Document 3x6qvOX1YwKv84gR81OjJ5Ga

July 30,1998 AR IIQ-0IH7 'ckn Fax to: Dr. Jack Moore Dr. Joe Rodricks Fax #202-289-8530 Fax #703.516.2393 From: John L. Butenhoff Fax #651.733-1773 Tel #651.733-1962 I have attached our rationale for dose setting on the six m onth chronic oral study o f PFOS in Cynomolgus Monkeys. Y ou will also find attached the report from the environmental laboratory o f serum levels for the four w eek range finder animals. Serum levels are reported for days -7 ,2 , 3, 14 and 29. W e have done an analysis o f the slopes o f the cumulative dose vs. serum concentration for these range finder animals at the low dose o f 0.02 mg per kg per day. This is also attached. By the end o f the day, we will have an analysis o f the slopes for the tw o high dose animals. I will fax that to you when available. The interesting thing to know from this analysis is the extreme linearity o f the cumulative dose vs. serum concentration. W e will need to produce new charts because o f a slight error in the data analysis. F or example: I f a serum value is reported for day two, there has only been one dose since collection o f serum is always prior to dosing on the day o f serum collection. The value that I state in th e rationale (5.8 ppm PFOS in serum per 1 mg per kg o f PFO S dose), has taken that fact into consideration. W e look forward to discussing this dose-setting exercise with you on August 3, at 5:30 pm (Eeastem ) during our conference call (Dial-In # (303)633-6052). ^'SOCs'fdsn^i 003576 Dose-Setting Rationale for Six-M onth Chronic O ral Study in Cynom olgus M onkeys Prepared by John Butenhoff July 29, 1998 R ecom m ended Dose G roups: 0.0, 0.02, 0.15 and 0.75 mg/kg/day R atio n ale: Results o f analyses for serum concentrations of PFOS from the four-week rangefinder (attached) suggest a highly linear relationship between total accumulated dose and serum concentration. A significant male/female difference is not apparent. The consistency o f the ratio o f serum [PFOS] to total accumulated dose at either dose level and all tim e points gives confidence in using this preliminary data to set dose levels for the six-month definitive study based on estimated serum concentrations. The data provided by the Environmental Lab suggest that serum PFOS concentration increases incrementally with each daily dose o f PFOS in direct proportion to the magnitude o f the dose. The data give us a good degree o f confidence in deriving a factor relating accumulated dose in mg/kg to ppm PFOS serum. This relationship suggests that 1 mg/kg results in a serum PFOS concentration increase o f approximately 5.8 0.39 ppm. Assuming that this zero-order accumulation kinetic will hold at the higher dose over the course o f a six-month study, this allows us to predict serum concentrations that would be attained with chronic dosing. The apparent precision o f this is invaluable in setting dose levels that will result in serum PFOS concentrations which can be compared directly to known human serum PFOS concentrations for risk characterization. In addition, we now have the data to estimate the serum concentrations that were associated with effects observed in the previous 90-day rhesus studies. Going back to the original 90-day rhesus studies, the LOEL, which we believe to be close to a N O EL, o f 0.5 mg/kg/day would have resulted in an estimated serum PFOS concentration o f 261 ppm. A t the 1.5 m g/kg/day dose, 783 ppm would have been attained after 90 days. D eath resulted w ithin seven weeks o f dosing at 4.5 mg/kg/day, which is now estim ated to have produced serum PFOS concentrations o f approximately 