Document XOL53M8VEoezqzeKQGjKkmYDG

$ M . OgOO. 3 73 4RtZZ6-O06O A - / DT 21; T-6316.9; FC Levels of Naive Rats CODE DRAFT 5/14/99 3M MEDICAL DEPARTMENT, TOXICOLOGY SERVICES Report for Study No. T-6316.9; DT21 Fluorochemical (FC) Levels in Naive Rats In-Life Start Date; July 8, 1998 In-Life End Date: July 28, 1998 pr,~. Background: .Cantata Mo *> In an attempt to determine the source of low-level perfluorooctanesulfonate (PFOS) body burden found in control rats involved in some 3M contract dietary studies, a comprehensive plan with the following objectives and responsibilities was designed: Objective 1 - To investigate potential sources o f contamination within the study housing area of the current dietary studies on perfluorinated test compounds at Covance Madison. Dr. Andrew Seacat was appointed study director with Dr. Marv Case as alternate. Jim Wolters, 3M Environmental, is responsible for coordinating and conducting direct air monitoring and wipe samples. > Objective 2 - To determine if contamination o f feed is leading to the low levels of PFOS seen in control rats in the two-year dietary study of N-Ethyl - Pertluorooctanesulfonamido ethanol (N-Ethyl FOSE) conducted at Covance Madison. Dr. Andrew Seacat was appointed study director with Dr. M arv Case as C\I alternate. co *-c Objective 3 - To investigate the background serum and liver PFOS levels in naive o rats of different age groups from different sources. Information on the various CD diets supplied by the different breeders is to be obtained. Deanna Nabbefeld was appointed study director with Dr. Marv Case as alternate. CrCnDD u p" Objective 4 - To investigate the possibility that PFOS exposure is stemming from t tainted feed, exposure in rat rooms or a combination o f both. Dr. M arv Case was appointed study director. 3M Environmental Analytical Laboratory is responsible for chemical analysis o f samples gathered in objectives 1-4. rcon The study to examine objective 3 is complete and is the focus o f this report. Once data addressing each objective are available, reports o f the various studies will be generated. Study Objective: This study was designed to determine what perfluorooctanesulfonate (PFOS) levels, if any, can be detected in untreated Sprague Dawley rats o f three different age groups acquired from three different breeders. In addition, information on the rodent feed provided by the various animal suppliers was obtained. A EPA-OTS 000811797X Page 1 of 19 0039S4 DT 21: T-6316.9; FC Levels of Nave Rats CODE DRAFT 5/14/99 Research Client: 3M Specialty Chemicals Division 3M Center Building 236 Saint Paul MN 55133-3220 Sponsor: 3M Toxicology Services Building 220-2E-02, 3M Center St. Paul MN 55144-1000 Study Location: 3M Strategic Alternative Toxicology Laboratory 3M Center, Building 270-SB-181 Saint Paul, MN 55133-3220 Study Director: Deanna Nabbefeld, MS Advanced Toxicologist 3M Medical Dept. /Toxicological Services 3M Center Budding 220-2E-02 Saint Paul, MN 55233-3220 Telephone No.: 651-737-1374 Facsimile No.: 651-733-1773 Alternate Study Director: Dr. Marv Case Corporate Scientist 3M Medical Dept. /Toxicological Services 3M Center Building 220-2E-02 Saint Paul, MN 55233-3220 Telephone No.: 651-733-5180 Facsimile No.: 651-733-1773 Summary: The livers o f male and female untreated Sprague Dawley rats o f three different age groups (6-8 weeks old, 10-14 weeks old and old retired breeders (ORB) / = 9-12 months old) from three different breeders (Breeder A, Breeder B and Breeder C) were examined for PFOS and other known fluorocheraical (FC) metabolites. Each breeder was contacted to provide information on the feed provided to their rats while at their facilities. Livers from Breeder A and Breeder C rats contained detectable levels o f PFOS (>15ppb). Male rat livers from both sources increased significantly (p = 0.05) in PFOS concentration with age, (slope = 51 Breeder A male; slope = 1 3 3 Breeder C male). The concentration o f PFOS in livers from female Breeder A and Breeder C rats, however, did not increase with age (slope = 0.6 Breeder A female; slope = -3.7 Breeder C female). The livers o f Breeder C male ORBs had the highest PFOS levels (average = 327 1 1 5 ppb), significantly higher than the levels found in Breeder A male ORBs (average = 145 12 ppb). No other FC metabolites could be detected in the Breeder A or Breeder C rat livers. No PFOS or other FC metabolites could be detected in the livers of Breeder B rats. Page 2 of 19 C039S5 DT 21: T-6316.9; FC Levels of Naive Rats CODE DRAFT 5/14/99 Breeder A rats are fed feed A supplied by Company A (City A, State A). Breeder B rats are fed feed B supplied by Company B (City B, State B). Breeder C rats are fed feed C supplied by Company C (City C, State C). Fish meal is the primary ingredient in feed C, the fifth listed ingredient in feed A and not present in feed B. From these data, it appears as though PFOS greatly bioaccumulates in male rats, while in female rats it does so to a lesser degree. Possible explanations for this difference are that PFOS is released in the milk and/or transferred in utero to the pups in females that have bore and nursed multiple litters. Also, female rats may more readily excrete PFOS through urinary excretion than male rats. It is postulated that fish meal, an