DEQ-CFW_00074751

C,4,,-A.;X Feb - q IO?
Toxicology Reports 2 (2015) 939-949
Contents lists available at ScienceDirect
Toxicology Reports '
ELSMER journal homepage: www.elsevier.com/locate/toxrep
Evaluation of chronic toxicity and carcinogenicity of ammonium &J �sMk
2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate in
Sprague-DaWley rats
J.M. Caverly Rae a, %, Lisa Craig b, Theodore W. Slone b, Steven R. Frame a, L,William Buxton c
, Gerald L Kennedyd
8 E L du Pont de Nemours and Company, Inc, Haskell Global Centers for Health &Environmental Sciences, Newark, DE 19714, USA
b MPI Research, Inc, Mattawan, MI49071, USA
c EL Want de Nemours and Company, Inc, Chemicals and FluoroproducM Wilmington, DE 19805, USA
d Consultant to DuPont Company, Wilmington, DE 19805,USA
ARTICLE I NFO ABSTRACT
Article history:
Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate, developed for use as a polymeriza-
Received 30 March 2015
tion processing aid in the manufacture of fluoropolymers, was tested for its potential chronic toxicity
Received in revised form 4 May 2015
and carcinogenicity in a 2-year oral dosing study in Sprague-Dawley rats. Male rats were given daily
Accepted 1 June 2015
doses of either 0, 0.1,1 or 50 mg/kg; females were given either 0,1, 50 or 500 mg/kg. Body weights, food
Available online 30 June 2015
consumption and clinical signs were monitored daily; clinical pathology was conducted at designated
Intervals and animals were given a complete pathological evaluation after 12 months and 24 months of
Keywords:
dosing. Normal survival was seen in all groups, no abnormal clinical signs were seen, and body weight
Fluoropolymers
gain was reduced only in female rats at 500 mg/kg. Both sexes at the high dose had mild decreases in
Chronic toxicity and carcinogenicity study
Sprague-Dawley rats
red cell mass which were somewhat more pronounced in females. Clinical pathology indicative of liver
PPARot agonist
injury was present in males that received 50 mg/kg and correlated with histomorphological liver changes
that included both hypertrophic and degenerative/necrotic lesions. Similar histomorphological lesions
were seen in the livers of females at 500 mg/kg. Previous shorter term toxicity studies have identified
this chemical as a PPARoe agonist and the finding of benign tumors of the liver, pancreas and/or testes in
males at 50 mg/kg and females at 500 mg/kg is consistent with the rat response to peroxisome prolifera-
tors and is of questionable human relevance. Changes in the kidney, tongue, and stomach were observed
only at the highest dose of 500 mg/kg In females. The no -observed -adverse -effect -level in this study lies
between 1 and 50 mg/kg for males and between 50 and 500 mg/kg for females.
© 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/),
1. Introduction
Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-
propanoate (CAS 62037-80-3) is a white/colorless solid with
a sublimation point of 130-140 °C, a decomposition point of
150-160 °C, a density of approximately 1.7 g/cm3 at 20 °C, and a
very low vapor pressure of approximately 0.01 Pa at 20°C [101.
The chemical has been developed for use as a polymerization
processing aid in the manufacture of fluoropolymers. The acute
toxicity profile includes both an oral LD50 of 1750 and 3129 mg/kg
in male and female rats, respectively, and 1030 mg/kg in mice
(females). The dermal ID50 in rats is greater than 5000 mg/kg. By
the inhalation route, the chemical has an acute LC50 of greater
* Corresponding author. Tel.: [phone].
than 5200 mg/m3 in rats exposed for 4 h. As tested in rabbits, it
is highly irritating to the eye but is not a skin irritant nor is it a
sensitizer as tested by the mouse local lymph node assay. The
material is not genotoxic based on a battery of tests including
Ames and chromosome aberration in -vitro studies and mouse
micronucleus, mouse bone marrow chromosomal analysis and rat
unscheduled DNA synthesis in -vivo studies [101.
The compound is rapidly and completely absorbed following
oral administration in both rats and mice, is not metabolized, and
is eliminated almost exclusively in the urine. In the cynomolgus
monkey given a single intravenous dose, the chemical was rapidly
eliminated, no sex differences were seen, and a biphasic pattern
of elimination was present. Based on oral studies in rats and mice,
and intravenous studies in rats and primates, elimination of the
test substance is most similar and more rapid in primates and rats,
with mice having a comparatively longer clearance time. Clearance
http://dx.dol.org/l0.1016/J.toxrep.2015.06.001
2214-7500/® 2015 The Authors. Published by Elsevier Ireland Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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J.M. Caverly Rae et al, / Toxicology Reports 2 (2015) 939-949
times in rats and primates are generally less than 12-24 h while
those in mice are 60-140 h. Plasma elimination kinetics were simi-
lar following both single and multiple dose studies indicating a low
propensity for accumulation in the body. Based on the pharmacoki-
netic curve following an intravenous dose of the compound, male
rats have a slightly slower elimination rate than females, resulting
in males having a higher plasma concentration than females after
the same dose [ 111.
In repeated dose toxicity studies, the test material induces hep-
atic peroxisomal ]3-oxidation activity and produces microscopic
changes in the livers of rodents consistent with the activity of
peroxisome proliferators. Thus, the test material is a member of
a class of compounds known as PPARot agonists, which include
endogenous long chain fatty acids, as well as many industrial chem-
icals and pharmaceuticals. These compounds produce effects in the
liver to which rodents have been shown to be more sensitive than
other species, including humans. In a series of repeated oral dos-
ing studies of 28-90 days duration in rats and mice, the sentinel
effects of this chemical were consistent with those of a PPAR(x ago-
nist [ 12,13 ]. These included increased liver beta -oxidation activity,
increased liver weights, microscopic hepatocellular hypertrophy,
and clinical pathology changes of decreases in red cell parameters,
changes in serum proteins, and decreases in serum lipids.
