Document XOZXGraM49dvVmrpYQwRpb4pJ
AR226-1527
S R P T T -fcS Ifc
Study 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 NCIC
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: 651-737-1374 FAX: 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
T l F l2-fe&0
Page 1 o f 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
P urpose o f R evision:
The purpose o f this revision is to incorporate all data available to date on the Naive Rat Study (T-6316.9) and the rat chow/fishm eal analysis. This includes data reported in the initial final N aive R at Study report written by Corporate Toxicology in 1998 (1), a summary report on the liver PFOS levels w ritten by 3M Environm ental in 1998 (Appendix 1), and a summary report o f the liver PFOS levels and rat chow/fishm eal analyses written by 3M Environm ental in 2001 (Appendix 3).
Study B ackground:
In an attem pt to determ ine the source o f low-level PFOS body burden found in control rats involved in some 3M contract dietary studies, a comprehensive plan w ith the following objectives and responsibilities was designed:
Objective 1 - To investigate potential sources o f contam ination within the study housing area o f the current dietary studies on perfluorinated test compounds at Covance M adison. Andrew Seacat was appointed study director w ith M arv Case as alternate. Jim W olters, 3M Environm ental, is responsible for coordinating and conducting direct air m onitoring and wipe samples. O bjective 2 - To determ ine if contam ination o f feed is leading to the low levels of PFOS seen in control rats in the two-year dietary study o f N-Ethyl FOSE conducted at Covance M adison. Andrew Seacat was appointed study director with M arv Case as alternate. O bjective 3 - To investigate the background serum and liver PFOS levels in naive rats o f different age groups from different sources. Inform ation on the various diets supplied by the different vendors is to be obtained. Deanna (Nabbefeld) Luebker was appointed study director w ith M arv Case as alternate. Objective 4 - To investigate the possibility that PFOS exposure is stemming from tainted feed, exposure in rat rooms or a com bination o f both. M arv Case was appointed study director. 3M Environm ental Analytical Laboratory was deemed responsible for chem ical analysis of samples gathered in objectives 1-4.
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 w ill be generated.
R eport Sum m ary:
The purpose of this study was to assess "endogenous" levels o f fluorochem icals (FCs) in untreated Sprague Dawley rats o f three different age groups acquired from three different breeders to determ ine what perfluorooctane sulfonate (PFOS) levels, if any, were present. Each vendor was contacted to provide inform ation on the feed provided to their rats while at their facilities. Levels o f PFOS in the rodent feed supplied by the different vendors and in fishm eal were determined.
Page 2 o f 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
The livers o f 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. M ale rat livers from Charles River and Taconic Farms test animals showed significant endogenous levels of PFOS that roughly correlated w ith age. Livers collected from the oldest group of male rats from Taconic Farms contained significantly more PFOS than any other group in the study. The livers o f the fem ale rats from Charles River and Taconic Farms contained very consistent levels o f PFOS, showing no correlation w ith age. The livers o f test animals from Harlan did not contain PFOS above the lim it o f detection (15 ng/g). Based on these data, PFOS had bioaccum ulated to a much great degree in male Charles River and Taconic Farms than in fem ale rats from the same vendors. This difference may have been a result o f PFOS being released in the m ilk and/or transferred in utero to the pups in females that had bore and nursed m ultiple litters. Female rats may also more readily excrete PFOS through urinary excretion than male rats. Results o f the vendor feed/diet history survey revealed that fishm eal was the prim ary 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 H arlan rats. Detectable levels of PFOS were found in samples o f the chow fed to both Charles River and Taconic Farms rats. No PFOS could be detected in chow provided to H arlan rats (fishm eal free). Analyses o f fishm eal (six types arising from a minimum o f 3 kinds o f fish) suggest that it may be the source o f PFOS in the chow. To definitively make this conclusion, analysis of the actual fishm eal incorporated into each of the chow samples analyzed is required. Another possible source o f PFOS is the paper coating used in feed bags. Further inform ation on feed packaging may be gathered in a future study.
M ethods:
This study was perform ed 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 fem ale from each o f 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, H arlan, and Taconic Farms. Each vendor was asked to provide the name o f the feed provided to their rats, name and location o f feed vendor, feed ingredient list and inform ation on feed packaging. All anim als rem ained in their shipping containers between arrival at 3M and euthanization. No food or water, other than that provided in the shipping containers, was furnished. W ithin one horn: o f arrival at 3M, rats were weighed, grossly examined, and euthanized by C 0 2asphyxiation. Sera and liver were harvested and sent to Kris Hansen, 3M Environm ental Laboratory - Fluorine Analytical Chem istry Team (FACT), for PFOS analysis.
Liver specimens were analyzed as described in the Experim ental Section o f Appendix 1. Sera samples have not been analyzed but w ill be retained for possible future analysis. Statistical analysis to detect significant differences (p<0.05) in liver PFOS concentration
Page 3 o f 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
between age groups, sexes, and vendors in the Charles River and Taconic Farms rats was perform ed using a one-tailed, homoscedastic T-test. The rate o f change in PFOS levels (ppb/week) was estim ated by dividing the change in PFOS concentration by the num ber of weeks evaluated (approxim ately 38.5 weeks).
