Document BXJwDBN110Rg5BovnoMVkJm8
FOR DU PONT USE ONLY AR226-2790
Approximate Lethal Concentration by Inhalation (ALC) of
Ammonium Perfluorononanoate (C-9)
Haskell Laboratory Report No. 293-85
Haskell
E. I. du Font de Nemours and Company
Laboratory for Toxicology and Industrial P. 0. Box 50, Elkton Road
Newark, Delaware 19714
Medicine
Date Issued: June 3, 1985
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HLR 293-85
Approximate Lethal Concentration by Inhalation (ALC) of Ammonium Perfluorononanoate (C-9)
Summary
To determine an ALC, groups of 6 male Crl;CD*(SD)BR rats were exposed to dust atmospheres of C-9 for a single* 4-hour period, followed by a 14-day recovery period. After determining an ALC, 2 groups of 10 male rats were exposed for 4 hours to a marginally lethal and approximately 1/lOth of a lethal concentration of C-9. For each test group, a control group of 10 male
rats was exposed simultaneously to air only. Five rats per group were killed on the 5ttr day of recoverjr.~and~5~raty per group'were killed on the' t2th day
of recovery for a gross examination of the liver.
Under the conditions of this test, the ALC for C-9 was 590 mg/m . material is considered moderately toxic by inhalation.
This
Rats exposed to 67 mg/m had significantly elevated mean liver weights and liver-to-body weight~rat1os on the 5th and 12th days after exposure. Rats exposed to 590 mg/m had significantly elevated liver-to-body weight ratios on the 12th day after exposure. " Liver weights for these rats were not significantly different than controls on the 5th day of recovery, and mean liver weights were significantly depressed on the 12th day of recovery.
However, these seemingly Inconsistent changes were due to severe body weight
loss in rats exposed to 590 mg/m .
Gross pathologic examination of rats exposed to 590 mg/m revealed discolored livers with prominent 'iobular patterns in 4/5 rats killed on the 5th day of recovery, and similar gross liver lesions in 2/5 rats killed on the 12th day of recovery.
Work by:
Study Director:
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Robert
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Technician
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Laura A. KTnney
Chemist
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Approved by:
N^CC^^A,!. "fllaanScyf <C. Chromey, Bh^O.
Section Supervisor Ac.u.4t-e ITn_v..e-sti:ga-*t.i;oAn-s-
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Haskell Laboratory Report No, 293-85
Material Tested;
Haskell No. 15,437
Nonanolc acid, 2,2,3,3,4,4,5,5,6,6,7,7,8.8,9,9hexadecafluoro-, afiBnonium salt
Sponsor:
Polymer Products Department
E. I. du Pont de Nemours and Company
Milmington, Delaware
Material Submitted by;
Test Facility:
Polymer Products Department
E. I. du Pont de Nemours and Company
Parkersburg, West Virginia
E. I. du Pont de Nemours and Company
Haskell Laboratory for Toxicology and Industrial Medicine
P. 0. Box 50, Elkton Road Newark, Delaware 19714
Study Initiated/Completed; 1/3/85 - 2/3/85
C'Notebook E-38738, pp.55-110. _^
There are 15 pages in this report.
Distribution:
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INTRODUCTION
HLR 293-85
The purpose of this study was to determine a 4-hour inhalation ALC for C-9 In male rats. The ALC was defined as the lowest atmospheric concentra tion tested that caused the death of 1 or more rats either on the day of exposure or within 14 days post exposure. In addition, following the determination of the ALC,additional exposures were conducted to monitor the
effects of C-9 Inhalation on the liver. Except as documented in the study records, this study was conducted according to the applicable Good Laboratory
Practice Regulations.