Specifically, in a 90-day repeated dose toxicity study in which
male rats were given daily gavage doses of either 0.1, 10, or
100 mg/kg, findings included most of the clinical pathology and
anatomic pathology effects noted above, as well as increased kid-
ney weights [13]. Similar finding were seen in female rats given
daily gavage doses of either 10,100, or 1000 mg/kg. These included
kidney weight increases at 10 mg/kg, PPARa.-related clinical chem-
istry changes at 100 mg/kg, and red blood cell effects, increased
liver weights, and hepatocellular hypertrophy at 1000 mg/kg. Find-
ings similar to these were seen in an earlier 28-day dosing study
with liver effects seen in males following dosing at 3 mg/kg and
in females at 300mg/kg [12]. These studies formed the basis for
dose level selection for the chronic toxicity/carcinogenicity study
reported here.
To characterize the chronic toxicity profile and to evaluate the
potential carcinogenic effects of this chemical, a 2-year chronic tox-
icity and carcinogenicity study was conducted in rats. This paper
covers the information derived from this experiment. The results of
this study are reported herein and will be compared to those found
in other chronic/carcinogenicity studies with similar fluorochemi-
cals to place the findings in perspective.
2. Materials and methods
2.1. Chemicals
The ammonium salt of 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)-propionic acid (84%; CAS number
62037-80-3; C6Ii4F11NO3; molecular weight: 347) was pro-
vided by E.I. du Pont de Nemours and Company (Wilmington,
DE). The test article was an 84% aqueous solution and doses were
adjusted accordingly and are expressed in terms of 100% test
chemical. Formulations of the test article were prepared weekly
and stored at room temperature.
2.2. Animals
All rat treatments, toxicity assessments and data acquisitions
were performed at MPI Research, Inc. (Mattawan, Michigan). The
protocol was reviewed and approved by the Institutional Animal
Care and Use Committee (IACUC), in compliance with the Animal
Welfare Act (AWA). Male and female CD® [Cr1:CD(SD)] rats were
received at approximately 5 weeks of age from Charles River Labo-
ratories (Portage, Michigan). Rats were acclimated for 14 days prior
to being randomly assigned to treatment groups using a standard,
by weight, measured value randomization procedure. Rats assigned
to study had body weights within f20% of the mean body weight
for each sex. The animals were pair -housed (same sex) in polyboxes
with non -aromatic bedding in an environmentally -controlled room
and were provided environmental enrichment. All animals were
maintained in accordance with the Guide for the Care and Use of
Laboratory Animals in the animal facilities at MPI Research, Inc.
(American Association for the Accreditation of Laboratory Animal
Care [AAALAC] accredited). Block Lab Diet® (Certified Rodent Diet
*5002, PMI Nutrition International, Inc., Shoreview, MN, USA) was
provided ad libitum. Tap water was available ad libitum via an auto-
matic watering system. Room temperature and humidity controls
were maintained to the maximum extent possible between 64 and
79 "C and 30-707., respectively. A 12 h light/dark photoperiod was
maintained.
2.3. Treatment
Four treatment groups of 80 rats/sex were formed. Control
group males and females received deionized water (vehi-
cle control). Male rats in test groups received either 0.1, 1
or 50mg ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-
propanoate/kg body weight/day. Female rats in test groups
received either 1, 50 or 500 mg ammonium 2,3,3,3-tetrafluoro-
2-(heptafluoropropoxy)-propanoate/kg body weight/day. Ten ani-
mals/sex/group were designated for the 12 month interim
necropsy. The remaining surviving animals were designated for the
terminal necropsy.
Dose selection was based on a previous subchronic (90-day) tox-
icity study [13] and a previous 28-day study [12]. The vehicle and
test article were administered once daily for up to 104 weeks in
males and up to 101 weeks in females via oral gavage at a dose
volume of 10 mL/kg/dose. The control group received the vehicle
in the same manner as the treated groups. Individual doses were
based on the most recent body weights. All animals were observed
for morbidity, mortality, injury, and availability of food and water
twice daily. Beginning atweek 53, a third daily cageside observation
was added. A detailed clinical examination, including a physical
exam and palpation of masses, was performed weekly. Ophthalmo-
scopic examinations were conducted on all animals pretest and on
all surviving animals prior to the interim and terminal necropsies.
2.4. Body weight and food consumption
Body weights were recorded weekly starting day 1(prior to dos-
ing) until week 14, and then every two weeks thereafter. Body
weight changes were calculated and reported weekly the first 3
months (weeks 1-13), the first year (weeks 1-52), and for the entire
study (weeks 1-102 for males and weeks 1-100 for females). Food
consumption was recorded pretest (week -1), weekly during the
first 13 weeks, and then every two weeks starting on week 14. Food
consumption was measured for the cage and divided by the num-
ber of surviving animals. Food consumption and efficiency were
calculated weekly the first 3 months (weeks 1-13), the first year
(weeks 1-52), and for the entire study (weeks 1-102 for males and
weeks 1-100 for females).
2.5. Clinical pathology
Clinical pathology evaluations were conducted on 10 ani-
mals/sex/group at 3, 6 and 12 months (hematology and clinical
chemistry) and 10 animals/sex/group at 6 and 12 months (coag-
ulation and urinalysis). The first ten animals in each group were
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941
selected for bleeding and these same animals were used for all
time points. Blood samples were collected via the vena cava or car-
diac puncture after carbon dioxide inhalation. The order of bleeding
was by alternating 1 animal from each dose group then repeating
to reduce handling and time biases. Blood samples for peripheral
blood smears were collected at 12 and 18 months and prior to ter-
minal necropsy. The animals designated for a full clinical pathology
evaluation had access to drinking water but were fasted overnight
prior to scheduled sample collection. The animals designated for
only blood smears had access to drinking water and food prior to
sample collection. Urine was collected after animals were housed
in stainless steel metabolism cages for at least 12 h.
