVG 6.0 13.1 28.6 SD 0.5 1.1 2.6 n 10 10 10 Group 3: 25% fat diet Mouse# Cage# PFOA dose in body (% of PFOA intake) t=1 t=2 t=4 21 9 28% 33% 32% 22 9 34% 35% 39% 23 9 35% 40% 41% 24 9 31% 41% 40% 25 10 35% 40% 43% 26 10 40% 39% 38% 27 11B 32% 32% 33% 28 11A 38% 41% 42% 29 12 35% 38% 38% 30 12 33% 35% 40% AVG 34% 38% 39% SD 3% 3% 4% n 10 10 10 Estimated PFOA or PFOS dose in body (ng/kg)= 200x PFOA or PFOS dose in plasma (ng/mL) (Estimation based on internal data from the Sponsor) 50 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 4: 35% fat diet Mouse# Cage# Estimated PFOA dose in body (g/kg) t=1 t=2 t=4 31 13 5.7 13.2 23.9 32 13 5.5 13.2 25.8 33 14 4.2 12.0 26.6 34 14 6.5 17.8 26.9 35 15 4.9 11.3 21.1 36 15 4.7 12.2 23.4 37 16 6.7 15.2 29.1 38 16 5.7 12.4 26.4 39 17 7.0 13.7 22.9 40 17 6.3 13.1 30.6 AVG 5.7 13.4 25.7 SD 0.9 1.9 2.9 n 10 10 10 Group 1: 5% fat diet Mouse# Cage# Estimated PFOS dose in body (g/kg) t=1 t=2 t=4 1 1 3.2 7.3 13.7 2 1 2.4 5.6 16.9 3 1 2.7 31.5 35.6 4 2 4.3 9.4 17.4 5 2 4.2 8.3 14.4 6 3 3.3 9.3 17.7 7 3 3.7 8.4 21.8 8 4 3.6 8.6 15.1 9 4 3.6 11.7 20.4 10 4 10.0 13.7 22.0 AVG 4.1 11.4 19.5 SD 2.2 7.4 6.4 n 10 10 10 Group 2: 15% fat diet Mouse# Cage# Estimated PFOS dose in body (g/kg) t=1 t=2 t=4 11 5 3.3 6.0 19.2 12 5 4.7 6.4 15.0 13 5 2.6 5.7 13.4 14 6 2.3 7.1 16.5 15 6 2.3 6.1 12.7 16 7 3.2 8.1 14.4 17 7 3.0 5.3 11.1 18 8 4.0 9.1 16.5 19 8 2.5 5.9 27.6 20 8 6.8 6.4 15.4 AVG 3.5 6.6 16.2 SD 1.4 1.2 4.6 n 10 10 10 Group 4: 35% fat diet Mouse# Cage# PFOA dose in body (% of PFOA intake) t=1 t=2 t=4 31 13 31% 36% 32% 32 13 30% 37% 35% 33 14 22% 32% 33% 34 14 38% 54% 39% 35 15 29% 33% 29% 36 15 28% 36% 33% 37 16 34% 41% 36% 38 16 26% 28% 27% 39 17 35% 33% 26% 40 17 32% 31% 35% AVG 31% 36% 32% SD 5% 7% 4% n 10 10 10 Group 1: 5% fat diet Mouse# Cage# Estimated PFOS dose in body (% of PFOS intake) t=1 t=2 t=4 1 1 25.2% 29.3% 26.3% 2 1 19.8% 23.1% 32.6% 3 1 22.2% 134.1% 72.0% 4 2 29.6% 33.5% 29.7% 5 2 26.5% 27.5% 22.2% 6 3 28.2% 41.2% 36.1% 7 3 31.6% 36.6% 45.5% 8 4 23.4% 27.7% 23.2% 9 4 24.2% 39.1% 32.1% 10 4 74.3% 50.0% 36.7% AVG 30.5% 44.2% 35.6% SD 15.8% 32.6% 14.5% n 10 10 10 Group 2: 15% fat diet Mouse# Cage# Estimated PFOS dose in body (% of PFOS intake) t=1 t=2 t=4 11 5 30.0% 27.3% 42.0% 12 5 45.9% 32.0% 34.4% 13 5 28.0% 31.6% 34.7% 14 6 22.1% 34.2% 38.4% 15 6 27.9% 37.5% 39.1% 16 7 42.1% 50.9% 45.1% 17 7 37.3% 31.3% 33.7% 18 8 41.8% 47.4% 40.4% 19 8 26.8% 32.4% 69.0% 20 8 70.4% 33.8% 37.8% AVG 37.2% 35.8% 41.5% SD 14.1% 7.5% 10.3% n 10 10 10 Estimated PFOA or PFOS dose in body (ng/kg)= 200x PFOA or PFOS dose in plasma (ng/mL) 51 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 3: 25% fat diet Mouse# Cage# Estimated PFOS dose in body (g/kg) t=1 t=2 t=4 21 9 3.6 8.0 14.0 22 9 2.2 4.6 9.6 23 9 3.9 5.8 12.8 24 9 5.8 6.4 14.2 25 10 2.0 4.2 13.1 26 10 3.3 5.9 28.7 27 11 3.4 5.7 12.2 28 11 3.0 4.7 10.7 29 12 2.0 6.1 13.2 30 12 2.1 3.8 10.7 AVG 3.1 5.5 13.9 SD 1.2 