Document O3n5RM9zqLQZpDJOOgoJzy651
3M Strategie Toxicology Laboratory
Study Title: Acute Inhalation Limit Toxicity Study of Perfluoroctanesulfonyl Fluoride (POSF) T-7098.3
Test Number (sample):
T-7098.3 (Perfluoroctanesulfonyl
Laboratory Project Identification: ST64
In-Life Initiation Date:
5/1/2001
In-Life Completion Date: 5/14/2001
Fluoride(POSF)
Research Client:
3M Specialty Materials Division 3M Center Building 236 Saint Paul, MN 55133-3220
Testing Laboratory: 3M Strategic Toxicology Laboratory 3M Center Building 270-SB-181 1 Saint Paul, MN 55133-3220
Study Director:
Andrew M. Seacat, Ph.D. Toxicologist Specialist 3M Medical Department/Toxicology Services . 3M Center Building 220-2E-02 Saint Paul, MN 55133-3220
Study Toxicologist:
Tommie Yvette Turner, Ph.D. Post Doctoral Research Fellow 3M Medical Department/Toxicology Services 3M Center Building 220-2E-02 Saint Paul, MN 55133-3220
T-7098.3 Acute POSF Inhalation Limit Test. Final Report
Summary:
An inhalation toxicity screen was conducted using 3 male rats for control (aironly) and 3 male rats exposed to perfluoroctanesulfonyl fluoride (POSF) at a nominal concentration of 1000 ppm for 4 hours. Overnight urines were collected on day zero to analyze for perfluorooctane sulfonate (PFOS) in the urine. Animals were observed for any adverse effects for up to 14 days after cessation of exposure. At necropsy, serum was collected for PFOS determination.
The control animals significantly gained weight (p<0.05) during the recovery period and showed no clinical pathology.
The POSF treated animals tolerated the 4 hour inhalation exposure, had significant (p<0.05) body weight gain over the 14 day recovery period. Organ weights were normal and histological evaluation of the liver, lungs, bladder, kidney and brain revealed no significant findings. The amount of PFOS in urine collected overnight on the day of dosing, and the amount of PFOS in serum on day 14 post-dose were 3.08 +-1.60 ppm (N=3) and 4.57 +- 0.70 ppm (N=3), respectively. These data indicate that POSF was absorbed from the lungs, metabolized to PFOS and found present in the serum then excreted in urine as PFOS. The average percentage of inhaled POSF in the serum or urine, as PFOS, was <1%
Conclusion: The 4-hour LC50 of POSF is greater than 1000 ppm..
I. Study Objective:
This study was done to establish data on acute inhalation toxicity and to
approximate the 4-hour LC50 value of POSF. The results of this range finder study will
be used as guidance for a 13-week inhalation study.
.
II. Test Articles:
A. Identification:
T# Name
7098.3
POSF
Structure MW (g/mole) Density (g/ml)
CgFi7S02F 502
1.69
B. Purity and Stability: Responsibility of research client. The POSF sample has been lab-fractionated and acid/water washed. Chemical characterization by 'H-NMR and 19F-NMR Spectroscopy was conducted by 3M Specialty Adhesives & Chemicals Analytical Laboratory/ SMD to determine the purity of the nominal product and to characterize as many impurity components as possible. The sample was found to be a high purity form of the nominal POSF product (1).
C. Handling and Storage: Upon receipt, the test article was stored tightly sealed at room temperature.
D. Disposition of Test Material: Any remaining unused portion of the test article was returned to the research client after completion of the study.
III. Test System:
2
T-7098.3 Acute POSF Inhalation Limit Test. Final Report
Male rats were exposed to test compound in air for 4 hours for one day in 13-liter glass exposure chambers (3 rats/chamber/compound). The flow-through atmosphere was generated by mixing test compound from a syringe pump into a stream of fresh air flowing at 3.0 L/min into the chamber and exhausted through a vent. The syringe flow rate was calculated based on using the Ideal Gas Law equation: ((target concentration)(MW)/24.79) x (lg/106 pg) x (airflow rate) x (1/density) x (1000 pl/mL)
Given: target concentration (ppm) MW = g/mole) conversion factor: (1 gram)/(l x 106pg) airflow rate: (L/min) 1/density: (mL/g) conversion factor: 1000 pl/mL
l-
The target concentrations of each test compound, the syringe flow rate and the
airflow are provided in the following table:
Dose Group
C
Syringe Flow Rate Airflow Rate
(ppm)
(pl/min)
(L/min) ,
Treated: POSF
1000
30
2.0-3.0
Control: Air only
2.0 - 3.0
Standard target concentrations for each compound were prepared and analyzed by Fourier Transform Infrared Spectrometry (FTIR). Chamber air was monitored by FTIR for concentrations of test compound and oxygen (2). The day of dosing was designated day zero of the study. On day zero, an atmosphere of appropriate concentration was generated by adding a calculated amount of the test material to a test chamber of known volume containing the animals. Airflow was increased when necessary to maintain a level of 1000 ppm. Control animals were exposed to room air under equivalent chamber conditions as the POSF dose group animals.
