April
4,
2003
Background
Information
and
Questions
for
Letter
Review
of
the
PFOA
Preliminary
Risk
Assessment.

As
part
of
the
effort
by
the
Office
of
Pollution
Prevention
and
Toxics
(
OPPT)
to
understand
health
and
environmental
issues
presented
by
fluorochemicals
in
the
wake
of
unexpected
toxicological
and
bioaccumulation
discoveries
with
respect
to
perfluorooctane
sulfonates
(
PFOS),
OPPT
has
been
investigating
perfluorooctanoic
acid
and
its
salts
(
PFOA).
OPPT
released
a
preliminary
Draft
Hazard
Assessment
of
Perfluorooctanoic
Acid
and
Its
Salts,
dated
February
20,
2002,
on
March
28,
2002,
and
issued
a
minor
correction
to
that
document
on
April
15,
2002.
That
draft
assessment
indicated
that
the
compound
is
persistent
in
the
environment
and
in
humans
with
a
half
life
of
years.
The
assessment
noted
the
potential
systemic
toxicity
and
carcinogenicity,
and
observed
that
blood
monitoring
data
suggested
exposure
to
the
general
population,
albeit
at
low
levels.
The
Agency
has
since
received
considerable
additional
toxicology
data
that
suggest
a
potential
for
developmental/
reproductive
toxicity
and
immunotoxicity,
and
additional
human
biomonitoring
data
that
indicate
low
level
exposures
to
the
general
population
that
are
unexplained
at
this
time.

On
September
27,
2002,
the
Director
of
OPPT
issued
a
memorandum
announcing
that
OPPT
would
initiate
a
priority
review
to
determine
whether
PFOA
meets
the
criteria
for
action
under
section
4(
f)
of
the
Toxic
Substances
Control
Act.
As
part
of
the
priority
review,
the
hazard
assessment
was
revised
and
released
on
September
30,
2002.
In
addition,
OPPT
conducted
a
preliminary
risk
assessment
of
PFOA.
OPPT
recognizes
that
there
are
a
wide
range
of
toxicological
endpoints
associated
with
exposure
to
PFOA,
but
restricted
the
analysis
to
examine
the
endpoints
that
are
included
in
section
4(
f).
These
include
only
cancer,
mutations,
and
birth
defects.
PFOA
is
not
mutagenic
so
mutagenicity
was
not
considered
in
the
preliminary
risk
assessment..
In
addition,
PFOA
is
a
PPARa­
agonist
and
through
this
mode
of
action
leads
to
the
formation
of
liver
tumors
in
rodents.
The
relevance
of
this
mode
of
action
for
humans
is
currently
under
scientific
debate,
and
the
Agency
is
engaged
in
activities
to
resolve
this
issue.
Therefore,
at
this
time,
OPPT
has
narrowly
restricted
the
analysis
to
examine
the
potential
risks
of
developmental
toxicity.

OPPT
welcomes
comments
on
any
aspect
of
this
preliminary
risk
assessment.
Expertise
pertinent
to
the
review
of
this
assessment
includes
knowledge
of
pharmacokinetics,
developmental
toxicology,
and
risk
assessment.
Specifically,
we
would
appreciate
comments
on
the
questions
that
follow.
2
Issue
1.
Developmental
Endpoints
The
Agency's
Developmental
Toxicity
Risk
Assessment
Guidelines
state
that
the
period
of
exposure
for
developmental
toxicity
is
prior
to
conception
to
either
parent,
through
prenatal
development
and
continuing
until
sexual
maturation.
In
contrast,
the
period
during
which
a
developmental
effect
may
be
manifested
includes
the
entire
lifespan
of
the
organism.
Based
on
this
definition
of
developmental
exposure,
OPPT
considered
developmental
effects
to
include
reductions
in
F1
mean
pup
body
weight
(
sexes
combined)
on
lactation
days
1,
5
and
8,
an
increase
in
mortality
during
the
first
few
days
after
weaning
(
both
sexes),
a
delay
in
the
timing
of
sexual
maturation
(
both
sexes),
and
a
reduction
in
mean
body
weight
postweaning
(
F1
males
only).

Question
1.1
Please
comment
on
the
choice
of
developmental
endpoints
from
the
2­
generation
reproductive
toxicity
study
for
this
preliminary
risk
assessment.

Issue
2.
Use
of
Human
Biomonitoring
Data
Margins
of
exposure
(
MOE)
in
this
preliminary
risk
assessment
were
calculated
using
serum
levels
from
the
rat
two­
generation
reproductive
study
and
human
serum
levels
obtained
from
biomonitoring
data.
Approximately
600
individuals
were
involved
in
each
of
the
U.
S.
samples
of
adults
and
children
from
which
the
serum
data
were
derived
for
the
general
population
exposures.

Question
2.1
Please
comment
on
the
adequacy
of
the
human
exposure
data
for
use
in
calculating
a
MOE.

Issue
3.
Use
of
Serum
Data
as
a
Measure
of
Internal
Dose
Margins
of
exposure
(
MOE)
in
this
preliminary
risk
assessment
were
calculated
using
serum
levels
from
the
rat
two­
generation
reproductive
study
and
human
serum
levels
obtained
from
biomonitoring
data.

Question
3.1
Please
comment
on
the
use
of
serum
data
as
a
measure
of
internal
dose
for
rats
and
humans
for
calculation
of
the
MOE.

Issue
4.
Use
of
F0
serum
levels
as
an
estimate
of
serum
levels
of
F1
animals
The
rat
serum
levels
measured
in
the
2­
generation
reproductive
toxicity
study
were
used
as
an
estimate
of
internal
dose.
However,
serum
levels
were
measured
only
in
the
F0
animals
and
only
measured
in
the
control,
10,
and
30
mg/
kg/
day
groups,
not
in
the
1
and
3
mg/
kg/
day
groups.
The
concern
for
developmental
toxicity
is
for
the
F1
animals.
The
assumption
has
been
made
that
the
serum
levels
measured
in
the
F0
animals
are
the
best
available
dose
estimate
for
the
F1
animals.
3
Question
4.1
Please
comment
on
the
use
of
F0
serum
values
as
a
dose
estimate
for
the
F1
rats.

Question
4.2
Please
comment
on
the
use
of
serum
values
for
F0
males
as
an
upper
estimate
of
peak
exposure
for
F0
females.

Question
4.3
Recent
studies
suggest
that
three
organic
anion
transporters
(
OAT),
OAT1,
OAT2,
and
OAT3,
are
involved
in
the
renal
transport
of
PFOA.
A
study
of
developmental
and
gender­
specific
influences
on
the
expression
of
rat
OAT
in
the
kidney
has
shown
that
at
birth
all
OAT
mRNA
levels
were
low.
Renal
OAT1
expression
approaches
adult
level
at
30
days,
where
at
day
40
and
45
OAT1
levels
were
greater
in
males
than
females.
OAT2
expression
was
minimal
through
day
30
but
increased
dramatically
only
in
females
at
day
35.
OAT3
expression
matured
the
earliest
and
reached
adult
levels
at
10
days.
Please
comment
on
the
significance
of
OAT
data
for
estimating
internal
dose
on
the
postweaning
rat.
EXTERNAL
PEER
REVIEWERS
FOR
PRELIMINARY
RISK
ASSESSMENT
OF
THE
DEVELOPMENTAL
TOXICITY
ASSOCIATED
WITH
EXPOSURE
TO
PERFLUOROOCTANOIC
ACID
AND
ITS
SALTS
Steve
Cragg,
Ph.
D.

Dr.
Cragg
has
25
years
experience
as
a
toxicologist
working
for
industry
with
industrial
chemicals
and
as
an
expert
with
consulting
firms.
During
this
time,
he
has
reviewed
and
monitored
all
types
and
manner
of
toxicity
studies
including
metabolism,
reproductive
toxicity
and
developmental
toxicity
studies.
In
addition
to
animal
studies,
Dr.
Cragg
has
reviewed
and
summarized
many
industrial
biomonitoring
surveys,
case
studies,
and
human
epidemiological
studies.
Dr
Cragg
summarized
the
complete
toxicology
and
fate
and
environmental
effects
literature
in
several
separate
chapters
for
a
wide
variety
of
chemicals
in
"
Patty's
Toxicology"
(
5th
Ed).
More
recently,
he
has
written
extensive
reviews
for
the
entire
spectrum
of
health
and
environmental
studies,
as
"
robust
summaries,"
under
the
EPA
High
Production
Volume
(
HPV)
Chemicals
Program.
This
requires
intimate
knowledge
of
the
procedures
used
to
conduct
such
studies.

Henry
Spencer,
Ph.
D.

Dr.
Spencer
has
over
30
years
of
experience
working
as
a
toxicologist
for
the
US
Government.
He
has
written
and
reviewed
several
toxicological/
risk
assessment
documents
while
at
the
Office
of
Pesticides
Program
of
the
EPA.
He
is
very
familiar
with
the
process
and
regulatory
requirements
of
producing
a
complete
toxicological
document
that
meets
the
needs
of
EPA.