1.2 5.4 n 10 10 10 Group 4: 35% fat diet Mouse# Cage# Estimated PFOS dose in body (g/kg) t=1 t=2 t=4 31 13 2.6 6.2 11.6 32 13 2.4 8.7 16.9 33 14 1.6 5.0 14.2 34 14 4.4 8.2 13.8 35 15 5.0 6.5 12.8 36 15 2.1 7.0 16.7 37 16 3.9 7.6 17.4 38 16 3.0 7.2 15.1 39 17 4.6 14.1 18.0 40 17 3.3 11.8 19.6 AVG 3.3 8.2 15.6 SD 1.2 2.8 2.5 n 10 10 10 Group 3: 25% fat diet Mouse# Cage# Estimated PFOS dose in body (% of PFOS intake) t=1 t=2 t=4 21 9 31.7% 34.9% 29.3% 22 9 20.8% 22.0% 22.3% 23 9 35.7% 26.7% 27.4% 24 9 52.9% 29.3% 30.4% 25 10 21.2% 21.8% 31.5% 26 10 32.0% 27.8% 63.1% 27 11B 30.1% 26.7% 27.2% 28 11A 29.8% 23.2% 23.9% 29 12 18.5% 28.2% 29.4% 30 12 18.5% 16.9% 22.3% AVG 29.1% 25.7% 30.7% SD 10.4% 5.0% 11.9% n 10 10 10 Group 4: 35% fat diet Mouse# Cage# Estimated PFOS dose in body (% of PFOS intake) t=1 t=2 t=4 31 13 12.6% 15.3% 13.9% 32 13 12.0% 22.3% 20.9% 33 14 7.8% 12.1% 16.1% 34 14 23.4% 22.8% 18.3% 35 15 27.1% 17.2% 16.0% 36 15 11.2% 18.6% 21.2% 37 16 18.4% 18.7% 19.6% 38 16 12.1% 15.1% 13.9% 39 17 21.1% 30.6% 18.3% 40 17 15.0% 25.3% 20.3% AVG 16.1% 19.8% 17.8% SD 6.2% 5.5% 2.7% n 10 10 10 Estimated PFOA or PFOS dose in body (ng/kg)= 200x PFOA or PFOS dose in plasma (ng/mL) 52 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix VII Plasma total cholesterol Group 1: 5% fat diet Mouse# Cage# 1 1 2 1 3 1 4 2 5 2 6 3 7 3 8 4 9 4 10 4 AVG SD n cholesterol (mM) in time t=-4 t=0 t=2 t=4 2.43 11.35 13.74 13.92 2.40 14.46 17.58 14.00 2.60 10.42 9.01 10.42 2.60 9.62 12.08 11.86 2.47 9.33 14.56 12.15 2.71 16.43 22.71 16.77 3.20 13.16 16.15 12.82 2.95 16.55 16.26 14.58 1.49 5.41 4.66 12.03 2.47 12.85 12.91 5.14 2.5 12.0 14.0 12.4 0.4 3.5 4.9 3.1 10 10 10 10 Group 2: 15% fat diet Mouse# Cage# 11 5 12 5 13 5 14 6 15 6 16 7 17 7 18 8 19 8 20 8 AVG SD n cholesterol (mM) in time t=-4 t=0 t=2 t=4 3.20 6.85 7.80 5.79 3.11 6.46 7.13 5.61 2.90 10.46 13.00 9.03 2.08 10.39 9.10 7.49 1.88 5.40 6.76 5.06 2.00 3.62 2.79 3.18 2.73 9.52 8.96 8.04 2.13 7.47 7.41 7.46 1.72 8.39 9.96 10.97 2.78 6.95 13.07 5.24 2.5 7.5 8.6 6.8 0.5 2.2 3.0 2.3 10 10 10 10 Group 3: 25% fat diet Mouse# Cage# 21 9 22 9 23 9 24 9 25 10 26 10 27 11 28 11 29 12 30 12 AVG SD n cholesterol (mM) in time t=-4 t=0 t=2 t=4 2.32 5.09 3.99 4.53 2.65 5.98 5.37 6.11 2.05 7.40 5.51 5.30 2.18 5.91 4.48 4.51 2.12 6.46 6.39 5.66 2.35 4.27 5.01 5.95 2.78 4.27 5.79 5.85 3.53 5.94 8.04 5.85 2.23 4.59 5.07 5.69 1.95 5.34 4.78 6.26 2.4 5.5 5.4 5.6 0.5 1.0 1.1 0.6 10 10 10 10 Group 4: 35% fat diet Mouse# Cage# 31 13 32 13 33 14 34 14 35 15 36 15 37 16 38 16 39 17 40 17 AVG SD n cholesterol (mM) in time t=-4 t=0 t=2 t=4 2.50 4.63 4.57 4.22 3.31 5.16 5.24 5.26 2.33 4.39 4.70 4.74 2.55 4.30 4.92 5.09 3.21 4.37 4.59 4.38 2.62 4.22 4.63 4.74 1.64 7.23 6.23 6.41 2.17 4.40 4.10 3.51 2.55 6.01 5.19 4.90 2.70 4.32 4.71 4.46 2.6 4.9 4.9 4.8 0.5 1.0 0.6 0.8 10 10 10 10 53 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix VIII Plasma triglycerides Group 1: 5% fat diet