R at chow from each o f the vendors and fish meal (six types arising from a minimum o f 3 kinds o f fish) were analyzed for PFOS concentrations as described in Appendix 2.
R esults:
In Life D ata & Liver PFOS Analysis
In life data including body weights, liver weights, and liver/body weight ratios as well as liver PFOS concentrations can be found in Tables 1-3. D etailed results o f the FC analysis, including graphs and a full data table, can be found in Appendix 1.
Liver PFOS levels in Charles River male and fem ale rats at 6-8 weeks o f age averaged 43.0 4.07 and 66.6 19.27 ppb, respectively. A t 10-14 weeks o f age, liver PFOS levels in male and fem ale Charles River rats had increased to 103.0 21.33 and 79.9 12.06 ppb, respectively. Liver PFOS levels in Charles River male and fem ale ORBs were 145.0 12.42 and 67.8 29.63 ppb, respectively (See Table 1 and Appendix 1).
Liver PFOS levels in all Harlan rats were below the method detection lim it o f 15 ppb (See Table 2 and Appendix 1).
Liver PFOS levels in Taconic Farms male and fem ale rats at 6-8 weeks o f age averaged 60.2 12.21 and 73.0 12.29 ppb, respectively. A t 10-14 weeks o f age, liver PFOS levels in male and female Taconic Farms rats had increased to 92.2 22.75 and 75.7 24.40 ppb, respectively. Liver PFOS levels in Charles River male and fem ale ORBs were 327.0 115.37 and 65.6 27.24 ppb, respectively (See Table 3 and Appendix 1).
The average rate o f change in PFOS liver concentration for male and female Charles River rats from week 7 to week 45.5 was estim ated to be approximately 2.6 and 0.031 ppb/week, respectively. The average rate o f change in PFOS liver concentration for male and female Taconic Farms rats over the same tim e-period was estim ated to be approximately 6.9 and 0.19 ppb/week, respectively.
Results o f 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 significant at all tim epoints (p < 0.05). Livers from Charles River male rats contained significantly more PFOS at all age groups than did livers o f Charles River fem ale rats o f the same age group. Livers o f male Taconic Farms ORBs contained significantly more PFOS than livers of fem ale Taconic Farms ORBs. Livers o f 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 o f 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Vendor Feed/Diet Survey
Charles River rats are fed PM I5L 79 Rat and M ouse D iet supplied by PM I N utrition International Inc., Brentwood, M O. PM I feed is packaged in paper bags coated w ith a paper coating m aterial produced by 3M. The feed ingredients, as listed by PM I N utrition International Inc., are as follows:
ground yellow com , wheat m iddlings, soybean meal, anim al fat preserved with BHA, fish m eal, alfalfa meal, cane molasses, calcium carbonate, salt, cyanocobalamin (source o f vitam in B-12), biotin, DL m ethionine, calcium
pantothenate, folic acid, riboflavin, cholecalciferol (source o f vitamin D-3), vitamin A acetate, di-alpha tocopheryl acetate (source o f vitam in E), thiam in, magnesium oxide, sodium selenite, nicotinic acid, pyridoxine hydrochloride, menadione dimethylprim idinol bisulfite (source o f vitam in 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 -485 M ouse/Rat Sterilizable D iet supplied by Harlan Teklad, M adison, 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 m aterial. The Teklad feed ingredients, as listed by Teklad, are as follows:
ground com , soybean meal, ground oats, w heat m iddlings, alfalfa meal, soybean oil, com gluten meal, calcium carbonate, dicalcium phosphate, brewers dried yeast, iodized salt, L-lysine, D l^m ethionine, vitam in A-acetate, D -activated anim al sterol (source o f vitam in D3), vitamin E supplement, niacin, calcium pantothenate, riboflavin, thiam in m ononitrate, pyridoxine hydrochloride, menadione sodium bisulfite complex (source o f vitam in K), folic acid, biotin, vitam in B i2supplement, calcium carbonate, manganous oxide, ferrous sulfate, copper sulfate, zinc oxide, calcium iodate, cobalt carbonate.
Taconic Farms rats are fed TAC #31 Rodent D iet supplied by Zigler Co., Gardner, PA. TAC #31 is packaged in a "commercially acceptable 3 ply lam inated paper bag". It is unknown at this tim e whether or not a 3M m aterial is used in these bags. The feed ingredients in TAC #31, as listed by Zigler Co., are as follows:
fish m eal, soybean meal, alfalfa meal, com gluten meal, ground whole wheat, ground #2 yellow com , ground whole oats, w heat m iddlings, Brew er's dried yeast, soybean oil, salt, dicalcium phosphate, ground limestone, vitam in and m ineral prem ixes.
Rodent Feed PFOS Analyses
Page 5 o f 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
PM I 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 o f PFOS. Teklad rat/rodent diet, fed to rats obtained from Harlan (fishm eal free), did not contain PFOS above the lim it o f quantitation (LOQ) (see Appendix 2).