Katherine
Squibb,
Ph.
D.

Dr.
Squibb
has
over
20
years
experience
as
a
researcher
and
educator
in
the
field
of
toxicology.
She
currently
is
the
Interim
Director
of
Program
in
Toxicology,
Environmental
Epidemiology
and
Toxicology
at
the
University
of
Maryland
School
of
Medicine
and
also
works
with
Tetrahedron
preparing
IRIS
documents
for
the
HED
of
OPP,
EPA.
Dr.
Squibb
has
also
served
as
the
President
of
the
Metals
Specialty
Section
of
the
Society
of
Toxicology
and
currently
is
a
Councilor.
She
is
also
a
Councilor
for
the
National
Capitol
Area
Chapter
of
Society
of
Toxicology.
Her
major
research
areas
of
interests
are:
mechanisms
of
trace
metal
metabolism
and
cellular
toxicity
and
toxicity
of
ambient
air
particles.
She
has
contributed
to
hundreds
of
technical
books
and
journals.
Dr.
Squibb
has
also
written
IRIS
documents
for
the
USEPA
Office
of
Pesticides
Program.
Peer
Review
of
the
April
4th,
2003
United
States
EPA
Document
Titled
"
Preliminary
Risk
Assessment
of
the
Developmental
Toxicity
Associated
with
Exposure
to
Perfluorooctanoic
Acid
and
Its
Salts"

Prepared
by
Tetrahedron,
Inc.
April
8,
2003
Review
of
Risk
Assessment
for
PFOA
April
2003
Page
2
of
6
REVIEWER:
Steve
T.
Cragg,
Ph.
D.,
DABT
EPA
Issue
1.
Developmental
Endpoints
The
Agency's
Developmental
Toxicity
Risk
Assessment
Guidelines
state
that
the
period
of
exposure
for
developmental
toxicity
is
prior
to
conception
to
either
parent,
through
prenatal
development
and
continuing
until
sexual
maturation.
In
contrast,
the
period
during
which
a
developmental
effect
may
be
manifested
includes
the
entire
lifespan
of
the
organism.
Based
on
this
definition
of
developmental
exposure,
OPPT
considered
developmental
effects
to
include
reductions
in
F1
mean
pup
body
weight
(
sexes
combined)
on
lactation
days
1,
5
and
8,
an
increase
in
mortality
during
the
first
few
days
after
weaning
(
both
sexes),
a
delay
in
the
timing
of
sexual
maturation
(
both
sexes),
and
a
reduction
in
mean
body
weight
postweaning
(
F1
males
only).

EPA
Question
1.1
Please
comment
on
the
choice
of
developmental
endpoints
from
the
2­
generation
reproductive
toxicity
study
for
this
preliminary
risk
assessment.

Reviewer
Answer
to
Issue
1,
Question
1.1:

The
choice
of
endpoints
from
the
2­
generation
reproductive
toxicity
study
used
for
this
preliminary
risk
assessment
are
appropriate.
Reductions
in
mean
pup
body
weight,
mortality
until
weaning,
timing
of
sexual
maturation,
and
body
weight
postweaning
are
now
and
have
been
for
a
long
time,
appropriate
endpoints
monitored
for
reproductive
toxicity
studies.
In
fact,
these
endpoints
are
described
explicitly
in
the
harmonized
OECD
&
FIFRA
testing
protocol
guidelines
for
reproductive
toxicity
studies.

EPA
Response:
The
reviewer
agrees
with
EPA's
choice
of
developmental
endpoints.

EPA
Issue
2.
Use
of
Human
Biomonitoring
Data
Margins
of
exposure
(
MOE)
in
this
preliminary
risk
assessment
were
calculated
using
serum
levels
from
the
rat
two­
generation
reproductive
study
and
human
serum
levels
obtained
from
biomonitoring
data.
Approximately
600
individuals
were
involved
in
each
of
the
U.
S.
samples
of
adults
and
children
from
which
the
serum
data
were
derived
for
the
general
population
exposures.

EPA
Question
2.1
Please
comment
on
the
adequacy
of
the
human
exposure
data
for
use
in
calculating
a
MOE.

Reviewer
Answer
to
Issue
2,
Question
2.1:

The
human
data
seem
adequate
given
the
large
numbers
of
subjects
monitored.
The
similarity
in
levels
despite
age
or
sex
further
argues
that
results
do
not
reflect
an
atypical
cohort.

EPA
Response:
The
reviewer
states
that
the
human
biomonitoring
data
are
adequate.

EPA
Issue
3.
Use
of
Serum
Data
as
a
Measure
of
Internal
Dose
Review
of
Risk
Assessment
for
PFOA
April
2003
Page
3
of
6
Margins
of
exposure
(
MOE)
in
this
preliminary
risk
assessment
were
calculated
using
serum
levels
from
the
rat
two­
generation
reproductive
study
and
human
serum
levels
obtained
from
biomonitoring
data.

EPA
Question
3.1
Please
comment
on
the
use
of
serum
data
as
a
measure
of
internal
dose
for
rats
and
humans
for
calculation
of
the
MOE.

Reviewer
Answer
to
Issue
3,
Question
3.1:

Using
serum
data
as
a
measure
of
internal
dose
for
both
rats
and
humans
is
better
than
using
an
oral
dose
in
mg/
kg­
day
and
trying
to
calculate
a
dose
in
humans
with
similar
units
from
a
combined
inhalation
and
dermal
exposure.
The
latter
exercise
would
require
the
use
of
several
assumptions
fraught
with
uncertainty
and
therefore
requiring
perhaps
an
overly­
conservative
approach.
Use
of
serum
levels
"
normalizes"
any
absorption
differences
that
might
exist
between
an
oral
exposure
(
in
the
animal
study)
and
dermal
and
inhalation
exposures
(
the
routes
most
likely
in
humans),
rendering
moot
differences
in
absorbed
dose
caused
by
the
route
of
administration.
Moreover,
using
plasma
levels
precludes
having
to
account
for
possible
differences
in
subsequent
distribution
and
elimination
of
PFOA
that
might
exist
between
rats
and
humans.
Even
metabolism
differences
tend
to
be
minimized
if
humans
and
the
animal
model
metabolize
PFOA
similarly.
Since
PFOA
apparently
is
not
metabolized
to
a
proximate
toxin
but
is
excreted
unchanged,
it
is
reasonable
to
assume
that
the
parent
compound
is
toxic
itself
and
that
this
is
true
in
animals
or
humans.
However,
there
is
not
confirmatory
information
that
in
humans,
metabolism
does
not
occur.
All
the
human
studies
discussed
in
the
report
appear
to
monitor
PFOA
levels
and
not
investigate
possible
metabolites.
Thus,
plasma
levels
of
the
parent
PFOA
are
a
good
reflection
of
dose.

EPA
Response:
The
reviewer
agrees
with
the
use
of
the
serum
data.

EPA
Issue
4.
Use
of
F0
serum
levels
as
an
estimate
of
serum
levels
of
F1
animals
The
rat
serum
levels
measured
in
the
2­
generation
reproductive
toxicity
study
were
used
as
an
estimate
of
internal
dose.
However,
serum
levels
were
measured
only
in
the
F0
animals
and
only
measured
in
the
control,
10,
and
30
mg/
kg/
day
groups,
not
in
the
1
and
3
mg/
kg/
day
groups.
The
concern
for
developmental
toxicity
is
for
F1
animals.
The
assumption
as
been
made
that
the
serum
levels
measured
in
the
F0
animals
are
the
bast
available
dose
estimate
for
the
F1
animals.

EPA
Question
4.1
Please
comment
on
the
use
of
F0
serum
values
as
a
dose
estimate
for
the
F1
rats.

Reviewer
Answer
to
Issue
4,
Question
4.1:

Since
F1
serum
values
were
not
collected,
the
F0
values
must
serve
as
surrogates.
Even
though
exposure
to
the
test
substance
was
for
a
greater
portion
of
their
lives,
it
does
seem
reasonable
to
assume
that
a
second
generation
would
metabolize
the
ammonium
salt
of
PFOA
in
a
manner
similar
to
their
parents.

EPA
Response:
The
reviewer
agrees
with
the
use
of
the
serum
data
from
the
F0
animals.
Review
of
Risk
Assessment
for
PFOA
April
2003
Page
4
of
6
EPA
Question
4.2
Please
comment
on
the
use
of
serum
values
for
F0
males
as
an
upper
estimate
of
peak
exposure
for
F0
females.

