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Studs NO.T-6316.9: DT21 Fluorochemical (FC) Levels in Naive Rats Final Report, Revision A January 09,2002 In-Life Start Date: July 8,1998 In-Life End Date: July 28, 1998 Study Location: 3M Strategic Toxicology Laboratory Corporate Toxicology 3M Medical Department 3M Center, Building 270-SB-314 St. Paul, MN 55144 RECEIVED OPPT NClC Study Director: Deanna J. Luebker, M.S., Advanced Research Toxicologist 3M Medical Dept. / Corporate Toxicology 3M Center, Building 220-2E-02 Saint Paul, MN 55144 Ph: 651-737-1374FAX:651-733-1773 2003 OCT-6 PM 12:27 Sponsor: 3M Corporate Toxicology 3M Center Building 220-2E-02 St. Paul, MN 55144 Research Client: 3M Specialty Chemicals Division 3M Center Building 236 St. Paul, MN 55144 Page 1 of 23 ` DT21;T-6316.9Final Study Report Revision A 01/09/02 Purpose of Revision: The purpose of this revision is to incorporate all data available to date on the Nave Rat Study (T-6316.9) and the rat chow/fishmeal analysis. This includes data reported in the initial final Nave Rat Study report written by Corporate Toxicology in 1998 (l), a summary report on the liver PFOS levels written by 3M Environmental in 1998 (Appendix l), and a summary report of the liver PFOS levels and rat chow/fishmeal analyses written by 3M Environmental in 2001 (Appendix 3). Study Background: In an attempt to determine the source of low-level PFOS body burden found in control rats involved in some 3M contract dietary studies, a comprehensiveplan with the following objectives and responsibilities was designed: Obiective 1 - To investigate potential sources of contamination within the study housing area of the current dietary studies on perfluorinated test compounds at Covance Madison. Andrew Seacat was appointed study director with Marv Case as alternate. Jim Wolters, 3M Environmental, is responsible for coordinatingand conducting direct air monitoring and wipe samples. Objective 2 - To determine if contamination of feed is leading to the low levels of PFOS seen in control rats in the two-year dietary study of N-Ethyl FOSE conducted at Covance Madison. Andrew Seacat was appointed study director with Marv Case as alternate. Obiective 3 - To investigatethe background serum and liver PFOS levels in nave rats of different age groups from different sources. Information on the various diets supplied by the different vendors is to be obtained. Deanna (Nabbefeld) Luebker was appointed study director with Marv Case as alternate. Obiective 4 - To investigate the possibility that PFOS exposure is stemming from tainted feed, exposure in rat rooms or a combination of both. Marv Case was appointed study director. 3M Environmental Analytical Laboratory was deemed responsible for chemical analysis of samples gathered in objectives 1-4. The study to examine objective 3 is complete and is the focus of this report. Once data addressing each objective are available, reports of the various studies will be generated. Report Summary: The purpose of this study was to assess "endogenous" levels of fluorochemicals (FCs) in untreated Sprague Dawley rats of three different age groups acquired from three different breeders to determine what perfluorooctane sulfonate (PFOS) levels, if any, were present. Each vendor was contacted to provide information on the feed provided to their rats while at their facilities. Levels of PFOS in the rodent feed supplied by the different vendors and in fishmeal were determined. Page 2 of 23 DT21;T-6316.9Final Study Report Revision A 01/09/02 The livers of male and female untreated Sprague Dawley rats 6-8 weeks old, 10-14 weeks old, and approximately 9-12 months old (old retired breeders, ORB)from Charles River, Harlan, and Taconic Farms Laboratories were examined for liver PFOS levels. Male rat livers from Charles River and Taconic Farms test animals showed significant endogenous levels of PFOS that roughly correlated with age. Livers collected from the oldest group of male rats from Taconic Farms contained significantlymore PFOS than any other group in the study. The livers of the female rats from Charles River and Taconic Farms contained very consistent levels of PFOS, showing no correlation with age. The livers of test animals from Harlan did not contain PFOS above the limit of detection (15 nglg). Based on these data, PFOS had bioaccumulated