Mouse# Cage# 1 1 2 1 3 1 4 2 5 2 6 3 7 3 8 4 9 4 10 4 AVG SD n triglycerides (mM) in time t=-4 t=0 t=2 t=4 3.78 4.92 3.57 2.91 4.42 5.58 4.84 2.41 4.43 3.96 3.32 3.03 3.59 3.54 3.97 3.88 3.53 2.89 3.65 2.93 3.56 4.55 3.44 3.73 4.23 3.38 4.25 3.22 3.44 4.48 3.41 3.16 0.91 1.58 1.36 3.23 3.48 4.23 4.15 1.46 3.5 3.9 3.6 3.0 1.0 1.1 0.9 0.7 10 10 10 10 Group 2: 15% fat diet Mouse# Cage# 11 5 12 5 13 5 14 6 15 6 16 7 17 7 18 8 19 8 20 8 AVG SD n triglycerides (mM) in time t=-4 t=0 t=2 t=4 3.98 3.62 1.97 3.30 4.83 3.18 3.35 3.62 4.26 4.23 4.97 3.91 2.66 4.78 3.95 3.38 3.14 3.67 3.20 2.25 1.09 1.70 1.72 1.42 4.63 4.57 2.97 3.13 3.64 3.60 3.57 2.97 2.54 4.41 3.65 4.60 4.78 3.66 0.58 2.47 3.6 3.7 3.0 3.1 1.2 0.9 1.3 0.9 10 10 10 10 Group 3: 25% fat diet Mouse# Cage# 21 9 22 9 23 9 24 9 25 10 26 10 27 11 28 11 29 12 30 12 AVG SD n triglycerides (mM) in time t=-4 t=0 t=2 t=4 3.81 2.21 1.35 2.11 4.03 4.09 1.83 2.70 3.05 4.47 2.48 2.36 2.90 2.86 1.97 1.81 3.22 2.44 1.58 1.59 2.85 1.24 1.16 1.20 4.75 1.04 0.70 1.08 5.58 2.48 0.81 1.70 3.22 2.34 1.72 2.12 3.39 1.92 1.59 1.96 3.7 2.5 1.5 1.9 0.9 1.1 0.5 0.5 10 10 10 10 Group 4: 35% fat diet Mouse# Cage# 31 13 32 13 33 14 34 14 35 15 36 15 37 16 38 16 39 17 40 17 AVG SD n triglycerides (mM) in time t=-4 t=0 t=2 t=4 2.72 1.82 1.22 1.29 4.72 2.15 1.64 1.24 3.21 1.67 1.06 1.39 3.98 2.00 1.36 1.38 4.29 1.47 1.38 0.90 4.19 1.65 1.52 1.25 2.68 2.23 2.41 2.19 3.48 1.87 1.13 1.10 3.36 2.91 1.33 1.52 3.47 1.42 1.03 1.40 3.6 1.9 1.4 1.4 0.7 0.4 0.4 0.3 10 10 10 10 54 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix IX Plasma HDL-cholesterol Group 1: 5% fat diet Mouse# Cage# 1 1 2 1 3 1 4 2 5 2 6 3 7 3 8 4 9 4 10 4 AVG SD n HDL-cholesterol (mM) in time t=0 t=2 t=4 0.51 0.41 0.74 0.60 0.50 0.81 0.45 0.41 0.56 0.53 0.32 0.34 0.24 0.61 0.24 0.46 0.68 0.73 0.78 0.62 0.78 0.23 0.27 0.41 0.28 0.24 0.51 0.21 0.24 0.23 0.43 0.43 0.54 0.19 0.16 0.22 10 10 10 Group 2: 15% fat diet Mouse# Cage# 11 5 12 5 13 5 14 6 15 6 16 7 17 7 18 8 19 8 20 8 AVG SD n HDL-cholesterol (mM) in time t=0 t=2 t=4 0.95 1.20 0.78 0.85 0.67 0.63 0.55 0.59 0.61 0.56 0.88 0.73 0.50 0.58 0.88 0.26 0.26 0.33 0.82 0.61 0.86 0.75 0.67 0.63 0.34 0.27 0.43 0.68 0.91 0.81 0.63 0.66 0.67 0.22 0.28 0.18 10 10 10 Group 3: 25% fat diet Mouse# Cage# 21 9 22 9 23 9 24 9 25 10 26 10 27 11 28 11 29 12 30 12 AVG SD n Group 4: 35% fat diet Mouse# Cage# 31 13 32 13 33 14 34 14 35 15 36 15 37 16 38 16 39 17 40 17 AVG SD n HDL-cholesterol (mM) in time t=0 t=2 t=4 1.19 1.11 1.01 0.92 1.30 1.26 0.61 0.47 0.90 0.88 0.73 0.96 1.03 1.42 1.36 0.88 1.14 1.77 1.12 2.00 1.48 0.85 1.21 1.50 0.83 1.01 1.18 0.83 0.80 1.18 0.91 1.12 1.26 0.16 0.42 0.27 10 10 10 HDL-cholesterol (mM) in time t=0 t=2 t=4 1.39 1.24 1.21 1.02 1.45 1.46 1.03 0.47 1.43 0.88 1.36 1.50 1.12 1.11 1.41 1.52 1.77 1.65 0.48 0.89 0.61 1.86 1.23 1.13 0.48 0.86 1.20 0.80 1.24 1.26 1.06 1.16 1.29 0.44 0.36 0.29 10 10 10 55 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix X Lipoprotein profiles Lipoprotein profiles t=-4 weeks 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 Sum TC % Recovery Group 1 0.00 0.00 0.01 0.10 0.21 0.15 0.11 0.12 0.14 0.14 0.12 0.12 0.10 0.07 0.05 0.02 0.02 0.03 0.07 0.11 0.10 0.07 0.04 0.02 1.9 2.5 76% Cholesterol (mM) Group 2 Group 3 0.02 0.00 0.01 0.00 0.02 0.01 0.15 0.14 0.22 0.21 0.15 0.16 0.12 0.11 0.14 0.11 0.12 0.09 0.15 0.12 0.12 0.11 0.13 0.12 0.11 0.09 0.07 0.07 0.05 0.04 0.03 0.01 0.03 0.00 0.04 0.02 0.07 0.05 0.09 0.07 0.10 0.07 0.05 0.04 0.04 0.01 0.03 0.00 2.1 1.6 2.5 2.4 84% 68% Group 4 0.00 0.00 0.00 0.14 0.20 0.13 0.11 0.12 0.11 0.13 0.12 0.11 0.09 0.05 0.03 0.01 0.01 0.02 0.05 0.08 0.07 0.04 0.01 0.00 1.6 2.6 64% Lipoprotein profiles t=0 weeks 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 Sum TC % Recovery Group 1 0.00 0.00 0.05 1.69 2.59 1.64 1.02 0.72 0.66 0.54 0.48 0.42 0.31 0.21 0.15 0.09 0.10 0.09 0.17 0.21 0.17 0.12 0.06 0.04 11.52 12.0 96% Cholesterol (mM) Group 2 Group 3 0.02 0.008 0.02 0.014 0.07 0.04 0.86 0.364 1.45 0.562 0.92 0.394 0.62 0.3 0.47 0.252 0.43 0.258 0.41 0.294 0.42 0.314 0.33 0.294 0.25 0.232 0.17 0.176 0.12 0.12 0.07 0.102 0.09 0.114 0.12 0.188 0.21 0.364 0.28 0.456 0.24 0.37 0.13 0.182 0.07 0.102 0.03 0.058 7.8 5.6 7.5 5.5 103% 101% Group 4 0.020 0.020 0.046 0.258 0.326 0.226 0.188 0.188 0.208 0.244 0.258 0.282 0.244 0.170 0.126 0.108 0.140 0.232 0.468 0.544 0.376 0.226 0.102 0.064 5.1 4.9 103% 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 Group 1 0.22 0.47 0.22 2.96 6.44 4.70 3.45 3.45 3.70 4.45 3.45 3.45 2.71 2.46 1.22 0.72 0.97 1.46 1.96 3.95 3.70 2.46 1.46 0.97 Phospholipids (mg/dL) Group 2 Group 3 0.72 0.37 0.72 0.62 0.47 0.62 4.45 4.08 6.44 6.07 4.70 4.83 3.70 3.34 3.95 3.34 3.70 3.09 4.45 3.84 3.70 3.34 3.70 3.59 3.45 2.35 1.96 1.85 1.71 1.11 0.97 0.86 1.22 0.62 1.71 1.11 2.71 2.10 3.45 3.34 3.95 2.85 2.21 2.10 1.46 1.11 1.22 0.86 Group 4 0.37 0.37 0.37 3.84 6.81 4.08 3.34 3.84 3.34 4.08 3.59 3.09 2.60 1.85 1.11 1.11 0.62 1.36 2.60 3.84 3.59 2.35 1.36 1.11 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 Group 1 0.00 0.10 0.34 17.35 28.94 20.31 12.67 9.46 9.71 8.48 7.49 6.75 4.78 3.06 2.32 1.58 1.58 2.32 5.03 7.74 6.51 4.54 2.32 1.82 Phospholipids (mg/dL) Group 2 Group 3 0.10 0.00 0.10 0.00 0.59 0.22 11.19 5.39 21.05 9.33 14.15 7.12 9.46 5.15 8.23 4.41 7.74 5.15 7.74 6.13 8.23 6.13 6.26 5.64 4.54 4.41 3.55 3.18 2.07 2.19 1.58 2.44 1.82 2.68 3.80 6.38 7.74 14.01 11.68 18.94 10.70 15.49 5.52 7.61 3.55 4.65 1.60 1.94 Group 4 0.00 0.00 0.22 4.90 6.38 4.41 3.42 3.67 4.16 5.15 5.15 6.13 4.90 2.93 2.68 2.68 4.16 9.09 19.68 24.61 17.22 11.30 4.65 2.19 56 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Lipoprotein profiles t=2 weeks 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 Sum TC % Recovery Group 1 0.00 0.01 0.14 2.19 2.74 1.75 0.98 0.80 0.64 0.60 0.48 0.38 0.28 0.16 0.10 0.07 0.07 0.07 0.14 0.16 0.11 0.06 0.04 0.01 11.98 14.0 