Hematology parameters evaluated included: leukocyte count
(total and absolute differential), erythrocyte count, hemoglobin,
hematocrit, mean corpuscular hemoglobin, mean corpuscular vol-
ume, mean corpuscular hemoglobin concentration (calculated);
absolute reticulocytes, platelet count, and platelet morphology.
Coagulation parameters evaluated included prothrombin time and
activated partial thromboplastin time.
Clinical chemistry parameters evaluated included alanine
aminotransferase, alkaline phosphatase, sorbitol dehydrogenase,
total protein, albumin, globulin and A/G (albumin/globulin) ratio
(calculated), urea nitrogen, creatinine, total cholesterol, triglyc-
erides, total bilirubin (with direct bilirubin if total bilirubin
exceeded 1 mg/dL), aspartate aminotransferase, total bile acids,
glucose, calcium, phosphorus, electrolytes (sodium, potassium and
chloride), and gamma glutamyl transferase.
Urinalysis parameters evaluated included volume, specific grav-
ity, pH, color and appearance, protein, glucose, bilirubin, ketones,
blood, urobilogen, and microscopy of centrifuged sediment.
2.6. Necropsy and histopathological evaluation
Postmortem study evaluations were performed on all animals
euthanized in extremis, animals found dead, and on all surviv-
ing animals scheduled and sacrificed at the interim (12 months)
and terminal (101 and 104 weeks - approximately 24 months)
necropsies. The animals were euthanized by carbon dioxide inhala-
tion followed by exsanguination via the abdominal vena cava. At
necropsy, external surfaces and abdominal, thoracic, and cranial
cavities were examined for abnormalities including masses. Tissues
from all organ systems (digestive, urinary, respiratory, cardio-
vascular, hematologic, nervous, endocrine, musculoskeletal, and
reproductive systems, as well as skin and gross observations) were
removed, examined, and, where required by protocol, placed in fix-
ative (neutral buffered formalin, except for the eye [including the
retina and optic nerve] and testes, which were fixed using a mod-
ified Davidson's fixative). Body weights and organ weights (brain,
adrenals, heart, kidneys, liver, spleen, thyroid/parathyroid, and/or
epididymides, testes, ovaries with oviducts, and uterus with cervix)
were recorded for all surviving animals at the scheduled necrop-
sies and appropriate organ weight ratios were calculated (relative
to body and brain weights). Paired organs were weighed together.
Microscopic examination of fixed hematoxylin and eosin -stained
paraffin sections was performed on tissues by a board -certified
veterinary pathologist. A four -step grading system was utilized to
define gradable lesions for comparison between dose groups. For
fatal and incidental neoplasms, the onset date was considered to be
the fate date of the affected animal. For mortality independent neo=
plasms, the onset date was considered to be the first appearance
of a related abnormality (e.g., abrasion, nodule, and/or swelling). A
second board -certified pathologist performed a formal peer review
of the histopathological findings and the results reported here rep-
resent the consensus.
2.7. Statistical analysis
For continuous data (body weight and weight gain, food con-
sumption, hematology, coagulation, clinical chemistries, and organ
weights), the statistical procedures were generally as follows: Lev-
ee's test [20] was used to assess homogeneity of group variances
for each specified endpoint and for all collection intervals. If Lev-
ee's test was not significant (p > 0.01), a pooled estimate of the
variance (mean square error) was computed from a one-way anal-
ysis of variance (ANOVA) and utilized by a Dunnett's comparison
[91 of each treatment group with the control group. If Levene's test
was significant (p < 0.01), comparisons with the control group were
made using Welch's t-test [281 with a Bonferroni correction.
Intercurrent mortality data were analyzed using the
Kaplan -Meier product -limit method. An overall test compar-
ing all groups was conducted using a log -rank test [2]. Tumor
incidence data were analyzed using both survival adjusted and
unadjusted tests. The Cochran -Armitage trend test [11 was cal-
culated and Fisher's exact test [291 was used to compare each
treatment group with the control group. The survival adjusted test
was conducted according to the prevalence/mortality methods
described by Peto et al. [241.
All endpoints were analyzed using two -tailed tests with results
of all pair -wise comparisons reported at the 0.05 and 0.01 signifi-
cance levels.
3. Results
This experiment was conducted in accord with the Toxic Sub-
stance Control Act (TSCA) Good Laboratory Practice Standards. All
system suitability tests, performance checks, and calibration stan-
dards met analysis acceptance criteria and the dose formulation
samples, analyzed for homogeneity and concentration, met the cri-
teria for acceptance based on accuracy and precision. On quality
assurance check, no significant deviations from the protocol which
could impact the quality or integrity of the study were found.
3.1. Survival
Mean survival data for male and female rats are illustrated in
Fig. 1. The test substance had no effect on survival. Females were
sacrificed during week 101, prior to scheduled termination atweek
104, due to low survival in all female dose groups (especially con-
trol and 50 mg/kg groups). However, even though survival among
all female groups was low, there were no statistically significant
differences between groups as survival was essentially the same.
Seven of the 500 mg/kg early death females had test substance -
associated papillary necrosis and inflammation of the kidney. All
other causes of death/morbidity were considered incidental and
common in rats of this strain and age.
3.2. In -life observations
There were no test substance -related clinical observations; all
observations were transient or common in this species. In addi-
tion, there were no test substance -related increases in masses
identified during physical examination nor were there any test
substance -related findings at the interim or terminal ophthalmo-
scopic examinations.
The test substance had no effect on body weight, body weight
gain, or food consumption and efficiency in males. These values
were generally comparable to those of control animals throughout
the study.