to a much great degree in male Charles River and Taconic Farms than in female rats from the same vendors. This difference may have been a result of PFOS being released in the milk and/or transferred in uferoto the pups in females that had bore and nursed multiple litters. Female rats may also more readily excrete PFOS through urinary excretion than male rats. Results of the vendor f d d i e t history survey revealed that fishmealwas the primary ingredient in feed supplied to Taconic Farms rats and the 5th listed ingredient in feed provided to Charles River rats. Fishmeal was not listed as an ingredient feed supplied to Harlan rats. Detectable levels of PFOS were found in samples of the chow fed to both Charles River and Taconic Farms rats. No PFOS could be detected in chow provided to Harlan rats (fishmeal free). Analyses of fishmeal(six types arising from a minimum of 3 kinds of fish) suggest that it may be the source of PFOS in the chow. To definitively make this conclusion, analysis of the actual fishmealincorporated into each of the chow samples analyzed is required. Another possible source of PFOS is the paper coating used in feed bags. Further information on feed packaging may be gathered in a future study. Methods: This study was performed in the 3M Strategic Toxicology Laboratory under a defined protocol (2). All in-life procedures were approved by the 3M Laboratory Animal Review Committee (3M LARC). - Thirty Sprague Dawley rats, five male and five female from each of the following age groups: 6-8 weeks old, 10-14 weeks old, and 9-12 weeks old (old retired breeder, ORBS) were ordered from Charles River, Harlan, and Taconic Farms. Each vendor was asked to provide the name of the feed provided to their rats, name and location of feed vendor, 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 of arrival at 3M, rats were weighed, grossly examined, and euthanized by CO, asphyxiation. Sera and liver were harvested and sent to Kris Hansen, 3M Environmental Laboratory - Fluorine Analytical Chemistry Team (FACT), for PFOS analysis. Liver specimens were analyzed as described in the Experimental Section of Appendix 1. Sera samples have not been analyzed but will be retained for possible future analysis. Statistical analysis to detect significant differences ( a . 0 5 )in liver PFOS concentration Page 3 of 23 DT21;T-6316.9 Final Study Report RevisionA 01/09/02 between age groups, sexes, and vendors in the Charles River and Taconic Farms rats was performed using a onetailed, homoscedastic T-test. The rate of change in PFOS levels (ppb/week) was estimated by dividing the change in PFOS concentration by the number of weeks evaluated (approximately 38.5 weeks). Rat chow from each of the vendors and fish meal (six types arising from a minimum of 3 kinds of fish) were analyzed for PFOS concentrations as described in Appendix 2. Results: In Life Data & Liver PFOS Analysis In life data including body weights, liver weights, and liverlbody weight ratios as well as liver PFOS concentrations can be found in Tables 1-3. Detailed results of the FC analysis, including graphs and a full data table, can be found in Appendix 1. Liver PFOS levels in Charles River male and female rats at 6-8 weeks of age averaged 43.0 f 4.07 and 66.6 f 19.27 ppb, respectively. At 10-14 weeks of age, liver PFOS levels in male and female Charles River rats had increased to 103.0 f 21.33 and 79.9 f 12.06 ppb, respectively. Liver PFOS levels in Charles River male and female ORBs were 145.0 f 12.42 and 67.8 f 29.63 ppb, respectively (See Table 1 and Appendix 1). Liver PFOS levels in allHarlan rats were below the method detection limit of 15 ppb (See Table 2 and Appendix 1). Liver PFOS levels in Taconic Farms male and female rats at 6-8 weeks of age averaged 60.2 f 12.21 and 73.0 f 12.29 ppb, respectively. At 10-14 weeks of age, liver PFOS levels in male and female Taconic Farms rats had increased to 92.2 f 22.75 and 75.7 f 24.40 ppb, respectively. Liver PFOS levels in Charles River male and female ORBs were 327.0f 115.37 and 65.6 f 27.24ppb, respectively (SeeTable 3 and Appendix 1). The average rate of change in PFOS liver concentration for male and female Charles River rats from week 7 to week 45.5 was estimated to be approximately 2.6 and 0.031 ppb/week, respectively. The average rate of change in PFOS liver concentration for male and