86% Cholesterol (mM) Group 2 Group 3 0.00 0.006 0.00 0.012 0.07 0.054 0.86 0.182 1.27 0.314 0.70 0.192 0.46 0.152 0.39 0.152 0.37 0.164 0.34 0.198 0.36 0.234 0.26 0.228 0.23 0.192 0.14 0.146 0.08 0.122 0.06 0.128 0.06 0.164 0.14 0.304 0.26 0.548 0.32 0.506 0.23 0.35 0.10 0.176 0.06 0.088 0.01 0.06 6.8 4.7 8.6 5.4 79% 86% Group 4 0.018 0.006 0.030 0.118 0.186 0.122 0.094 0.118 0.122 0.164 0.192 0.234 0.182 0.152 0.128 0.140 0.198 0.344 0.652 0.646 0.430 0.222 0.112 0.076 4.7 4.9 96% 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 Group 1 0.18 0.43 1.67 21.59 29.80 19.84 12.13 10.13 8.89 9.39 7.40 5.90 4.16 2.91 1.67 1.42 1.42 2.42 5.65 6.40 4.66 2.91 1.92 1.42 Phospholipids (mg/dL) Group 2 Group 3 0.43 0.33 0.43 0.33 0.92 0.82 9.89 2.07 15.61 4.82 9.39 3.07 6.40 2.57 6.15 2.32 6.15 2.82 6.15 3.32 6.40 4.07 4.91 4.07 4.16 3.32 2.42 2.57 1.67 2.32 1.42 2.32 1.67 4.07 4.66 8.81 9.39 16.29 10.88 15.29 8.39 11.05 4.16 5.56 2.67 3.07 1.67 2.07 Group 4 0.58 0.08 0.58 2.07 3.57 2.32 1.82 2.07 2.57 3.32 4.07 4.57 3.82 3.07 2.82 3.07 5.31 10.30 21.03 20.03 14.54 7.81 3.82 3.57 Lipoprotein profiles t=4 weeks 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 Sum TC % Recovery Group 1 0.00 0.01 0.09 1.75 2.31 1.52 0.91 0.65 0.56 0.53 0.42 0.37 0.28 0.18 0.12 0.07 0.07 0.07 0.13 0.16 0.13 0.08 0.04 0.02 10.48 12.4 85% Cholesterol (mM) Group 2 Group 3 0.01 0.006 0.01 0.012 0.06 0.048 0.70 0.262 1.09 0.412 0.65 0.292 0.42 0.226 0.33 0.19 0.31 0.202 0.31 0.244 0.32 0.268 0.24 0.244 0.18 0.214 0.12 0.16 0.08 0.12 0.05 0.108 0.06 0.136 0.10 0.244 0.19 0.424 0.25 0.482 0.18 0.346 0.09 0.166 0.05 0.096 0.02 0.06 5.8 5.0 6.8 5.6 85% 89% Group 4 0.018 0.012 0.036 0.178 0.238 0.154 0.126 0.142 0.154 0.184 0.208 0.238 0.196 0.154 0.114 0.114 0.154 0.268 0.524 0.530 0.358 0.208 0.096 0.066 4.5 4.8 94% 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 Group 1 0.07 0.31 0.80 17.27 26.61 18.75 11.37 8.42 8.67 8.42 6.46 5.47 4.00 2.77 1.79 1.30 1.30 2.03 4.74 6.70 5.23 3.26 2.03 1.54 Phospholipids (mg/dL) Group 2 Group 3 0.31 0.06 0.31 0.06 0.56 0.56 7.93 3.52 14.57 6.73 9.65 4.76 6.21 3.77 5.72 3.27 5.72 3.77 5.72 4.51 5.47 5.00 4.25 4.76 3.26 3.77 2.53 2.78 1.54 2.29 1.05 2.29 1.30 3.27 3.26 7.23 6.70 14.89 9.16 15.88 7.44 12.42 4.00 6.49 2.53 3.77 1.30 1.79 Group 4 0.31 0.06 0.31 3.27 4.76 3.03 2.53 2.53 3.03 3.77 4.02 5.00 4.02 2.78 2.53 2.78 4.51 8.71 18.10 19.83 15.14 8.46 4.02 2.53 57 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix XI Average plasma cholesterol levels and relative PFOA/PFOS doses Group 1: 5% fat diet Mouse# Cage# 1 1 2 1 3 1 4 2 5 2 6 3 7 3 8 4 9 4 10 4 AVG SD n Average Cholesterol (mM) t=0-4 weeks 13.00 15.35 9.95 11.19 12.02 18.63 14.04 15.80 7.37 10.30 12.8 3.3 10 Group 2: 15% fat diet Mouse# Cage# 11 5 12 5 13 5 14 6 15 6 16 7 17 7 18 8 19 8 20 8 AVG SD n Average Cholesterol (mM) t=0-4 weeks 6.81 6.40 10.83 8.99 5.74 3.20 8.84 7.44 9.78 8.42 7.6 2.2 10 Average relative PFOA