Document a1YyyJ8MRjVkOV27Dz5GOK6B
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D O&06- OO
June 19, 2006
06 JUN20 AM 6*09
DuPont Haskell Laboratory
M
for Health and'Environmental Sciences
Elkton Road, P.O. Box 50
Newark, DE 19714-0050
aa6 . 3662
Via Federal Express
Document Processing Center (Mail Code 7407M) CONTAIN NO CBI
Room 6428
Attention: 8(e) Coordinator
Office o f Pollution Prevention and Toxics
U.S. Environmental Protection Agency
1201 Constitution Ave., NW Washington, DC 20460
89060000355
S ' V if "6 0 0 0 0 3 5
Dear 8(e) Coordinator:
8EHO-Q381-0394 Ammonium Perfluorooctanoate (APFO Linear)
This letter is to inform you of the results of a recently conducted 28-day immunotoxicity study in male rats and mice with the above referenced substance.
The immunotoxicity study in male rats and mice was conducted to evaluate the potential of Ammonium Perfluorooctanoate (APFO-linear) to suppress the primary humoral immune response to sheep red blood cells (SRBC) when administered by oral gavage for at least 28 days. The study was conducted according to EPA OPPTS 870.7800 Immunotoxicity, Health Effects Test Guidelines (1998). Additional hematological, clinical chemistry, organ cell counts, and histopathological endpoints were included as well. The oral route of administration was selected because it is a potential route of human exposure.
Six groups of 10 male Crl:CD (SD)IGS rats or 20 male CrhCDl(ICR) mice were dosed by intragastric intubation at a dose volume of 10 mL/kg body weight for 28 consecutive days with 0 (control), 0.3, 1, 10, 30, or 30 (satellite recovery group) mg/kg APFO. NANOpure water was used as the diluting vehicle. The recovery group was dosed with 30 mL/kg APFO for 22 (rat) or 23 (mice) consecutive days. Following injection of SRBC on test day 23 (rat) or 24 (mice), these groups were dosed with NANOpure water at a dose volume of 10 mL/kg body weight until sacrifice.
The overall conclusions are 1) the rat study was negative for immunosuppression and 2) in the mouse, a number of immune-related findings, that only occurred at 10 and 30 mg/kg, likely represent secondary responses to the systemic toxicity and stress observed at these two doses.
Rat
> No suppression of the ability to make anti-sheep RBC antibody was observed at any dose
> 10 and 30 mg/kg resulted in systemic toxicity as evidenced by the following:
Decreases in body weight gain of 74 and 37%, respectively.
Serum corticosterone levels increased to 135 and 196% of control in the 10 and 30 mg/kg treatment groups, respectively. [This effect was reversed in the 30 mg/kg (recovery) group.]
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Mouse > The top two doses administered in this study, 10 and 30 mg/kg, resulted in marked general toxicity and stress, as evidenced by the following: a loss in body weight of 3.8 and 6.6g, respectively. -230% increase in serum corticosterone and increases in absolute numbers of blood neutrophils and monocytes with an accompanying decrease in absolute lymphocyte numbers.
> A number of immune-related findings occurred at 10 and 30 mg/kg that likely represent secondary responses to the systemic toxicity and stress observed at these doses: Statistically significant suppression of the ability to make anti-sheep RBC antibody was observed at 10 mg/kg (20% suppression) and 30 mg/kg (28% suppression). Serum corticosterone levels increased to about 230% of control in the 10 and 30 mg/kg groups. Spleen and thymus weights (relative to body weight) declined to 55-65% of control values after exposure to 10 and 30 mg/kg. Spleen cell numbers declined to 53 and 37% of control and thymocytes declined to 44 and 18% of control in the 10 and 30 mg/kg groups, respectively. Microscopic depletion/atrophy of lymphoid tissue was considered to be treatment related starting at 10 mg/kg in the thymus and 30 mg/kg in the spleen.
> In addition, liver weight relative to body weight in mice increased to 350 and 373% of control, respectively.