female Taconic Farms rats over the same timeperiod was estimated to be approximately 6.9 and - 0.19 ppb/week, respectively. Results of the T-test are included in Table 4. The change in liver PFOS concentration between age groups in male Charles River and Taconic Farms rats was significantat all timepoints (p 5 0.05). Livers from Charles River male rats contained significantly more PFOS at all age groups than did livers of Charles River female rats of the same age group. Livers of male Taconic Farms ORBs contained significantly more PFOS than livers of female Taconic Farms ORBs. Livers of male Taconic Farms 6-8 week and ORB rats contained significantly higher PFOS concentrations than did male Charles River rats of the same age groups. Page 4 of 23 DT21;T-6316.9 Final Study Report Revision A 01/09/02 Vendor Feed/Diet Survey Charles River rats are fed PMI 5L79 Rat and Mouse Diet supplied by PMI Nutrition International Inc., Brentwood, MO. PMI feed is packaged in paper bags coated with a paper coating material produced by 3M. The feed ingredients, as listed by PMINutrition International Inc., are as follows: ground yellow corn, wheat middlings, soybean meal, animal fat preserved with BHA, fish meal, alfalfa meal, cane molasses, calcium carbonate, salt, cyanocobalamin (source of vitamin B-12), biotin, DL methionine, calcium pantothenate, folic acid, riboflavin, cholecalciferol (source of vitamin D-3), vitamin A acetate, di-alpha tocopheql acetate (source of vitamin E), thiamin, magnesium oxide, sodium selenite, nicotinic acid, pyridoxine hydrochloride, menadione dimethylprimidinolbisulfite (source of vitamin K activity), silicon dioxide, calcium iodate, manganous oxide, copper sulfate, cobalt carbonate, ferrous carbonate, zinc sulfate, zinc oxide. Harlan rats are fed Harlan Teklad LM-485MoudRat Sterilizable Diet supplied by Harlan Teklad, Madison, WI. Teklad feed 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 Teklad feed ingredients, as listed by Teklad, are as follows: ground corn, soybean meal, ground oats, wheat middlings, alfalfa meal, soybean oil, corn gluten meal, calcium carbonate, dicalcium phosphate, brewers dried yeast, iodized salt, Llysine, DLmethionine, vitamin A-acetate, D-activated animal sterol (source of vitamin D3), vitamin E supplement, niacin, calcium pantothenate, riboflavin, thiamin mononitrate, pyridoxine hydrochloride, menadione sodium bisulfite complex (source of vitamin K), folic acid, biotin, vitamin BI2supplement, calcium carbonate, manganous oxide, ferrous sulfate, copper sulfate, zinc oxide, calcium iodate, cobalt carbonate. Taconic Farms rats are fed TAC #31 Rodent Diet supplied by Zigler Co., Gardner, PA. TAC #3 1 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 TAC #31, as listed by Zigler Co., are as follows: fish meal, soybean meal, alfalfa meal, corn gluten meal, ground whole wheat, ground ##2yellow corn, ground whole oats, wheat middlings, Brewer's dried yeast, soybean oil, salt, dicalcium phosphate, ground limestone, vitamin and mineral premixes. Rodent Feed PFOS Analyses Page 5 of 23 DT21;T-6316.9Final Study Report Revision A 0 1/09/02 PMI Lab diet, fed to rats obtained from Charles River contained approximately 18 ng/g PFOS. Rodent diet from Zigler, fed to rats purchased from Taconic Farms contained approximately 12 ng/g of PFOS. Teklad ravrodent diet, fed to rats obtained from Harlan (fishmealfree), did not contain PFOS above the limit of quantitation (LOQ) (see Appendix 2). Fish Meal PFOS Analyses Six types of fishmeal arising from at least three types of fish were analyzed for PFOS (see Appendix 2). PFOS was detected in 50% of the fishmeal samples analyzed. It is not known, however, what type of fishmeal was incorporated into the various rat chows. Therefore, it cannot be definitively concluded that fishmeal is the source of PFOS in the rat chow. Analysis of the actual fishmeal incorporated into the chow is required to provide a direct link. Conclusions: Based on these data,PFOS had bioaccumulated to a much great degree in male rats from Charles River and Taconic Farms than in female rats from these same vendors. This differencemay have been a result of PFOS being released in the milk andor transferred in utero to the pups in females that had bore and nursed multiple litters. Female rats may also more readily excrete PFOS through urinary