Exposure of female rats to 500 mg/kg produced reductions in
body weight, body weight gain, and food efficiency. Mean body
weight for this group was 13% below that for control animals at
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942 J.M. Coverly Rae et al. / Toxicology Reports 2 (2015) 939-949
(A) Summary of Survival — Male Rats
Study Week
• 0 mglkg/day ' 0.1 mg/kglday
1 mglkglday * 50 mg/kglday
(B) Summary of Survival — Female Rats
20 40 60 au 1 w I &0
Study Week
0 mg/kg/day ' 1 mglkglday
50 mg/kg/day * 500 mg/kglday
Fig. 1. Kaplan -Meier survival curves for rats treated with ammonium 2,3,3,3-tetraFluoro-2-(heptaFluoropropoxy)-propanoate for 104 weeks (male) or 101 weeks (female).
(A) Survival curve of male rats treated with either 0, 0.1, 1 or 50 mg/kg, p> 0.05. (B) Survival curve of female rats treated with either 0, 1, 50 or 500 mg/kg, p> 0.05.
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Study Week
■ 0 mglk0/day A 1 nV&iYday
50 nvyk91day * 500 Mg*gfday
Fig. 2. Mean body weights for female rats treated with either 0, 1, 50 or 500 mg/kg ammonium 2,3,3,3-tetrafluoro-2-(heptaFluoropropoxy)-propanoate for 101 weeks.
week 52 (statistically significant), but was comparable to controls
at study termination (Fig. 2). Consequently, mean body weight gain
was 20% below controls over weeks 1-52 in this group, but only
slightly lower than controls over the entire 2 year study (not sta-
tistically significant). There were no effects on food consumption;
the reduced body weight gain was associated with lower mean
food efficiency over the first year (although food efficiency over
the entire 2 year study was comparable to controls).
3.3. Clinical pathology
At the 3, 6, and 12 month intervals, the test substance
caused mild but adverse decreases in red cell mass (erythrocytes,
hemoglobin, and hematocrit; up to 28% below controls) in females
receiving 500 mg/kg (Fig. 3 ). These changes were associated with an
appropriate increase in reticulocytes (up to 106% above controls).
There were no effects on erythrocyte morphology.
Statistically significant decreases in erythrocytes, hemoglobin,
and hematocrit were also present in males receiving 50 mg/kg at
the 3 and 6 month intervals. However, the decreases were small,
transient (no statistically significant difference at 12 months), and
did not induce a statistically significant increase in reticulocytes.
Increases in enzymes indicative of liver injury, including mild
increases in alkaline phosphatase, alanine aminotransferase, aspar-
tate aminotransferase, and sorbitol dehydrogenase (aspartate
aminotransferase was not statistically significant) were observed
at 12 months in males at 50 mg/kg (Fig. 4) and were correlated with
microscopic findings of minimal cystic degeneration and minimal
to mild focal necrosis in the liver. Minimal increases in alkaline
phosphatase, unassociated with increases in other liver injury -
specific enzymes, were also present in males at this dose level at
3 and 6 months. There were no test article -related liver enzyme
943
changes in males receiving either 0.1 or I mg/kg or in females at
any dose.
Test substance -associated increases in albumin (up to 16%
above controls), decreases in globulin (up to 17% below con-
trols), and increases in the albumin/globulin ratio were present in
males at 50 mg/kg and females at 500 mg/kg at various intervals
with variable statistical significance. Albumin was also statis-
tically significantly increased in males receiving 1 mg/kg at 12
months (8% above controls) and globulin was statistically signif-
icantly decreased transiently (only at 6 months, 6% below controls)
in females receiving 50 mg/kg. Minimal, statistically -significant
increases in the albumin/globulin ratio were also present in the
1 mg/kg males and the 50 mg/kg females at various intervals.
A minimal diuresis was present at 6 and 12 months in females
that received 500 mg/kg, as evidenced by statistically significant
increases in urine volume and decreases in urine specific gravity.
There were no changes in kidney -related chemistry parameters.
This diuresis may be correlated to an increase in the incidence and
severity of chronic progressive nephropathy (CPN) observed in this
group at the 1-year sacrifice (Table I ).
There were no other test article related clinical pathology
effects. All other mean and individual clinical pathology parame-
ters were considered within an acceptable range for biological and
procedure -related variation.
3.4. Organ weights and anatomic pathology observations
No test -substance associated anatomic pathology findings
occurred in male rats administered either 0.1 or I mg/kg or in
female rats administered either I or 50 mg/kg. Test substance -
associated findings observed in males at 50 mg/kg and in females
at 500 mg/kg are described in Section 3.4.1, 3.4.2 and 3.4.3. All
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944
50
45
40
35
30
25
20
15
10
5
0
J.M. Caverly Rae et al. / Toxicology Reports 2 (2015) 939-949
■ 0 mg/kg/day
1 mg/kg/day
50 mg/kg/day
■ 500 mg/kg/day
RBC hemo HCT
Fig. 3. Mean red cell mass parameters for female rats treated with either 0 (control), 1, 50 or 500 mg/kg ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate
for 12 months. RBC = erythrocytes (106iµL); hemo = hemoglobin (gidL); HCT= hematocrit (%). "= Significantly different from control (p < 0.05); #= significantly different from
control (p < 0.01), N =10.
other organ weight, macroscopic and non-neoplastic and neoplas-
tic microscopic observations were of the type typically seen in
rats of this strain and age, and were considered incidental and not
related to compound administration.
3.4.1. Organ weights and macroscopic findings
At the interim necropsy, test substance -associated organ weight
effects were limited to increased liver weights (Table 1) in the
50 mg/kg male group (16% above controls relative to body weight)
250
WX
016111
100
50
C
and in the 500 mg/kg female group (69% above controls relative to
body weight). In females, liver weight changes correlated with cen-
trilobular hypertrophy microscopically. In one 500 mg/kg female,
the macroscopic finding of irregular surface of the kidney correlated
with CPN in this animal and was likely test substance -related.