Animals were handled in accordance with an animal usage protocol approved by the 3M LARC. Body weights were determined immediately prior to dosing and immediately prior to euthanasia. At the termination of the study, all animals were humanely euthanized by CO2 asphyxiation and necropsies were performed.
Serum was collected at necropsy for PFOS analysis. Analysis of PFOS levels by highpressure liquid chromatography/tandem electrospray mass spectroscopy (HPLC-MS/MS) was performed according to published methods (3). Urine was collected overnight on day zero from rats of the limit test group. Urine PFOS levels were determined following a validated extraction procedure (4), with a slight modification in that no.surrogate standard (THPFOS) was added.
3
T-7098.3 Acute POSF Inhalation Limit Test. Find Report
Lungs, liver, kidney, bladder and brain were collected at necropsy on day 14 from the limit test group. A portion of each tissue was immediately fixed in a 10% formalin solution for subsequent histological evaluation. The remaining sections were placed directly into propylene tubes filled with liquid nitrogen for possible future evaluation.
IV. Results and Discussion:
Test Atmosphere
The POSF atmosphere generated in the exposure chamber averaged 912 91 ppm
for the limit test group (5). The measured concentration in the chamber was 91% of the
target concentration for the limit test group. Factors which may account for this include
the fact that the calculated concentration based on the Ideal Gas Law and the actual
concentration in the flow-through chambers when the system reaches equilibrium may
not be the same. The airflow was increased to 3 liters/ minutes to adjust chamber
concentration.
A
Control Group: The three control group male rats had no adverse clinical observations noted
during the 4 hr air-only exposure. The average body weight was 3 7 8 1 2 g a t t h e initiation of the study. The control group gained an average of 22 g (5.5%) of body
"Weight during the 14 day study period. Liver weights averaged 14.1 1.0 g and the
average liver to body weight ratio was 0.035 0.001 (Tablel). Necropsy findings and the histopathology report by Dr. Elden Lamprecht, 3M
Surgical and Histopathology Services, is found in Appendix 1. All rat lungs (both control and treated groups) had mild to moderate congestion of alveoli with occasional extravasation of blood around arterioles and bronchioles. This was possibly an agonal change associated with CO2 euthanasia. It was noted that some of the lungs were not well insufflated. Other than these artifacts, histological analysis revealed no significant changes in lungs and urinary bladder in all three of the controls.
POSF Limit Group:
The three POSF group male rats were exposed to 912 91 ppm POSF measured continuously for four hours. There were no biologically significant effects over a 14-day recovery period after exposure for the POSF treated group. As a gross observation, the POSF treated animals had an increase of urine and feces output on day one post-exposure as compared to control. The average body weight was 404 31 g (N =3) fourteen days post exposure. The limit group gained an average of 19g (4.9%) of body weight during the 14-day study period. Liver weights averaged 14.2 2.0g and the average liver to body weight ratio was 0.035 0.002 (Table 2), similar to controls.
Day zero, overnight urine samples were collected and extracted, to measure the levels of PFOS. Each urine sample was prepared in duplicate. Independent dilutions of the urine were done with each value calculated from the average of the duplicate determination. The average amount of PFOS in urine was 3.08 1.60 ppm and the
average percentage of PFOS in urine was 0.05 0.03% (Table 3). The standard curve
4
T-7098.3 Acute POSF Inhalation Limit T est Final Report
used to calculate the levels of PFOS in extracted urine samples is shown in Figure 1, and the area under the curve (AUC) for these extracted urine standards are in Table 4.
Serum samples were collected at necropsy on day 13 post-dose to measure the concentration of PFOS (Table 5). The average amount of PFOS in serum was 0.058 0.004 mg. The average percentage of inhaled POSF in serum at necropsy was 0.101 0.007% (Table 5).
These findings are similar to the PFOS levels observed in an accompanying toxicokinetic study of POSF (T-7098.4) in which a small percentage of PFOS was also found in the serum and urine (4). V. Conclusion:
The apparent inhalation 4-hour LC50 in rats for POSF is greater than 1000 ppm.