Reviewer
Answer
to
Issue
4,
Question
4.2:

Using
the
male
plasma
values,
which
are
50
­
100
fold
higher
than
females,
profoundly
increases
the
MOE.
The
fact
that
male
serum
levels
are
"
saturated"
may
actually
mean
they
are
more
representative
of
toxic
dose
levels
particularly
when
serum
levels
were
measured
so
long
after
dosing
(
i.
e.,
24
hours).
The
toxic
manifestations
of
a
chemical,
when
expressed
as
a
plasma
"
dose"
level,
are
more
likely
incurred
at
a
peak
level
than,
as
in
the
females,
when
rapid
excretion
has
reduced
that
level
drastically.
This
would
argue
that
the
male
levels
are
more
representative
of
doses
that
actually
caused
the
toxic
injury.

EPA
Response:
The
reviewer
agrees
with
the
approach
used
by
EPA.

EPA
Question
4.3
Recent
studies
suggest
that
three
organic
anion
transporters
(
OAT),
OAT1,
OAT2,
and
OAT3,
are
involved
in
the
renal
transport
of
PFOA.
A
study
of
developmental
and
gender­
specific
influences
on
the
expression
of
rat
OAT
in
the
kidney
has
shown
that
at
birth
all
OAT
mRNA
levels
were
low.
Renal
OAT1
expression
approaches
adult
level
at
30
days,
where
at
day
40
and
45
OAT2
levels
were
greater
in
males
than
females.
OAT2
expression
was
minimal
through
day
30
but
increased
dramatically
only
in
females
at
day
35.
OAT3
expression
matured
the
earliest
and
reached
adult
levels
at
10
days.
Please
comment
on
the
significance
of
OAT
data
for
estimating
internal
dose
on
the
postweaning
rat.

Reviewer
Answer
to
Issue
4,
Question
4.3:

The
authors
of
the
report
assume
that
OAT2,
which
levels
are
dramatically
higher
in
female
rats
only,
accounts
for
the
sex
difference
in
the
more
rapid
excretion
of
PFOA
by
females
(
and
consequent
lower
plasma
levels).
If
true,
and
if
OAT2
doesn't
develop
until
approximately
day
40,
then
young
females
might
have
been
subject
to
higher
plasma
levels
than
after
40
days
of
age.
This
supports
the
argument
that
adult
female
plasma
levels,
being
measured
24
hours
after
dosing
and
so
rapidly
excreted,
are
low
estimates
of
rat
exposure
to
PFOA.
All
this
discussion
of
OAT
notwithstanding,
it
seems
such
rapid
elimination
by
females,
coupled
with
sampling
so
long
after
dosing,
would
surely
tend
to
underestimate
dose
levels.
It
would
be
important
if
parallels
could
be
established
in
humans
but
such
human
studies
have
not
been
conducted.

EPA
Response:
The
reviewer
agrees
that
the
OAT
data
suggest
that
serum
PFOA
levels
may
be
higher
in
prepubescent
female
rat
than
in
the
adult
rat,
but
the
actual
difference
cannot
be
quantitated
at
this
time.
Review
of
Risk
Assessment
for
PFOA
April
2003
Page
5
of
6
General
Comments:

1.
The
reproductive
toxicity
study
that
serves
as
the
animal
extrapolation
basis
for
this
risk
assessment
appears
to
have
been
well
conducted.
Evaluation
of
the
experimental
design
and
monitored
endpoints
shows
that
the
study
satisfies
the
testing
requirements
of
OPPTS
870.3800
"
Reproduction
and
Fertility
Effects,"
as
well
as
the
harmonized
OECD
Guideline
for
Testing
of
Chemicals
Protocol
416
"
Two­
Generation
Reproduction
Toxicity
Study."

EPA
Response:
The
reviewer
agrees
that
the
two
generation
reproductive
toxicity
study
was
an
appropriately
designed
and
conducted
study.

2.
When
the
rat
reproductive
toxicity
study
is
discussed,
serum
levels
with
units
of
µ
g/
ml
are
used.
When
human
survey
data
are
discussed
later
in
the
report,
units
of
ppm
are
used,
without
explicit
definition
in
Tables
2
and
3
(
or
in
the
narrative),
that
these
units
are
equivalent.
Suggest
using
µ
g/
ml
(
nanog/
ml)
or
ppm
(
ppb)
throughout
the
report.

EPA
Response:
The
text
of
the
preliminary
risk
assessment
has
been
changed
so
that
all
serum
levels
are
expressed
as
ppm
or
ppb.

3.
In
the
reproductive
toxicity
study
in
rats,
the
authors
of
the
report
concluded
that
the
delayed
maturation
seen
in
the
F1
subjects
was
due
to
"
decreased
gestational
age."
What
exactly
this
meant
was
not
clearly
explained
in
the
report.
If
it
means
that
the
time
of
gestation
was
shorter
in
the
30
mg/
kg­
day
group
than
in
the
controls,
then
the
time
between
birth
and
sexual
maturation
could
indeed
be
longer
simply
because
the
F1
rats
were
born
less
mature.
If
so,
the
delay
would
be
explained
merely
because
of
the
more
immature
status
of
the
high
dose
rats
at
birth
and
not
an
effect
of
PFOA.
If
on
the
other
hand,
the
authors
of
the
report
are
defining
"
decreased
gestational
age"
not
as
a
result
of
premature
birth
but
some
sort
of
lessened
maturity
at
birth
due
to
the
toxic
effects
of
PFOA,
perhaps
as
reflected
in
decreased
birth
weight,
then
this
may
be
reaching.
However,
the
critique
by
EPA
of
the
authors
statistical
evaluation
of
this
question
seemed
a
little
dismissive.
What
additional
data
would
have
satisfied
EPA?
Suggest
this
issue
be
clarified
in
the
final
draft.

EPA
Response:
The
reviewer
indicates
that
EPA
has
dismissed
the
argument
that
the
delayed
sexual
maturation
of
the
F1
pups
in
the
two
generation
reproductive
toxicity
study
is
due
to
"
decreased
gestational
age"
for
statistical
reasons.
The
reviewer
states
that
EPA
did
not
provide
a
definition
of
gestational
age
and
also
indicates
that
EPA
should
state
what
additional
data
would
need
to
be
provided
to
support
this
hypothesis.

EPA
has
provided
a
sufficient
discussion
of
this
issue
as
well
as
an
indication
of
the
data
that
would
be
needed
to
support
the
hypothesis
in
section
3.5.
As
stated
in
the
discussion,
the
authors
of
the
study
defined
gestational
age
as
the
time
in
days
from
evidence
of
mating
in
the
F0
generation
until
evidence
of
sexual
maturation
in
the
F1
generation.
EPA's
analysis
of
the
data
is
as
follows:

"
While
it
is
known
and
commonly
accepted
that
changes
in
the
body
weights
of
offspring
can
affect
the
time
to
sexual
maturation,
whether
or
not
gestational
age,
as
defined
by
the
authors,
also
affects
the
time
of
sexual
maturation
is
purely
speculative,
especially
since
there
were
no
data
provided
by
the
authors
to
support
this
relationship.
Additionally,
covarying
gestational
age
with
time
to
sexual
maturation
is
problematic
from
a
statistical
standpoint.
Since
there
was
no
significant
change
in
the
length
of
gestation
Review
of
Risk
Assessment
for
PFOA
April
2003
Page
6
of
6
at
30
mg/
kg/
day,
based
on
the
authors'
definition
of
`
gestational
age',
the
decreases
in
gestational
age
would
have
to
be
due
mostly
to
changes
in
time
to
sexual
maturation.
Therefore,
sexual
maturation
is
essentially
being
covaried
with
itself.
Still,
even
if
a
relationship
between
gestational
age
and
time
to
sexual
maturation
were
shown,
it
merely
offers
an
explanation
for
the
observed
delays
in
sexual
maturation
in
high­
dose
animals,
but
does
not
diminish
its
significance."

4.
The
report
is
well
written
and
generally
clear
in
presenting
the
data
and
explaining
conclusions.
The
following
are
a
few
specific
comments
that
focus
mostly
on
issues
of
clarity.

Specific
Comments:

Page
11,
second
full
paragraph
Were
non­
ionic
fluorine
levels
assumed
to
be
PFOA
absorbed
to
plasma
proteins?
The
review
of
this
study
should
so
state
even
though
other
studies
indicate
that
PFOA
is
not
metabolized.

EPA
Response:
Yes.
The
text
has
been
clarified.

Page
43,
Table
2
Don't
force
the
reader
to
peruse
the
narrative
to
determine
the
units
in
the
table.
The
table
should
be
able
to
"
stand­
alone."
Also
define
ppm
as
µ
g/
ml
serum.

EPA
Response:
The
text
has
been
changed
so
all
serum
values
are
reported
as
ppm
or
ppb.

Page
45,
Table
3
Same
comment.
Add
footnote
denoting
units
and
define
ppb
as
nanograms
per
ml
serum.

EPA
Response:
The
text
has
been
changed
so
all
serum
values
are
reported
as
ppm
or
ppb.