At the terminal sacrifice, no test compound -associated organ
weight or macroscopic effects were observed in males. In females,
test substance -associated organ weight changes were limited to
increased liver weights (Table 1) at 500mg/kg (43% above con-
■ 0 mg/kg/day
0.1 mg/kg/day
1 mg/kg/day
50 mg/kg/day
Alk Phos ALT AST SDH
Fig. 4. Selected mean clinical chemistry parameters for male rats treated with either 0 (control), 0.1, 1 or 50 mg/kg ammonium 2,3,3,3-tetrafuoro-2-(heptafiuoropropoxy)-
propanoate for 12 months. Alk Phos= alkaline phosphatase (UiL); ALT= alanine aminotransferase (U/Q; AST= aspartate aminotransferase (UiL); SDH = sorbitol dehydrogenase
(U/Q. "=Significantly different from control (p<0.05); # = significantly different from control (p<0.01). N=10.
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Table 1
Mean liver weight parameters for male rats treated with either 0, 0.1, 1 or 50 mg/kg ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate
and female rats
treated with either 0, 1, 50
or 500 mg/kg ammonium 2,3.3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate for 12 (interim sacrifice) and 24 (terminal sacrifice) months.
Mean liver weights at interim and terminal sacrifice in male and female rats
Males
Interim sacrifice
Terminal sacrifice
Dose level (mg/kg)
0 0.1 1
50 0 0.1
1
50
N
10 10 10
10 15 20
18
18
Absolute (g)
26.9 f 4.9* 26.1 t 43 25.5: 1"
29.7 f 5.0 24.313.6 26.61 6.4
24.1 t 5.6
27.1 t 4.8
%
-3% -5%
10% 9%
-1%
12%
Liver/body weight (%)
3.210.3 3.310.3 3310.2
3.7 f 0.40 2.9 f 0.4 3311.3
3.2 t0.7
3.210.5
%
3% 3%
16% 14%
10%
10%
Females
Interim sacrifice
Terminal sacrifice
Dose level (mg/kg)
0 1 50
Soo 0 1
50
500
N
10 10 10
10 16 22
15
18
Absolute (g)
132 t 2.5 12.011.6 14.8 f 3.6
17.6 f 2.61' 16A d:3.8 15.8 t43
17213.0
232 f 82"
%
-9% 12%
33% -4%
5%
41%
Liver/body weight (%)
2.9 f 03 3.0 103 3.31 0.4
4.910.60 3.5f 0.4 33 f 0.7
33 10.6
5.0 t 0.9e
%
3% 14%
69% -6%
-6%
43%
a Significantly different from control (p <0.01). %Indicates difference from controls.
Mean f standard deviation.
trols relative to body weight). Macroscopic findings in this group
included tan focus/foci in the liver (8/70 versus 1/70 in controls),
mass/nodule in the liver (14/70 versus 0/70 in controls) and irregu-
lar surface of the kidney (16/70 versus 0/70 in controls). The organ
weight and macroscopic observations in females were correlated
to non-neoplastic and neoplastic histological observations at this
dose.
3.42. Non-neoplastic histological findings
At the interim necropsy, non-neoplastic test substance -
associated effects were present in the liver of males at 50 mg/kg
and in the liver and kidneys of females at 500 mg/kg. In the
liver of males administered 50 mg/kg, histological lesions included
minimal focal cystic degeneration (3/10 versus 0/10 in controls)
and minimal to mild focal necrosis (5/10 versus 1/10 in con-
trols). In females, histological changes in the liver were limited
to minimal to mild hepatocellular hypertrophy in all 10 females
receiving 500 mg/kg. A slightly increased incidence and severity of
CPN was also observed in interim sacrifice females at 500 mg/kg
(Table 2).
At the terminal sacrifice, non-neoplastic test substance -
associated effects were observed histologically at the highest dose
level in the liver of both sexes (Table 3) and, additionally, in the
kidney (Tables 4), glandular stomach (limiting ridge) and tongue of
females at the highest dose (Table 5).
In 50 mg/kg males, histopathological effects were limited to
the liver and included statistically significant increases in the
incidences of focal cystic degeneration, centrilobular hepatocellu-
lar hypertrophy, and centrlobular hepatocellular necrosis. These
changes were also present in 500 mg/kg females, in addition
to low incidences of panlobular hepatocellular hypertrophy and
Individual cell hepatocellular necrosis (Table 3). Cystic degener=
ation was characterized by the presence of multilocular cystic
spaces containing finely granular or flocculent material without
endothelial or epithelial cells lining the spaces. Centrilobular hyper-
trophy, morphologically consistent with peroxisome proliferation,
was characterized by hepatocytes with red granular cytoplasm
sometimes containing small amounts of pigment compatible with
lipofuscin. Centrilobular hepatocellular necrosis was typically of
the coagulative type with strongly eosinophilic-staining cytoplasm
and pyknotic nuclei. Panlobular hepatocellular hypertrophy was
characterized by enlargement of hepatocytes throughout the entire
liver. Individual cell necrosis was characterized by the presence of
scattered single hepatocytes with features characteristic of apopto-
sis.
Kidney changes in females at 500 mg/kg included tubular dila-
tion, edema of the renal papilla, transitional cell hyperplasia in
the renal pelvis, tubular mineralization, renal papillary necrosis
and CPN. Tubular dilation frequently occurred in an ascending pat-
tern extending from the papilla to the outer cortex, while at other
times it was present only in the papilla. Edema of the papilla was
characterized by increased rarefaction or myxomatous change in
the papillary interstitium, sometimes with polypoid protrusions
from the lateral surface of the papilla. The edema and tubular dila-
tion were often associated with hyperplasia of the transitional cell
epithelium lining the papilla and pelvis. Small foci of tubular min-
eralization were often present and, in some animals, necrosis of the
tip of the papilla was present.