excretion than male rats. The livers of test animals from Harlan did not contain PFOS above the limit of detection (15 ng/g). Results of the feed analyses revealed detectable levels of PFOS in samples of the chow fed to both Charles River and Taconic Farms rats. No PFOS could be detected in chow provided to Harlan rats (fishmeal free). Analyses of fishmeal (six types arising from a minimum of 3 kinds of fish) suggest that it may be the source of PFOS in the chow. To definitively make this conclusion, analysis of the actual fishmeal incorporated into each of the chow samples analyzed is required. Another possible source of PFOS is the paper coating used in feed bags. As stated in the results section, 3M supplies paper coating to PMI, the manufacturer of Charles River PMI chow. It is not known, however, if Harlan and Taconic Farms feed suppliers also use 3M material in their bags. Further information on feed packaging may be gathered in a future study. Page 6 of 23 DT21;T-6316.9FinalStudy Reporl Revision A 01/09/02 List of Tables: 1) Charles River In-Life and Liver PFOS Data 2) Harlan In-Life and Liver PFOS Data 3) Taconic Farms In-Life and Liver PFOS Data 4) T-TestResults @ values) - Charles River and Taconic Farms Age, Sex, and Vendor Liver PFOS Comparision. rage 7 of 23 DT21;T-6316.9 FinalStudy Report Revision A 01/09/02 'able 1:Charles River In-Life and Liver PFOS Data rendor liD lSex IAge Grp. IBody ILiver liiver wt/ IPFOS Liver Conc. (nglg or ppb wt(g) wt(g) bodywt individual Ave Std D e k :HARLES W E R CR1 M 6-8wkS 171.7 8.30 0.048 **305 43.0 4.0 CR2 M 6-8wkS 179.6 8.30 0.046 48.8 CR3 M 6-8wks 161.5 7.05 0.044 42.6 CR4 M 6-8wkS 151.6 6.60 0.044 39.5 CR5 M 6-8wks 166.8 8.06 0.048 41.1 CR6 F CR7 F CR8 F CR9 F CR10 F 6-8wks 6-8wks 6-8wks 6-8wks 6-8wks 173.7 8.69 168.3 7.13 165.1 7.46 141.7 4.91 159.7 6.47 0.050 0.042 0.045 0.035 0.041 54.9 66.6 19.2 62.0 65.4 99.6 51.2 CR11 M CR12 M 10-14W ~ S324.3 10.55 10-14wk~ 367.9 12.66 0.033 0.034 100.0 103.0 21.3 140.0 CR13 M 10-14W ~ S356.7 13.10 0.037 96.8 CR14 M 10-14W ~ S358.5 13.23 0.037 87.6 CR15 M 10-14W ~ S365.6 13.65 0.037 90.1 CR16 F CR17 F CR18 F CR19 F 10-14 wks 226.1 10-14W ~ S199.5 10-14wks 203.4 10-14wk~ 196.4 10.49 6.32 6.13 7.14 0.046 0.032 0.030 0.036 72.2 79.9 12.0 93.9 71.6 92.2 CR20 F 10-14wks 209.8 7.08 0.034 69.6 CR21 M CR22 M CR23 M cR24 M CR25 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 0.030 131.0 145.0 12.4 133.0 151.0 149.0 160.0 CR26 F ORB 394.2 12.70 CR27 F ORB 412.5 16.09 CR28 F ORB 482.9 14.80 .CR29 F CR30 F ORB ORB ORB = old retired breeders Outlier - not used in calculations. 485.3 424.2 14.96 11.34 lethod detection limit (MDL): PFOS = 15ng/g or ppb 0.032 0.039 0.031 0.031 0.027 1 19.0 67.1 45.0 55.5 53.0 67.8 29.6 Page 8 of 23 DT21; T-6316.9 Final Study Report Revision A 0 1/09/02 able 2: Harlan in-Life and Liver I I I I 3ody Liver liver wt/ PFOS Liver Conc. (ng/g or ppb) vt(g) wt(g) bodywt individual Ave Std Dev 198.4 9.45 201.7 9.34 203.4 8.50 203.9 8.38 204.2 9.17 0.048 0.046 0.042 0.041 0.045 <MDL <MDL <MDL <MDL <MDL <MDL H6 F H7 F H8 F - H9 F H10 F H11 M H12 M H13 M - H14 M H15 M H16 F H17 F H18 F - H19 F H20 IF H21 M H22 iM H23 M - H24 M H25 M H26 F H27 F H28 F H29 F H30 F 6-8wkS 6-8wks 6-8wks 6-8wkS 6-8wks 214.3 208.0 212.8 209.3 209.0 10-14wk~ 300.6 10-14wk~ 10-14wk~ 10-14wk~ 10-14wk~ 10-14wk~ 10-14wk~ 10-14wk~ 10-14wk~ 10-14 wkS *ORB ORB ORB ORB ORB 470.7 462.8 458.2 ORB ORB ORB ORB ORB 326.2 285.7 286.2 306.9 7.62 7.19 7.14 7.30 8.08 10.69 17.00 14.90 15.55 14.52 11.19 10.45 10.27 11.oo 0.036 0.035 0.034 0.035 0.039 0.036 f0.040 0.037 0.037 0.028 0.030 0.030 0.036 0.031 0.036 0.032 0.035 0.032 <MDL <MDL <MDL <MDL <MDL <MDL <MDL cMDL <MDL <MDL <MDL cMDL cMDL <MDL <MDL 0.031 0.030 0.031 0.036 cMDL cMDL <MDL ORB = old retired breeders lethod detection limit (MDL): PFOS = 15nglg or ppb Page 9 of 23 DT21;T-6316.9 Final Study Report Revision A 01/09/02 - lendor TACONl =ARMS 7 :onicFarms in-Lifeai Body Liver wt(g) wt(g) liver wt/ PFOS Liver Conc. (ng/g or ppb: bodywt individual Ave Std Dev TF1 M TF2 M TF3 M TF4 M TF5 M TF6 F TF7 F TF8 F TF9 F TF10 F TF11 M TF12 M TF13 M TF14 M TF15 M TF16 F TF17 F TF18 F TF19 F TF20 F TF21 M TF22 M TF23 M TF24 M TF25 M TF26 F TF27 F TF28 F TF29 F TF30 F 6-8wkS 6-8wks 6-8wks 6-8wks 6-8wks 200.2 202.4 184.1 187.5 183.6 6-8wkS 6-8wks 6-8wks 6-8wks 6-8wks 201.2 189.9 172.5 207.9 199.5 10-14wks 454.3 10-14W ~ S334.6 10-14W ~ S459.9 10-14wks 407.5 10-14wks 317.8 10-14wks 214.4 10-14W ~ S226.1 10-14W ~ S216.1 10-14W ~ S241.0 10-14 WkS 246.5 *ORB ORB ORB ORB ORB 687.8 561.2 456.0 525.7 586.6 ORB ORB ORB ORB ORB 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 .o 379.0 390.0 276.0 59.5 105.0 71.3 63.0 29.0 60.2 12.2. 