5
T-7098.3 Acute POSF Inhalation Limit Test Final Report
VI. References:
1. Kestner, T. Chemical Characterization of POSF (FM-3270) by 'H-NMR & 19F-NMR Spectroscopy. 3M Specialty Materials & Manufacturing Division Analytical Laboratory. Request No. 6370. May 15, 2001.
2. Modified EPA Method 320. Pace Analytical. (2001).
3. Hansen, K.J., Clemen, L. A., Ellefson, M. E., Johnson, H.O. (2001). CompoundSpecific, Quantitative Characterization of Organic Fluorochemicals in Biological Matrices. Environ. Sci. and Tech..35, 766-770.
4. Method Number: ETS-8-96.0. Extraction of potassium perfluorooctanesulfonate or other fluorochemical compounds from urine for analysisiusing HPLCelectrospray/mass spectrometry/mass spectrometry. 3M Environmental Laboratory.
5. Gutzkow, T. (2001) POSF Animal Exposure Test Report. Pace Analytical Services,
Inc. 3M Laboratory Request No. E01-0648. 3M ET&SS Field Analytical Services
and Technologies.
-
6. Seacat, A. and Turner, T. (2001) Acute Inhalation Toxicokinetic Study of Pefluooctanesulfonyl Fluoride (POSF) T-7098.4.
6
T-7098.3 Acute POSF Inhalation Limit T est Final Report
VI. Signature Page Authors:
Study Director
Reviewed by:
John L. Butenhoff, Ph.D, CIH, DABT
Toxicologist Specialist
Study Director
.
.
Date
7
Table 1: Control Group Biological Data
Exposur
Control 1 Control 2 Control 3
av9 SD
animal number
R01462 R01463 R01464
5/1/01 Animal weight
Pre exposure Day 0 (g)
5/2/01 Animal weight Post
exposure Day 1 (g)
5/3/01 Animal weight Post
exposure Day 2 (g)
5/7/01 Animal weight Post
exposure Day 6 (g)
5/14/01 Animal weight (g)
374 372 369.1
368
364.5
360.1
392 376 386
376 373 401
378 371 372 383 12 6 13 15
391 386 422
400 20
T-Test p-value Predose vs day 6 BW
(1)
5/14/01 Necropsy
Liver weights
(9)
0.04
14.1 13.1 15
14.1 1
Liver/BW ratio
0.036 0.034 0.036 0.035 0.001
(1) p-value for a 2-tailed Student's T-test, assuming equal variance. Predose vs. day 6 body weight. Statistically significant if P<0.05.
notes
(overnight fast)
T-7098.3 Acute POSF Inhalation Limit Test. Final Report
Table 2: POSF Limit Dose Group Liver Weights
Exposure animal
POSF
number
(ppm)
1000
R01465
1000
R01466
1000
R01467
avg
SD
P-value H I_______
5/1/01 Animal weight Pre exposure Day 0 (g)
5/2/01 Animal weight Post exposure Day
1(9)
5/3/01 Animal weight Post exposure Day 2 (g)
5/7/01 Animal weight Post exposure Day 6 (g)
5/14/01 T-Test Liver weights Animal p-value (g) weight (g) Prexpo 5/14/01
vs day Necropsy
6 BW
Liver/BW ratio
(1)
374
372 370.7
379 390 0.01
12.8 0.033
364
355 360.7
368 383
13.6 0.036
416
392.6
409.8
429 440
16.1 0.037
385 373 380 392 404
14.2
0.035
28 19 26 33 31
2 0.002
0.72
0.85
0.63
0.70
0.84
0.93 .
0.880
(1) p-value for a 2-tailed Students' T-test, assuming equal variance. Predose vs. body weight on day 6. Statistically significant if p<0.05 (2) p-value comparing control vs. treated group body weights. Statistically significant if p<0.05
9
T-7098.3 Acute POSF Inhalation Limit Test. Final Report
Table 3: Lim it Group PFOS In Urine
Area under Concentrata
the curve n of PFOS in
(AUC)
urine (ppb)
Samples MeOH Solvent Blank
Abundance x min
8888
R01645 d1PD R01645 d1PD
avg SD
1154557 949883 1052220 144726
R01646 d1PD R01646 d1PD
avg SD
394606 460644 427625 46696
R 01647 d1PD R01647 d1PD
avg SD
Overall AVG Overall SD
1384246 1238595 1311420.5 102991
(PPb) -94
1936 1573 1755 256
590 707 648 83
2343 2085 2214 182
ppm PFOS
1.94 1.57
2 0.26
0.59 0.71
1 0.08
2.34 2.08
2 0.18
Dilution factor
Collected
Amount Average
Amount of PFOSin
Overnight Urine Average
Undiluted urine PFOS Volume of (ppm) Urine (ml)
Total PFOS
(ug)
Total PFOS (mg)
% PFOS in urine (3)
2 3.88 2 3.14
3.51 10 35.1 0.52
0.04
2 1.18 2 1.42
1.3 17 22.1 0.17
0.02
2 4.68
2 4.16
4.42
18 79.56
0.08
0.37
3.08
0.05
1.60
^-0.03
0.06
0.04
0.14 0.08 0.05
(1) Standard curve equation where Y= area under the curve (AUC) in units of relative abundance multiplied by time (minutes), and X = ppb of PFOS in urine. X = (Y-61687)/564.53
(2) Mean + SD of PFOS in urine are shown for N=3, where each of the 3 values are the mean-of duplicate determinations.