MATERIALS AND METHODS
A. Animal Husbandry
Young adult male Crl:CD(SD)BR rats were received from Charles River Breeding Laboratories, Kingston, New York. Each rat was assigned a
unique 6-digit identification number which corresponded to a numbered card affixed to the cage. Rats were quarantined for one week prior to testing, and were weighed and observed twice during the quarantine period. During the test, rats were housed in pairs in 8" x 14" x 8" suspended, stainless steel wire-mesh cages. The rat assigned the lower number in each cage was identified by a slash in the right ear. Prior to exposure, rats' tails and cage cards were color-coded with waterinsoluble markers so that individual rats could be identified after exposure. Except during exposure, Purina Certified Rodent Chow* f500Z and water were available ad libitum.
8. Exposure Protocol
To detbnnine an ALC, groups of 6 rats, 8 weeks old and weighing between 234 and 298 grams, were restrained in perforated, stainless steel cylinders with conical nose pieces. Each group was exposed nose-only for
a single, 4-hour period to a dust atmosphere of C-9 in air. Rats were
weighed prior to exposure, and were observed for clinical signs of toxicity du ing exposure. Surviving rats were weighed and observed daily for 14 days post exposure, weekends excluded except when' deemed necessary by the rats' condition.
To monitor the effects of C-9 inhalation on the liver, 2 groups of 10 rats, 8 weeks old and weighing between 237 and 277 grams, were restrained and exposed to a marginally lethal and approximately 1/lOth of a lethal exposure concentration of C-9, respectively. Two groups of 10 rats, 8
weeks old and weighing between 231 and 267 grams, were restrained and
exposed to air only. Each control group was exposed concurrently with
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HLR 293-85
one of the test groups. Five rats per group were killed 5 days after exposure, and 5 rats per group were killed 12 days after exposure for pathologic examination of the liver.
C. Test Material
Physical Form: Purity: Synonyms:
Other Codes:
Stability:
uMBRHHy-* White solid
^
^^Mnibnium pert! uorononanoate
W^W^HB^R^B^B The test material was assumed to be stable throughout
the test.
D. Atmosphere Generation
For most exposures, dust atmospheres of C-Q were generated with a K-Tron Bin Feeder equipped with twin feed screws. The feed rate was regulated with a K-Tron* Volumetric Feed Controller. The bin feeder metered test material into a glass transfer tube. Air introduced at the tube swept the test material through a size-reducing cyclone and into the exposure chamber. The cyclone removed large particles by inertia! impaction, while aerodynamic particles passed through the cyclone and into the exposure chamber. The atmospheric concentration of C-9 was controlled by varying the feed rate.
For the lowest exposure concentration, the atmosphere was generated
by passing pressurized air through a 2-stage glass generator. A round
flask at the bottom of the generator served as a dust reservoir. A cyclone elutriator was inserted above the reservior. A motorized stirring rod with plastic paddles agitated dust in the generator. Air introduced at the bottom of the reservoir and at the cyclone elutriator swept dust particles into the exposure chamber. The atmospheric concentration of C-g was controlled by varying the 2 airflows.
E. Analytical
The atmospheric concentration of C-9 was determined at approximately 30-minute intervals by drawing calibrated volumes of chamber atmosphere
through preweighed, Gelman glass fiber filters. Filters were weighed on
a Cahn model 26 Automatic Electrobalance. The atmospheric concentration
of particulate was determined from the filter weight differential before
and after sampling.
Particle size (mass median aerodynamic diameter and percent respirable) was determined with a Sierra model 210 cascade impactor
during each exposure. During most exposures, chamber temperature was
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HLR 293-85
measured with a mercury thermometer, relative humidity was measured with
a Bendix model 566 psychrometer, and chamber oxygen content was measured with a BloMarlne model 225 oxygen analyzer.
F. Pathology
Two groups of 10 rats were exposed for 4 hours to 67 or 590 mg/m of
C-9 in air, and 2 groups of 10 rats were exposed to air only. Five rats per group were arbitrarily selected and killed by chloroform anesthesia
and exsanguination 5 days after exposure. The remaining 5 rats/group were killed 12 days after exposure. Rats were necropsied and the livers were weighed. Mean liver weights and liver-to-body weight ratios for test rats were compared to their respective controls by Least Significant Difference and Dunnett's tests. Significance was judged at the 0.05 probability level.