In addition, in female rats given 500 mg/kg, statistically sig-
nificant increases in hyperplasia of squamous epithelium were
observed in the nonglandular stomach (limiting ridge only) and
tongue (in association with subacute/chronic inflammation in the
tongue).
A statistically significant increase (42/70 or 61%) in alveolar his-
tiocytosis was present in females at 500 mg/kg and was at the
upper end of the historical control range of 9.2-61.7% [221. The
increased incidence of this common background lesion may be
secondary to aspiration of dosing formulation at this high concen-
tration; however, a definitive mechanism for this increase could
not be determined.
3.43. Neoplastic histological findings
Neoplastic test substance -associated effects were observed his-
tologically at the highest dose level in the liver of females and,
equivocally, in the pancreas and testes of males at the highest dose
(Table 6).
Compound -related neoplastic changes occurred in the livers
of females administered 500 mg/kg and included increased inci-
dences of hepatocellular adenoma and carcinoma (Table 6). These
tumors occurred in association with the degenerative and necrotic
liver lesions observed at this dose as described above. Hepatocellu-
lar tumors and test substance -associated degenerative and necrotic
lesions were not observed in females at lower doses and the inci-
dences of hepatocellular tumors were similar in all male groups. In
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Table 2
incidence of chronic progressive nephropathy in the kidneys of female rats treated with either 0 (control), 1, 50 or 500mg/kg of ammonium 2,3,3.3-tetratluoro-2-
(heptafiuoropropoxy)-propanoate for 12 months, N=10.
Incidences and severity of chronic progressive nephropathy In the kidneys of female rats: interim sacrifice
Dose level (mg/kg) 0 1 50 500
Nephropathy, chronic progressive 6 4 6 9
-Minimal 5 3 4 3
-Mild 0 1 2 6
-Moderate 1 0 0 0
Table 3
Test substance-assoclated non-neoplastic histological lesions In the liver of male rats treated with either 0, 0.1, 1 or 50 mg/kg ammonium 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)-propanoate and female rats treated with either0,1, 50or500 mg/kg ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate for 24 months,
N=70.
Summary of test substance -associated non-neoplastic findings in the liver of male and female rats: terminal sacrifice
Males
Dose level (mg/kg)
0
0.1
1
50
Degeneration, cystic focal
24
24
19
42'
Hypertrophy, hepatocyte, centrilobular
0
0
0
7'
Necrosis, hepatocytes, centrilobular
1
0
1
5'
Females
Dose level (mg/kg)
0
1
50
500
Degeneration, cystic focal
2
2
2
14'
Hypertrophy, hepatocyte, centrilobular
0
0
3
65'
Hypertrophy, hepatocyte, panlobular
0
0
0
3'
Necrosis, hepatocytes, centrilobular
1
1
4
7'
Necrosis, individual hepatocyte
0
0
0
3'
' Significantly different from control (p <0.05).
males administered 50 mg/kg, a statistically significant increase in
the combined incidence of pancreatic acinar cell adenomas and car-
cinomas was seen (Table 6), but neither the incidence of adenoma
or carcinoma alone was statistically increased, although the inci-
dence of carcinomas (2.9%) was slightly outside the historical range
of 0-1.7% [211. In addition, the incidence of acinar cell hyperplasia
was not significantly different from controls in any treated male
groups. However, based on the known PPAR(x agonist activity of
the test compound, the marginal increase in pancreatic acinar cell
tumors at this dose provides equivocal evidence of test substance -
associated effect Incidences of proliferative acinar cell lesions in
males receiving 0.1 and 1 mg/kg were similar to controls and no
acinar cell adenomas or carcinomas were present in females at any
dose.
The incidence of Leydig cell adenomas (11.4%) was increased
above historical control ranges for this tumor (0-8.3%) [211 in
males administered 50 mg/kg, although this increase was not stat-
ically significant compared to controls. In addition, a Leydig cell
adenoma was present in 1 male at the interim necropsy in the
50 mg/kg group. The incidence of Leydig cell hyperplasia was also
Table 4
Test substance -associated non-neoplastic histological lesions in the kidney of female rats treated with either 0, 1, 50 or 500mg/kg ammonium 2,3,3,3-tetrafluoro-2-
(heptafiuoropropoxy)-propanoate for 24 months, N=70.
Summary of test substance -associated non-neoplastic findings in the Iddneys of female rats: terminal sacrifice
Dose level (mg/kg) 0 1
50
500
Dilation, tubular 4 2
5
28'
Edema, papilla 4 1
2
43'
Hyperplasia, transitional cell 6 3
12
33'
Mineralization, tubular 25 32
28
42'
Necrosis, papillary 0 0
0
16'
Nephropathy, chronic progressive 39 40
41
64'
Significantly different from control (p<0.05).
Table 5
Test substance -associated non-neoplastic histological lesions In the nonglandular stomach and tongue of female rats treated with either 0. 1, 50 or 500 mg/kg ammonium
2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate for 24 months, N- 70.
Summary of test substance -associated non-neoplastic findings in the nonglandular stomach and tongue of female rats: terminal sacrifice
Dose level (mg/kg) 0 1 50 Soo
Stomach, nonglandular
Hyperplasla, epithellal, limiting ridge 0 0 0 9'
Tongue
Hyperplasla, squamous cell 2 8 4 13'
Inflammation, subacute/chronic 3 8 4 13'
Significantly different from control (p <0.05).
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J.M. Caverly Rae et al. /Toxicology Reports 2 (2015) 939-949
Table 6
Test substance -associated neoplastic histological lesions in the liver, pancreas and testes of male rats treated with either 0, 0.1,1 or 50 mg/kg ammonium 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)-propanoate and the liver and pancreas of female rats treated with either 0,1, 50 or 500 mg/kg ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-
propanoate for 24 months, N= 70.