Fish M eal PFOS Analyses
Six types of fishm eal arising from at least three types o f fish were analyzed for PFOS (see Appendix 2). PFOS was detected in 50% o f the fishm eal samples analyzed. It is not known, however, w hat type o f fishm eal was incorporated into the various rat chows. Therefore, it cannot be definitively concluded that fishm eal is the source of PFOS in the rat chow. Analysis o f the actual fishm eal incorporated into the chow is required to provide a direct link.
C onclusions:
Based on these data, PFOS had bioaccum ulated to a much great degree in male rats from Charles River and Taconic Farms than in fem ale rats from these same vendors. This difference may have been a result o f PFOS being released in the m ilk and/or transferred in utero to the pups in females that had bore and nursed m ultiple litters. Female rats may also more readily excrete PFOS through urinary excretion than male rats. The livers o f test animals from Harlan did not contain PFOS above the lim it o f detection (15 ng/g). Results o f the feed analyses revealed detectable levels o f PFOS in samples of the chow fed to both Charles River and Taconic Farms rats. N o PFOS could be detected in chow provided to Harlan rats (fishm eal free). Analyses o f fishm eal (six types arising from a minimum o f 3 kinds o f fish) suggest that it may be the source o f PFOS in the chow. To definitively make this conclusion, analysis o f the actual fishm eal incorporated into each o f the chow samples analyzed is required. 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 PM I, the m anufacturer o f Charles River PM I chow. It is not known, however, if Harlan and Taconic Farms feed suppliers also use 3M m aterial in their bags. Further inform ation on feed packaging may be gathered in a future study.
Page 6 o f 23
DT21; T-63I6.9 Final Study Report Revision A 01/09/02
List of Tables: 1) Charles River ln-Life and Liver PFOS Data 2) Harlan In-Life and Liver PFOS Data 3) Taconic Farms In-Life and Liver PFOS Data 4) T-Test Results (p values) - Charles River and Taconic Farms Age, Sex, and Vendor
Liver PFOS Comparision.
Page 7 of 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Table 1: C harles R iver In-L ife an d L iver PFGIS D ata
Vendor ID Sex Age Grp. Body Liver liver wt/ PFOS Liver Cone, (ng/j3 or ppb)
w t(g) w t(g) body wt Individual Ave
Std Dev
CHARLES
RIVER
CR1 M 6-8 wks 171.7 8.30 0.048
**305 43.0 4.07
CR2 M 6-8 wks CR3 M 6-8 wks
179.6 8.30 161.5 7.05
0.046 0.044
48.8 42.6
CR4 M 6-8 wks CR5 M 6-8 wks
151.6 6.60 166.8 8.06
0.044 0.048
39.5 41.1
CR6 F CR7 F CR8 F
6-8 wks 6-8 wks 6-8 wks
173.7 168.3 165.1
8.69 7.13 7.46
0.050 0.042 0.045
54.9 66.6 19.27 62.0 65.4
CR9 F 6-8 wks CR10 F 6-8 wks
141.7 4.91 159.7 6.47
0.035 0.041
99.6 51.2
CR11 M 10-14 wks 324.3 10.55 0.033
100.0 103.0 21.33
CR12 M 10-14 wks 367.9 12.66 0.034
140.0
CR13 M 10-14 wks 356.7 13.10 0.037
96.8
CR14 M 10-14 wks 358.5 13.23 CR15 M 10-14 wks 365.6 13.65
0.037 0.037
87.6 90.1
CR16 F CR17 F CR18 F CR19 F
10-14 wks 10-14 wks 10-14 wks 10-14 wks
226.1 199.5 203.4 196.4
10.49 6.32 6.13 7.14
0.046 0.032 0.030 0.036
72.2 79.9 12.06 93.9 71.6 92.2
CR20 F 10-14 wks 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
131.0 133.0 151.0 149.0 160.0
145.0
12.42
CR26 F CR27 F CR28 F CR29 F CR30 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 8 of 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Table 2: Hartan In-Life and Liver PFOS Data
Vendor
ID Sex Age Grp. Body Liver liver wt/ PFOS Liver Cone. (ng/ 3 or ppb)
w t(g) w t(g) body wt Individuai Ave
Std Dev
HARLAN
H1 M 6-8 wks H2 M 6-8 wks H3 M 6-8 wks H4 M 6-8 wks H5 M 6-8 wks