G. Records Retention
All raw data and the final reports will be stored in the archives of Haskell Laboratory for Toxicology and Industrial Medicine, Newark,
Delaware, or in the DuPont Hall of Records, E. I. du Pont de Nemours and
Company, Mllmlngton, Delaware.
RESULTS
A. Exposure Conditions and Associated Mortality
During most exposures, the chamber walls were coated with the test material. Chamber temperatures ranged between 23-27C, relative humidities ranged from 19-45X, and chamber oxygen contents were 21%. Atmospheric characterization and mortality data are summarized in the following table.
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Character!zatlon of C-9 Atmospheres and:^ssociated Rat Mortalit7
HLR 293-85
Concentration (mg/m3)
Mean S.D.
Range
620 910 1600 4600
180 760 420 600
490 10 -
920 4100 -
980 1700 1900 5900
67
35
590- 290
12 300 -
120 1200
% a
Respirable
91 94 93 90
95 75
b
MHD(uro)
3.4 3.2 3.4 4.2
3.5 3.7
Mortality ( deaths/^ exposed)
0/6 4/6 6/6 6/6
l'/^
a Percent by weight of particles with aerodynamic diameter less than 10 urn. Mass median aerodynamic diameter.
Exposures for subsequent pathologic examination. d Although this exposure was conducted for subsequent pathologic
examination of the rats, 1/5 rats designated to be killed 12 days after
exposure died on the 12th day.
B. Clinical Observations
Curing or immediately following exposure, some rats exposed to the 5 lowest exposure concent Rations had red or brown facial discharges. At
620 mg/iii and 1600 ing/m , rats had test material on their heads. Rats exposed to 4600 mg/m had labored breathing, profuse clear nasal and oral discharges, a diminished startle response and test,material on their heads. All rats died during exposure to 4600 mg/m .
^During the postexposure period, 1/5 remaining rats exposed to 590
mg/m died 12 days post exposure, 4/6 rats exposed ^910 mg/m died 9-11
days post exposure, and 6/6 rats exposed to 1600 mg/m died 4-8 days post
exposure. Rats exposed to 67 mg/m lost 1-9!6 of initial body weight 1
day post exposure, followed by normal weight gain. At concentrations
greater than 67 mg/m , rats lost approximately 6-15% of initial body
weight 1 day post exposure, and continued to lose weight either throughout the recovery period er until they died. Most surviving rats
- exposed to 590, 620 or 910 mg/m weighed only'54-711 of initial body
weight when they were sacrificed 12 days post exposure or at the end of the recovery period, respectively.
No adverse clinical signs were observed in rats exposed to 67 mg/m throughout the recovery period. Common clinical signs at higher concentrations included hunched posture, ruffled or discolored fur, red
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HLR 293-85
or brown facial discharges, wet or stained perineum, pallor, lung noise or labored breathing, lethargy, limpness and hair loss. Clinical signs were observed throughout the recovery period.
C. Pathology
Gross pathologic examination of rats exposed to 67 mg/m revealed large livers In 5/5 rats killed on the 5th day of recovery. After 12 days,of recovery, none of the rats' livers were noted as large. At 590 ing/m , 4/5 rats killed on the 5th day of recovery had discolored livers with prominent lobular patterns or markings. These patterns were probably attributable to diffuse fatty change with a zonal distribution. On the 12th day of recovery, gross liver lesions were noted in 2/5 rats.
The Pathology report Is attached as Appendix I.