1305 ppm. W e have the following prim ary objectives for the six-m onth study: 1) to establish a NOAEL for chronic exposure; 2) to determine threshold effects for health surveillance; 3) to provide a framework for interspecies comparison; and, 4) to provide a reference dose for human risk characterization. It is not our objective to study higher levels o f exposure which would result in serious illness in the test monkeys. Dose levels for the six-month study should be set to allow us to achieve the stated objectives: 003577 Low Dose: The low dose needs to be a confirmed NOEL. If we use the low dose from the rangefinder study, we expect to achieve approxim ately 21 ppm PFOS in the serum o f the low dose animals at the end o f six months. After the first two weeks o f dosing, the animals will be at and above the average serum concentration o f our workforce. A t study term, these animals will have serum concentrations that are approximately an order o f magnitude above average worker serum concentrations. We are suggesting that we keep 0.02 mg/kg/day dose as our low dose. M id Dose: The mid-dose is the m ost problematic from a dose-setting perspective. From the rhesus study, we know that m inor effects (questionable significance) occurred at 0.5 mg/kg/day over 90 days. W e estimate that serum concentrations at term were 261 ppm. If we use this serum concentration as a reference for setting dose on a six-month study, we would assume that sim ilar serum concentrations would be reached with 0.25 mg/kg/day. From a risk characterization perspective, 261 ppm serum PFOS is two orders o f magnitude above average worker serum [PFOS] o f 2-3 ppm. Achieving a NOAEL at this level o f exposure would be desirable; however, confidence that 0.25 mg/kg/day w ould be a NOAEL is not as high as confidence that 0.15 mg/kg/day would be a N O A EL. The S. C. Johnson W ax 90-day drinking water study in rats yielded reduced serum triglycerides in m ales at 0.3 mg/kg/day. Females had increased liver weights down to 0.06 mg/kg/day; however, we know that the rat liver responds more than the monkey to PFOS. Setting the mid-dose at 0.15 mg/kg/day would achieve an estimated serum PFOS concentration of 156 ppm at the end o f six months. This serum level is still reasonably close to two orders o f magnitude above average employee serum levels and close to four orders o f m agnitude above currently understood general population values. Therefore, the 0.15 m g/kg/day dose seems to be the best fit for the mid-dose. H igh Dose: The high dose should be designed to produce threshold effects. A fter one m onth o f dosing at 2.0 m g/kg/day (resulting in an average o f 306 ppm PFOS in serum), the male and female rangefinder monkeys (one per sex) demonstrated the expected reduction in serum cholesterol and triglycerides, which appears from rat and monkey studies to be an early marker o f biologic effect. There were no remarkable histopathologic effects. Even though there were no major disturbances after one m onth at 2.0 mg/kg/day, six-months o f exposure at this dose is now predicted to result in lethal body burdens. The rangefinder data tell us that threshold effects begin to occur at serum levels approximating 300 ppm. Using the serum concentrations estimated to have been produced in the 90-day rhesus study by 1.5 m g/kg/day (783 ppm ) as a reference, a 0.75 mg/kg/day high dose would seem appropriate to achieve marginal effects on body weight and general energy level o f the anim als. Raising the dose to 1.0 m g/kg/day w ould result in serum levels of approximately 1044 ppm by term, which would approach fatal serum