198.4 201.7 203.4 203.9 204.2
9.45 9.34 8.50 8.38 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
6-8 wks 6-8 wks 6-8 wks 6-8 wks 6-8 wks
214.3 208.0 212.8 209.3 209.0
7.62 7.19 7.14 7.30 8.08
0.036 0.035 0.034 0.035 0.039
<MDL <MDL <MDL <MDL <MDL
<MDL
H11 M H12 M H13 M H14 M H15 M
10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks
300.6 307.9 304.0 299.8 306.0
10.69 12.33 11.26 10.09 11.38
0.036 0.040 0.037 0.034 0.037
<MDL <MDL <MDL <MDL <MDL
<MDL
H16 F H17 F H18 F H19 F H20 F
10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks
237.7 241.1 239.7 236.3 233.4
6.65 7.20 7.14 8.54 7.33
0.028 0.030 0.030 0.036 0.031
<MDL <MDL <MDL <MDL <MDL
<MDL
H21 M H22 M H23 M H24 M H25 M
*ORB ORB ORB ORB ORB
470.7 462.8 446.6 458.2 478.9
17.00 14.90 15.55 14.52 11.19
0.036 0.032 0.035 0.032 0.023
<MDL <MDL <MDL <MDL <MDL
<MDL
H26 F H27 F H28 F H29 F H30 F
ORB ORB ORB ORB ORB
337.8 326.2 285.7 286.2 306.9
10.45 10.27
8.54 9.01 11.00
0.031 0.031 0.030 0.031 0.036
<MDL <MDL <MDL <MDL <MDL
<MDL
* ORB = old retired breeders Method detection limit (MDL): PFOS = 15ng/g or ppb
Page 9 o f 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Table 3: Taconic Farms In-Life and Liver PFOS Data
Vendor
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
Std Dev
TACONIC
FARMS
TF1 M TF2 M TF3 M TF4 M TF5 M
6-8 wks 6-8 wks 6-8 wks 6-8 wks 6-8 wks
200.2 202.4 184.1 187.5 183.6
9.64 10.18
9.28 10.26
9.34
0.048 0.050 0.050 0.055 0.051
63.6 63.0 47.7 49.2 77.5
60.2 12.21
TF6 F TF7 F TF8 F TF9 F TF10 F
6-8 wks 6-8 wks 6-8 wks 6-8 wks 6-8 wks
201.2 189.9 172.5 207.9 199.5
9.73 8.40 9.40 10.28 10.54
0.048 0.044 0.055 0.049 0.053
67.0 73.0 12.29 89.3 78.8 73.2 56.6
TF11 M TF12 M TF13 M TF14 M TF15 M
10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks
454.3 334.6 459.9 407.5 317.8
17.97 13.07 17.65 14.17 12.75
0.040 0.039 0.038 0.035 0.040
86.5 54.7 84.9 119.0 86.2
92.2 22.75
TF16 F TF17 F TF18 F TF19 F TF20 F
10-14 wks 10-14 wks 10-14 wks 10-14 wks 10-14 wks
214.4 226.1 216.1 241.0 246.5
9.33 8.94 8.47 11.21 8.53
0.044 0.040 0.039 0.047 0.035
52.9 71.5 72.6 64.4 117.0
75.7 24.40
TF21 M TF22 M TF23 M TF24 M TF25 M
*ORB ORB ORB ORB ORB
687.8 561.2 456.0 525.7 586.6
22.22 18.24 14.96 17.91 18.24
0.032 0.033 0.033 0.034 0.031
151.0 441.0 379.0 390.0 276.0
327.0 115.37
TF26 F TF27 F TF28 F TF29 F TF30 F
ORB ORB ORB ORB ORB
281.5 281.4 247.5 289.5 338.7
19.56 18.81 15.65 14.95 17.58
0.069 0.067 0.063 0.052 0.052
59.5 105.0
71.3 63.0 29.0
65.6 27.24
* ORB = old retired breeders Method detection limit (MDL): PFOS = 15ng/g or ppb
Page 10 o f 23
DT21; T-6316.9 Final Study Repon Revision A 01/09/02
Table 4: T-Test Results (p values) - Charles River and Taconic Farms Age, Sex, and Vendor Liver PFOS Comparision________________________________________________________
Vendor
Age/Sex Group Comparision
Male
Female
Male/Female
6-8 &
10-14wk 6-8wk & 6-8 &
10-14wk 6-8wk & 6-8 wk
10-14wk O R B &
10-14 wk & ORB ORB
10-14 wk & ORB ORB
& 6-8 wk & 10-14wk ORB
Charles
0.00
0.00 0.00
0.11
0.21 0.47
0.02
R iv e r
0.03 0.00
Taconic Farm s
0.03
0.00 0.00
0.42
0.28 0.30
Vendor Compar sion
Male
Female
Charles 6-8 wk 10-14wk ORB & River & 6-8 wk & 10-14wk ORB
6-8 wk & 6-8 wk
10-14wk
& 1014wk
ORB & ORB
vs Taconic Farm s
0.03
0.13 0.00
0.28
0.37 0.45
T-TEST - one-tailed distribution, homoscedastic
0.07 0.25 0.00 xxxxxxxxxxxxxxxxx xxxxx
Page 11 of 23
01/0MB2
ToxicologyServices Reportfor Study No. T-6316.9; DT21. Levelsis Naiverats. Sept, 1998. 3) SMMedicsl Department, Toxicology ServicesProtect for Study No. T-6316.9; DT2I. Rteoeocte Bcal(PC)Leve8m Naverats. July, 1998.