(3) Inhaled Dose of POSF: (2.0 ml/breath) x (120 breaths/min) x (1 mg PO SF/ L of air) x (240 min)/f( Body weight day 0 (g)/(1000q/Kg)______
10
T-7098.3 Acute POSF Inhalation Limit Test. Final Report
Table 4: Extracted Urine Combined Standard Curve for PFOS
Sample MeOH Solvent Blank
Milli Q water Control urine Matrix blank
MS 4 ppb MS 8 ppb MS 20 ppb MS 40 ppb MS 80 ppb MS 202 ppb MS 404 ppb MS 808 ppb Control urine Matrix blank MS 4 ppb .-MS 8 ppb MS 20 ppb MS 40 ppb MS 80 ppb MS 202 ppb MS 404 ppb MS 808 ppb
POSF AUC (Ml) 11952 36312 35330 f 32394 V53129 56366 77325 119932 147773 220143 442924 45441 71331 54846 70255 63918 197266 304118 293452 592511
11
T-7098.3 Acute POSF Inhalation Limit Test. Final Report
Dosa (ppm)
Predose Body Animal # weight Day 0
(9)
Inhaled Dose POSF(1 )
(mg/kg)
Table S: POSF Limit Dose Group Serum PFOS Data
Body weight
Body weight day
day 1 post Body weight day B.W. day 5 13 post dose (at
dose
2 post dose post dose necropsy)
(9) (9) (9) (9)
Serum volume (2)
(ml) ,
serum ppm (PFOS) (3)
(ug/ml)
;
Amount of
Available
PFOS In serum POSF in
(4) Inhaled air (5)
(mg)
(mg)
% of Inhaled POSF In serum
as PFOS at necropsy
serum)
1000 ppm POSF 1000 ppm POSF 1000 ppm POSF
Avg
STD
R01645 R01646 R01647
374
364
416 385 28
154.01
158.24
138.46 150.24 10.42
372
355
392.6 373 19
370.7
360.7
409.8 380 26
379
368
429 392 32.5
390
383
440 411.5
40
12.02
12.37
14.21 12.87 1.18
5.1
4.8
3.8 4.57 0.7
0.061
0.059
0.054 0.058 0.004
57.60
57.60
57.60 57.60 0.00
0.11
0.10
0.09 0.10 0.01
(1) Inhaled Dose of POSF: (2.0 ml/breath) x (120 breaths/min) x (1 mg POSF/ 1000ml air) x (240 min)/[( Body weight day 0 (g)/(1000g/Kg)]
(2) Given: (32.3 mis serum)/(kg of body weight). Serum volume =(BW day 13 (g)/1000g/Kg) x 32.3 ml/Kg
(3) Serum ppm: Analytical work done by Dave Ehresman PFOS in serum measured by HPLC/MS (see appendix 2).
(4) PFOS in serum (mg): [(Serum volume ml )(Serum PFOS ug/ml)] /1000 ug/mg
(5) POSF in inhaled air (mg): (2.0 ml/breath) x (120 breaths/min) x (1 mg/ 1000ml) x (240 min) = 57.6 mg POSF in inhaled air
(6) % inhaled POSF in serum: [PFOS in serum (mg)/POSF in inhaled air (mg)]*100
-
12
T-7098.3 Acute POSF Inhalation Limit Test. Final Report
AUC (Ml)
700000
Figure 1: extracted urine combined standard curV^e for PFOS
600000
500000
400000 300000
Series 1 -- Linear (Series!)