ingredient in some rodent chow, may contain FC and thus lead to the PFOS found in untreated rats. The possibility that 3M material is used to coat the feed bags and that this is a source of PFOS is also being considered. Methods: Thirty Sprague Dawley rats (five male, five female per age group; age groups = 6-8 weeks old, 10-14 weeks old and old retired breeder (ORB) / * 9-12 months old) were ordered from each o f the following breeders: Breeder A, Breeder B and Breeder C. Each breeder was asked to provide the name of the feed provided to their rats, name and location of feed breeder, feed ingredient list and information on feed packaging. All animals remained in their shipping containers between arrival at 3M and euthanization. No food or water, other than that provided in the shipping containers, was furnished. Within one hour o f arrival at 3M, rats were weighed, grossly examined and euthanized by CO2. Sera and liver were harvested and sent to Kris Hansen, 3M Environmental Laboratory - Fluorine Analytical Chemistry Team (FACT), for FC analysis. (A description o f the analysis method can be found in Appendix 1, Experimental section.) Statistical significance o f results was determined using the students T-Test (p = 0.05). The rate of change of PFOS levels was analyzed by plotting age in months versus PFOS concentration (ppb) and calculating the slope of the line. Results: Laboratory Data Raw data, including body weights, liver weights, liver/body weight ratios and PFOS concentrations can be found in Appendix 1. Results of the FC analysis, including graphs and a full data table, can be found in Appendix 2 - a Summary Report prepared by Kris Hansen. Liver samples were deemed most meaningful and thus analyzed first. Sera samples have not yet been analyzed. Depending on the outcomes o f objectives 1, 2 and 4 o f the overall study plan (see background section, pg. 1), sera PFOS analysis may not performed. Average PFOS liver levels standard deviation for the various age / sex / breeder groups Page 3 of 19 0039S6 included in this study are as follows: DT 21; T-6316.9; FC Levels of Nave Rats CODE DRAFT 5/14/99 Breeder A 6-8 week old Male Breeder A 6-8 week old Female Breeder A 10-14 week old Male Breeder A 10-14 week old Female Breeder A ORB Male Breeder A ORB Female 43.0 4.07 ppb 66.6 19.27 103.0 21.33 79.9 12.06 145.0 12.42 67.8 29.63 Breeder B all groups < method detection limit (15 ppb) Breeder C 6-8 week old Male Breeder C 6-8 week old Female Breeder C 10-14 week old Male Breeder C 10-14 week old Female Breeder C ORB Male Breeder C ORB Female 60.2 12.21 ppb 73.0 12.29 92.2 22.75 75.7 24.40 327.0 115.37 65.6 27.24 The rate o f change o f PFOS liver concentration with age is depict in Figures 1 and 2 for Breeder A and Breeder C rats respectively. The slope o f the line for male Breeder A rats is 51 while that for female Breeder A rats is 0.6. The slope o f the line for male and female Breeder C rats is 133 and -3.7 respectively. Page 4 of 19 003987 Figure 1 DT 2 1: T-6316.9; FC Levels of Nave Rats CODE DRAFT 5/14/99 Breeder A PFOS Liver Concentrations 7 12 45.5 ` Age (Weeks) * 7 weeks = 6-8 weeks old; 12 weeks = 10-14 weeks old, 45.5 weeks = ORB. Figure 2 Breeder C PFOS Liver Concentrations * 7 weeks = 6-8 weeks old; 12 weeks = 10-14 weeks old, 45.5 weeks = ORB. The increase in PFOS concentration found in the livers o f male Breeder A rats was significant between all three age groups (p = 0.05). The increase in PFOS levels in male Breeder C rats was significant between the 10-14 week old group and the ORB group, but not between the 6-8 and 10-14 week old groups. Comparing males to females at each age Page 5 o f l 9 < L '039S S DT 21; T-6316.9; FC Levels of Naive Rats CODE DRAFT 5/14/99 group the Breeder A 10-14 week old groups differed significantly and the ORB groups from both Breeder A and Breeder C differed significantly. Comparing Breeder A rats to Breeder C rats at each age/sex group, male Breeder C ORB rats had significantly higher PFOS liver concentrations than did male Breeder A rats o f the same age group. AD other age/sex groups did not significantly differ in PFOS levels between the two breeders. Results o f the T-Tests are shown in Table 1. Table 1 STATISTICS T-TEST - two-tailed distribution, paired, p = 04)5 , * = statistically significant Breeder Age/Sex Group Comparison Male 6-8 & 10-14wk 10-14 wk & O R B Female 6-8wk& 6-8 & 10-14wk ORB 10-14 wk & ORB Male/Female 6-8wk & 6-8 wk 10-14wk ORBA ORB & 6-8 wk & 10-14wk ORB A *0.0123 *0.0388 *0.0008 0.1139 0.4702 0.9459 0.1043 *0.0478 *0.0125 C 0.1213 *0.0112 *0.0079 0.8620 0.6474 0.3421 0.2564 0.5374 *0.0045 Breeder Comparison Male Female xxxxxxxxxxxxxxxxxxxxxx 6-8 wk 10-14wk ORB& 6-8 wk 10-14wk O R B ,S A & 6-8 wk & 10-14wk ORB & 6-8 wk & 10-14wk ORB vs. C 0.0997 0.4305 *0.0232 0.5157 0.7749 0.9004 Breeder Feed/Diet History Breeder A rats are fed feed A suppUed by company A (city A, state A). Feed A is packaged in paper bags coated with a paper coating material produced by 3M. The feed ingredients, as listed by company A, are as foUows: ground yeUow com , wheat middlings, soybean meal, animal fat preserved with BHA, fish m eal, alfalfa meal, cane molasses, calcium carbonate, salt, cyanocobalamin (source o f vitamin B-12), biotin, DL methionine, calcium