73.C k 5 5 92.2 22.7! - 75.7 24.4( 327.0 115.3 65.6 27.24 ORB = old retired breeders dethod detection limit (MDL):PFOS = 15ng/gor ppb Page 10 of 23 DT21;T-6316.9Final Study Report Revision A 01/09/02 ITabie4 T-Test Results (p values) - Charles River and Taconic Fa- Age, Sex, and Vendor I IVendor I Male 6-8& 10-14wk 10-14Wk&ORB Aaelsex G ~ U CDornmarision I Fernale MaielFemaie 6-8wk& 6-8& 10-14wk 6-8wk& 6-8wk 10-14wk ORB& ORB 10-14wk &ORB ORB &6-8wk & 10-14wk ORB Charles 0.00 0.00 0.00 0.1 1 0.21 0.47 0.02 River 0.03 0.00 I ITaconicI Farms I I I I 0.031 I 0.001 Male I 0.001 I I 0.421 I 0.281 I 0.301 Vendor Cornmrlslon Fernale 0.071 I I 0.251 0.001 vs Taconlc - I Farms p-TEST one-tailed distribution, hornoscedastlc P%e 11 of 23 &# " ' DY21; T-6316.9FinalStyReportRevision A OLON02 : SEM NitseeMfSy.TS . Pisano (FC)Level inNatverats.Sept. 1998. 2)3M Motil Department,Taxioology ServicesProtocolforStudy No. T-6316.9; DF21. Piuoso(cFC)hLeeveslsinniNatcvearalts.July, 1998. Page120f23 . Signatures: Prepared By: Study Director Reviewed By: John Butenhoff, PhD., DABT Corporate Toxicology Management Andrew Seacat, PhD., DABT Senior Research Toxicologist DT2l;T-6316.9 Fiaal Study Report Revision A 01/09/02 [/@/a>@ Date Date Date Page 13 of 23 DT21;T-6316.9 Fhal Study Report Revision A 01/09/02 Appendix 1 3M EnvironmentalLaboratory-Fluorine Analytical Chemistry Team Contact: Kris Hausen Fluorine Analytical Chemistry Team Building 2-3E-09 8-6018 Study of PFOS levels in Nave Rats Summary report Supplier Charles River Harh Taconic Farms Location Raleigh, NC Indianapolis,IN Germantown, NY # a t 6 4 #at 10- h14 chow wks 14wks wks Purina* 10 10 10 TEKlad 10 10 10 TAC #3 1 10 10 10 Ratio male:female 1:l 1:l 1:l Analytical Summary Liver samples were homogenized and extracted using an ion-paring reagent. The extracts were analyzedquantitativelyusing high-pressureliquid chromatography-electrospraytandem mass spectrometry (HPLC-ESMSMS) and evaluated versus an extracted curve. Analyticaldetailsare availablein the full report. The presence or absence of other known fluorochemicalcontaminantsand metaboliteswas ascertained by inspection. Results Summary Rat livers fromCharlesRiver and TaconicFarms test animalsshowed significantendogenous levels of PFOS. Livers collected from oldest group of male rats from Taconic Farms contained significantly more PFOS than any other group in the study. The livers of test animals from Harlan did not contain PFOS above the limit of detection (15 ppb). PFOS levels in the livers of male rats from Charles River and Taconic Farms 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 Charles River and Taconic Farms were determined to contain very consistent levels of PFOS, showing no correlation with age. No other known fluorochemicalcontaminantsor metaboliteswere identifiedin the liver samples analyzedin this study. Graphical results and a full table of results are attached. Page 14 of 23 i , ... . DT21; T-6316.9 Final Study Report Revision A 01/09/02 Appendix 1 Currently, methods are being developed for the analysis of low levels of PFOS and ethyl-FOSE alcohol in samplesof chow fromeach supplier. Experimental Samplepreparatlon-aqueoussamples, HPU-ESMS: Ion-pairing exftaction Analyte is extracted from a sample matrix with an ion-pairing reagent (tetrabutyl ammonium hydrogen sulfate (TBA)) in a pHantrolled 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 reconstitutedin methanolfor MS analysis. HPU-ESMS and HPU-ESMSMS: For dGtailed qualitativework In HPLC, an aliquot of extract is injected and passed through a reverse-phase liquid chromatographiccolumn. 