Summary tables for rats and mice detailing these and other findings are attached. Under these experimental conditions, the findings described above are being reported in accordance with the guidance given in the EPA TSCA Section 8(e) Reporting Guide (June 1991). A copy of the final report(s) will be sent to the Agency when available.
Sincerely,
A A. Michael Kaplan, Ph.D. Director - Regulatory Affairs and Occupational Health AMK/DMH: clp (302) 366-5260
JZ
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Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats
Male rats
III V VII IX XI
Dose (mg/kg):
0.3 1 10 30 30 (Recovery)
Results:
Mortality:
Comment: All rats survived to scheduled sacrifice.
Clinical Signs:
M: 0/10
M: 0/10
M: 0/10
M: 0/10
M: 1/10
Comment: One rat (1109) in 30 mg/kg recovery group was not dosed on d6-8 due to decreased body
weight observed on day 6. (64% decrease from day 0). Clinical Signs for rat 1109 on
days 6-8 included: lethargic, high carriage, feces absent, stained fur/skin (abdomen,
forepaw, inguen, perineum, ventral body, perinasal and perioral), wet fur ventral and not
eating.
Final Body Weights (day 28):
90% 75% 79%
Body Weight Gain:
74% 37% 50%
Daily Food Consumption:
83% 84%
Hematology RBC HGB HCT MCV MCH MCHC RDW ARET PLT WBC ANEU ALYM AMON AEOS
95%
91% 92%
97%
95%
111%
109%#
130% 114%
133%
132%
98%
91% 92% 95% 94%
115%
112%#
137%
123%
140%
140%
88% 86% 87%
99% 97%
123% 1977c#
111% 100% 114% 116%
ABAS
ALUC
133%
147%
127%
Comments: #- Microscopically, $onoeofthe rate)n.these3 groups had:increased nisocytosis (variation-hired.;'
cellsize; al^bb^ry
I m g/^dl^ rnerocytds!s'(iricreased #sofjpceIs);
polycluomasa(m crca^ftth .taming ufiMMOd &IM). and hypoduonuiu i paleisamng ot
redbk^mls)b=Thse'c^tes:'w^^nsTstCT(!w|f'inlntally ncres^^ticuloctiiibme -
aninSst'=;-! `
, - -, - -
Serum Chemistry CHOL TRIG TP ALB GLOB HDL LDL SCORT
Comment:
64% 69% 69% 75%
106%
75% 79% 58% 63%
FlemoK/ed serum al M). 30. and 30R
81%
68%
112% 89% 75%
85% 135%
84%
66%
115% 89%
79% 88% 196%
69%
107%
112%
96%
2
J
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Male rats Dose (mg/kg):
Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Rats
III V VII IX 0.3 1 10 30
XI 30 (Recovery)
Gross Pathology:
Liver, Discoloration, tan
12
1
Comment: Underlined values were interpreted to be test-substance related increases, as compared to
control values.
Absolute Organ Weight:
Liver Spleen
131%
163%
142%
123%*
Thymus Brain
86% 113%*
Relative Organ Weight (% of body weight) Liver Spleen Thymus Brain
182% 112%
191%
115% 134%
156%*
144%* 121%*
Histopathology
Liver, Hypertrophy, hepatocellular
5(1.0)
10(1.7)
10 (3.0)
10 (3.0)
10 (3.0)
Liver, Necrosis, focal
4(1.0)
Spleen, EMH, increased
7(1.3)
Comment: ( ) = Number in parentheses is the average grade (grades 1 - 4) when lesion is present
(i.e., sum of grades -f- # animals with lesion). Grading scale: 1 = minimal; 2 = mild; 3 = moderate; 4 = severe.
EMH = Extramedullary hematopoiesis.
Underlined values were interpreted to be test-substance related increases, as compared to
control values.
Spleen Cell Count:
Thymus Cell Count:
IgM:
103% I
98%
* Group XV TI mean is sig__nificantly different from group IX.