concentrations. Therefore, 0.75 mg/kg/day is the suggested high dose that will clearly take us beyond threshold effects o f serum cholesterol and triglygeride reduction. C o m m en t: It is interesting to note that the consistent linearity o f increasing serum [PFOS] and apparent lack of significant elimination may make it possible to give much 003578 less frequent doses or even to give a single dose at the beginning o f the study and adjust that dose periodically to maintain a relatively constant serum level. W e will not reach a steady state in the study as it is currently designed and will be studying the effect of increasing body burden over time. Is there also value in attempting to hold animals at a relatively constant serum level over time? Also, it would be interesting to look at serum concentration at extremely low level exposure over tim e; e.g., 0.1 to 1.0 ug/kg/day. 003579 A M D T# 41598.1 Covane# 6329-222 Study: Product NumberfTest Substance): Matrix: Method/Revision: Analytical Equipment System Number Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Interccpt: Dates of Extnction/Anilyst: Dates of Analysis/Analyst: 4-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt in Cynomolgus Monkeys T-6295 (PFOS) Monkey Senim FACT-M-3,0 Sl FACT-M-4.0 Amelia 062498- Ma5sLynx3.l See Attachments See Attachments See Attachments See Attachments 7/16/98 RWW 7/22/98 RWW 7/17/98 HOJ 7/24/98 HOJ Group 1 Control 0.0 mg/kg/day Sample # I05349M I05350M 105366F I05367F Timepoint D a y -7 Day 2 Day 3 Day 7 Day 14 Day 29 Day -7 Day 2 Day 3 Day 7 Day 14 Day 29 Day -7 Day 2 Day 3 Day 7 Day 14 Day 29 D a y -7 Day 2 Day 3 Day 7 Day 14 Day 29 PFOS Cone. ug/mL 0.0173 0.0223 0.172 0.181 0.184 0.160 0.0121 0.0207 0.0202 0.0259 0.0178 0.0220 0.0118 0.0214 0.0232 0.0194 0.0183 0.0269 0.0135 0.0215 0.0175 0.0155 0.0333 0.118 Group 2 Low Dose 0.02 mg/kg/day Sample V Timepoint PFOS Cone. ug/mL I05344M D a y -7 Day 2 Day 3 Day 7 0.0120 0.157 0.174 0.449 Day 14 Day 29 1.37 2.78 105347M Day -7 0.0140 Day 2 0.147 Day 3 0.287 Day 7 0.776 Day 14 1.36 Day 29 2.56 I05348M Day-7 0.0100 Day 2 0.161 Day 3 0.289 Day 7 Day 14 Day 29 0.874 1.46 3.57 I05364F Day -7 0.0161 Day 2 0.158 Day 3 0.292 Day 7 Day 14 Day 29 0.736 1.58 5.76 I0536SF Day-7 0.0144 Day 2 0.117 Day 3 0.267 Day 7 0.628 Day 14 1.22 Day 29 2.65 I05370F Day-7 0.0111 Day 2 0.100 Day 3 0.213 Day 7 0.664 Day 14 Day 29 1.54 3.20 Practical Quantitation Limit (PQL): * PFOS = 25 ng/mL, PFOSA * 0.5 ng/mL. PFOSAA = 10 ng/mL PFOS = Perfluorooctanesulfonate Group 3 High Dose 2.0 mg/kg/day Sample# Timepoint PFOS Cone. ug/mL I05345M D ay-7 0.0128 Day 2 12.6 Day 3 29.7 Day 7 71.8 Day 14 Day 29 129 313 I05369F D ay-7 0.0139 Day 2 14.4 Day 3 18.3 D*y7 72.7 Day 14 Day 29 143 299 003580 G035S1 AMDT# 1)41598.1 Covaticci 6329-222 Method Detection U n it (MDL): PFOS 12.5 ng/ntL, PFOSA =0.25 ng/mL. PFOSAA - 5 ng/niL l ------ 105344M ------ 105347M Chart1 AMDT# 041598.1 105348M ----- 105364F ------ 105365F ------ 105370F O O CO en oo w Page 1 105344M Acc Dose mg/kg 0.00 0.04 0.06 0.14 0.28 0.58 [serum] ug/mL 0.0120 0.1570 0.1740 0.4490 1.3700 2.7800 105347M Acc Dose [serum] ..._53aft.a___ ug/mL 0.00 0.0140 0.04 0.1470 0.06 0.2870 0.14 0.7760 0.28 1.3600 0.58 2.5600 105348M Acc Dose mg/kg 0.00 0.04 0.06 0.14 0.28 0.58 [serum] ug/mL 0.0100 0.1610 0.2890 0.8740 1.4600 3.5700 105364F Acc Dose [serum] mg/xg____ ug/ml 0.00 0.0161 0.04 0.1580 0.06 0.2920 0.14 0.7360 0.28 1.5800 0.58 5.7600 Sheetl slope se (slope) r-square F-value ss (reg) 4.92 0.21 0.99 540.118 5.749 -0.08 0.06 0.10 4 0.043 y-int se (y-int) se (y-est) df ss (resid) slope se (slope) r-square F-value ss (reg) 4.43 0.17 0.99 667.749 