Statistical analyses showed significantly elevated mean liverg weights and liver-to-body weight ratios in rats exposed to 6Z ing/in on both the 5th and the 12th days of recovery. In the 590 mg/m exposure group, mean liver weights and liver-to-body weight ratios were not significantly different from controls on the 5th day of recovery. On the 12th day of recovery, mean liver weights were significantly lower than
controls; however, liver-to-body weight ratios were significantly elevated. The lack of significant liver weight changes on the 5th day of recovery, and the depressed mean liver weights on the 12th day of recovery were due to a large loss of body weight in rats exposed to 590 mg/m . Mean body weights for rats exposed to 590 mg/m were only 701 and 48X of controls on the 5th and 12th days of recovery, respectively. However, mean liver weights were 8336 and 72% of controls, respectively. Statistical analyses of mean liver weights and liver-to-body weight ratios are presented in Appendix II.
CONCLUSION
3
Under the conditions of this study, the ALC for C-9 was 590 ing/in. This material,is considered moderately toxic by inhalation (ALC between 200 and 800 fflg/m ). However, C-9 caused elevated liver weights in rats exposed to 67 mg/m on the 5th and 12th days after exposure, and gross liver lesions in rats exposed to 590 mg/m . '
Calculation described in Sierra Instruments, Inc., Bulletin 7-79-219IM, Instruction Manual: Series 210 Ambient Cascade Impactors and Cyclone
Preseparators.'
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HLR 293-85 Appendix I
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xa-ud rev in M a s
E. I. OU PONT DE NEMOURS S COMPANY IMCORM1MTBO HASKELL LJMORATOKY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE P.O. BOX 30, ELKTON ROAD NEWARK. DELAWARE 19711
CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT
HASKELL LABORATORY NO. 15437
NONANOIC ACID, 2,2,3,3,4,4,5,5,6,6,7,7.8,8,9,9 -HEXADECAFLUORO-, ____________________AMMONIUM SALT__________________
C-9
APPROXIMATE LETHAL CONCENTRATION (ALC) TOXICITY STUDY
___________IN MALE Ctl;CD*(SD)BR RATS__________
GROSS PATBOLOGY
POLYMER PRODDCTS DEPARTMENT
DATE ISSUED: MAY 16. 1985
10
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ALC TOXICITY STPPY IS MALE RATS WITH C'9
Introduction and Results
Male Crl;CD(SD)BR racs exposed to C-9 for 4 hours via Inhalation were necropsled and livers were weighed and examined at 5 and 12 days postexposure* Method of euthanasia was chloroform anesthesia and exsangulnatlon.
Table I Identifies the exposure groups and contains the gross observations made at necropsy.: The liver of only a few exposed animals was noted as large at necropsy, however, liver weights (weights and statistics
will not be presented in this report) Indicate that the liver of all exposed
aalBala was large The prominent Ibbular" pattern" noted^ in 4 of 5 alglr dose rats examined on day 5 post-exposure Is most likely attributable to diffuse fatty change with a zonal distribution (centrllobular or periportal). A
portion of the Increase In liver size may be due to fatty change, however, it
Is more likely that the liver functions as the primary sice of metabolism of the compound and that the Increase In size Is due to a proliferation of smooth
endoplasmic retlculum.
Acknowledgement
Joan A. Wolfe was pathology supervisor for this study.
Report by:
<2LX^u JZ^^^ \J^as^n^ Uh Theodore W. Slone, D.V.M. Diplomate A.C.V.P. Staff Pathologist
TWS/WCK/wfd
SLONE 3.13
Approved by;
William C. Kruasa, D.V.M. Manager, Pathology Division
n
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B-15437
TABLE I
Animal Number
Test
Days
Recovery Days
Group IA - Cont:rol
386616
6
5
386618
6
5
386620
6
5
386622
6
5
386624
6
5
386617
13
12
386619
13
12
386621
13
12
386623
13
12
386625
13
12
GROSS PATHOLOGY C-9
WLE RATS - INHALATION
Mode of*
Death
Observations
SD
Liver - discoloration, dark red area, (5
HUB), left lobe
SD
No abnormalities detected
SO
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
Ho abnormalities detected
SD
No abnormalities detected
Group IB - Control
386988
6
5
386990
6.