Page
53,
Table
6
The
left
column
is
titled
"
Human
Serum
Values"
yet
none
appear
below.
It
would
make
the
table
clear
if
this
title
were
deleted
and
the
corresponding
title
in
the
right
column
were
renamed
"
Rat
Serum
Values
Divided
by
Human
Serum
Values."
Or,
the
title
in
the
right
column
could
simply
be
titled
"
MOEs"
with
a
footnote
defining
MOE.
As
with
the
previous
tables,
a
footnote
should
be
adding
defining
ppm
as
micrograms
per
liter.

EPA
Response:
Table
6
has
been
clarified.
Peer
Review
of
the
April
4th,
2003
United
States
EPA
Document
Titled
"
Preliminary
Risk
Assessment
of
the
Developmental
Toxicity
Associated
with
Exposure
to
Perfluorooctanoic
Acid
and
Its
Salts"

Prepared
by
Tetrahedron,
Inc.
April
8,
2003
REVIEWER:
Henry
Spencer,
Ph.
D
QUESTIONS:
Issue
I:
The
Agency's
Developmental
Toxicity
Risk
Assessment
Guidelines
state
that
the
period
of
exposure
for
developmental
toxicity
is
prior
to
conception
to
either
parent,
through
prenatal
development
and
continuing
until
sexual
maturation.
In
contrast,
the
period
during
which
a
developmental
effect
may
be
manifested
includes
the
entire
lifespan
of
the
organism.
Based
on
this
definition
of
developmental
exposure,
OPPT
considered
developmental
effects
to
include
reductions
in
F1
mean
pup
body
weight
(
sexes
combined)
on
lactation
days
1,
5
and
8,
an
increase
in
mortality
during
the
first
few
days
after
weaning
(
both
sexes),
a
delay
in
the
timing
of
sexual
maturation
(
both
sexes),
and
a
reduction
in
mean
body
weight
postweaning
(
F1
males
only).

Response:

Developmental
toxicity
is
obviously
a
situation
which
may
well
have
its
beginning
in
the
time
frame
of
preconception
through
exposure
of
the
parents.
Exposure
throughout
the
gestational
period
and
weaning
to
the
end
of
not
only
sexual
maturation
but
to
include
the
end
of
development
to
a
mature
adult
animal.
I
personally
would
probably
limit
the
definition
to
that
and
not
include
the
entire
lifespan
of
the
organism.

EPA
Response:

EPA
has
used
the
definition
that
is
provided
in
the
Agency's
Developmental
Toxicity
Risk
Assessment
Guidelines.

Question
1.1:

Please
comment
on
the
choice
of
developmental
endpoints
from
the
2­
generation
reproductive
toxicity
study
for
this
preliminary
risk
assessment.

Response:

Generally,
the
data
in
the
document
do
not
provide
evidence
that
developmental
toxicity
is
seen
in
other
species
that
does
not
also
occur
in
the
rat
2­
generation
reproduction
study.
The
only
reason
that
I
can
see
that
we
would
use
the
reproduction
study
is
that
the
exposure
that
occurs
there
is
carried
thru
to
maturity,
precohabitation,
gestation,
lactation,
weaning
and
maturity
again.

We
see
that
for
the
most
part,
the
effects
occurring
at
the
lowest
dose
are
in
the
F1
generation
and
occurs
in
both
sexes.
Lactational
early
weight
losses
generally
are
a
consequence
of
milk
transfer
and
may
well
stem
from
a
high
serum
level
of
test
material.
NOAEL
=
10
mg/
kg
in
F1
females.
3
Mortality
in
the
F1
males
and
females
at
30
mg/
kg
in
early
days
after
weaning.
NOAEL=
10
mg/
kg
Delay
in
sexual
maturation
in
F1
females
and
F1
males
at
30
mg/
kg
NOTE:
This
was
a
gavage
study
with
very
high
serum
spikes
of
test
material
occurring
while
the
usual
study
is
an
oral
feeding
study
which
usually
does
not
provide
the
very
high
serum
levels
in
a
short
period
of
time.
This
type
of
serum
level
often
has
a
detrimental
effect
more
often
than
does
the
serum
levels
obtained
from
the
feeding
study.

EPA
Response:

EPA
has
stated
that
developmental
effects
were
noted
in
a
prenatal
developmental
toxicity
study
in
rabbits
and
in
the
two
generation
reproductive
toxicity
study
in
rats.
Both
studies
were
considered
important
for
the
assessment.
However,
only
the
two
generation
reproductive
toxicity
study
was
used
in
the
calculation
of
the
MOEs
as
serum
levels
were
not
measured
in
the
rabbit
study.
Uncertainties
associated
with
the
choice
of
the
rat
study
are
addressed
in
section
5.5.

Issue
2:
Use
of
Human
Biomonitoring
Data
Margins
of
exposure
(
MOE)
in
this
preliminary
risk
assessment
were
calculated
using
serum
levels
from
the
rat
two­
generation
reproductive
study
and
human
serum
levels
obtained
from
biomonitoring
data.
Approximately
600
individuals
were
involved
in
each
of
the
U.
S.
samples
of
adults
and
children
from
which
the
serum
data
were
derived
for
the
general
population
exposures.

Response:

The
use
of
human
biomonitoring
data,
if
relevant,
is
probably
the
best
that
can
be
obtained.
Because
this
is
a
preliminary
risk
assessment
one
should
use
the
most
reasonable
data
that
can
be
obtained
.

EPA
Response:

Reviewer
agrees
with
EPA
approach.
No
comment.

Question
2.1:

Please
comment
on
the
adequacy
of
the
human
exposure
data
for
use
in
calculating
a
MOE.
4
Response:

I
believe
that
the
data
are
probably
not
too
bad
if,
used
properly.
However,
the
data
from
the
blood
banks
with
stratification
to
sex
and
age
groups
and
covering
the
few
cities
most
likely
are
the
best
that
you'll
have
at
this
time.

I
think
that
since
you
are
producing
MOEs,
that
the
range
of
values
should
also
be
used
in
producing
the
MOEs
in
ranges.

I
also
believe
that
more
data
should
be
obtained
to
more
thoroughly
address
the
question
of
reality.
Without
seeing
the
study,
I
do
not
believe
that
the
Streptococcal
childrens
data
would
necessarily
represent
anymore
than
one
segment
of
the
population
of
children,
those
living
in
possibly
the
lower
economic
levels
of
the
District
of
Columbia.

EPA
Response:

In
general,
the
reviewer
appears
to
agree
with
the
use
of
these
data.
EPA
has
revised
section
5.4
to
clarify
issues
associated
with
the
use
of
the
mean
and
range.
EPA
is
not
clear
as
to
the
intent
of
the
last
paragraph
of
the
reviewers
comments
above.
However,
to
clarify,
the
sample
of
children
included
598
children
from
23
states
and
the
District
of
Columbia.

Issue
3:
Use
of
Serum
Data
as
a
Measure
of
Internal
Dose
Margins
of
exposure
(
MOE)
in
this
preliminary
risk
assessment
were
calculated
using
serum
levels
from
the
rat
two­
generation
reproductive
study
and
human
serum
levels
obtained
from
biomonitoring
data.

Response:

The
use
of
serum
as
a
measure
of
internal
dose
is
probably
as
good
an
indicator
of
internal
dose
without
actually
measuring
the
dose
given.
Naturally,
the
use
of
serum
is
only
as
good
as
the
limitations
that
are
set
upon
it
by
the
test
material.
In
this
case,
perfluorooctanoic
acid
is
a
reasonably
good
candidate
to
give
the
correct
results
in
serum.
However,
one
sex
does
not
handle
the
chemical
as
well
as
the
other
and
serum
results
must
be
evaluated
differently
for
each
sex.
The
female
rat
appears
to
handle
organic
acids
better
than
the
male
rat
with
altered
secretion
by
virtue
of
sex
hormones
with
the
result
that
the
serum
½
life
is
very
long
in
the
male
and
relative
short
in
the
female.

The
use
of
serum
values
in
humans
is
plausible,
but
little
is
known
of
the
excretion/
secretion
of
the
perfluorooctanoic
acid
moiety
in
this
species.
There
are
no
data
on
the
possible
sex
differences
on
the
excretion
rates
of
this
chemical.
5
EPA
Response:

In
general,
the
reviewer
agrees
with
the
MOE
approach.
No
comment.

Question
3.1:

Please
comment
on
the
use
of
serum
data
as
a
measure
of
internal
dose
for
rats
and
humans
for
calculation
of
the
MOE.

There
are
data
on
a
number
of
human
subjects
with
perflurooctanoic
acid
serum
values.
The
data
include
both
occupational
and
the
general
public.
These
data
only
provide
a
cursory
insight
into
the
possible
real
body
burden
of
the
chemical.
Further
studies
will
be
needed
to
find
the
real
activity
of
the
chemical
in
the
body.