pantothenate, foUc acid, riboflavin, cholecalciferol (source o f vitamin D-3), vitamin A acetate, di-alpha tocopheryl acetate (source of vitamin E), thiamin, magnesium oxide, sodium selenite, nicotinic acid, pyridoxine hydrochloride, menadione 0039S9 Page 6 of 19 DT 21; T-6316.9; FC Levels of Naive Rats CODE DRAFT 5/14/99 dimethylprimidinol bisulfite (source o f vitamin K activity), silicon dioxide, calcium iodate, manganous oxide, copper sulfate, cobalt carbonate, ferrous carbonate, zinc sulfate, zinc oxide. Breeder B rats are fed feed B supplied by company B (city B, state B). Feed B is also packaged in paper bags. It is unknown at this time, however, whether or not these bags are coated with a 3M material. The feed B ingredients, as listed by company B, are as follows: ground com , soybean meal, ground oats, wheat middlings, alfalfa meal, soybean oil, com gluten meal, calcium carbonate, dicalcium phosphate, brewers dried yeast, iodized salt, L-lysine, DL-methionine, vitamin A-acetate, D-activated animal sterol (source o f vitamin D3), vitamin E supplement, niacin, calcium pantothenate, riboflavin, thiamin mononitrate, pyridoxine hydrochloride, menadione sodium bisulfite complex (source o f vitamin K), folic acid, biotin, vitamin B 12 supplement, calcium carbonate, manganous oxide, ferrous sulfate, copper sulfate, zinc oxide, calcium iodate, cobalt carbonate. Breeder C rats are fed feed C supplied by company C (city C, state C). Feed C is packaged in a "commercially acceptable 3 ply laminated paper bag" . It is unknown at this time whether or not a 3M material is used in these bags. The feed ingredients in feed C, as listed by company C., are as follows: fish m eal, soybean meal, alfalfa meal, com gluten meal, ground whole wheat, ground #2 yellow com , ground whole oats, wheat middlings, Brewer's dried yeast, soybean oil, salt, dicalcium phosphate, ground limestone, vitamin and mineral premixes. Conclusions/Future Directions: The livers o f Breeder B rats appear free o f FC. Livers from male rats supplied by Breeder A and Breeder C increase in PFOS concentration with age. Livers from female rats supplied by Breeder A have detectable levels o f PFOS but these levels plateau, slightly increasing from 6-8 weeks old to 10-14 weeks old and decreasing slightly in old retired breeders. Female rats supplied by Breeder C actually decrease slightly, although not significantly, in PFOS liver concentration with age. From these data, it appears as though PFOS greatly bioaccumulates in male rats, while in female rats it does so to a lesser degree. One hypothesis is that PFOS is released in the milk and/or transferred in utero to the pups in females that have bore and nursed multiple litters. Another possibility is that female rats are more readily able to clear PFOS through urinary excretion than are male rats. Although the sources o f PFOS leading to the liver concentrations identified in this study are unknown, one hypothesis is that PFOS is contained in the rat chow (Objective 2). It is thought that fish meal may be an ingredient containing PFOS. While not present in the 003990 Page 7 of 19 DT 21; T-6316.9; FC Levels o f Nave Rats CODE DRAFT 5/14/99 Breeder B rat chow, fish meal is the primary ingredient listed for Breeder C rat chow and the 5th ingredient listed for Breeder A chow. Analysis of the FC content o f the rat chow used by each animal breeder is currently underway. In addition, samples o f the fish meal may be obtained and analyzed for FC content. Another possible source o f PFOS is the paper coating used in feed bags. As stated in the results section, 3M supplies paper coating to Company A, the manufacturer o f Feed A. It is not known at this time if 3M material is also used in bags for Feeds B and C. Further information on feed packaging will be gathered in a separate study. Signatures: Prepared By: Deanna Nabbefeld, MS Advanced Toxicologist Reviewed By: Date Dr. Marv Case Corporate Scientist Date Dr. Andrew Seacat Senior Research Toxicologist Date Page 8 of 19 003391 DT 21; T-6316.9; FC Levels of Nave Rats CODE DRAFT 5/14/99 Appendix 1 Breeder ID Sex Age Grp. Body Liver liver wt/ PFOS Liver Cone. (ng/9 or ppb) w t(g) w t(g ) body wt Individuai Ave/Grp Std/Grp A A1 M 6-8 wks A2 M 6-8 wks A3 M 6-8 wks A4 M 6-8 wks A5 M 6-8 wks 171.7 179.6 161.5 151.6 166.8 8.30 8.30 7.05 6.60 8.06 0.048 0.046 0.044 0.044 0.048 **305 48.8 42.6 39.5 41.1 43.0 4.07 A6 F A7 F A8 F A9 F A10 F 6-8 wks 6-8 wks 6-8 wks 6-8 wks 6-8 wks 173.7 168.3 165.1 141.7 159.7 8.69 7.13 7.46 4.91 6.47 0.050 0.042 0.045 0.035 0.041 54.9 62.0 65.4 99.6 51.2 66.6 19.27 A11 M A12 M A13 M A14 M A15 M A16 F A17 F A18 F A19 F A20 F 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 324.3 367.9 356.7 358.5 365.6 226.1 199.5 203.4 196.4 209.8 10.55 12.66 13.10 13.23 13.65 10.49 6.32 6.13 7.14 7.08 0.033 0.034 0.037 0.037 0.037 0.046 0.032 0.030 0.036 0.034 100.0 140.0 96.8 87.6 90.1 72.2 93.9 71.6 92.2 69.6 103.0 79.9 21.33 12.06 A21 M A22 M A23 M A24 M A25 M *ORB ORB ORB ORB ORB 406.2 342.3 395.1 363.7 460.2 9.75 10.09 10.83 9.24 13.93 0.024 0.029 0.027 0.025 0.030 131.0 133.0 151.0 149.0 160.0 145.0 12.42 A26 F A27 F A28 F A29 F A30 F ORB ORB ORB ORB ORB 394.2 412.5 482.9 485.3 424.2 12.70 16.09 14.80 14.96 11.34 0.032 0.039 0.031 0.031 0.027 119.0 67.1 45.0 