200000
100000
0 0 100 200 300 400 500 600 700 800 900
ppb PFOS in urine
13
T-7098.3 Acute POSF Inhalation Limit Test. Final Report
Appendix 1
Histopathologv Results
Necropsy 5/3/01
All rats lungs have generalized mild to moderate congestion of alveoli with occasional extravasation of blood around arterioles and bronchioles. This is possibly an agonal change associated with C02 euthanasia. Some lungs were not well insufflated.
Animal # Control R01462: No significant changes in lung and bladder.
Toxicokinetic R01468: No significant changes in lung and bladder.
'
Toxicokinetic R01471:
...
One macroscopic focus of atelectasis (collapsed alveoli) is present in one lung lobe. The change was chronic in duration. No
significant change is present is bladder.
Toxicokinetic R01472: No significant changes in lung and bladder
; ^
14
Appendix 2 Serum PFOS
To: Andrew Seacat/US-Corporate/3M/US@3M-Corporate
Tommie Tumer/US-Corporate/3M/US cc: John L. Butenhoff/US-Corporate/3M/US@3M-Corporate Paul Lieder/US-Corporate/3M/US@3M-Corporate Sue Tanaka/US-Corporate/3M/US@3M-Corporate Kathy Thompson/US-Corporate/3M/US Deanna J. Luebker/US-Corporate/3M/US@3M-Corporate Subject: PFOS Levels from rats exposed to POSF via inhalation T-7098.3
Andrew and Tommie:
The 1 to 100 estimate for the serum levels of PFOS was right on target. The following results are based on the rat serum being diluted 1:100 prior to extraction. The extraction was carried out at pH 3.0 using a single shake out in Ethyl Acetate. The EA was transferred to a clean polypropylene tube and blown to dryness using nitrogen gas. The resultant residue was re-dissolved in 100 uL of 25% acetonitrile and 75% 10 mm ammonium acetate. Of this solution 20 uL were injected onto a Betasil C18 reversed phase HPLC column (50 x 2 mm, 5 micron particle size). The flow rate was maintained at 0.5 ml/min. A gradient flow of acetonitrile was used to elute the compounds of interest starting with 15% acetonitrile and ending with 65% acetonitrile over 5.5 minutes. Return to initial operational parameters and column equilibration requires and additional 2.5 minutes for a run time cyble of 8.0 minutes per run.
The MS was operated in the Electro Spray negative ionization mode with a constant 3.0 kV potential applied to the source. The method used was an MS/MS method involving the transition of the following ions:
Compound Q-2 Offset Voltage
Base Peak
MS/MS Ion
Internal Standard (PFHS) 60 volts
299 amu
80 amu
60 volts
PFOS
499 amu
80 amu
The capillary heater was held at a constant 300 degrees C. All quantitative calculations were based on the MS/MS ion rations between the compound of interest (PFOS) and the internal standard (PFHS). The standard curve ranged from 5 ng/mL (ppb) to 250 ng/mL (ppb). A quadratic curve fit was used with the standard value weighted 1/X . This curve fit produced an R squared value of 0.9935.
The first tube of two available for control animal number 1R01464 was diluted and run with the initial extractions. This tube (1:100 dilution) had a PFOS level of 2.3 ppm. Repeat of this tube dilution produced comparable results (2.1 ppm). The second tube labeled 1R01464 was diluted 1:100 and extracted. The second tube on this control animal had a result of less than the lowest standard (5.0 ppb oh the diluted serum).
T-70983 Acute POSF Inhalation Limit Test. Final Report "
Only one serum tube was available for animal 1R01465, unfortunately this tube was labeled 1R0465 with the correct date and T # recorded. I diluted and extracted this tube and recorded results under the animal number 1R01465.
The sample for 1R01468 was noted as hemolyzed (2+ hemolysis). The initial result of ~r ~ 1 R 014 7Z ip eared to be significantly tower 1han 1R01468 and 1R01471. This sample was
also re::extracted and the analysis repeated. No significant difference was noted on re extraction.
Results:
R a t#
Control
PFOS Level
1R01462 1R01463 1R01464
yes yes yes
<5.0 <5.0 <5.0
ppb (on diluted serum)
N
II
1R01465 1R01466 1R01467 1R01468 1R01471 1R01472
no no no no no. no
5.1 ppm 4.8 ppm \ 3.8 ppm 8.6 ppm 9.3 ppm 5.0 ppm (5.1 ppm on duplicate)
dje
David J. Ehresman 3M Corporate Toxicology Bldg. 236-1B-22 Phone 651-733-5070 FAX 651-737-4754 djehresman@mmm.com
S * -' v i
T-7098.3 Acute POSF Inhalation Limit T est Final Report
17