The
data
on
rats
is
much
greater
than
found
for
humans.
However,
the
use
of
the
female
and
male
data
in
the
reproduction
study
must
be
tempered
with
caution.
The
male
data
appear
to
represent
a
real
level
of
relatively
high
constant
internal
exposure.
While
the
female
serum
when
taken
at
the
same
time
showed
very
low
levels
of
chemical.
Data
are
present
in
the
report
which
indicate
that
with
a
2
mg
dose
the
female
rat
has
the
initial
very
high
spike
at
4.5
hours
of
approximately
13
ppm.
By
8
hours
the
values
are
approximately
11
ppm
and
by
24
hours
the
value
has
dropped
to
the
normal
of
approximately
0.35
ppm
which
is
essentially
the
same
as
the
value
of
0.37
ppm
reported
in
reproduction
study.

It
seems
reasonable
that
there
might
be
enough
data
in
this
additional
study
to
be
able
to
use
it
as
the
values
for
MOEs
instead
of
using
the
lowest
dose
of
0.35
ppm.
I
don't
see
any
other
way
to
do
a
risk
assessment
with
MOEs
in
humans
without
some
data
and
human
serum
data
is
the
best
you
have
at
this
time.

EPA
Response:

In
general,
the
reviewer
agrees
with
the
use
of
serum
levels
as
a
measure
of
internal
dose.
The
reviewer
suggests
that
the
kinetics
data
from
the
Ophaug
and
Singer
(
1980)
study
could
be
used
to
better
delineate
the
serum
levels
in
the
female
rat.
EPA
has
addressed
the
uncertainties
associated
with
several
of
the
existing
studies
which
provide
kinetic
and
half
life
information
in
section
5.5.
Generally,
these
studies
were
not
used
as
there
was
a
wide
variation
in
the
rat
strains,
method
of
dosing,
method
of
measuring
serum
PFOA,
and
half
life.

Issue
4:
Use
of
F0
serum
levels
as
an
estimate
of
serum
levels
of
F1
animals
The
rat
serum
levels
measured
in
the
2­
generation
reproductive
toxicity
study
were
used
as
an
estimate
of
internal
dose.
However,
serum
levels
were
measured
only
in
the
F0
animals
and
only
measured
in
the
control,
10,
and
30
mg/
kg/
day
groups,
not
in
the
1
and
3
mg/
kg/
day
groups.
The
concern
for
developmental
toxicity
is
for
the
F1
animals.
The
6
assumption
has
been
made
that
the
serum
levels
measured
in
the
F0
animals
are
the
best
available
dose
estimate
for
the
F1
animals.

Question
4.1:

Please
comment
on
the
use
of
F0
serum
values
as
a
dose
estimate
for
the
F1
rats.

Response:

The
serum
levels
measured
in
the
2­
generation
reproduction
study
only
represent
a
part
of
the
real
story
in
the
quest
for
an
internal
dose
evaluation
and
subsequent
MOEs.
We
know
from
the
data
in
the
reports
presented
that:
1.
PFOA
is
well
and
relatively
rapidly
absorbed
from
the
stomach.
2.
The
report
by
Ophaug
and
Singer
indicates
a
high
spike
of
serum
PFOA
levels
following
2
mg
(
approx.
6.6
mg/
kg)
which
persist
for
at
least
close
to
12
hours
where
after
the
level
reaches
the
values
found
in
the
reproduction
study
of
0.37
ppm.
3.
The
reproduction
study
indicates
that
the
male
rat
serum
levels
remain
very
high
and
stable
for
considerable
time
and
that
a
value
that
stable
would
be
repeatable
time
after
time.
4.
Since
the
value
of
0.37
ppm
does
NOT
occur
immediately,
and
there
is
data
to
indicate
that
the
PFOA
levels
stay
elevated
for
at
least
one
half
the
24
hour
time
period,
the
0.37
ppm
value
can
not
represent
the
serum
internal
burden
for
females.

EPA
Response:

In
general,
the
reviewer
agrees
with
the
uncertainties
that
EPA
has
addressed
concerning
the
serum
levels
in
the
female
and
male
rats.
EPA
has
repeatedly
stressed
that
the
available
serum
data
for
the
females
in
the
two
generation
reproductive
toxicity
study
were
obtained
24
hours
after
dosing
and
therefore
represent
the
low
end
of
the
internal
dose
in
the
female
rat.
EPA
has
also
repeatedly
stressed
that
due
to
the
gender
differences
in
the
elimination
of
PFOA
in
the
rat,
the
serum
levels
in
the
male
rat
are
high
due
to
bioaccumulation
and
saturation.

Question
4.2:

Please
comment
on
the
use
of
serum
values
for
F0
males
as
an
upper
estimate
of
peak
exposure
for
F0
females.

Response:

With
regard
to
the
possible
use
of
serum
values
for
F0
males
as
an
upper
estimate
of
peak
exposure
for
F0
females
it
would
seem
inadvisable
without
a
study
or
data
to
support
it
as
reasonable.
Since
the
males
are
treated
each
day
by
gavage
and
tend
to
not
be
able
to
transport
the
organic
acid
as
does
the
females,
the
likelihood
that
levels
would
build
up
in
the
males
over
the
peak
single
exposure
in
a
24
hour
period.
The
females
which
are
normally
able
to
excrete
the
organic
acid
are
able
to
keep
the
levels
low.
However,
the
data
in
the
rat
study
support
that
the
females
at
30
mg/
kg
are
showing
an
increasing
concentration
of
organic
acid
when
compared
7
to
the
females
receiving
only
10
mg/
kg.
The
higher
doses
in
the
males
will
be
even
higher
and
shows
the
effects
of
being
saturated
in
the
transport
mechanism
at
the
10
and
30
mg/
kg
doses.
Since
these
effects
are
not
as
pronounced
in
the
females,
it
is
highly
unlikely
that
the
serum
data
of
the
F0
males
should
be
surrogates
for
the
F0
females
as
an
upper
estimate
of
peak
exposure.

EPA
Response:

In
general,
the
reviewer
agrees
with
the
descriptions
that
EPA
has
provided
regarding
the
serum
levels
in
the
male
rat.
EPA
has
stated
that
the
serum
levels
in
the
male
rats
are
probably
much
higher
than
the
peak
dose
in
the
female
rat
due
to
bioaccumulation
and
saturation.
EPA
has
also
stressed
that
the
serum
levels
for
the
F0
male
rats
simply
represent
an
upper
end
estimate
and
that
the
peak
levels
in
the
F0
female
rats
are
probably
lower.

Question
4.3:

Recent
studies
suggest
that
three
organic
anion
transporters
(
OAT),
OAT1,
OAT2,
and
OAT3,
are
involved
in
the
renal
transport
of
PFOA.
A
study
of
developmental
and
genderspecific
influences
on
the
expression
of
rat
OAT
in
the
kidney
has
shown
that
at
birth
all
OAT
mRNA
levels
were
low.
Renal
OAT1
expression
approaches
adult
level
at
30
days,
where
at
day
40
and
45
OAT1
levels
were
greater
in
males
than
females.
OAT2
expression
was
minimal
through
day
30
but
increased
dramatically
only
in
females
at
day
35.
OAT3
expression
matured
the
earliest
and
reached
adult
levels
at
10
days.
Please
comment
on
the
significance
of
OAT
data
for
estimating
internal
dose
on
the
postweaning
rat.

Response:

These
organic
anion
transporters
OAT,
thru
OAT3
are
involved
with
the
transport
of
PFOA.
The
times
frames
in
which
they
mature
in
the
postweaning
rat
are
significant
to
its
well
being
in
the
study
cases
in
the
documents.
As
we
can
see
the
OAT3
and
the
OAT2
in
females
show
expression
earlier
than
in
males
while
only
OAT1
is
at
greater
levels
in
the
male
than
in
females
at
the
same
time.
This
data
indicates
that
the
male
is
in
jeopardy
if
he
has
less
of
an
OAT
level
than
the
females
taking
in
high
levels
of
PFOA
at
an
earlier
age
such
as
10­
20
days
post
weaning.
In
addition,
the
OAT
2
and
OAT
3
transporters
are
the
most
active
in
the
female
rat.
I
don't
believe
that
the
OAT
data
are
useful
for
estimating
internal
dose
on
the
postweaning
rat.
Each
rat
will
have
its
own
complement
or
level
of
OAT
and
will
only
approximate
any
other
rat.

EPA
Response:

The
reviewer
agrees
that
maturation
of
the
OATs
will
influence
the
elimination
of
PFOA
in
the
prepubescent
rat.
However,
it
is
not
possible
to
quantify
the
impact
at
this
time.
EPA
agrees
and
has
addressed
this
in
section
5.5.
8
Comments
on
a
page­
by­
page
basis.

PP­
1
par.
2­
line2
It
might
be
better
to
indicate
the
numbers
of
males
and
females
in
this
sentence.
EPA
Response:
Correction
made.

PP­
1
par
2­
line
9
Please
indicate
that
there
are
also
dogs
and
there
appears
to
be
a
sex
difference.
EPA
Response:
The
dog
study
is
described
in
detail
in
section
3.2.
EPA
does
not
agree
that
this
study
should
be
included
in
the
executive
summary.