55.5 53.0 67.8 29.63 * ORB = old retired breeders ** Outlier - not used in calculations. Method detection limit (MDL): PFOS = 15ng/g or ppb Page 9 of 19 003992 DT 21; T-6316.9; FC Levels of Naive Rats CODE DRAFT 5/14/99 Appendix 1 Breeder B ID Sex Age Grp. Body Liver liver wt/ PFOS Liver Cone. (ng/9 or PPb) w t(g ) w t(g ) body wt Individuai Ave/Grp Std/Grp B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30 M M M M M F F F F F M M M M M F F F F F M M M M M F F F F F 6-8 wks 6-8 wks 6-8 wks 6-8 wks 6-8 wks 6-8 wks 6-8 wks 6-8 wks 6-8 wks 6-8 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks ` ORB ORB ORB ORB ORB ORB ORB ORB ORB ORB 198.4 201.7 203.4 203.9 204.2 214.3 208.0 212.8 209.3 209.0 300.6 307.9 304.0 299.8 306.0 237.7 241.1 239.7 236.3 233.4 470.7 462.8 446.6 458.2 478.9 337.8 326.2 285.7 286.2 306.9 9.45 9.34 8.50 8.38 9.17 7.62 7.19 7.14 7.30 8.08 10.69 12.33 11.26 10.09 11.38 6.65 7.20 7.14 8.54 7.33 17.00 14.90 15.55 14.52 11.19 10.45 10.27 8.54 9.01 11.00 0.048 0.040 0.042 0.041 0.046 0.036 0.035 0.034 0.035 0.039 0.036 0.040 0.037 0.034 0.037 0.028 0.030 0.Q3Q 0.036 0.031 0.036 0.032 0.035 0.032 0.023 0.031 0.031 0.030 0.031 0.036 <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL * ORB = old retired breeders Method detection limit (MDL): PFOS = 15ng/g or ppb Page 10 of 19 003993 DT 21; T-6316.9; FC Levels of Naive Rats CODE DRAFT 5/14/99 Appendix 1 Breeder C ID Sex Age Grp. Body Liver liver wt/ PFOS Liver Cone, (ng/g or ppb) w t(g ) w t(g ) body wt individuai Ave/Grp Std/Grp C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 M 6-8 wks M 6-8 wks M 6-8 wks M . 6-8 wks M 6-8 wks F 6-8 wks F 6-8 wks F 6-8 wks F 6-8 wks F 6-8 wks M 10-14 wks M 10-14 wks M 10-14 wks M 10-14 wks M 10-14 wks F 10-14 wks F 10-14 wks F 10-14 wks F 10-14 wks F 10-14 wks M *ORB M ORB M ORB M ORB M ORB F ORB F ORB F ORB F ORB F ORB 200.2 202.4 184.1 187.5 183.6 201.2 189.9 172.5 207.9 199.5 454.3 334.6 459.9 407.5 317.8 214.4 226.1 216.1 241.0 246.5 687.8 561.2 456.0 525.7 586.6 281.5 281.4 247.5 289.5 338.7 9.64 10.18 9.28 10.26 9.34 9.73 8.40 9.40 10.28 10.54 17.97 13.07 17.65 14.17 12.75 9.33 8.94 8.47 11.21 8.53 22.22 18.24 14.96 17.91 18.24 19.56 18.81 15.65 14.95 17.58 0.048 0.050 0.050 0.055 0.051 0.048 0.044 0.055 0.049 0.053 0.040 0.039 0.038 0.035 0.040 0.044 0.040 0.039 0.047 0.035 0.032 0.033 0.033 0.034 0.031 0.069 0.067 0.063 0.052 0.052 63.6 63.0 47.7 49.2 77.5 67.0 89.3 78.8 73.2 56.6 86.5 54.7 84.9 119.0 86.2 52.9 71.5 72.6 64.4 117.0 151.0 441.0 379.0 390.0 276.0 59.5 105.0 71.3 63.0 29.0 60.2 12.21 73.q 12.29 92.2 22.75 75.7 24.40 327.0 115.37 65.6 27.24 * ORB = old retired breeders Method detection limit (MDL): PFOS = 15ng/g or ppb Page 11 of 19 003994 DT 21; T-6316.9; FC Levels of Nave Rats CODE DRAFT 5/14/99 Appendix 2 3M Environmental Laboratory- Fluorine Analytical Chemistry Team Contact; Kris Hansen Fluorine Analytical Chemistry Team Building 2-3E-09 8-6018 Study of PFOS levels in Nave Rats Summary report Experimental Summary In order to assess "endogenous"levels of PFOS in test animals, the livers of ninety rats from three different suppliers were quantitatively analyzed for PFOS. Three distinct ages of rats were represented in the group of animals received from each supplier: 6-8 weeks old, 10-14 weeks old, and retired breeders (> 14 weeks). The test animals, received by the Toxicology Department at 3M, were sacrificed upon receipt; tissue samples were delivered to the 3M Environmental Lab for extraction and analysis by FACT. # at # at #>14 Ratio Supplier Location Chow 6-8 10-14 wks male:femal wks wks e Breeder A City A, State * Feed A 10 10 10 1:1 A Breeder B City B, State Feed B 10 10 10 1:1 B Breeder C City C, State Feed C 10 10 10 1:1 C *3M is/was a supplier of paper coating material to company A, supplier of feed A. Currently, it is not known if 3M supplies material to company B or C, suppliers of feeds B and C respectively. Analytical Summary Liver samples were homogenized and extracted using an ion-paring reagent. The extracts were analyzed quantitatively using high-pressure liquid chromatography-electrospray tandem mass spectrometry (HPLCESMSMS) and evaluated versus an extracted curve. Analytical details are available in the full report. The presence or absence of other known fluorochemical contaminants and metabolites was ascertained by inspection. Page 12 of 19 C03995 DT 21; T-6316.9; FC Levels of Naive Rats CODE DRAFT 5/14/99 Appendix 2 Results Summary Rat livers from Breeder A and Breeder C test animals showed significant endogenous levels of PFOS. Livers collected from oldest group of male rats from Breeder C contained significantly more PFOS than any other group in the study. The livers of test animals from Breeder B did not contain PFOS above the limit of detection (15ppb). PFOS levels in the livers of male rats from Breeder A and Breeder C roughly correlated with the age of the animals. That is, livers collected from the youngest male rats, 6-8 weeks old, contained the least PFOS, while the old, retired breeder male rats contained the highest concentration. The livers of the female rats from Breeder A and Breeder C were determined to contain very consistent levels of PFOS, showing no correlation with age. No other known fluorochemical contaminants or metabolites were