Document VjEb9m92aEa8zogq0Ba3EJm4Z
QW6
. i-~``3 _ ~~'+` r #f 06 JUN ~ ~ PM 6 : 0 9
p. 1
Eo ' 43 1 - 3 1 q
g ~~f~- G
DuPont Haskell Laboratory for Health and ' Environmental Sciences
Elkton Road, P .O . Box 5 0 Newark, DE 19714-0050
AR a a 6_ 3GGz
June 19, 200 6
Via Federal Express
CONTAIN NO CBI Document Processing Center (Mail Code 7407M)
Room 642 8 Att ention : 8(e) Coordinato r Office of Pollution Prevention and Toxics U .S . Environmental Protection Agency 1201 WashingDtCo2n04,60IIN~WI' 11 !Ell 111 1~1 IEi 1111 :1 (I' 111111 IIl [T 111~i 11~
F y 0 d C) () l) () 3 5 5 Dear 8(e) Coordinator:
BEHQ-0381-0394 Ammonium Perfl uorooctanoate (APFO Linear)
This lett er 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 prima ry 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 Cr1 :CD ( SD)IGS rats or 20 male Cr1 :CD1(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 recove ry 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 un ti l sacrifice .
The overall conclusions are 1) the rat study was negative for immunosuppression and 2) in the mouse, a number of immune-related fi ndings, that only occurred at 10 and 30 mg/kg, likely represent secondary responses to the systemic toxicity and stress obse rv ed at these two doses .
Rat
No suppression of the ability to make anti-sheep RBC antibody was obse rved at any dose
10 and 30 mg/kg resulted in systemic toxicity as evidenced by the following :
s Decreases in body weight gain of 74 and 37%, respectively .
s Serum cort icosterone 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 (recove ry) group .]
~
p. 2
Mous e 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 : s a loss in body weight of 3 .8 and 6 .6g, respectively. s -230% increase in serum corticosterone and s 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 : s 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) . s Serum corticosterone levels increased to about 230% of control in the 10 and 30 mg/kg groups . s Spleen and thymus weights (relative to body weight) declined to 55-65% of control values after exposure to 10 and 30 mg/kg . s 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. s 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,
Ll. - - - A . Michael Kaplan, Ph.D . Director - Regulatory Affairs and Occupational Health AMK/DMH : cl p (302) 366-5260
p. 3
Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rat s
Male rats
III V VII IX X I
Dose ( m kg) : Results :
0 .3 1 10 30 30 (Recovery)
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 Foo d Consumption:
83% 84%
Hematolog y
RBC HGB HCT MCV MCH MCHC RDW
ARET PLT
95%
98% 88%
91% 91% 86%
92% 92% 87%
97% 95%
99%
95% 94% 97%
111% 115% 123%
109%4 112%#
197%#
WBC 130%
137%
111%
ANEU 114% 123% 100%
ALYM
133% 140%
114%
AMON 132% 140% 116% AEO S
ABAS
ALUC 133% 147% 127%
Comments : # Mccroscopically, so 9t the rats an thes s had-increased anisocytosis (variapoR ui redcc I siz,e ; alsa ohser~ed ~= t dosetl ~t~fh I ~~~F rn~ctocytosis'~jin~reea5ect #s of7ar~excells) ; :
palyehrotnasia~incsea~; 1ce1Is);audliypelrr6masta(pale;~tainin'g'of '
rcd b~ovd celis~ Y I hes~ Ch .' ~ mttiiknatly increased ieticu1ocyte~, sri_~ome
animals: E
w
Serum Chemistry
CHOL TRIG
64% 69%
81% 84%
69% 75% 68% 66% 69%
TP
107%
ALB GLOB HDL LDL
106%
75% 79% 58% 63%
112% 115%
89% 89%
75%
79 %
85% 88%
112% 96%
SCORT 135% 196% Comment: Hemolvzed serum at 10, 30, and 30R ~
p. 4
Male rats Dose (mg/kg) :
Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Rat s
III 0 .3
V VII IX 1 10 30
XI 30 (Recovery)
Gross Patholo :
Liver, Discoloration, tan
1 2 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 weig h Liver Spleen Thymus Brain
182%
191%
156%*
115%
144%*
112% 134% 121%*
Histo atholo
Liver, Hypertrophy, 5(1 .0) 10 (1 .7) 10 (3 .0) 10 (3 .0) 10 (3 .0) hepatocellular
Liver, Necrosis, focal 40-0) 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 =# 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 :
95% 142%*
I gm :
103% 98fo g9% g8fo 95~a:
* Group XI mean is significantly different from group IX .