12 of 23
S ig n a tu re s: Prepared By:
Deanna J. Luebkef; MS Study D irector
DT21; T-6316.9 Final Study Report Revision A 01/09/02
rnfm/jcv^a
D ate
Reviewed By:
John Butenhoff, PhD ., DABT Corporate Toxicology M anagement
ftif f/ 2-0&5
D ate
__Qwk&J
Andrew Seacat, PhD ., DABT Senior Research Toxicologist
_____ _______ / A / o ~ _____
D ate
Page 13 o f 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Appendix 1
3M Environmental Laboratory- Fluorine Analytical Chemistry Team
Contact: Kris Hansen Fluorine Analytical Chemistry Team Building 2-3E-09 8-6018
Study o f PFOS levels in Naive Rats Summary report
E xperim ental Sum m ary In order to assess "endogenous" levels o f PFOS in test animals, the livers o f ninety rats from three
different suppliers were quantitatively analyzed for PFOS. Three distinct ages o f rats were represented in the group o f 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.
# a t 6-8 # a t 10- #>14
R atio
S u p p lie r
L o ca tio n
Chow
wks 14 wks wks
m aletfem ale
Charles River Raleigh, NC
Purina* 10 10 10
1:1
Harlan
Indianapolis, IN
TEKlad
10
10
10
1:1
Taconic Farms Germantown, NY TAC #31
10
10
10
1:1
*3M is/was a supplier o f paper coating material to Purina M ills, Inc. Currently, it is not known if 3M
supplies material to TEKlad or Taconic Farms.
A nalytical Sum m ary 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 (HPLC-ESMSMS) and evaluated versus an extracted curve. Analytical details are available in the full report. The presence or absence o f other known fluorochemical contaminants and metabolites was ascertained by inspection.
R esults Sum m ary Rat livers from Charles River and Taconic Farms test animals showed significant endogenous
levels of PFOS. Livers collected from oldest group o f male rats from Taconic Farms contained significantly more PFOS than any other group in the study.
The livers o f test animals from Harlan did not contain PFOS above the limit o f detection (IS ppb). PFOS levels in the livers o f male rats from Charles River and Taconic Farms roughly correlated with the age o f 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 o f the female rats from Charles River and Taconic Farms were determined to contain very consistent levels o f 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 o f results are attached.
Page 14 of 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Appendix 1
Currently, methods are being developed for the analysis o f low levels o f PFOS and ethyl-FOSE alcohol in samples of chow from each supplier.
Experimental Sim ple preparation-aqueous samples, HPLC-ESMS: Ion-pairing extraction
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 and HPLC-ESMSMS: For detailed qualitative work In HPLC, an aliquot o f extract is injected and passed through a reverse-phase liquid
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 groundwater in this analysis were matched to within 1% on the HPLC system.
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. M olecules are ionized, possibly fragmented, and detected.
ESMSMS adds an additional dimension o f certainty to compound identification. As in ESMS, a characteristic primary ion is selected. However, instead o f simply monitoring the primary ion, in ESMSMS the ion is bombarded with high-energy gas. As a result o f high- energy collisions, smaller secondary ionic fragments unique to the primary ion are created and detected.
For example, for PFOS (C7FisS(V) analysis, ion 499 is selected as the characteristic primary ion. This ion is fragmented into other ions such as 80 amu (corresponding to S03'), 99 amu (corresponding to FS03'), 130 amu (corresponding to CF2SO3), 180 amu (C2F4SO3), and 230 amu (C3F6S0 3 '). Each o f 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:
M ethanol
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
R untim e:
13.5 minutes
Electrospray Tandem Mass Spectrometer M icromass Quattro II API mass spectrometer mass spectrometer
Mass Lynx 3.1 software
Page 15 o f 23
DT21; T-6316.9 Final Study Report Revision A
01/09/02
Appendix 1
Cone voltage:
30-60V
Collision gas energy:
40 eV
M ode:
electrospray negative
Source block temperature: 115C
Desolvation temperature: 250 C
Primary Ion:
499
Daughter Ions:
8 0 ,9 9 ,1 3 0 ,1 8 0
Electrode:
Z-spray
Quality control summary All analyses were conducted with a moderate to high level o f quality control. Duplicate matrix
spike analyses were conducted for one animal from each group o f animals. Except as noted in the results table, recoveries were within the acceptable range o f 80-120%.
A calibration check standard was analyzed every 5-10 samples to m onitor instrumental drift. Quantiation was based on linear regression analysis o f two curves bracketing each group o f samples. Quantitation o f PFOS was based on the response o f 3-4 daughter ions o f the primary ion.
Results 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 16 o f 23
Appendix 1
DT21; T-6316.9 Final Study Report Revision A 01/09/02
ProductNu>ibeir*ftStortane*): __
____[___ __I....
Metho4R*vision:
__
.A n | iy tiri) F tp e jre n t S j^ H tM fe r.