identified in the liver samples analyzed in this study. Graphical results and a full table of results are attached. Currently, methods are being developed for the analysis of low levels of PFOS and ethyl-FOSE alcohol in samples of chow from each supplier. E xperim en tal Sam ple preparation-aqueous sam ples, HPLC-ESMS: Ion-pairing extrctction Analyte is extracted from a sample matrix with an ion-pairing reagent (tetrabutyl ammonium hydrogen sulfate (TBA)) in a pH-controlled environment. The cationic reagent selectively targets anionic fluorochemicals. Once the anion-TBA pair is formed, the analyte is transferred into a non-polar organic solvent (ethyl acetate), dried, and reconstituted in methanol for MS analysis. HPLC-ESMS an d HPLC-ESMSMS: For detailed qualitative w ork In HPLC, an aliquot of extract is injected and passed through a reverse-phase liquid chromatographic column. Based on the affinity of the analyte for the stationary phase in the column relative to the liquid mobile phase, the analyte is retained for a characteristic amount of time. For example, in a standard solution PFOS may elute at 10.5 minutes. Retention times between a standard PFOS solution and the analyte extracted from groundwater in this analysis were matched to within 1% on the HPLC system. Page 13 o f l 9 003996 DT 21; T-6316.9; FC Levels of Nave Rats CODE DRAFT 5/14/99 Appendix 2 Following HPLC separation, ESMS provides a rapid and accurate means for analyzing a wide range o f organic compounds, including fluorochemicals. Electrospray, an ionization technique used primarily for the detection o f molecular ions, is generally operated at relatively mild temperatures. Molecules are ionized, possibly fragmented, and detected. ESMSMS adds an additional dimension of certainty to compound identification. As in ESMS, a characteristic primary ion is selected. However, instead of simply monitoring the primary ion, in ESMSMS the ion is bombarded with high-energy gas. As a result of high- energy collisions, smaller secondary ionic fragments unique to the primary ion are created and detected. For example, for PFOS (C7F15S 0 3) analysis, ion 499 is selected as the characteristic primary ion. This ion is fragmented into other ions such as 80 amu (corresponding to S 0 3), 99 amu (corresponding to F S03), 130 amu (corresponding to CF^O/), 180 amu (CjF^SOj), and 230 amu (C3F6S 0 3). Each of these secondary fragments is detected and can be used to differentiate PFOS from other compounds that might have the same characteristic 499 amu primary ion but different chemical compositions and secondary ion fragmentation patterns. HPLC system: Hewlett-Packard Series 1100 Liquid Chromatograph Column: Keystone Betasil C l 8 column 2 X 100 mm, 5 pm particle size Flow rate: 300 pl/min Solvent A: 2.0 mM ammonium acetate Solvent B: Methanol Solvent Gradient: 40% to 90% B in 8.5 minutes Hold at 90% B for 3 minutes Return to 40% B in 1 minute Hold at 40% B for 1 minute Injection volume: 10 pL Run time: 13.5 minutes Electrospray Tandem Mass Spectrometer Micromass Quattro II API mass spectrometer mass spectrometer Mass Lynx 3.1 software Cone voltage: 30-60V Collision gas energy: 40 eV Mode: electrospray negative Source block temperature: 115C Desolvation temperature: 250 C Primary Ion: 499 Page 14 of 19 003997 DT 21; T-6316.9; FC Levels of Nave Rats CODE DRAFT 5/14/99 Appendix 2 Daughter Ions: Electrode: 80,99,130,180 Z-spray Quality control summary All analyses were conducted with a moderate to high level of quality control. Duplicate matrix spike analyses were conducted for one animal from each group of animals. Except as noted in the results table, recoveries were within the acceptable range of 80-120%. A calibration check standard was analyzed every 5-10 samples to monitor instrumental drift. Quantiation was based on linear regression analysis of two curves bracketing each group of samples. Quantitation of PFOS was based on the response of 3-4 daughter ions of the primary ion. R esu lts See spreadsheet attached to this report. FACT members participating: K. Hansen L. Clemen H. Johnson M. Ellefson G. Langenburg R. Wynne I. A. Smith S. Heimdahl Page 15 of 19 003998 Sufc Deduct NuaherfTwtUmanceh Matrix: Methcd/Revirion: Analytical EquipmentSyriem Number hrinxnent SoAware/Venion: Date of EktnduiVAnalyat Dale ofAnalyviriAnciyafc Date of Dria Reduction/Analyrt: SaxnpleDtria Appendix 2 DT 21; T-6316.9; FC Levels of Naive Rats CODE DRAFT 5/14/99 PC Levels uring Nriva BataT-6316.9 T -6316(B FO Sli-O H l Rat Liver FACT-M- LOA FACT-M-20 Amalia 063498 Madeline 041088 M tdeline 041098 MareLynx 3.0 Maariyrut 3.0 Maadynx 3.0 7/14/96 IASSAJ7/3tt96 SAH & y98 IAS 7/27/98 K H 7/31/96M EE W O W S K H 7/2898 KJH 3A 98 K H WM/98 K H FI lename: Su: Attachment* full report only R-Squered Value See Attachment* full report only Hope: St* Attachment* lull report only Y-lntercepC See Attachment* lull report only HAT LIVER Banff # p ro s Amnxk Deae Calc. Cone. cfPTOS Method Blk MedwdWk Matrix Blk Matrix Blk Q C -100 ppb ___ * * lEOBlk-l <MDL H Blk-2 <MDL RatLiverBlk-1 <MDL Retliver Blk-2 5.82 A-1M-MS to? A-1M-MSD 9&0 a <MDL <MDL <MDL 0.00682 90% 82% A-1M 305 0.306 Week 6-8 A-2M 48.8 0.0488 A-3M 42.6 0.042S A-4M 39.6 0.0396 A-BM 4L1 0.0411 A-6F 54.9 0.05 A-7F 62.0 0.0620 A-8P 65.4 0.06B4 A-9F 99.6 