Document 0J2eZp0wE1oyVqLoa3O1Z1kJn
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3M Strategic Toxicology Laboratory
Study Title: Acute Inhalation Limit Toxicity Study of Perfluoroctanesulfonyl Fluoride (POSF) T-7098.3
rest Number (sample):
T-7098.3 (Perfluoroctanesulfonyl Fluoride (POSF)
Laboratory Project Identification: ST64
[n-Life Initiation Date:
5/1/2001
[n-Life Completion Date: 5/14/2001
Research Client:
3M Specialty Materials Division 3M Center Building 236 Saint Paul, MN 55 133-3220
Testing Laboratory:
3M Strategic Toxicology Laboratory 3M Center Building 270-SB-181 Saint Paul, h8N 55133L3220
Study Director:
Andrew M. Seacat, Ph.D. Toxicologist Specialist 3M Medical DepartmentIToxicology 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 DepartmentIToxicology Services 3M Center Building 220-2E-02 Saint Paul, MN 55133-3220
T-7098.3Acute 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 perfluoroctanesulfonylfluoride (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 sigdicant @<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 4 %
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.
11. Test Articles:
A. Identification:
T#
Name
7098.3
POSF
Structure MW (@mole) Density (g/ml)
CgF17S02F 502
1.69
B. Purity and Stability: Responsibility of research client. The POSF sample has been lab-fiactionated and acidwater washed. Chemical characterization by 'H-NMR and "F-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:
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T-7098.3Acute POSF Inhalation Limit Test. F i l 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 h m 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/106pg) x (airflow rate) x (Vdensity) x (1000
NmL)
Given:
target concentration (ppm)
MW = g/mole)
conversion factor: (1 gram)/( 1 x 106pg)
airflow rate: (L/min)
l/density: (mug)
conversion factor: 1000 pl/mL
I
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
Treated: POSF
@Pm> 1000
(pumin) 30 .-'-
(L/min) ,
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 1 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.
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T-7098.3Acute POSF Inhalation Limit Test. Final Report
Lungs, liver, kidney, bladder and brain were collected at necropsy on day 14 fiom the limit test group. A portion of each tissue was immediately fixed in a 10% fomalin solution for subsequent histological evaluation. The remaining sections were placed directly into propylene tubes filled with liquid nitrogen for possible future evaluation.
W . Results and Discussion:
Test Atmosphere
The POSF atmosphere generated in the exposure chamber averaged 912 k 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.
I
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 378 k 12 g at the initiation of the study. The control group gained an average of 22 g (5.5%) of body
*-Weightduring the 14 day study period. Liver weights averaged 14.1 f 1.O g and the
average liver to body weight ratio was 0.035 k 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 COz 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 Grow:
The three POSF group male rats were exposed to 912 k 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 f 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 k 2.0g and the average liver to body weight ratio was 0.035 f 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 k 1.60 ppm and the average percentage of PFOS in urine was 0.05 k 0.03% (Table 3). The standard curve
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T-7098.3Acute POSF Inhalation Limit Test. Fmal bpoxt 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 standardsare in Table 4. Senun samples were collected at necropsy on day 13postdose to measure the
concentrationof PFOS (Table 5). Theaverage amount of PFOS in senim was 0.058 k 0.004 mg.The average percentage of inhaled POSF in serum at necropsy was 0.101 f 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 +hour LC50 in rats for POSF is greater than 1000 ppm.
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T-7098.3Acute POSF Inhalation Limit Test.
Final Report
M. References:
1. Kestner, T. Chemical Characterization of POSF (FM-3270) by 'H-NMR & "F-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. -MethodNumber: ETS-8-96.0. Extraction of potassium perfluorooctanesulfonate or other fluorochemical compounds from urine for analysiswing HPLCelectrospray/massspectrometry/massspectrometry. 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 PefluooctanesulfonylFluoride (POSF) T-7098.4.
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T-7098.3 Acute POSFMalation limit Test.