dose (%) t=1-4 weeks 42% 50% 50% 42% 39% 52% 56% 33% 50% 43% 46% 7% 10 Average relative PFOA dose (%) t=1-4 weeks 27% 32% 34% 34% 46% 53% 37% 37% 33% 35% 37% 8% 10 Average relative PFOS dose (%) t=1-4 weeks 27% 25% 76% 31% 25% 35% 38% 25% 32% 54% 37% 16% 10 Average relative PFOS dose (%) t=1-4 weeks 33% 37% 31% 32% 35% 46% 34% 43% 43% 47% 38% 6% 10 Group 3: 25% fat diet Mouse# Cage# 21 9 22 9 23 9 24 9 25 10 26 10 27 11 28 11 29 12 30 12 AVG SD n Average Cholesterol (mM) t=0-4 weeks 4.53 5.82 6.07 4.97 6.17 5.08 5.30 6.61 5.12 5.46 5.5 0.6 10 Average relative PFOA dose (%) t=1-4 weeks 31% 36% 38% 38% 39% 39% 32% 41% 37% 36% 37% 3% 10 Average relative PFOS dose (%) t=1-4 weeks 32% 22% 30% 38% 25% 41% 28% 26% 25% 19% 29% 7% 10 58 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 4: 35% fat diet Mouse# Cage# 31 13 32 13 33 14 34 14 35 15 36 15 37 16 38 16 39 17 40 17 AVG SD n Average Cholesterol (mM) t=0-4 weeks 4.47 5.22 4.61 4.77 4.45 4.53 6.62 4.00 5.37 4.50 4.9 0.7 10 Average relative PFOA dose (%) t=0-4 weeks 33% 34% 29% 44% 31% 32% 37% 27% 31% 32% 33% 5% 10 Average relative PFOS dose (%) t=1-4 weeks 14% 18% 12% 22% 20% 17% 19% 14% 23% 20% 18% 4% 10 59 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix XII PFOA and PFOS liver levels Absolute PFOA and PFOS liver levels Group 1: 5% fat diet + PFOA/PFOS Mouse# Cage# PFOA liver levels ng/g liver ng per liver 1 1 386 660 2 1 237 493 3 1 459 973 4 2 492 886 5 2 435 766 6 3 457 754 7 3 461 784 8 4 447 769 9 4 174 214 10 4 450 842 AVG 400 714 SD 107 218 n 10 10 PFOS liver levels ng/g liver ng per liver 394 674 216 449 978 2073 773 1391 664 1169 500 825 564 959 596 1025 231 284 612 1144 553 999 234 507 10 10 Group 2: 15% fat diet + PFOA/PFOS Mouse# Cage# PFOA liver levels ng/g liver ng per liver 11 5 378 510 12 5 349 586 13 5 375 713 14 6 409 479 15 6 244 417 16 7 293 483 17 7 431 806 18 8 413 653 19 8 437 734 20 8 494 726 AVG 382 611 SD 73 134 n 10 10 PFOS liver levels ng/g liver ng per liver 534 721 379 637 414 787 472 552 201 344 324 535 392 733 527 833 956 1606 579 851 478 760 202 336 10 10 Group 3: 25% fat diet + PFOA/PFOS Mouse# Cage# PFOA liver levels ng/g liver ng per liver 21 9 351 470 22 9 404 614 23 9 395 569 24 9 334 461 25 10 334 458 26 10 284 366 27 11B 402 611 28 11A 374 561 29 12 309 392 30 12 326 378 AVG 351 488 SD 41 95 n 10 10 PFOS liver levels ng/g liver ng per liver 388 520 304 462 398 573 366 505 332 455 591 762 414 629 291 437 294 373 245 284 362 500 97 134 10 10 60 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 4: 35% fat diet + PFOA/PFOS Mouse# Cage# PFOA liver levels ng/g liver ng per liver 31 13 353 477 32 13 333 440 33 14 397 464 34 14 337 431 35 15 378 548 36 15 368 563 37 16 266 362 38 16 351 442 39 17 411 530 40 17 339 458 AVG 353 471 SD 40 61 n 10 10 PFOS liver levels ng/g liver ng per liver 371 501 469 619 453 530 347 444 476 690 495 757 382 520 449 566 652 841 453 612 455 608 85 124 10 10 PFOA and