;bMliu^ 3 c^w w yVigk>q: _ _j ___ _
D^(^E^ctioc^A xM ^rt:
Date ofAaalyns/AiMiyst
Date ofDataReduction/Analyst
FCUvei* usingNaive RateT-6316.9
lf^l6(^bSEIO! "311-11.11..1.111
^RatLiver
1FACT-M4I1 ft FACT-M-20...1 .........1 ' ....... T~~
Agw^QQ4W
MasiljrnxXO _ M uaLynxio ;MeeaLyg30
7/1498 1A3/SAH 7/30/98 SAH 84B/98 IAS
7/27/98 KJH
7/31/98MEE 0/0408 KJH
7/28/98 KJH
803/98 KJH 8/D5/W KJH
See A ttechttent*,ftjlreport only
R-Squered VelueSee Attachment#,fu irepoctoniy
Slope:
J im i*itte rh riin t s .flill rap oil^iu
Y-Intercept SeeAttarkaente, faftreport onty_
B A T L IV E R
............ ........ -............ h .............-
Gkvap Dean ! Method BSc Method Bflc M atoB k M etm Bft QC-100ppb
S a ile d
HSOBflc-l H20 BSe-2 Rtf Liver B fe! RatLiverB8c-2 CRrlM-MS CRrlM-MSD
pros
Cak. Cans.
ilk <MDL <MDL <MDL
582 107 980
A m rart afPFOS
<MDL <MDL <MDL 000582 90% 82%
_ ..A H H R ... pros v rt 0 <MDL 0 000582
88%
RSD SU.Dot.
0 <MDL
0 NA
00552
Sanale#
H20B&-1 H20 Bflc-2 RetLiverBHg-1 R1LiyB0c-2 S39S-178-MS S398-178-MSD
PFOS Cak. Cene.
m /k <MDL <MDL <MDL <MDL
860 105
AwOT afPFOS
* it <MDL <MDL <MDL <MDL 72% 88%
PFOS MfK <MDL
<MDL
80%
RSD S ii. Dot.
PFOS Cak. Cana.
<MDL <MDL 0.114
H20 BBs-1
H20B&J RetLiverBIfclet Liver BOc-
TF-1M-MS TF-1M-MSD
<MDL <m dl <MDL <MDL 87.7
803
A nm t afPFOS
efe <MDL <MDL <MDL <MDL
73% 67%
- - A " * ... RSD PFOS M i.Dot. w fe
<MDL <MDL
<MDL <MDL
70% 00439
G nn 1 W eek 6 8
G n^2 Week 10-14
CR-IM CR-2M CR-3M CR44 CR-3M CRr6F CR-7F CR8F CRr9F CR-10F
CR11M CR-12M CR-13M CR-14M CR-15M CR-ltfF CR-17F
305 488 428 39.5 41.1 54.9 620 65.4 998 312
100 140 968 878 90.1 722 939
0205 00488 00426 00395 00411 00549 00820 00654 00996 00512 00999 0.140 00968 00876 00901 00722 00939
00430** 00954
930** 000408**
123 0.117
00666
280 00193
0.103
208 00212
H-1M H-2M H-3M H-4M H-5M H-6P H-7F H-8F H-9F H-10F
H -llM H-12M H-13M H-14M H-15M H-16F H-17F
<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 <MDL
<MDL <MDL
<MDL <MDL
<MDL <MDL
TF-1M TF-2M TF-3M TF-4M TF-5M TF-6F TF-7F TF-8F TF-9F TF-10F TF-11M TF-12M TF43M TF-I4M TT-5M TF-16F TF-17F
838 830 47.7 49.2 773 870 893 780 732 568 863 84.7 840 119 862 520 71.5
00636 00630 00477 00492 00775 00670 00893 00788 00733 00566 00865 00847 00849 0.119 00862 00529 00715
00602
203 00122
00730
160 00123
00922
162 00149
CR-18F CR-19F CR-20F
718 922 898
00716 00922 00696
00799
15.1 00121
H-18F H.19F H-20F
<MDL <MDL <MDL
<MDL <MDL <MDL
<MDL
<MDL <MDL
TF-18F TF-19F TF-20F
728 64.4 117
0.0726 00644 0.117
00757
323 00344
(k n f3
CR.21M
31dRetiredBreeder CR22M
131 133
0.131 0.133
H-21M H-22M
<MDL <MDL
<MDL <MDL
TF-21M TF-22M
151 441
0.151 0.441
CR-23M
151
0.151
H-23M
<MDL
CR24M
149
0.149
889
H-24M
<MDL
CR-25M
180
0.160 0.145 00126 H-25M <MDL
CR-2SF
119
0.119
H-26F
<MDL
CR-27F
87.1
00671
H-27F
<MDL
CR-28F
458
00450
H-28F
<MDL
CR-29F
553
00555
43.4 H-29F <MDL
CR-30F
538
00530
00678
00295
H-3QF
<MDL
PracticalQuart#rtioo Lind(PQL) -PFO S- 30nrfgPFO 3A -10nrt,PFO SA A -60n^gEiFO B E-I CR-ChatferRivei___
MethodPetectonLBBM|ilDI: PFOS" 13nffgtPFQSA" 3np/gPF0BAA* 3Qng/g,EtP08El" dpng/H - HadanGroup