4.671 0.04 0.05 0.08 4 0.028 y-int se (y-int) se (y-est) df ss (resid) slope se (slope) r-square F-value ss (reg) 6.13 0.24 0.99 642.745 8.944 -0.06 0.07 0.12 4 0.056 y-int se (y-int) se (y-est) df ss (resid) slope se (slope) r-square F-value ss (reg) 9.85 1.06 0.96 86.129 23.092 -0.38 0.29 0.52 4 1.072 y-int se (y-int) se (y-est) df ss (resid) Page 1 0035S3 105365F Acc Dose mg/kg 0.00 0.04 0.06 0.14 0.28 0.58 [serum] ug/mL 0.0144 0.1170 0.2670 0.6280 1.2200 2.6500 105370F Acc Dose mg/kg 0.00 0.04 0.06 0.14 0.28 0.58 [serum] ug/mL 0.0111 0.1000 0.2130 0.6640 1.5400 3.2000 Sheetl slope se (slope) r-square F-value ss (reg) 4.58 0.08 1.00 3683.682 4.989 -0.02 0.02 0.04 4 0.005 y-int se (y-int) se (y-est) df ss (resid) slope se (slope) r-square F-value ss (reg) 5.66 0.13 1.00 1886.988 7.623 -0.08 0.04 0.06 4 0.016 y-int se (y-int) se (y-est) df ss (resid) Page 2 003584 Dose-Setting Rationale for Six-Month Chronic Oral Study in Cynomolgus Monkeys Record o f August 3, 1998 Conference Call Participants: Dr. John Butenhoff, 3M Dr. Marv Case, 3M Dr. Jeff Mandel, 3M Dr. John Moore, IEHR Dr. Geary Olsen, 3M Dr. Joe Rodricks, ENVIRON Dr. Andrew Seacat, 3M Outcome: Data from the rangefinder was discussed including the strong relationship between cumulative dose and serum PFOS concentration (avg. of 5.27 +/- 0.61 ppm serum PFOS concentration per mg/kg PFOS dose). Discussion verified July 29,1998 written rationale for setting doses with one change. Dr. Moore suggested that the low dose be raised from 0.02 mg/kg/day to 0.03 mg/kg/day to keep doses at multiples o f 5. The suggestion was accepted. The study will proceed on August 12th, 1998 with the following dose levels: Control Low-Dose Mid-Dose High-dose 0.00 mg/kg/day 0.03 mg/kg/day 0.15 mg/kg/day 0.75 mg/kg/day Copies: Participants Dr. Larry Zobel Dr. Bill Weppner Study File (T-6295.7) 0035S5 6-m onth toxicity study with PFOS in C ynom olgus Monkeys Date: T-6295.7 Covance 6329-223 3/16/99 Urobilinogen (UB) Analysis Results from Dr Joseph P. M cConnell., Porphyrins and nutritional Biochem istry lab, Dept of Laboratory Medicine and Pathology. Mayo Clinic, Rochester MN. F em ale Average SD n Average SD n T -TEST M ale Average SD n Average SD n T-TEST mq UB/q Control 0.078 0.017 6.000 0.75 mq/kq/d 0.112 0.075 6.000 0.357 mq UB/q Control 0.097 0.031 6.000 0.75 mg/kg/d 0.064 0.018 5.000 0.059 (n=6) 0035S6 6-month toxicity study in Cynomolgus Monkeys T-6295.7 Covance 6329-223 Date: 3/16/99 Urobilinogen Analysis Results from, Dr Joseph P. McConnell., Porphyrins and nutritional Biochemisry lab, Dept of Laboratory Medicine and Pathology. Mayo Clinic, Rochester MN. Animal Dose number Level Water Stool Weight Added Total Weight mg UB per gram specified I05529 I05530 105531 I05535 I05544 105549 Average SD n 1F 1F 1F 1F 1F 1F 10.0 10 20.0 8.0 10 18.0 9.0 10 19.0 4.0 10 14.0 8.0 10 18.0 6.0 10 16.0 7.5 10 17.5 2.2 0 2.2 6.0 6 6.0 0.07 0.11 0.07 0.08 0.06 0.08 0.08 0.02 6.00 105533 105534 I05536 I05540 I05542 105551 Average SD n T -TEST 4F 4F 4F 4F 4F 4F 13.0 10 23.0 9.0 10 19.0 12.0 10 22.0 10.0 10 20.0 6.0 10 16.0 5.0 10 15.0 9.2 10 19.2 3.2 0 3.2 6.0 6 6.0 0.16 0.08 0.24 0.09 0.05 0.05 0.11 0.07 6.00 0.36 I05508 105517 105519 105520 I05526 I05527 Average SD n 1M 1M 1M 1M 1M 1M 6.0 10 16.0 10.0 10 20.0 8.0 10 18.0 10.0 10 20.0 7.0 10 17.0 8.0 10 18.0 8.2 10 18.2 1.6 0 1.6 6.0 6 6.0 0.15 0.08 0.10 0.11 0.07 0.07 0.10 0.03 6.00 0035S7 105506 105507 105511 105512 I05522 Average. SD n T-TEST 4M 4M 4M 4M 4M 8.0 3.0 10.0 6.0 7.0 6.8 2.6 5.0 10 18.0 10 13.0 20 30.0 10 16.0 10 17.0 12 18.8 4 6.5 5 5.0 0.09 0.04 0.06 0.06 0.07 0.06 0.02 5.00 0.059492809 (n=6) 003588