Summary of test substance -associated neoplastic findings in male and female rats: terminal sacrifice
Males
Dose level (mg/kg)
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Pancreas
Acinar cell adenoma
Acinar cell carcinoma
Combined adenoma/carcinoma
Testes
Leydig cell tumor
Females
Dose level (mg/kg)
Liver
Hepatocellular adenoma
Hepatocellular carcinoma
Pancreas
Acinar cell adenoma
Acinar cell carcinoma
Combined adenoma/carcinoma
Significantly different from control (p <0.05).
increased above historical control range in this group at terminal
sacrifice (also 0-8.3%; 1221; although again, this incidence was not
statistically significant versus controls. However, comparison to
within -study controls was complicated by the fact that controls had
a relatively high incidence of Leydig cell hyperplasia (10%). Based
on the above considerations and the known activity of PPARtx ago-
nists to produce Leydig cell hyperplasia and adenomas in rats, the
relationship to the test compound for these lesions was considered
equivocal in this study.
All other findings were considered to represent the spontaneous
occurrence of neoplasms commonly seen in rats of this strain and
age.
4. Discussion
Doses employed in this study were designed to and did meet the
expectations stated below based on information gathered from pre-
vious shorter -term repeated dose studies. The high dose (50 mg/kg
in males, 500mg/kg in females) did produce adverse effects on a
number of parameters without producing excessive toxicity which
might affect survival. Seven females in the 500 mg/kg group died
from papillary necrosis of the kidney but overall survival in this
group was similar to that of the other test groups and the controls.
The intermediate dose (1 mg/kg in males, 50 mg/kg in females) was
selected to either produce slight to minimal effects on target tis-
sues and organs (liver and kidney) and/or to be a no -observed
adverse effect level (NOAEL), which it was found to be. The low
dose (0.1 mg/kg in males, 1 mg/kg in females) was projected and
also found to be a NOAEL in both sexes.
No unusual clinical signs were observed in any of the test groups
during the course of the study. Body weights among females in the
500 mg/kg group were significantly reduced, being up to 20% lower
than those of the controls. No body weight differences were seen
among the male test groups. Food consumption in all test groups
was unchanged; however, because of the weight gain depression,
females in the 500 mg/kg group showed lower food efficiencies.
Females receiving 500 mg/kg had mild but adverse decreases in
red cell mass parameters at all time points tested. These changes
0.1
50
50
500
11
4
were associated with an appropriate increase in reticulocytes and
were not accompanied by changes in erythrocyte morphology. Sim-
ilar decreases in red cell mass parameters were also present in
males receiving 50 mg/kg at the 3 and 6 month intervals. How-
ever, in males, the decreases were small, transient (there was no
statistically significant difference at 12 months), did not induce a
statically significant increase in reticulocytes, and values in indi-
vidual animals were similar to controls. Thus, the changes observed
in 50 mg/kg males were considered to be related to treatment but
were not considered adverse. This collection of findings suggests
red cell loss or hemolysis, although the exact mechanisms involved
are unknown. Peroxisome proliferators have been shown to affect
iron metabolism, and this mechanism has been hypothesized to be
the possible cause of the mild anemia sometimes seen in humans
and rats that receive these compounds [ 14,151. No changes in leuko-
cyte numbers or differential counts were seen and coagulation
parameters were normal in all test groups.
Clinical chemistry evaluations among males receiving 50 mg/kg
revealed mild but adverse increases in enzymes indicative of liver
injury, including alkaline phosphatase, alanine aminotransferase,
aspartate aminotransferase, and sorbitol dehydrogenase. These
enzyme changes correlated with microscopic findings in the liver
which consisted of minimal cystic degeneration and minimal to
mild focal necrosis. Transient, minimal increases in alkaline phos-
phatase, unassociated with increases in other liver injury -specific
enzymes, were also present in males at this dose level at 3 and 6
months; however, this increase was likely due to hepatic enzyme
induction as the test substance has been demonstrated previously
to increase total P450 enzyme activity in male rats at 30 mg/kg
[ 121. These liver enzyme changes were not seen in the male groups
receiving either 0.1 or 1 mg/kg or in any of the female test groups.
In addition, a small increase in serum albumin and a decrease
in serum globulin were seen in males in the 50 mg/kg group
and females in the 500 mg/kg group which led to increases in
the albumin/globulin ratio. Slight changes in these serum protein
components were also seen in males given 1 mg/kg and females
given 50 mg/kg. The test chemical is a peroxisome proliferator
and the pattern of change in serum proteins is a well -established
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948
J.M. Caverly Rae et d / Toxicology Reports 2 (2015) 939-949
response to PPAR activation [8]. Peroxisome proliferators are
anti-inflammatory, producing decreases in acute phase proteins
(which contribute to the globulin fraction) and increases in the neg-
ative acute phase protein, albumin. No adverse biological outcomes
have been associated with such changes in these serum proteins.
Therefore, the statistically significant changes in albumin/globulin
ratio in these groups were considered to be test article -related but
nonadverse based on the minimal nature of the changes and lack
of association with known adverse outcomes.
The minimal diuresis observed in females that received
500 mg/kg at 6 and 12 months was not associated with changes
in kidney -related chemistry parameters. This diuresis may be cor-
related to an increase in the incidence and severity of CPN observed
in this group at the 1-year sacrifice (Table 1).
Liver weights were increased in high dose male and female rats
at the interim sacrifice and in high dose females only at terminal
sacrifice. Microscopic evaluation of the liver revealed changes only
in the high dose males and females. These changes occurred at both
the terminal sacrifice and, in a less progressed form, at the interim
sacrifice and consisted of microscopic observations including focal
cystic degeneration, centrilobular (and panlobular in females only)
hepatocellular hypertrophy, centrilobular hepatocellular necrosis,
and individual cell necrosis (females only). In addition, the inci-
dences of both adenoma and carcinoma in the liver exceeded
the historical control range in females administered 500 mg/kg
(only a few hepatocellular tumors occurred in males with the inci-
dence being essentially the same between the controls and the test
groups) and the increased incidences of hepatocellular tumors in
females occurred in association with degenerative/necrotic liver
changes. This battery of liver changes is consistent with PPARa
activation.