PP­
1
par
2­
last
line
This
gender
difference
has
not
been
studied
in
humans.
EPA
Response:
This
has
been
clarified
in
the
text.

PP­
1
par
3­
last
line
Suggesting
exposure
from
some
low
level
source.
EPA
Response:
Interesting
speculation.
No
comment.

PP­
2
par­
1­
line1
The
large
majority
of
production
workers­­­­
are
males
EPA
Response:
This
comment
appears
to
state
that
proper
grammar
would
dictate
the
use
of
the
plural
for
males.
EPA
disagrees
as
the
subject
of
the
sentence
(
ie.
majority)
is
singular
not
plural.
PP­
2
par­
5­
last
line
Determined
24
hours
after
the
last
dosing
EPA
Response:
The
details
regarding
the
serum
levels
are
provided
in
the
preliminary
risk
assessment
section
of
the
executive
summar.

PP­
3
par­
last
The
study
should
note
it
was
by
oral
gavage,
and
the
length
of
the
dosing
is
80
days.
EPA
Response:
These
details
are
provided
in
section
3.5
and
do
not
need
to
be
repeated
in
the
executive
summary.

PP­
8
par­
2
The
MP
is
45­
?
C.
Is
this
55
C?
EPA
Response:
The
text
was
corrected.

PP­
8
par­
last
APFO
did
not
recently
report....
Authors......
recently
reported.
EPA
Response:
The
text
was
corrected.

PP­
10
par­
2
Only
provided
1/
3
of
the
information.
How
about
the
other
retirees?
EPA
Response:
The
information
was
added
to
the
text.

PP­
10
par­
3­
line
5
It
is
stated
that
the
data
cannot
be
pooled
or
averaged
unless
the
decay
curves
show
first­
order
kinetics.
But
in
the
par.
above
the
data
is
averaged.
Wouldn't
it
be
better
to
show
the
data
in
a
table
to
give
us
an
idea
of
the
variation.
9
EPA
Response:
The
text
was
clarified.
The
data
are
too
preliminary
to
present
in
a
table
at
this
time.

PP­
11
par­
2
This
Ophaug
and
Singer
study
certainly
appears
to
have
been
overlooked
or
totally
neglected
in
finding
the
high
serum
levels
of
the
female
rats
at
as
early
as
4.5
hours
after
a
single
2
mg
gavage
dose.

EPA
Response:
The
limitations
in
using
the
kinetic
information
from
this
study
as
well
as
the
other
kinetic
studies
were
addressed
in
section
5.5.

PP­
12
par­
1
line3
At
a
doses
of
10
­
doses
should
be
singular.
EPA
Response:
The
text
was
corrected.

PP­
21
par­
2
line­
2
Please
state
whether
this
hormone
testing
was
for
males
or
females?
EPA
Response:
The
text
was
clarified.

PP­
23
par­
2
line­
5
Spell
the
95%
or
change
the
sentence.
EPA
Response:
The
text
was
corrected.

PP­
28
par
last
line­
1
What
was
the
%
or
grams
of
weight
reduction?
EPA
Response:
This
comment
refers
to
a
transient
weight
reduction
in
the
pregnant
animal.
EPA
does
not
believe
that
this
level
of
detail
is
needed.

PP­
29
par­
3
line­
8
After
the
1
ml/
kg
please
add
"
of
test
material"
EPA
Response:
EPA
disagrees
as
this
refers
only
to
the
dose
volume,
not
specifically
to
the
volume
of
APFO.

PP­
30
par­
1
Was
the
increase
%
of
total
pups
or
by
%
litters?
EPA
Response:
The
study
authors
did
the
analysis
on
a
per
pup
basis.

PP­
30
par­
2
In
the
inhalation
study
is
it
possible
to
put
the
mg/
m3
into
mg/
kg/
day?
EPA
Response:
EPA
does
not
think
this
is
appropriate.

PP­
32
In
the
York
study,
were
the
litters
culled
or
were
they
left
to
litter
as
they
could?

EPA
Response:
The
litters
were
not
culled.
The
text
was
clarified.

PP­
34
par­
1
line­
5
How
far
from
the
normal
weighing
dates
were
the
dates
of
sexual
maturation/
Would
it
make
difference
in
the
reporting
of
body
wt.
values?
EPA
Response:
The
covariate
analysis
of
the
day
of
sexual
maturation
and
body
weight
was
conducted
and
is
presented
in
the
text.
10
PP­
35
par­
5
line­
4
Treated
is
still
misspelled.
EPA
Response:
The
text
was
corrected.

PP­
36
par­
3
line­
3
There
is
a
slash
mark
missing
after
the
ug
.
EPA
Response:
The
text
was
corrected.

PP­
36
par­
5
line­
1
Is
it
possible
to
put
back
in
the
30mg/
kg/
day
here?
EPA
Response:
The
text
was
clarified.

PP­
39
par­
5
line­
3
Was
there
data
available
to
indicate
the
sex
of
the
pups
found
dead
with
no
milk
in
their
stomachs?
EPA
Response:
EPA
does
not
agree
that
this
information
is
necessary.

PP­
40
par­
2
line­
1
Were
the
increases
in
dead
pups
on
a
litter
basis?
EPA
Response:
Yes,
the
prenatal
and
lactational
deaths
were
analyzed
on
a
litter
basis
by
the
study
authors,
as
well
as
by
EPA
statisticians.

PP­
46
par­
2
What
were
the
ages
that
were
included
in
calculations
for
child­
bearing?
If
so,
then
the
data
from
the
different
blood
banks
and
programs
should
be
culled
to
note
that
and
use
only
that
data.
Please
not
that
this
has
happened
here.
We
didn't
use
the
2­
8
year
females
in
the
.
or
the
69
year
females
in
the
calculations.
EPA
Response:
EPA
had
gender
specific
data
for
the
geometric
mean
and
range,
but
not
for
the
arithmetic
mean.
Since
the
geometric
means
for
the
males
and
females
were
very
similar
(
4.2
ppb
for
females
and
4.9
ppb
for
males),
the
value
used
in
the
MOE
calculation
was
the
mean
for
sexes
combined
which
was
4.6
ppb.
EPA
does
not
think
that
this
significantly
impacts
the
calculations.

PP­
49
par­
11ine­
14
At
10
mg/
kg
don't
the
data
indicate
that
the
body
wts
started
before
day
36
postweaning?
EPA
Response:
The
text
was
clarified.

PP­
49
par­
1
line­
16
Please
explain
this
sentence
"
No
treatment
related
effects
were
observed
at
any
doses
test
in
the
study."?
EPA
Response:
This
sentence
was
referring
to
the
F2
pups.

PP­
55
par­
4
line­
10
Is
the
10
mg/
kg
correct
or
is
it
2
mg/
kg?
EPA
Response:
Actually
they
administered
a
total
dose
of
2
mg
to
the
female
rats
that
weighed
250
g.
This
is
actually
a
dose
of
8
mg/
kg.
The
text
was
corrected.

A
General
comment
for
the
document
is
that
it
is
fairly
complete.
I'm
not
sure
just
why
the
writer
appears
to
be
not
aware
of
a
piece
of
data
that
would
give
a
better
estimate
of
a
high
value
and
shows
the
decrease
in
serum
F
over
a
period
of
almost
24
hours
which
gives
the
almost
identical
value
at
that
point
as
the
one
in
the
rat
reproductive
toxicity
study.
11
EPA
Response:
This
comment
has
been
addressed
above.

Please
allow
the
reader
to
see
the
real
data
numbers
interspersed
in
the
individual
study
writeups
This
would
give
a
better
support
to
the
statements
such
as
statistically
significantly
increased
or
decreased.

EPA
Response:
Where
appropriate,
these
details
have
been
provided.
Peer
Review
of
the
April
4th,
2003
United
States
EPA
Document
Titled
"
Preliminary
Risk
Assessment
of
the
Developmental
Toxicity
Associated
with
Exposure
to
Perfluorooctanoic
Acid
and
Its
Salts"

Prepared
by
Tetrahedron,
Inc.
April
8,
2003
1
REVIEWER:
Katherine
Squibb,
Ph.
D.

INTRODUCTION
On
April
4,
2003,
the
U.
S.
EPA's
Office
of
Pollution,
Prevention,
and
Toxics
released
a
preliminary
risk
assessment
document
on
perfluorooctanoic
acid
and
its
salts.
As
part
of
the
review
process,
the
U.
S.
EPA
requested
comments
addressing
four
separate
issues
evaluated
in
the
preliminary
risk
assessment.
Tetrahedron,
Inc.
has
carefully
reviewed
the
preliminary
risk
assessment,
and
has
generated
responses
to
each
of
the
issues
requiring
comment.
Responses
to
each
issue
appear
below.