PFOS liver levels as % of food intake Group 1: 5% fat diet Mouse# Cage# Liver PFOA/PFOS (% of PFOA/PFOS intake) PFOA PFOS 1 1 28% 45% 2 1 21% 30% 3 1 41% 139% 4 2 33% 83% 5 2 29% 70% 6 3 35% 62% 7 3 37% 72% 8 4 24% 52% 9 4 7% 14% 10 4 27% 58% AVG 28% 63% SD 10% 34% n 10 10 Group 2: 15% fat diet Mouse# Cage# Liver PFOA/PFOS (% of PFOA/PFOS intake) PFOA PFOS 11 5 18% 59% 12 5 21% 52% 13 5 26% 64% 14 6 22% 56% 15 6 19% 35% 16 7 20% 49% 17 7 33% 67% 18 8 23% 67% 19 8 26% 130% 20 8 26% 69% AVG 23% 65% SD 4% 25% n 10 10 61 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Group 3: 25% fat diet Mouse# Cage# Liver PFOA/PFOS (% of PFOA/PFOS intake) PFOA PFOS 21 9 22% 40% 22 9 29% 35% 23 9 27% 44% 24 9 21% 39% 25 10 25% 41% 26 10 20% 69% 27 11B 28% 46% 28 11A 26% 33% 29 12 19% 30% 30 12 18% 23% AVG 24% 40% SD 4% 12% n 10 10 Group 4: 35% fat diet Mouse# Cage# Liver PFOA/PFOS (% of PFOA/PFOS intake) PFOA PFOS 31 13 23% 22% 32 13 21% 27% 33 14 23% 24% 34 14 21% 20% 35 15 25% 28% 36 15 25% 31% 37 16 14% 18% 38 16 17% 20% 39 17 22% 31% 40 17 19% 23% AVG 21% 24% SD 4% 5% n 10 10 62 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Appendix XIII 10/17/11 Report on statistical analysis of associations among effects resulting from feeding fat, PFOA, and PFOS in the diet of mice for four weeks by David W. Gaylor, Ph.D. Statistical Analysis of Associations among Effects Resulting from feeding Fat, PFOA, and PFOS in the diet of Mice for Four Weeks David W. Gaylor, Ph.D. Background A study was conducted at TNO Biosciences in which male mice were fed 5%, 15%, 25%, or 35% fat in the diet for four weeks plus approximately 0.03 ppm of PFOA and PFOS in the diet. PFOA and PFOS concentrations in plasma, HDL and non-HDL cholesterol, and triglycerides were measured on 10 mice per dietary fat group (40 mice total) after various periods of exposure. Several effects due to dietary fat were noted in the TNO study. The purpose of the following statistical analysis is to exam associations among the endpoints (PFOA and PFOS plasma concentration, HDL and non-HDL cholesterol, and triglycerides) apart from correlations due to dietary fat. Approach In this analysis, the results after four weeks of exposure were used. Since dietary fat intake affected several endpoints, the calculation of correlation coefficients between endpoints across the 40 mice is not appropriate. For example, if dietary fat affects two endpoints a correlation between the two endpoints will necessarily occur and it is not possible to determine from the overall correlation coefficient if the two endpoints are associated in addition to the effects due to dietary fat. Hence, in order to remove the confounding introduced by fat intake, correlation coefficients between endpoints were calculated from the 10 animals within each fat intake group and then averaged across the four fat dietary groups. Results Average correlation coefficients, apart from the effects of dietary fat, among pairs of the measured effects at four weeks are listed in Table 1. There was a strong positive correlation between triglycerides and non-HDL cholesterol levels. There was a negative correlation between triglycerides and HDL cholesterol levels. That is, HDL cholesterol levels decreased as the triglyceride levels increased. All other correlations were relatively insignificant. 