Other non-neoplastic microscopic observations included find-
ings in the kidneys of females at 500 mg/kg at both the interim (in a
less severe form) and terminal sacrifices, and included tubular dila-
tion, edema of the renal papilla, transitional cell hyperplasia in the
renal pelvis, tubular mineralization, renal papillary necrosis, and
CPN. In addition, the nonglandular stomach (limiting ridge only)
and the tongue had statistically significantly increased incidences
of hyperplasia of squarnous epithelium in females at 500 mg/kg at
the terminal sacrifice only. The lesion in the tongue was associated
with subacute/chronic inflammation.
In males administered 50 mg/kg, the incidences of pancreatic
acinar cell adenoma/carcinoma combined, but not adenoma or
carcinoma alone, were statistically significantly increased and the
incidence of carcinoma was slightly outside the historical control
range. Since pancreatic acinar cell hyperplasia and adenoma in
rats occur along a continuum, the incidence of acinar cell hyper-
plasia would have been expected to be increased if a test -article
related increase in acinar cell adenoma was found. However, the
incidences of acinar cell hyperplasia were not significantly differ-
ent from controls in any of the treated male groups. Based on these
considerations, and the known PPAR(x agonist activity of the test
article, the marginal increase in pancreatic acinar cell tumors in this
group provides equivocal evidence of a test article -related effect
The incidences of Leydig cell adenoma of the testes were
Increased above historical control range in the 50 mg/kg group at
terminal sacrifice, although this finding was not statistically signif-
icant. In addition, a single Leydig cell adenoma was also present in
1 male from this group at the interim sacrifice. Incidences of Leydig
cell hyperplasia were also elevated in this group above historical
control range, although this, too, was not statistically significant
compared to controls. Since PPAR(x agonists are known to produce
proliferative Leydig cell lesions (hyperplasia and adenoma) in the
testes of rats, a relationship to treatment for these findings in the
50 mg/kg group cannot be ruled out.
The test chemical belongs to a class of compounds known
as peroxisome proliferators (PPARa agonists) which are known
to produce liver, pancreatic, and testicular tumors in rats and
liver tumors in mice [3,161. However, these compounds have not
been shown to be carcinogenic in other species including humans
[16,7]. Based on the extensive research into the comparative biol-
ogy of peroxisome proliferator-induced hepatic carcinogenesis, the
induction of liver tumors in rodents by non-genotoxic peroxisome
proliferators (this compound was shown to be inactive in a bat-
tery of genotoxicity assays) is not considered relevant to humans
123,7,17 ]. While there is less definitive mechanistic data on the role
PPARac plays in the induction of pancreatic acinar cell tumors in
rats, the available data involving altered bile flow and increased
cholecystokinin suggests that this mode of action is also likely to be
non -relevant for humans [161. While less robust, research consid-
ering comparative biology and mechanism of action of Leydig cell
tumor induction in rodents by a wide variety of chemical classes
suggests these tumors most likely have low relevance to humans
[25,6].
The findings of this study with this fluorochemical polymeriza-
tion processing aid can be compared to the results from similar
studies with other fluorochemical polymerization processing aids
(and PPAR(x agonists). The perfluorinated C6 chain chemical, per-
fluorohexanoic acid (PFHxA), showed no evidence of an adverse
effect in male rats given oral doses of up to 100 mg/kg for 2
years [18]. Females given either 5, 30, or 200mg/kg showed a
dose -related decrease in survival and, at 200 mg/kg, had kidney
lesions characterized by papillary necrosis and tubular degener-
ation. No evidence for a tumorigenic response was found. For
a sulfonated fluorochemical, perfluorooctane sulfonate (PFOS),
when fed in the diet of rats for 2 years at doses equivalent
to approximately 0.02, 0.1, 0.2, and 1 mg/kg, the primary organ
affected was the liver with changes consisting of hepatocellular
centrilobular hypertrophy, eosinophilic granules, and vacuolation
[27]. Neoplastic lesions consisted of an increase of hepatocellu-
lar adenomas in male rats; females showed no increase in the
incidence of neoplasia. With the ammonium salt of perfluorooc-
tanoic acid (APFO), when fed for 2 years to male and female
rats at doses approximately equivalent to 1.5 and 15 mg/kg, the
liver was the target organ with an increase in the incidence of
hepatocellular hypertrophy, monocellular infiltration, and hepato-
celltrlar vacuolation [5]. The neoplastic changes in this study were
restricted to Leydig cell adenomas of the testes. In a follow-up
mechanistic study, male rats treated at 300 ppm of APFO for 2
years (equivalent to a daily dose of 15 mg/kg) showed increased
tumor incidences in the liver (hepatocellular adenomas), pan-
creas (acinar cell adenomas), and testes (Leydig cell adenomas)
[4].
The neoplastic changes found with this fluorochemical are con-
sistent with many of those seen with the compounds above and
with other peroxisome proliferating chemicals, such as clofibrate
[26], HCFC-123 [19], gemfibrozil, and diethyl-hexyl phthalate [6],
when tested in rodents. Although hepatocellular adenomas were
not identified in males in this study, the neoplastic pattern is sim-
ilar to that seen in rats administered APFO but was seen at a
dose somewhat higher (50 versus 15 mg/kg). In female rats, no
increase in liver neopaasia was reported with APFO while this
chemical produced an increase in both adenomas and carcinomas,
but the response was seen only when the dose was consider-
ably higher (500 versus 15 mg/kg) than the dose used in the APFO
study.
Acknowledgement
This work was funded by E. I. duPont de Nemours and Company.
DEQ-CFW 00074760

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949
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