<MDL
TF-23M
379
0379
<MDL
<MDL TF-24M
390
0390
353
<MDL <MDL <MDL TF-25M
276
0276
0327 0.116
<MDL
TF-26F
393
00595
<MDL
TF-27F
105
0.105
<MDL
# .2 8 F
713
00713
<MDL
<MDL TF-29F
63.0
00630
418
<MDL <MDL <MDL TF-3QF
290
00290
00656
00273
_____ ___ ___ ...__ _____ _ _________ ___......... ___ ______ _____ ___ ____ _
FFOS-
T F -fe c o n ic F n
PFOSA*
PFOSAA PedfaoioocianeeutfonwadgacetaU
**CRI irai a confirmed outlier and not chided in th en ceteulehone; datais OK.
NenowRange N>EthylPexftuorooctaneeutfbnaaido etfaytalcohol
Drte Entered/By: 7/2908 LAC 80308 LAC 805/98 LAC 3 ^ ? e ^ E 8 0 W 8 JOT JO T / KJH 807/98 KJH
Page 17 of 23
Appendix 1
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Male Naive Rats-T6316.9, FACT070998.1
500.0
Charles River Harlan Group Taconic Farms
Week 6-8
Week 10-14
Age Group
Old Retired Breeders
Charles River Rats (Raleigh, North Caroline) were fed PM I (Purina Mills) Chow Harlan Group Rats (Indianapolis, Indiana) were fed TEKIad Chow Taconic Farms Rats (Germantown, New York) were fed TAC#31 Chow
Average PFOS levels determined from population n = 5 (per Age Group, per Source), except Charles River 6-8 Week Male Rats This data set used only 4 samples in calculation of average PFOS levels due to an outlier. Harlan Group PFOS results were all < PFOS MDL (15 ppb)
Page 18 of 23
Appendix 1
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Charles River Rats (Raleigh, North Caroline) were fed PMJ (Purina Mills) Chow Harlan Group Rats (Indianapolis, Indiana) were fed TEKIad Chow Taconic Farms Rats (Germantown, New York) were fed TAC#31 Chow Average PFOS levels determined from population n = 5 (per Age Group, per Source), except Charles River 6-8 Week Male Rats This data set used only 4 samples in calculation of average PFOS levels due to an outlier. Harlan Group PFOS results were all < PFOS MDL (15 ppb)
Page 19 of 23
Appendix 1
DT21; T-6316.9 Final Study Report Revision A 01/09/02
N aive Rat S tu d y-T 6 3 1 6 .9 , FA C T070998.1
350
300
250 Female
200 Male
150
100
CR-Week 6-8 CR-Week 10-14
Cf-Otd Retired
Breeders
H-Week 6-8
H-Week 10-14
H-Old Retired Breeders
Age Group/Source of Rats
Male TF-CXd pe m a |e Retired
Breeders
Sex
Charles River Rats (Raleigh, North Caroline) were fed PMI (Purina Mills) Chow Harlan Group Rats (Indianapolis, Indiana) were fed TEKIad Chow Taconic Farms Rats (Germantown, New York) were fed TAC#31 Chow Average PFOS levels determined from population n = 5 (per Age Group, per Source), except Charles River 6-8 Week Male Rats This data set used only 4 samples in calculation of average PFOS levels due to an outlier. Harlan Group PFOS results were all < PFOS MDL (15 ppb)
Page 20 of 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Appendix 2
SUM M ARY O F NAIVE RA T/RA T C H O W /FISH M EA L STUDY ANALYSES
Conducted by the 3M Environmental Lab (prepared 03/01/01)
Nave Rats
(Taken from summary report issued in 1998.) In order to assess "endogenous" levels o f PFOS in test animals, the livers o f ninety rats from three different suppliers were quantitatively analyzed for PFOS. Three distinct ages o f rats were represented in the group o f animals received from each supplier: 6-8 weeks old, 10-14 weeks old, and retired breeders (> 14 weeks).
Rat livers from Charles River and Taconic Farms test animals showed significant endogenous levels of PFOS. liv ers collected from oldest group o f male rats from Taconic Farms contained significantly more PFOS than any other group in the study.
The livers o f test animals from Harlan did not contain PFOS above the limit o f detection (IS ng/g).
PFOS levels in the livers o f male rats from Charles River and Taconic Farms roughly correlated with the age o f 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 o f the female rats from Charles River and Taconic Farms were determined to contain very consistent levels o f PFOS, showing no correlation with age.
Limits o f Data:
Screening for other known metabolites o f Et-FOSE-OH (e.g. PFOSA, PFOSAA) was performed. The original summary report document stated that "No other known fluorochemical contaminants or metabolites were identified in the liver samples analyzed in this study." However, based on the levels o f 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% o f PFOS levels; PFOSA concentrations would be expected to be approximately 5% o f PFOS levels. Back calculated, the expected concentrations o f the metabolites are near the 1998 LOQ. Data from the relatively high-dose 2 year feeding study may or may not provide an acceptable model o f this low-level exposure.)
Spike recovery studies were conducted for PFOS in rat livers and were acceptable. M atrix spike studies conducted for other analytes were, for the most part, acceptable.