MRePort
VI. SignaturePap
Authors:
Study Toxicologist
Y
Andrew Sacat, Ph.D. Toxicologist Specialist
Study Director
c
._.-
Reviewed by:
John L. Butenhoff, Ph.D, CIH, DABT Toxicologist Specialist Study Director
ate Date
1
I
Date
7
i
Exposurr2
Control I Control 2 Control 3
avg SD
animal number
R01462 R01463 R01464
. Table 1: Control G r o w Biolopical Data
5/1/01Animal weight
Pre exposure Day 0 (9)
5/2/01Animal weight Post exposure Day 1 (9)
5/3/01Animal weight Post exposure
Day 2 (9)
5/7/01Animal weight Post exposure Day 6 (9)
374
372
369.1
376
368
364.5
360.1
373
392
376
386
401
378
371
372
383
12
6
13
15
5/14/01 Animal weight (9)
391 386 422
400 20
T-Test p-value Predose vs day 6 BW
(1)
0.04
511 4/01 Necropsy
Liver weights (9)
14.1 13.1 15
14.1 1
LwerlBW ratio
0.036 0.034 0.036 0.036 0.001
notes
(overnight fast)
T-7098.3 Acute POSF Inhalation Limit Test. Final Report
Table 2: POSF Limit Dose Grow Liver Webhts \
Exposure
SD
P-value
1%
`28 0.72
19
26
33
31
0.85
0.63
0.70
0.84
2
0.93 ,.
0.001 0.88C
(1) p-value for a 2-tailed Students' T-test, assumingequal 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
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T-7098.3 Acute POSF Inhalation Limit Test. Final Report
Table 3: Limit Group PFOS in Urine
Area under Concentratio the curve n of PFOS in
(AUC) urine (ppb)
Dilution factor
Collected
Amount Average
A m o y t of PFOSJn Overnight Urine Average
Samples
MeOH Solvent Blank
I R01645dlPD
R01645 d l P D avg
I SD R01646dlPD R01646 d l P D avg SD
Abundance x min 8888
1154557 949883 1062220 144726
394606 460644 427625 46696
(PPb) -94
I936 1573 1766 266
590 707 648 83
Ppm PFOS
1.94 1.57
2 0.26
0.59 0.71
1 0.08
Undiluted urine PFOS Volume of (ppm) Urine (mi)
Total PFOS (ug)
Total PFOS (mg)
% PFOS in urine (3)
2
3.88
2
3.14
3.61
10
35.1
0.04
0.06
0.62
2
1.18
2
1.42
I.3
17
22.1
0.02
0.04
0.17
R01647 dlPD
1384246
2343
2.34
2
4.68
R01647 d l P D
1238595
2085
2.08
2
4.16
avg
1311420.6 2214
2
4.42
18
79.56
0.08
0.14
SD
102991
182
0.18
0.37
Overall AVG
3.08
0.05
0.08
Overall SD
1.60
,-0.03
0.06
I
(1) Standard curve equationwhere 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 meanof duplicate determinations.
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(3) Inhaled Dose of POSF: (2.0 mllbreath)x (120 breathshin) x (Img POSFlL of air) x (240 min)/l( Body weight dav 0 (g)l(lOOOc~lKg)
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T-7098.3 Acute POSF Inhalation Limit Test. , Final Report i
Table 4: Extracted Urine Combined Standard Curve for PFOS
i '
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T-7098.3 Acute POSF Inhalation Limit Test. Final Report
I
DOSE
(PPm)
Anknal t
Predose Body weight Day 0
(9)
Inhaled Dose POSF(1)
(mgW
Table 5 POSF Limit Dose Group Serum PFOS Data
Body wight day 1 post
dose
(0)
Body wight day B.W. day 5 2 pod dose post dose
(0)
(g)
Body weight day 13 post dose (at
necmpry)
Serum volume (2)
(ml)
serum ppm (PFOS) (3)
(Wml)
Amount of PFOS'In s m m
(4)
(ma
Avallable POSF In
Inhaled ak (5)
% of lrhdod
POSF In m m at PFOS at
nocmpay
serum)
1oaJ PPm
POSF
R01645
374
1000 ppm
POSF
R01646
364
lo00 ppm
POSF
R01647
416
Avg
385
STD
28
154.01
158.24
138.46 150.24 10.42
372
355
392.6 373 19
370.7
379
360.7
368
409.8
429
380
392
26
32.S
390
383
440 411.S
40
12.02
5.1
12 37
4.8
14.21
3.8
12.87
4.57
1.18
0.7
0.061
0.059
0.054 O.OS8 0.004
57.60
57.60
57.60 S7.80 0.00
0.11
0.10
0.09 0.10 0.01
(1) InhaledDose of POSF: (2.0 mWbreath)x (120 breathslmin) x (1 mg POSF/ 1OOOmlair) x (240 min)4( Body weighj day 0 (g)/(lOoOg/Kg)] (2) Gwen: (32.3 mls serum)/(kg of body weight). Serum volume =(BW day 13 (g)/lOOOg/Kg) x 32.3 mVKg (3) Serum ppm: Analyticalwork done by Dave EhresmanPFOS in serum measured by HPLWMS (see appendix 2). (4) PFOS in serum (mg): [(Serum volume ml )(Serum PFOS ug/ml)]/lo00 ug/mg (5) POSF in inhaled air (mg): (2.0 mllbreath) x (120 breathdmin)x (1 mgl1000ml) x (240 min) = 57.6 mg POSF in inhaledair (6)% inhaled POSF in serum: [PFOS in Serum (mg)/POSF in inhaled air (mg)l'l00
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T-7098.3 Acute POSF Inhalation Limit Test. Final Report
700000
Figure 1: extracted urine combined standard cudp for PFOS
600000
500000
- 400000
h
E.