RESPONSES
Issue
1:
Developmental
Endpoints
The
Agency's
Developmental
Toxicity
Risk
Assessment
Guidelines
state
that
the
period
of
exposure
for
developmental
toxicity
is
prior
to
conception
to
either
parent,
through
prenatal
development
and
continuing
until
sexual
maturation.
In
contrast,
the
period
during
which
a
developmental
effect
may
be
manifested
includes
the
entire
lifespan
of
the
organism.
Based
on
this
definition
of
developmental
exposure,
OPPT
considered
developmental
effects
to
include
reductions
in
F1
mean
pup
body
weight
(
sexes
combined)
on
lactation
days
1,
5
and
8,
an
increase
in
mortality
during
the
first
few
days
after
weaning
(
both
sexes),
a
delay
in
the
timing
of
sexual
maturation
(
both
sexes),
and
a
reduction
in
mean
body
weight
postweaning
(
F1
males
only).

U.
S.
EPA
question
1.1
requested
comment
on
the
choice
of
developmental
endpoints
from
the
two­
generation
reproductive
toxicity
study
that
served
as
the
basis
of
the
preliminary
risk
assessment.

Tetrahedron
concurs
with
the
U.
S.
EPA's
decision
to
base
the
preliminary
risk
assessment
of
perfluorooctanoic
acid
(
PFOA)
upon
developmental
effects
reported
in
the
York
(
2002)

twogeneration
study.
Among
the
various
types
of
toxicity
studies
presented
in
the
U.
S.
EPA's
April
4,
2003
preliminary
risk
assessment
and
November
4,
2002
draft
hazard
assessment
of
perfluorooctanoic
acid
(
U.
S.
EPA
2002,
2003),
developmental
effects
appear
to
be
the
most
sensitive
critical
effect
among
those
examined
to
date.
The
developmental
effects
reported
in
the
York
study,
including
significant
decreases
in
body
weights,
body
weight
gains,
terminal
body
weights,
and
delays
in
sexual
maturation
among
F1
generation
offspring,
are
relevant
endpoints
2
to
be
considered
when
assessing
potential
developmental
health
risks
(
U.
S.
EPA
1991).

Although
the
delays
in
sexual
maturation
reported
among
high­
dose
(
30
mg/
kg/
day)
F1
generation
offspring
may
be
partially
explained
by
differences
in
gestational
age
or
body
weights,
such
explanations
cannot
completely
account
for
the
occurrence
of
this
developmental
effect,
and
fail
to
discount
its
biological
significance.
Developmental
effects
reported
at
lower
dose
levels
among
F1
generation
offspring
(
e.
g.,
decreased
body
weights)
are
also
considered
biologically
significant,
and
as
such,
result
in
the
assignment
of
low
NOAELs
and
LOAELs
compared
to
other
toxicity
studies
performed
on
PFOA.
In
the
absence
of
additional
data
to
explain
the
occurrence
of
developmental
effects
occurring
at
these
low
dose
levels,
their
biological
significance
should
not
be
discounted.

EPA
Response:
The
reviewer
agrees
with
the
use
of
the
endpoints
from
the
two
generation
reproductive
toxicity
study.
However,
it
should
be
noted
that
EPA
chose
these
endpoints
because
this
preliminary
assessment
focuses
only
on
developmental
toxicity;
these
endpoints
were
not
necessarily
the
"
critical"
endpoints
in
the
entire
toxicological
data
base.

Issue
2:
Use
of
Human
Biomonitoring
Data
Margins
of
exposure
(
MOE)
in
this
preliminary
risk
assessment
were
calculated
using
serum
levels
from
the
rat
two­
generation
reproductive
study
and
human
serum
levels
obtained
from
biomonitoring
data.
Approximately
600
individuals
were
involved
in
each
of
the
U.
S.
samples
of
adults
and
children
from
which
the
serum
data
were
derived
for
the
general
population
exposures.

U.
S.
EPA
question
2.1
requested
comment
on
the
adequacy
of
the
human
exposure
data
for
use
in
calculating
margins
of
exposure
in
adults
and
children.

Human
exposure
data
used
by
U.
S.
EPA
are
considered
inadequate
for
MOE
derivation.
The
PFOA
serum
levels
sampled
from
blood
donated
by
645
adults
(
age
20­
69
years)
are
derived
from
males
and
females.
Ideally,
the
mean
PFOA
serum
concentration
used
in
the
MOE
estimation
for
adults
should
have
been
calculated
entirely
from
blood
samples
collected
from
females
since
women
of
child­
bearing
age
are
considered
the
potential
population
at
risk.
It
should
be
noted,
however,
that
adult
males
in
this
sample
had
higher
serum
PFOA
levels
than
females,
so
inclusion
of
serum
PFOA
concentration
data
from
males
most
likely
resulted
in
a
1It
appears
as
if
there
is
a
typographical
error
on
page
46
relating
to
the
mean
serum
PFOA
concentrations
in
blood
samples
collected
from
645
adult
males
and
females.
The
U.
S.
EPA
preliminary
risk
assessment
document
identifies
the
geometric
mean
of
PFOA
in
male
and
female
adults
as
37.8
ppb
and
32.1
ppb,
respectively.
How
can
these
sex­
specific
concentrations
be
so
high
when
the
preliminary
risk
assessment
document
states
(
on
page
45)
that
the
combined
geometric
mean
of
serum
PFOA
in
male
and
female
adults
from
this
same
population
is
only
4.6
ppb?

3
lower
MOE
estimation,
and
likely
overestimated
potential
health
risks
from
PFOA
exposure.
1
It
is
not
clear
why
U.
S.
EPA
used
the
mean
PFOA
concentration
calculated
from
645
male
and
female
blood
samples
instead
of
using
a
mean
PFOA
concentration
calculated
from
only
313
female
blood
samples
from
this
data
set.
Secondly,
it
is
impossible
to
determine
whether
this
data
set
is
an
accurate
representation
of
the
American
population.
Such
a
criticism
also
applies
to
the
598
blood
samples
collected
from
2­
12
year
old
children,
although
the
data
set
from
which
the
children's
blood
was
sampled
appears
to
be
fairly
comprehensive
(
23
states
plus
the
District
of
Columbia).
Although
the
MOE
estimation
includes
adequate
caveats,
including
a
discussion
of
data
weaknesses,
it
is
recommended
that
additional
resources
be
devoted
to
improving
the
estimates
of
average
serum
PFOA
concentrations,
particularly
in
adults.

EPA
Response:
For
the
adults,
EPA
had
gender
specific
data
for
the
geometric
mean
and
range,

but
not
for
the
arithmetic
mean.
Since
the
geometric
means
for
the
males
and
females
were
very
similar
(
4.2
ppb
for
females
and
4.9
ppb
for
males),
the
value
used
in
the
MOE
calculation
was
the
mean
for
sexes
combined
which
was
4.6
ppb.
EPA
does
not
think
that
this
significantly
impacts
the
calculations.
With
reference
to
the
issue
of
whether
the
samples
are
representative
of
the
US
population,
it
should
be
noted
that
the
adult
serum
samples
came
from
several
cities
including
Los
Angeles,
Minneapolis,
Charlotte,
Boston,
Portland,
Oregon,
and
Hagerstown,

Maryland.
The
children's
serum
samples
came
from
23
states
and
the
District
of
Columbia.

These
samples
were
not
from
areas
in
the
vicinity
of
manufacturing
facilities.
EPA
believes
that
this
is
a
fairly
wide
sampling
of
the
US
population,
and
is
appropriate
to
use
at
this
time.
In
reference
to
the
footnote
below,
EPA
has
corrected
the
text.
4
Issue
3:
Use
of
Serum
Data
as
a
Measure
of
Internal
Dose
Margins
of
exposure
(
MOE)
in
this
preliminary
risk
assessment
were
calculated
using
serum
levels
from
the
rat
two­
generation
reproductive
study
and
human
serum
levels
obtained
from
biomonitoring
data.

U.
S.
EPA
question
3.1
requested
comment
on
the
use
of
serum
data
as
a
measure
of
internal
PFOA
dose
for
rats
and
humans.
These
data
were
used
in
calculation
of
the
MOE.

In
theory,
the
use
of
serum
data
as
a
measure
of
internal
dose
is
a
viable
method
in
which
to
approximate
internal
doses
of
PFOA
in
both
rats
and
humans.
Following
oral
and
inhalation
exposures,
PFOA
distributes
to
plasma,
in
addition
to
major
organs
such
as
the
liver
and
kidney.

In
practice,
however,
use
of
serum
data
is
associated
with
numerous
shortcomings
that
significantly
reduces
the
accuracy
of
internal
dose
estimations.
Pharmacokinetic
studies
in
rats
have
failed
to
consistently
demonstrate
a
significant
correlation
between
administered
PFOA
dose
and
serum
PFOA
concentration
(
e.
g.,
Ylinen
et
al.
1990).
This
may
be
due
in
part
to
saturation
of
plasma
proteins
at
PFOA
concentrations
equal
to
or
greater
than
30
mg/
kg/
day.
In
addition,
animals
administered
repeat
doses
of
PFOA
(
e.
g.,
Ylinen
et
al.
1990)
have
demonstrated
that
there
are
gender
differences
in
serum
PFOA
concentrations
that
should
be
taken
into
account
when
evaluating
uncertainties
associated
with
the
MOE
estimation.
Because
of
these
shortcomings,
serum
data
in
rats
should
only
be
considered
extremely
rough
approximations
of
internal
dose.