0.0996 A-11 6L2 0.0612 A-11M too 0.0999 Week 10-14 A-12M 140 0.140 A-13M A-14M 96.8 87.6 0.0968 0.0876 A-IBM - - M l . . .. ftMPl A-16F 722 0.0722 A-17F 93.9 0.0939 A-18F 716 0.0716 A-19F 922 0.0922 A-20F 69.6 0.0696 A-21M >idRetired Breeds A-22M 131 133 am 0.133 A-23M 151 0.151 A-24M A*2hM 149 160 a 149 0.160 A-26F 119 aii9 A-27F 67.1 0.0671 A-28F 45.0 0.0460 A-29F 55.5 0.066S A-30F 53.0 0.0630 Average p ro s *0* <MDL 0 0.00682 86% 0.0430** 0.0954 0.0666 ____ 0103 0.0799 0.145 00678 RtD S td.D e.. 0 <MDL 0 NA 0.0652 9.50** 0.00408** 123 0.117 2a 0.0193 20.6 00212 ia i 0.0121 8.69 0.0126 4a4 0.0295 Banff# H 20B IH H B lk2 Rat Liver Blk-1 Ratliver Blk-2 S398-178-MS 3398.178-1*91 B-1M B-2M B-3M B-4M DBM B-6F B-7F D8F DBF B-10F B-UM B-12M B-13M D14M B-16M &16F B-17F &18F B-19F B-20F B-21M B-22M D23M B-24M B-2SM B-26F B-Z7P B-28F B-20F B-S0F p ro s Calc. Cane. *aA <MDL <MDL <MDL <MDI. 86.0 105 <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDL <MDI, <MDL <MDL <MDL <MDL <MDL A im it A m p RSD Sample# 8cfPFOB p ro s td>Der wA <MDL I t Blk 1 <MDL <MDL <MDL LfcJDRik-*2 <MDL Ratliver Blk 1 <MDL <MDL 8872% C-lMMS % 80% a 114 C-lM-M <MDL <MDL C-1M C-2M <MDI. C*9M <M13L <MDL C-4M <MUL <MDL C-6M <MDL C- <MDL C-7F <MDL C-8F <MDL <MDL c-ae <MDL <MDL <MDL C-10F cMDL C-llM <MDL C-12M <MDL C-13M <M13L <MDL C-14M <MD!, <MIM. <MDL C-lfiM <MDL C-18F <MDL C-17F <MDL C-18F <MDL <MDL <MDI, <MDL <MDL C-19F C-20F <MDL C-21M <MDL C-22M <MDL C-23M <MDL <MDL C-24M <MDL <MDL <MDL C-25M <MDL C-28F <MDL C-27F <MDL C-28F <M1)L <MDL C-29F <MDL <MDL <MDL C-30F ^v *** * y | e * e v u i u x * w l y g i L M \/> J C t * W i y g A riH W U C T A MethodDetectionlimit (MDL): PTO8- 15 r^g, FEOSA*5 itfg, ffOSAA * 30 itfg, EtflOflB 60 ttfg B-breederB iTOS fferfluorooctanBsdfbnate C* breederC W JSA ftfOuDiooctenewifonAnide FTOSAA IW!u>rooctaneadfonamidoacetate NarrowRange N-Etfyi ftrfluorooctaneulfonimido et%l alcohol * A1 wasconfirmed anoutlierand not included in therecakdadong is OK PFOS Calc. Cane. M it <MDL <MDL <MDL <MDL 87.7 80.3 63.6 63.0 47.7 49.2 77.5 67.0 89.3 ?&s 73,2 56.6 86.5 84.7 84.9 119 86.2 529 7L6 726 64.4 117 lfil 441 379 390 276 59.5 106 7L3 63.0 29.0 Amount tkPFOS ta g <MDL *MD1. <MDL <Mni. 73% 67% 0.0636 0.0630 0.0477 0.0492 a 0775 0.0670 0.0893 0.0788 a0732 0.0566 00865 0.0847 0.0849 am 00629 0.0715 0.0728 0.0644 au7 0.151 0441 0379 0.390 0J276 0.0695 0.106 0.0713 00630 0.0290 Date&iteroVBy: 7/2^98 LAC W03/98 LAC &W981AC DateVeriffcd/ By 8/97/98 K H 07/98 K H 8W/96 KJH 003999 Page 16 of 19 BEST COPY AVAILABLE 004000 Appendix 2 DT 21; T-6316.9; FC Levels of Naive Rats CODE DRAFT 5/14/99 Male Naive Rats-T6316.9, FACT070998.1 500.0 > 450.0 c 400.0 o 350.0 "(0 ? S ' 300.0 S Q d> 250.0 o 'S </> & 200.0 O a 150.0 a> S 100.0 a> < 50.0 0.0 Week 6-8 El Breeder A Breeder B Breeder C Week 10-14 Age Group Old Retired Breeders Breeder A rate (City A, State A) were fed Feed A (Company A) Breeder B rats (City B, State B) were fed Feed B Breeder C rats (City C, State C) were fed Feed C Average PFOS levels determined from population n = 5 (per Age Group, per Source), except Breeder A 6-8 Week Male Rats Breeder B Group PFOS results were all < PFOS MDL (15 ppb) Page 17 of 19 Appendix 2. DT 21; T-6316.9; FC Levels of Naive Rats CODE DRAFT 5/14/99 Female Naive Rats-T6316.9, FACT070998.1 BEST COPY AVAILABLE 004001 Week 6-8 Week 10-14 Age Group Old Retired Breeders Breeder A rats (City A, State A) were fed Feed A (Company A) Breeder B rats (City B, State B) were fed Feed B Breeder C rate (City C, State C) were fed Feed C Average PFOS levels determined from population n = 5 (per Age Group, per Source), except Breeder A 6-8 Week Male Rate Breeder B Group PFOS results were all < PFOS MDL (15 ppb) Page 18 of 19 BEST COPY AVAILABLE 004002 Appendix 2 DT 21; T-6316.9; FC Levels of Naive Rais CODE DRAFT 5/14/99 Naive Rat Study-T6316.9, FACT070998.1 El Female EBMale Breeder A rats (City A, State A) were fed Feed A (Company A) Breeder B rats (City B, State B) were fed Feed B Breeder C rats (City C, State C) were fed Feed C Average PFOS levels determined from population n = 5 (per Age Group, per Source), except Breeder A 6-8 Week Male Rats Breeder B Group PFOS results were all < PFOS MDL (15 ppb) Page 19 of 19 DT21; T-6316.9; FC Levels in Naive Rais - Further Investigation of Rat Chow CODE. DRAFT 5/14/99 3M MEDICAL DEPARTMENT, TOXICOLOGY SERVICES Report for Study No. T-6316.9; DT21 Fluorochemical (FC) Levels in Naive Rats - Further Investigation o f Rat Chow Background: In an attempt to determine the source of low-level perfluorooctanesulfonate (PFOS) body burden found in control rats involved in some 3M contract dietary studies, a comprehensive plan with the following objectives and responsibilities was designed: Objective 1 - To investigate potential sources of contamination within the study housing area of the current dietary studies on perfluorinated test compounds at Covance Madison. Dr. Andrew Seacat was appointed study director with Dr. Marv Case as alternate. Jim W olters, 3M Environmental, is responsible for coordinating and conducting direct air monitoring and wipe samples. Objective 2 - To determine if contamination of feed is leading to the low levels o f PFOS seen in control rats in the two-year dietary study of N-Ethyl Perfluorooctanesulfonamido ethanol (N-Ethyl FOSE) conducted at Covance Madison. Dr. Andrew Seacat was appointed study director with Dr. M arv Case as alternate. Objective 3 - To investigate the background serum and liver PFOS levels in naive rats o f different age groups from different sources. Information on the various diets supplied by the different vendors is to be obtained. Deanna Nabbefeld was appointed study director with Dr. Marv Case as alternate. Objective 4 - To investigate the possibility that PFOS exposure is stemming from tainted feed, exposure in rat rooms or a combination o f both. Dr. M arv Case was appointed study director. 