63 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Table 1. Correlation coefficients at 4 weeks of dietary exposure to fat, PFOA, and PFOS. ________________________________________________________________________ Endpoints 5% fat 15% fat 25% fat 35% fat Average ________________________________________________________________________ PFOA / PFOS 0.38 PFOA / Triglyc.a -0.46 PFOA / HDL chol.b 0.38 -0.32 -0.75 -0.07 -0.06 0.18 -0.19 0.50 0.56 -0.42 0.125 -0.118 -0.075 PFOA / non-HDL -0.37 -0.63 0.25 0.39 -0.090 PFOS / Triglyc. -0.10 0.57 -0.51 0.44 0.100 PFOS / HDL chol. 0.03 0.45 0.52 -0.23 0.192 PFOS / non-HDL -0.46 Triglyc. / HDL 0.23 Triglyc. / non-HDL 0.71 0.54 -0.05 0.87 -0.31 -0.68 0.23 0.36 -0.73 0.90 0.032 -0.308* 0.678** HDL / non-HDL 0.56 -0.16 0.23 -0.65 -0.005 ______________________________________________________________________ a Triglycerides b Cholesterol * P0.10 ** P0.01 In addition, multiple regression analyses were employed in order to simultaneously examine the effects of dietary fat, PFOA, PFOS, triglycerides, plasma HDL cholesterol, and plasma non-HDL cholesterol. For example, for plasma triglycerides the following model was obtained: Triglycerides (mmol / L) = -1.13 + 0.173(% dietary fat) - 0.0036(%dietary fat)2 -0.4915(HDL mmol / L)2 + 0.5253( non-HDL mmol / L) -0.0178(non-HDL mmol / L)2 In this study, triglyceride concentrations were associated with dietary fat, HDL and non-HDL cholesterol. Quadratic (squared) terms were considered for each endpoint in order to account for non-linear relationships. The relationship between triglycerides and HDL cholesterol was adequately described by the quadratic term without a linear term. Note the absence of PFOA and PFOS in the resulting regression model. The PFOA and PFOS plasma levels in the study did not have a significant effect on triglyceride concentrations. 64 3M-06: Effect of increasing amounts of dietary fat on plasma concentrations of cholesterol, PFOS, and PFOA plasma in male APOE*3-Leiden.CETP mice fed approximately 0.03 ppm of PFOS and PFOA - 07-Apr-2016 Similarly, the multiple regression analysis indicated that HDL cholesterol was affected only by dietary fat: HDL cholesterol (mmol / L) = 0.369 + 0.0284(% dietary fat) Also, non-HDL cholesterol was only associated with dietary fat: non-HDL cholesterol (mmol /L) = 15.17 - 0.758(% dietary fat) + 0.0122(%dietary fat)2 In conclusion, the levels of PFOA and PFOS in this study were not significantly associated with triglycerides, HDL cholesterol, or non-HDL cholesterol levels. 65