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 analyteextractedfromgroundwaterin this analysiswere matched to within 1% on theHPLC system. Following HPLC separation, ESMS provides a rapid and accurate means for analyzing a wide range of organic compounds, including fluorochemicals. Electrospray, an ionization technique used primarily for the detection of molecular ions, is generally operated at relatively mild temperatures. Moleculesare ionized,possibly fragmented,and detected. ESMSMS adds an additional dimension of certainty to compound identification. As in ESMS, a characteristicprimary ion is selected. However, instead of simply monitoring the primary ion, in ESMSMS the ion is bombarded with highenergy 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 (C,FlsSOi) analysis, ion 499 is selected as the characteristic primary ion. This ion is fragmented into other ions such as 80 amu (correspondingto SOi),99 amu (correspondingto FSOi), 130amu (correspondingto CF2SO;), 180amu (CzF4SOi),and 230 amu (C3F,jS033.Each of these secondary fragments is detected and can be used to differentiate PFOS from other compounds that might have the stlme characteristic 499 amu primary ion but different chemical compositions and secondary ion fragmentation patterns. HPLC system:Hewlett-PackardSeries 1100Liquid Chromatograph Column: Keystone Betasil C18 column 2X100mm,5pparticlesize Flow rate: 300 clymin 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 to40% B in 1 minute Hold at 40% B for 1 minute Injection volume: 10 pL Run tinae: 13.5 minutes ElectrosprayTandemMass Spectrometer MicromassQuattro I1API mass spectrometermass spectrometer Mass Lynx 3.1 software Page 15 of 23 DT21; T-6316.9 Final Study Report Revision A 01/09/02 Appendix 1 Cone voltage: 30-60v Collision gas energy: Mode: 40 eV electrospray negative Source block temperature: 115C Desolvation temperatm: 250"C primary Ion: 499 Daughter Ions: 80,99,130,180 Electrode: %spray Quality control summary All analyses were conducted with a m0der;gteto high level of qualitycontrol. Duplicate matrix spike analyses were conducted for one animal from each group of animals. Except as noted in the results table, recovefieswere within the acceptable range of 8O-120%. A calibration check standard was analyzed every 5-10 samplesto monitorinstnunentaldrift. Quantiation was based on linear regression analysis of two curves bracketing each group of samples. Quantitationof PFOS was based on the response of 3-4 daughterions of the primary ion. R?SUltS See spreadsheet attached to this report. FACT members participating: KHansen L.Clemen H. Johnson M.Ellefson G. Langenburg R. Wynne LA. Smith S. Heimdahl Page 16 of 23 Appendix 1 DT21; T-6316.9 Final Study Report Revision A 01/09/02 ........ n? _ _ a??? :. . . . . . . . . . . r .- ... w e . arms . . . . . . . . CR!?.. .-* ..-*.: : :3%! b ... CR!F . . . . ...... .cR!Ip . . . . 939.. CR? . . . '114. . . . . . m.a . .. CRPP 696 ...oqm . . . . . . . . . . . . . . . . . . . . . . . .. .om.. .. .......... O.rn!S . . . . . . . . . . . . . O.........m............ .. ?!-.! 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E:?'?? ..Oes..~ ; cR.?!F ................... . . . . ~ . ! ! 9 , ..~. . . . . 0.119.......... _ _ .................... -..H-?%... .. .*.x w:???. .*L. _**LL... .=.- e.0~ ..?o.rn.!e?..L. ;... ..u?pX OM!. - A 9 v 5 . ~ . . . . . . . . . ..- :., .............. . . . . . . ..rn.l. . . . . . . . . . . . . . . . . . . . . . . . . . . . . H-?Ip... ... ..... -, . . . . . . . . . . . .O. .w. . .0. . . . . . . . . . . . . . . . . . . . . . . . -. .............H..-.2..o.p ..% . . . . . . . . . . . . . . . . . . C. .R. .l.D.C. . . . . . om m om om5 CIWlp ..... ..................... Om q.4 ........ ...... QLDL H-rP QalL .?@L ..... ...................... . ... - .. . ..... .*. .... . . -. ......... .*.. -cMDL & .... .. . OlDL .TP-?!K . 1% ... OJ? ..Tp;lpa_.. -.%. . . . . O ! ' . ~ rp-F. . . . . .?? ....... 0 - e . . . . .Y . . . . 276 on6 -. ... - .......... .F:?F .... ................ . . . .+?L. .............................. ..... ..?? ..... . ..lo!. . . . . . O.! .T:?!.. ... ?I?.. ..... F : P ... ...~ ..... ..... OdDL . .- ....... ...... n-pa 1pD ..................... ................ . . . . . . -. . . . . . .. ..... ' .'-. .. 3 . 3 . 