3M Environmental Analytical Laboratory is responsible for chemical analysis o f samples gathered in objectives 1-4. The focus o f this report is to cover the data to date examining the possibility o f rat chow contamination. T he study to examine objective 3 is complete and is covered in a separate report. Once further data addressing each objective are available, reports o f the various studies will be generated. The 3M Environmental Laboratory - Fluorine Analytical Chemistry Team (FACT) is currently in the process o f analyzing rat chow samples for PFOS and its metabolites. Attached is a preliminary summary report generated by 3M Environmental Laboratory in August o f 1998 (Appendix 1). Samples o f rat chow from Covance Laboratories, Harlan Laboratories and NIH were analyzed for 2-(N-ethylperfluorooctanesulfonamido)-ethyl alcohol (EtFOSE-OH) by GCMS. Traces o f EtFOSE-OH were found in a sample of Covance chow. This prompted the 3M Environmental Lab Field Group to collected air, airborne particle and wipe samples on site. Preliminary conclusions drawn by the Environmental Lab based on the data to date are that "Contamination in control animals may be due to respiration of airborne EtFOSE-OH, from transfer of test material from one animal to another during sample handling and possibly from sporadic contamination of the food source". Page 1 of 5 004003 DT21 ; T-6316.9; FC Levels in Nave Rats - Further Investigation of Rat Chow CODE. DRAFT 5/14/99 Appendix 1 3M Environmental Laboratory - Fluorine Analytical Chemistry Team Lisa Dick / Kris Hansen Fluorine Analytical Chemistry Team Building 2-3E-09 612-778-7540 / 612-778-6018 ladick@mmm.com / kjhansen@mmm.com Preliminary summary report: Further Investigation of Rat Chow Summary: In June 1998, several rat chow samples from Covance Laboratories and 3 from NIH were supplied to the Environmental Lab for characterization of fluorochemicals that are being monitored in animal studies conducted by 3M Toxicology. A single side o f the bag chow sample from Covance was determined to be of significantly higher contamination than the samples from NIH. Because contaminants were found, air and airborne particle and wipe samples were collected on site by the 3M Environmental Lab Field Group. So that results could be based on more than a single sample, additional bags o f chow from Covance and Vendor B were also analyzed and are reported in this summary. In the chow samples, 2-(N-ethylperfluorooctanesulfonamido)-ethyl alcohol (EtFOSE-OH) was quantitated by GCMS. EtFOSE-OH was not present above the detection limit o f 1 ng/g chow in the triplicate analyzed samples from three Covance bags (labeled meals for June 9, June 15, and June 21, respectively) or from the single Vendor B bag. Triplicate chow samples have not been analyzed for PFOS. Air and airborne particle samples were collected at Covance by Jim W olter and Kurt Oldenburg o f the 3M Environmental Lab Field Group. Air was passed through charcoal and glass fiber filters. Details of the sampling procedure can be found in a report by Jim W olter and Kurt Oldenburg. The largest volume air samples from the backs o f rooms were collected on glass fiber filters and analyzed while samples from the front o f each room were collected on charcoal and analyzed. Wipe samples from all rat backs were analyzed but wipes from cages, doorhandles, and floors were not. In air samples taken from Room 3045, EtFOSE-OH was detected by HPLC/ESMS at levels below the practical quantitation limit (approximately 0.17 pph/L) but above the method detection limit (approximately 0.02 ppb/L) in some samples. Perfluorooctanesulfonate (PFOS) was not detected at levels above the blanks in any room. Analysis o f wipes from the backs o f rats in Room 3045 contained a measurable amount o f EtFOSE-OH, whereas wipes from rats in other rooms did not. Due to variations in sampling technique, wipe samples were not quantifiable. Wipes from animal backs were not analyzed for PFOS. Page 2 of 5 004004 DT21; T-6316.9; FC Levels in Nave Rats - Further Investigation of Rat Chow CODE. DRAFT 5/14/99 Appendix 1 Chow source Vendor B Covance - June 9 Covance - June 15 Covance - June 21 EtFOSE-OH Levels in Rat Chow Number of Matrix Spike EtFOSE- samples Pass OH 3 2/sample Non-detect 3 2/sample Non-detect 3 2/sample Non-detect 3 2/sample Non-detect EtFOSE-OH Levels in Covance Lab Environment Covance Sample Location Number of EtFOSE-OH detection samples Room 3045 Air Samples 4 out of 8 0.02 ppb/L < detect < 0.17 (GF and charcoal) ppb/L Room 3004, 349 Air 16 Non-detect Samples (GF and charcoal) Room 3045 Rat Backs 6 Detect (8 ppb/wipe average) Room 3004, 349 Rat Backs 12 Non-detect