5
386992
6
5
386994
6
5
386996
6
5
386989
13
12
386991
13
12
386993
13
12
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities dececced
* SD Sacrificed by design; FD = Found dead
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154
TABLE I
Part 2
Animal Number
Test Days
Recovery Days
GROSS
:
MALE RATS
"""P""--
Mode of Death
PATHOLOGY
C-9 - INHALATION
Observations
386995
13
12
386997
13
12
SD
No abnormalities detected
SD
No abnormalities detected
Group II -
386545
"ffh Dose (0.59 Bff/L)
6
5
SD
Liver - discoloration, lobular pattern
prominent
386546
6
5
SD
Liver - discoloration, lobular pattern
prominent
386550
6
5
SO
No abnormalities detected
386552
6
5
SD
Liver - discoloration, lobular pattern
prominent
^
386554
6
5
SD
Liver - discoloration, lobular markings
prominent, tan streaks scattered
386547
13
12
SO
Liver - foci, white, scattered, 2 mm
In diameter), all lobes
386549
13
12
PD
No abnormalities detected
386551
13
12
SO
No abnormalities detected
386553
13
12
386555
13
12
SD
Liver - discoloration, small, white area,
left lobe, 2 mm In diameter)
Perineum - alopecia, moderate
'SD
Dorsum - alopecia, right, moderate
Group III - Low Dose (0.067 mg/L)
387033
6
5
SD
387035
6
5
SD
"
Liver - large Liver - large
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TABLE I
Animal
Number
Test Days
Recovery Days
GROSS PATHOLOGY
C-9 HALE RATS - INHALATION
Mode of Death
Observations
387037
6
5
387039
6
5
387041
6
5
387034
13
12
387036
13
12
387038
13
12
387040
13
12
387042
13
12
SD
Liver - large
SD
Liver - large
SD
Liver - large
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
SD
No abnormalities detected
Part 3
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APPENDIX II
HLR 293-85
C9 - MEAN LIVER WEIGHTS. AND LIVER-TO-BODY WEIGHT RATIOS RATS SACRIFICED ON THE 5TH DAY OP RECOVERY
GROUP
CONTROL,!
67 MG/M-
II CONTROL
590 MG/M-
FINAL WT.
275.6 (0.000) 276.6 (0,912)
293.4 (0.000) 204.0 (0.000)^
LIVER
11.306 (0.000) 14.501 (0.002))?
14.511 (0.000) 12.010 (0.164)
LIVER-TO-BODY WEIGHT RATIO
4.090 (0.000) 5.243 (0.000)<
4.946 (0.000) 5.803 (0.053)
Values in parentheses - P value of Student's t test comparison of treatment mean to control mean.
+ - Significantly different (p<0.05) from control group by LSD # - Significantly different (p<0.05) from control group by LSD and
Dunnett's test
C9 - MEAN LIVER WEIGHTS AND LIVER-TO-BODY WEIGHT RATIOS RATS SACRIFICED ON THE 12TH DAY OF RECOVERY
GROUP
CONTROL-1 67 MG/M"
II CONTROL
590 MG/M-'
FINAL WT.
315.4 (0.000) 325.0 (0.374)
335.4 (0.000) 160.0 (0.000))?
LIVER
14.806 (0.000) 20.371 (O.OOl)iJt
15.557 (0.000) 11.198 (0.002))?
LIVER-TO-BODY WEIGHT RATIO
4.678 (0.000) 6.268 (0.000))?
4.649 (0.000) 7.003 (0.000))?
Values in parentheses - P value of Student's t test comparison
of treatment mean to control mean. + - Significantly different (p<0.05) from control group by LSD - Significantly different (p<0.05) from control group by LSD and
Dunnett's test
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