0.116 ...................... ................... ...................... om .................. .416 .. om73 Page 17 of 23 Appendix 1 Mz1; T6316.9Rerd S t o d y ~ R c v i s i o Ao OI/09/02 500.0 - r, 450.0- Male Naive Rats-T6316.9, FACT070998.1 --II I Charles River IHarlan Group llaconic Farm Week MI W 1M4 Age Group cbartes River Rats (Raleigh,North Caroline)were fed P M I (PurimMills) Chow Hprlan Group Rats (laaiaanpdis,lndisnn)were fed TEKhd Cbow Taco& Farms Rats (Germpotown,New Yo&) were fed TAM31 Chow Average PFOS levels determinedfmm populationn =S (perAge GNJR~p,er Source), except cberksRiver 6-8Week Male Rats Tbis data set used only 4 semptes in uleolstion of average PFOS lev& due to an outlier. Harlan GroupPFOS d t s were ell <PFOS MDL (ISppb) Page 18 of 23 Appendix I Female Naive Rats-T6316.9, FACT070998.1 Week 6-8 Week 10-14 Old Retired Breeders Age Group Charles River Rats (Raleigh, North CaroUne)were fed PMI (Purina Mills) Chow Harlan Group Rats (Indianapolis, Indiana) were fed TEKlad Chow T m n i c FarmsRats (Germantown, New Yo&) were fed TAC#31 Chow Average PFOS levels determinedfrom populationn = 5 @er Age Group, per Source), except Charles River 6-8Week Male Rats This data set used only 4 samples in calculation of average PFOS levels due to an outlier. Harlan Group PFOS results were aU < PFOS MDL (15 ppb) Page 19 of 21 Appendix 1 h i v e Rat Shrdy-T6316.9, FACTO70998.1 DT21; Td316.9F d Study Report Revision A 01/09/02 50 0 Sex CharlesRiver Rats (RaMgh, North C d i )were fed PMI (Purina Mills) Chow Harlnn Grwp Rats (Miannpdis,Miann) were fed TEKIad Chow Taconk Farms Rats (Germantown,New York)were fed TAC#31 Chow Average PFOS levelsdeterminedfrom popolatbn n =5 (perAge Group,per Source),except CharlesRiver 6-8Week Male Rats This data & med only 4 samples in enleolstiwof average PFOS levels due to an outlier. Harlan Grwp PFOS refiults were ell < PFOS MDL (15 ppb) Page 20 of 23 DT21; T-6316.9 Final Study Report Revision A 01/09/02 Appendix 2 SUMMARY OF N A h E RAT/RAT CHOWIFISH MEAL STUDY ANALYSES Conducted by the 3M Environmental Lab (prepared 03/01/01) Nave Rats (Taken from summaryreport issued in 1998.) In order to assess "endogenous"levels of PFOS in test animals, the livers of ninety rats fromthreedifferent supplierswere quantitativelyanalyzedfor 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). Rat livers fromCharlesRiver and TaconicFarms test animals showed significantendogenouslevels of PFOS. Livers collectedfrom oldest groupof male rats fromTmnic Farms contained significantlymore PFOS than any other group in the study. The livers of test animals from Harlan did not contain PFOS above the limit of detection (15 ng/g). PFOS levels in the livers of male rats from Charles River and Taconic Farms 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 fromCharlesRiver and TaconicFarms were determid to contain very consistentlevelsof PFOS, showing no correlationwith age. Limits of Data: Screeningfor other known metabolitesof Et-FOSEOH (e.g. PFOSA, PFOSAA) was performed. The originalsummaryreport document stated that "No other known fluorochemicalcontaminantsor metabolites were identifiedin the liver samplesanalyzed in this study." However, based on the levels of PFOS observed in the liver samples, the analytical LOQ may not have been sufficient to see expected metabolite levels. (Based on the 2 years Et-FOSE-OH feeding study, PFOSAA levels would be expected to be approximately 9%of PFOS levels; PFOSA concentrationswould be expected to be approximately5 % of PFOS levels. Back calculated,the expected concentrationsof the metabolitesare near the 1998LOQ.Data fromthe relativelyhighdose 2 year feeding study may or may not provide an acceptable model of this low-level exposure.) Spike recovery studies were conducted for PFOS in rat livers and were acceptable. Matrix spike studies conducted for other analytes were, for the most part, acceptable. Rat Chow Rat chow from 4 separate vendors was investigated. Two of the sources of rat chow were determined to contain PFOS above the limit of quantitation. NIWPMI Lab diet, fed to rats obtained fromCharlesRiver was determined to contain approx 18ng/g PFOS. Small animaldiet fromZeigler, fed to rats purchased fromTaconicFarms was determined to contain approximately 12 ng/g of PFOS. Teklad rat/rodent diet, fed to rats obtained from Harlan, did not contain PFOS above the LOQ. Limits of the Data: Page 21 of 23 DT21;T-63 16.9 Final Study Report Revision A 01/09/02 Appendix 2 Spike recoveries studies were conducted on each type of chow tested and all results were acceptable in all cases (77-98%). The LOQ for PFOS in rat chow is approximately2-10 ng/g. Fish Meal There is great variabilityin both the source and types of fishmeal availablecommercially. This variability complicatesthe analysisof fishmealand the interpretationof