~
p. 5
Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mic e
Male mice Dose (me/kg) : Itesults :
III V VII IX X I 0.3 1 10 30 30 ( Recovery)
Mortali :
Comment : No treatment related deaths occurred .
Clinical Signs :
M : 0/20 M : 0/20 M : 0/20 M : 2/20 M : 4/20
Comment : 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
Final Body Weights
(Day 28) :
86% 78% 88%*
Body Weight Gain :
1 .5g 1 .5g
(Day 29)
Comment : Control group gained 0 .9 rams (g)
Daily Food
Consumption :
-3.8g -6.6g -3 .3g
108%
98% 100%
Hematology
RBC
94% 86%
HGB 88% 82%
HCT MCH
91%
85%
MCHC
RDW ARET PLT
109 % 148%
WBC
ANEU
236% 296% 256%
ALYM AMON
59% 74%
285% 254%
177%
AEOS 57% 64% 64% ABAS
ALUC
Serum Chemistry
CHOL TRIG
69%
51%
80%
47% 32% 57%
TI' ALB GLOB
HDL
71%
125% 145%
61%
109%
134%
131% 148%
119%
44% 69%
LDL 85% 65% 103%
SCORT
230% 232%
154%
Comment : Icteric serum at 10, 30, and 30R
Gross Pathology :
Liver, Large 17 16 17
Liver, Discoloration
1
Spleen, Small
8
Thymus, Small
3
Comment : Control group exhibited Liver, Large incidence of 1
5 8
2
1 12
2
~
p. 6
Ammonium Perfluorooctanoate : 28-Day Immunotoxicity Study in Male Mic e
Male mice Dose (mg/kg) : Absolute Organ Weight:
III V VII IX XI 0 .3 1 10 30 30 (Recovery)
Liver Spleen Thymus
162% 301% 293% 317% 56% 44% 65% 50% 50% 54%
Brain 95% 93% 94%
Relative Organ Weight (% of body weight) Liver
Spleen Thymus Brain
Histo atholo
160% 350% 373% 350% 86% 65% 55% 70% 57% 61% 58% 110% 119% 103%*
Liver, Hypertrophy, 20 (2 .0) 20 (3 .0) 20 (4 .0) 19 (4 .0) 19 (4 .0) hepatocellular
Liver, Necrosis, 11 individual cel l Liver, Necrosis, focal Liver, Mitotic figures, increase d
(1 .1) 3 (1 .0)
20
(1 .9)
4 (1 .8) 10(i .0)
19 (2 .0)
7 (1 .7) 15 (1 .0)
19 19
(1 .7)
3 (1 .7) (1 .4)
Liver, Hyperplasia, bile 6(1 .0) 17 (1 .2) duc t
12(l .0)
Liver, Fatty change, 9(1 .0) 14 (1 .0) nonzona l
4 (1 .0)
Thymus, Depletion/Atrophy ,
6(l .2) 7(2 .9) 4 (2 .8)
lymphoid
Spleen, Depletion/Atrophy ,
8
(1 .1) 7 (1 .1)
lymphoid
Spleen, EMH, increased 15 (2 .1) Bone Marrow, 3(1 .7) 4(1 .0) 3(1 .7) H e lasia, granulocyti c
Bone Hyperplasia, erythrocyti c
Marrow,
1
(2 .0)
Comment : ()= Number in parentheses is the average grade (grades 1- 4) when lesion is present (i .e ., sum of grades =# 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%
I gm :
98%
92% 80% 72% 70%
* Group XI mean is significantly different from group IX .