0 3 4
300000
+ Series1
-Linear (Series11
200000
100000
0
0
100
200
300
400
500
600
700
800
900
-_
ppb PFOS in urine
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T-7098.3 Acute POSF Inhalation Limit Test. Final Report
Appendix 1 Histomtholow 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 RO1462: No significant changes in lung and bladder.
Toxicokinetic RO 1468: No significant changes in lung and bladder.
Toxicokinetic RO1471: 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 RO 1472: No significant changes in lung and bladder
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Amendix 2 Serum PFOS
To:
Andrew SeacatlUS-Corporate/3MIUS@3M-Corporate
Tommie Tumer/US-Corporate/3MIUS
cc:
John L. Butenhoff/US-Corporate/3M/US@3M-Corporate
Paul Lieder/US-Corporate/3M/US@3M-Corporate
Sue TanakalUS-Corporatel3M/US@3M-Corporate
Kathy Thompson/US-Corporatel3MlUS
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:lOO prior to extraction. The extraction was carried out at pH 3.0 using a single shake out in Ethyl Acetatq. The EA was transferred to a clean polypropylenetube and blown to dryneqs using nitrogen:gas. The resultant residue was redissolved in 100 ULof 25% acetonitrile and 75% 10 mm ammonium acetate. Of this solution 20 ULwere 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 cdumn equilibration requires and additional 2.5 minutes for a run time cycle 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 (2-2Offset Voltage
Base Peak
MS/MS Ion
internal Standard (PFHS) 60 volts
299 amu
80 amu
1 ~"
60 volts
PFOS
499 amu
80 amu
I
The capillary heater was held at a constant 300 degrees C. All quantitative calculations were
based on the MSlMS 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 initialextractions. This tube (1:lo0 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 on the diluted serum).
b
._.-
SF Inhalation L i t Test.
m tube was available for animal 1R01465, unfortunately this tube was labeled
correct date and T # recorded. I diluted and extracted this tube and
under the animal number lR01465.
ed (2+ hernolysis). The initial result of
n lR014s8 and lR01471. This sample was No significant difference was noted on re-
Results: dje
Rat ##
1R01462 1R01463 lR01464
1R01465 1R01466 1R01467 1R01468 1ROi471 1R01472
Control
Yes Ye= Yes
no no no no
no.
no
PFOS Level
< 5.0 4.0 6.0
ppb (on diluted serum)
N
*I
5.1 PPrn \ 4.8 pprn 3.8 pprn
8.6 ppm
9.3 ppm
5.0 pprn (5.1 pprn on duplicate)
David J. Ehresrnan 3M Corporate Toxicology Bldg. 236-18-22 Phone 651-733-5070 FAX 651-737-4754 djehresrnan@mrnrn.com
.._~ _
16
T-70983ACU~PEOSFL a h r l R t i ~L~im~it Test.
Repoa
11.O
-- . .
Y = 0 . 0 8 5 5 5 * 0 . 0 0 5 2 9 5 3 c B . 2 7 ~ 0 ~P2 2 = 0.8835
W. 1K
, ' , , , , , , , , 0.0: 0
I , , I I II I II II
I I I I I1.
50
100
150
200
250
ndmL
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