Experimental summary: Sample preparation: Methylene chloride extraction Analytes were extracted from chow by addition o f ether. Samples were weighed and then covered with 20 mL ether. Non-polar organic analytes transfer from the chow to the organic layer. Samples were shaken for 1 hour and then centrifuged for 30 minutes. Fifteen mL of ether was removed and blown down to 1 to 2 mL. Glass filter fiber and charcoal adsorbates were split and then prepared by extraction with methanol or ether. All of the charcoal inside the sterile tubes was extracted. Final sample volumes were 1 mL. GC: Characteristic retention times In gas chromatography, an aliquot o f sample is injected and vaporized onto a chromatographic column. Individual components o f the sample adsorb to the stationary phase o f the column. As the temperature is raised, components are eluted from the column based on physical and chemical characteristics. An inert gaseous mobile phase carries the components through the column. Carrier gas flow rate, column temperature and gas pressure are adjusted to optimize chromatographic separation. MS: Electron Impact One method for producing ions for mass spectra is by bombardment with energetic electrons. In addition to the formation of a molecular ion, a series of reactions leads to the formation o f other fragment ions that may be larger or smaller than the molecular ion and are useful for compound identification and quantitation. Page 3 of 5 004005 DT21; T-6316.9; FC Levels in Naive Rats - Further Investigation o f Rat Chow CODE. DRAFT 5/14/99 Appendix 1 HPLC: Characteristic retention times In HPLC, an aliquot of extract is injected and passed through a liquid phase chromatographic column. Based on the affinity o f the analyte for the stationary phase in the column relative to the liquid mobile phase, the analyte is retained for a characteristic amount o f time. For example, in a standard solution PFOS may elute at 10.5 minutes. Retention times between a standard PFOS solution and the analyte extracted from filter fibers in this analysis were matched to within 1% on the HPLC system. ES/MS: Detecting and monitoring molecular ions Following HPLC separation, ES/MS provides a rapid and accurate means for analyzing a wide range o f organic compounds, including fluorochemicals. Electrospray, one of the softest ionization techniques available, is generally operated at relatively mild temperatures. Molecules are ionized, fragmented, and detected. Initially, the mass to charge range m/z = 100 to 1210 is monitored following direct flow injection o f the samples. Ions characteristic of known fluorochemicals were observed. These results are used to select ions that can be monitored selectively for quantitative results. Analysis o f organic fluorine standard compound indicates that the primary ion characteristic o f EtFOSE-OH is m/z = 630 amu, corresponding to the mass o f the compound complexed to acetate anion from the running buffer: C8Fi7S02N(C2H5)(CH2C H 20H )/C H 3C 00\ Single ion monitoring was used to determine the concentration o f this ion in the samples. Quality control summary: Methanol blanks were analyzed periodically to ensure complete isolation o f the sample. Charcoal and glass fiber filter blanks collected on site were also analyzed and found to be blank. Quantitation of HPLC-ESMS data for fluorochemicals is based on the linear regression o f 5 point standard curves from 10 ppb to 1000 ppb or matrix spike recovery comparisons. Quantitation of GC-MS peaks is based on the recovery o f known spike amounts in the same sample matrix and on the linear regression of 5 point standard curves. Instrumental specifics: GC/Mass Spectrometers Hewlett-Packard ATD 400 Gas Chromatograph and Mass Spectrometer Column: J& W DB-624 30m Temperature ramp: 50C to 250C @ 20C/min 250C hold 5 min Carrier gas: Helium Spike volume: 25 p.L Oven temperature: 180C Trap low temp.: -30C Desorb time: 10 min. Trap fast: Yes Trap high temp.: 250C Page 4 of 5 004006 U 2 1; T-6316.9; FC Levels in Nave Rats - Further Investigation of Rat Chow CODE. Appendix 1 DRAFT 5/14/99 Line temperature: 225C Pressure: 18.4 psi Valve temperature: 225C Trap hold: 5 min. Desorb flow: 25 mL/min Ions monitored: 540,448 amu Source temperature: 250C Quad temperature: 125C EM volts: 2598 V Interface temperature: 250C HPLC system Hewlett-Packard Series 1100 Liquid Chromatograph Column: Keystone Betasil C18 column, 2 X 100 mm, 5 firn particle size Flow rate: 300 (il/min Solvent A: 2.0 mM ammonium acetate Solvent B: Methanol Solvent Gradient: 40% to 90% B in 8.5 minutes Hold at 90% B for 3 minutes Return to 40% B in 1 minute Hold at 40% B for 1 minute Injection volume: 10 pL Run time: 13.5 minutes Electrospray Mass Spectrometer Micromass Platform II atmospheric pressure ionization (API) mass spectrometer Mass Lynx 2.1 software Cone voltage: -60V Mode: electrospray negative Source temperature: 90C Analyzer pressure: 9.2 X 10'5 mBar Ions: 630, 526,499 Electrode: cross-flow Conclusions: Low levels o f PFOS found in control animals may be due to respiration o f airborne EtFOSE-OH, from transfer of test material from one animal to another during sample handling, and possibly from sporadic contamination o f the food source. 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