Rat Chow
Rat chow from 4 separate vendors was investigated. Two o f the sources o f rat chow were determined to contain PFOS above the limit o f quantitation.
NIH/PMI Lab diet, fed to rats obtained from Charles River was determined to contain approx 18 ng/g PFOS. Small animal diet from Zeigler, fed to rats purchased from Taconic Farms was determined to contain approximately 12 ng/g o f PFOS. Teklad rat/rodent diet, fed to rats obtained from Harlan, did not contain PFOS above the LOQ.
Limits o f the Data:
Page 21 o f 23
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Appendix 2
Spike recoveries studies were conducted on each type o f chow tested and all results were acceptable in all cases (77-98%). The LOQ for PFOS in rat chow is approximately 2-10 ng/g.
Fish M eal
There is great variability in both the source and types o f fishmeal available commercially. This variability complicates the analysis o f fishmeal and the interpretation o f data concerning fishmeal.
Fishmeals used for animal and fish diet are typically generated from what are referred to as "industrial fish". Industrial fish, including menhaden and herring, are usually quite high in oil content and thus not fit for human consumption. Approximately 90% o f the world's fishmeal is produced from industrial fish. Fishmeal is processed in factories or directly on the fishing vessel and results in the production o f several separate fishmeal products. 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 soluble protein, is "stickwater". Subsequent to pressing, the oil is separated from the remaining components o f the stickwater by centrifugation. A fter further distillation, this fish oil may be directly incorporated into animal diet. The remaining stickwater is evaporated down to a thick syrup o f 30-50% solids and may be sold as "condensed fish solubles". The syrup may also be added back to the presscake and dried and sold as "whole meal". Presscake without the syrup is sold as "presscake meal." All o f these components may be incorporated into animal feed.
According to the University o f Florida Corporate Extension Service, good quality fishmeal averages 6070% protein; the oil content ranges from 2-15%.
Fishmeal is incorporated into food for a variety o f animals and fish including farm-raised salmon, poultry, and cows (particularly dairy cows).
In this study, six types o f fishmeal, arising from at least 3 types o f fish, were analyzed.
Menhaden travel in large schools and migrate up the coast each year. They are found in warmer, near shore waters and in the US, are fished primarily o ff Atlantic Coast. Menhaden constitute 98% o f the fishmeal produced in the US.
Capelin, also a schooling fish, spend much o f their lives off shore in the deep waters o f the Northern Pacific and the Arctic and move in-shore only to spawn.
The habitat for different classes o f herring is much more far ranging than that o f either menhaden or capelin. Different types o f herring can be found from the Northern Pacific waters off the coast o f Canada and down to the southeastern Atlantic coast. Like menhaden and capelin, herring are small, schooling fish and thus are targeted for large-scale netting.
Type of Fishm eal A nalyzed H erring Capelin Protein Concentrate (type o f fishmeal not specified) Omega Protein FAQ (type o f fishmeal not specified) Omega Protein Sealac (presumed to be menhaden) Menhaden Fishmeal
PFO S (ng/g) < LOO (approx 3 ng/g) < LOO (approx 3 ng/g) < LOQ (approx 3 ng/g)
15.7 9.87 6.10
Page 22 o f 23
Limits o f the data:
DT21; T-6316.9 Final Study Report Revision A 01/09/02
Appendix 2
M atrix spike studies were conducted on a single type o f fishmeal (menhaden). PFOS spiked into the menhaden fishmeal was recovered at approximately 66%. M atrix spike studies were not conducted for the remaining types of fishmeal tested.
The LOQ for PFOS determination in these fishmeal samples was approximately 3.5 ng/g.
Summary of the 3 components of this study;
V endor of R ats Charles River
R ats w ith endogenous PFOS?
Yes
R at Chow used by V endor C ontains PFO S?
Yes
R at Chow used by Vendor C ontains fishm eal? Yes
Taconic Farms
Yes
Yes
Yes
Harlan
No (LOQ=30 ng/g)
No (LOQ=2 ng/g)
No
Limits o f the study:
Because it is not known what type o f fishmeal was incorporated into the various rat chows, it cannot be definitively concluded that fishmeal is the source o f PFOS in the rat chow. Although indirect evidence indicates is probability, because PFOS was only detected in 50% o f the fishmeal samples analyzed, analysis o f the actual fishmeal incorporated into the chow is required to provide a direct link.
Other variables affecting the rats prior to shipment to 3M (such as PFOS levels in water and in other components o f the feed) have not been tested. To the best o f my memory, this chow has not been tested for the potential o f EtFOSE-OH transfer from packaging.
Should further work be conducted on the tissue samples, a lower level screen for metabolites and other known fluorochemicals is recommended. This information would provide additional evidence concerning the origin o f PFOS identified in rat livers.
The motivation to pursue any o f these additional investigations depends on the corporation's end use o f this data. The original study objective: "... to determine what PFOS levels, if any, are present in Sprague Dawley rats o f three different ages from three different suppliers" (M edical Dept, protocol for study # T6319.9) was completed upon issuance o f the Environmental Lab summary report in 1998.
Page 23 o f 23