data concerohg fishmeal. Fishmeals used for animal and fish diet are typically generated from what are referred to as "industrial fish". Industrialfish, includingmenhaden and herring, are usually quitehigh in oil content and thus not fit for human consumption. Approximately 90%of the world`s fishmealis producedfrom industrial fish. Fishmeal is processed in factories or directly on the fishing vessel and results in the production of several separatefishmealproducts. After the fish are cooked, a liquor (oil,water, and protein) is separated from the solids by "pressing". The solid that remains after pressing is called the "presscake", the liquid component, oil and solubleprotein, is "stickwater". Subsequentto pressing, the oil is separatedfrom the remaining components of the stickwaterby centrifugation. After furtherdistillation,this fish oil may be directly incorporated into animal diet. The remaining stickwater is evaporated down to a thick syrup of 30-50% solidsand may be sold as "condensed fish solubles". The syrupmay also be added back to the presscake and dried and sold as `'whole meal". Presscake without the syrupis sold as "presscakemeal." All of these componentsmay be incorporatedinto auimalfeed. According to the Universityof FloridaCorporateExtension Service,good qualityfishmealaverages6070%protein; the oil content ranges from 2-15%. Fishmeal is incorporatedinto food for a variety of animals and fish includingfarm-raisedsalmon,poultry, and cows (particularly dairy cows). In this study, six types of fishmeal,arising from at least 3 types of fish, were analyzed. 0 Menhaden travel in large schoolsand migrateup the coast each year. They are found in warmer, near shorewaters and in the US, are fished primarily offAtlanticCoast. Menhaden constitute98%of the fishmeal produced in the US. 0 Capelin,also a schoolingfish, spend much of their lives offshore in the deep waters of the Northern Pacific and the Arctic and move in-shore only to spawn. 0 The habitat for different classes of herring is much more far ranging than that of either menhaden or capelin. Different types of herring can be found from the Northern Pacific waters off the coast of Canadaand down to the Southeastern Atlanticcoast. Like menhaden and capelin,herring are small, schoolingfish and thus are targeted for large-scalenetting. Omega Protein Sealac (presumed to be menhaden) 9.87 Page 22 of 23 Limits of the data: DT21;T-6316.9 Final StudyReport RevisionA 0 1/09/02 Appendix 2 Matrix spike studies were conducted on a single type of fishmeal (menhaden). PFOS spiked into the menhaden fishmeal was recovered at approximately 66%. Matrix spike studies were not conducted for the remaining types of fishmeal tested. The LOQfor PFOS determinationin these fishmeal sampleswas approximately3.5 ng/g. Summarv of the 3 comwnentsof this studv: . Vendor of Rats Charles River Rats with endogenous PFOS? Yes Rat Chow used by Vendor Contains PFOS? Yes Rat Chow used by Vendor contains &heal? Yes Taconic Farms Yes Yes Yes Harlan No -30 ng/g) No (LoQ=2 ndg) No I I I I J Limits of the studv: Because it is not known what type of fishmeal was incorporatedinto the various rat chows, it cannot be definitivelyconcluded that fishmealis the sourceof PFOS in the rat chow. Although indirectevidence indicatesthis probability,because PFOS was only detected in 50% of the fishmeal samplesanalyzed, analysisof the actual fishmealincorporatedinto the chow is required to provide a direct link. Other variablesaffectingthe rats prior to shipmentto 3M (such as PFOS levels in water and in other components of the feed) have not been tested. To the best of my memory, this chow has not been tested for the potential of EtFOSE-OH transfer from packaging. Shouldfurther work be conducted on the tissue samples,a lower level screen for metabolitesand other knownfluorochemicalsis recommended. This informationwould provide additionalevidenceconcerning the origin of PFOS identified in rat livers. .. The motivationto pursue any of these additionalinvestigationsdependson the corporation'send use of this data. The original study objective: ". to determine what PFOS levels, if any, are present in Sprague Dawley rats of threedifferent agesfrom threedifemtsuppliers"(MedicalDept. protocol for study# T6319.9) was completedupon issuance of the EnvironmentalLab summary report in 1998. Page 23 of 23