99% '
95% 98%
142%* 95%
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Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice
Male mice Dose (mg/kg):_________
Mortality: Comment:
Clinical Signs: Comment:
Final Body Weights (Day 28):
III V VII
0.3 -----------1---------- , 10 '1 Vs-'
IX
30 -
XI 30 (Recovery)
No treatment related deaths occurred.
M: 0/20
M: 0/20
M: 0/20
M: 2/20
M: 4/20
2 mice were not dosed on study days 8-10 due to decrease body weight observed on
day 8. (66% and 72% decrease from day 0)
4 mice were not dosed on study days 9-11 due to decrease body weight observed on
day 9. (70-75% decrease from day 0)
Lethargy: 1 mouse in 0, 30, 30R groups; Abnormal gait: 1 mouse in 0, 1, 10 groups
Prostrate: 1 mouse in 30R
86% 78% 88%*
Body Weight Gain: (Day 29)
Comment: Daily Food Consumption:
l-5g 1.5g Control group gained 0.9grams (g)
-3.8g -6.6g
108%
98%
-3.3g
100%
Hematology
RBC
HGB
HCT
MCH
MCHC
RDW
ARET
PLT
WBC
ANEU ALYM AMON
236% 285%
AEOS ABAS
57%
ALUC
Serum Chemistry CHOL TRIG TP ALB GLOB HDL LDL SCORT
69% 47% 125% 145%
71% 61%
85%
230%
Comment: Icteric serum at 10, 30, and 30R
Gross Pathology:
Liver, Large
17
Liver, Discoloration
1
Spleen, Small
8
Thymus, Small
3
Comment: Control group exhibited Liver, Large incidence of 1
94% 88% 91%
296% 59%
254% 64%
51% 32%
109%
131% 44%
65% 232%
16 5 8 2
86% 82% 85%
109% 148%
256%
74% 177% 64%
80% 57%
134% 148% 119% 69%
103% 154%
17 1
12 2
r
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Ammonium Perfluorooctanoate: 28-Day Immunotoxicity Study in Male Mice
Male mice Dose (mg/kg): Absolute Organ Weight: Liver Spleen Thymus Brain
III 0.3
V 1
162%
VII 10
301% 56% 50% 95%
IX XI 30 30 (Recovery)
293% 317% 44% 65% 50% 54% 93% 94%
Relative Organ Weight (% of body weight) Liver Spleen Thymus Brain Histopathology
160% 86%
350% 65% 57% 110%
373% 350% 55% 70% 61% 58% 119% 103%*
Liver, Hypertrophy, hepatocellular
20 (2.0)
20 (3.0)
20 (4.0)
19 (4.0)
19 (4.0)
Liver, Necrosis, individual cell
11(1-1)
20(1.9)
19 (2.0)
19(1.7)
Liver, Necrosis, focal Liver, Mitotic figures, increased
2(1.0)
4(1.8) 10(1.0)
7(1.7) 15 (1.0)
3(1.7) 19(1.4)
Liver, Hyperplasia, bile duct
6(1.0)
12(1.2)
12(1.0)
Liver, Fatty change, nonzonal
9(1.0)
14(1-0)
4(1.0)
Thymus, Depletion/Atrophy,
6(1.2)
2 (2.9)
4(2.8)
lymphoid
Spleen, Depletion/Atrophy,
8(1.1)
2(1-1)
lymphoid
Spleen, EMH, increased Bone Marrow, Hyperplasia, granulocytic
3(1.7)
4(1.0)
15(2.1) 2(1.7)
Bone Marrow, Hyperplasia, erythrocytic
3(2.0)
Comment: ( ) = Number in parentheses is the average grade (grades 1 - 4) when lesion is present
(i.e., sum of grades H- # animals with lesion). Grading scale: 1 = minimal; 2 = mild; 3 =
moderate; 4 = severe.
EMH = Extramedullary hematopoiesis.
Spleen Cell Count:
Underlined values were interpreted to be test-substance related increases, as compared to
control values.
53% 37% 56%
Thymus Cell Count:
44% 18% 49%
IgM: 98% 92% 80% 72% 70%
* Group XI mean is significantly different from group IX.
Q