EPA
Response:
The
reviewer
agrees
with
the
use
of
the
data,
and
the
caveats
that
EPA
has
provided.
With
reference
to
the
issue
regarding
the
correlation
of
the
administered
dose
and
the
serum
levels,
this
relationship
is
linear
in
male
rats,
but
saturation
had
occurred
at
the
time
the
serum
samples
were
measured
in
many
of
the
studies.

Issue
4:
Use
of
F0
serum
levels
as
an
estimate
of
serum
levels
of
F1
animals
The
rat
serum
levels
measured
in
the
2­
generation
reproductive
toxicity
study
were
used
as
an
estimate
of
internal
dose.
However,
serum
levels
were
measured
only
in
the
F0
animals
and
only
measured
in
the
control,
10,
and
30
mg/
kg/
day
groups,
not
in
the
1
and
3
mg/
kg/
day
groups.
The
concern
for
developmental
toxicity
is
for
the
F1
animals.
The
assumption
has
been
made
that
the
serum
levels
measured
in
the
F0
animals
are
the
best
available
dose
estimate
for
the
F1
animals.
5
U.
S.
EPA
question
4.1
requested
comment
on
the
use
of
F0
serum
levels
as
internal
dose
estimates
among
F1
animals.

Use
of
F0
serum
levels
to
estimate
internal
doses
among
F1
animals
from
the
York
(
2002)

twogeneration
study
introduces
a
false
sense
of
certainty
in
the
MOE
estimates.
Instead
of
estimating
serum
levels
of
F1
rats
based
upon
from
F0
serum
levels,
it
would
be
much
better
for
U.
S.
EPA
to
contact
York
and
determine
whether
blood
samples
from
the
1
and
3
mg/
kg/
day
dose
groups
were
frozen
as
part
of
the
archival
process,
and
have
the
archived
F1
blood
samples
analyzed
to
determine
actual
serum
PFOA
concentrations.
The
U.
S.
EPA
preliminary
risk
assessment
does
not
explain
why
serum
PFOA
measurements
were
only
conducted
on
F0
rats,

and
also
fails
to
explain
why
PFOA
measurements
were
only
conducted
among
rats
from
the
0,

10,
and
30
mg/
kg/
day
dose
groups.
The
preliminary
risk
assessment
identifies
numerous
shortcomings
associated
with
use
of
F0
serum
data
that
cannot
be
addressed
through
an
evaluation
of
PFOA's
pharmacokinetics.
These
shortcomings
include
(
1)
uncertainties
associated
with
estimating
serum
PFOA
levels
in
blood
sampled
24
hours
post­
dosing;
and
(
2)

uncertainties
associated
with
estimating
serum
PFOA
levels
among
3
mg/
kg/
day
F1
rats
based
upon
serum
PFOA
levels
among
10
mg/
kg/
day
F0
rats.
Uncertainties
associated
with
issue
(
1)

primarily
affect
the
estimation
of
serum
PFOA
levels
among
F1
female
rats.
Serum
data
in
F0
rats
are
based
upon
blood
samples
collected
24
hours
following
the
last
dose.
The
serum
half­
life
of
PFOA
in
female
rats
is
less
than
24
hours.
Therefore,
serum
PFOA
concentrations
in
female
rats
do
not
represent
peak
PFOA
serum
concentrations,
resulting
in
an
underestimation
of
the
internal
PFOA
dose.
The
risk
assessment
attempts
to
account
for
this
underestimation
by
stating
that
peak
serum
PFOA
levels
in
females
are
likely
to
be
lower
than
measured
in
male
rats
due
to
differences
in
elimination
rates.
While
this
may
be
true,
the
statement
is
speculative
in
nature,

and
introduces
additional
uncertainty
into
the
MOE
estimate.
As
a
consequence
of
issue
(
1),

MOE
estimates
will
be
too
low
for
females,
and
may
overstate
the
potential
health
risk.

Uncertainties
associated
with
issue
(
2)
may
have
a
profound
effect
on
the
MOE
estimate
in
light
of
the
nonlinear
relationship
between
PFOA
dose
and
PFOA
serum
concentration.
Use
of
serum
PFOA
levels
from
10
mg/
kg/
day
F0
rats
overestimates
PFOA
serum
concentrations
in
3
mg/
kg/
day
F1
rats,
and
results
in
MOE
estimates
that
are
too
high,
in
effect
understating
the
potential
health
risk.
The
magnitude
of
uncertainty
associated
with
the
MOE
estimates
is
6
evidenced
by
the
wide
range
of
MOE
values
(
88­
11,109).
Due
to
these
shortcomings,
the
EPA
should
to
determine
whether
archived
blood
samples
can
be
obtained
from
the
1
and
3
mg/
kg/
day
dose
groups
in
order
to
measure
serum
PFOA
concentrations.
This
would
improve
the
MOE
estimates
and
will
ensure
that
the
MOEs
are
not
underestimated.

EPA
Response:
EPA
has
already
contacted
the
sponsor
of
the
study
to
determine
whether
serum
samples
had
been
collected
for
the
F1
animals
or
for
other
dose
groups.
There
are
no
archived
samples
to
analyze.
In
lieu
of
this
information,
the
reviewer
agrees
with
the
approach
EPA
has
used
for
the
preliminary
risk
assessment.
In
addition,
EPA
has
addressed
the
uncertainties
raised
by
the
reviewer.

U.
S.
EPA
question
4.2
requested
comment
on
the
use
of
serum
values
for
F0
males
as
an
upper
estimate
of
peak
exposure
for
F0
females.

There
are
sufficient
uncertainty
underlying
differences
between
male
and
female
PFOA
serum
concentrations
in
repeat­
dose
rat
studies
to
justify
using
serum
values
for
F0
males
to
provide
upper
estimates
of
peak
exposure
for
F0
females.
The
Ylinen
et
al.
(
1990)
repeat­
dose
study
does
indicate
that
male
rats
administered
repeated
doses
of
3,
10,
and
30
mg/
kg/
day
PFOA
have
consistently
higher
PFOA
serum
concentrations
than
females
at
all
three
dose
levels.
This
provides
some
assurance
that
F0
male
serum
values
are
higher
than
F0
females.
However,

without
additional
pharmacokinetic
data,
it
is
extremely
difficult
to
know
whether
serum
values
in
F0
males
are
entirely
accurate
predictors
of
peak
serum
values
in
F0
females.
There
are
apparently
no
absorption
studies
that
have
examined
differences
between
the
rate
and/
or
percent
of
absorption
in
males
verses
females.

EPA
Response:
All
kinetic
studies
show
that
serum
values
in
the
male
rat
are
higher
than
in
the
female
rat
due
to
the
gender
differences
in
elimination.
EPA
has
therefore
appropriately
presented
the
serum
values
for
the
F0
males
as
representing
a
high
end
estimate
of
the
peak
values
in
the
F0
females.
The
uncertainties
associated
with
this
approach
have
been
addressed
in
section
5.5.
7
U.
S.
EPA
question
4.3
requested
comment
on
the
significance
of
OAT
data
for
estimating
internal
doses
among
postweanling
rats.

Pharmacokinetic
data
presented
in
the
preliminary
risk
assessment
provide
evidence
to
suggest
that
OAT
data
may
be
more
significant
for
estimating
internal
doses
of
PFOA
among
female
postweanling
rats
than
male
postweanling
rats.
Three
rat
studies
cited
in
the
preliminary
risk
assessment
provide
data
suggesting
that
female
rats
excrete
PFOA
or
AFPO
to
a
much
greater
extent
than
male
rats
via
an
active
secretion
process.
Specifically,
Hanhijarvi
et
al.
(
1982)

investigated
the
effect
of
probenecid
administration
among
male
and
female
Holtzmann
rats.

Probenecid
strongly
inhibits
active
renal
secretion
of
organic
acids.
Hanhijarvi
et
al.
reported
that
intraperitoneal
injection
of
probenecid
was
not
found
to
significantly
influence
the
excretion
of
APFO
among
male
rats,
while
in
female
rats,
probenecid
had
a
significant
affect
on
APFO
excretion.
As
part
of
a
study
in
rats
designed
to
examine
the
effects
of
adrogens
and
estrogens
upon
PFOA
excretion,
Vanden
Heuvel
et
al.
(
1992a)
administered
probenecid
(
dose
not
identified)
to
male
and
female
rats
(
strain
not
identified).
They
found
that
probenecid
decreased
the
high
rate
of
renal
PFOA
excretion
among
castrated
male
rats
but
had
no
effect
on
male
rats
with
intact
testis.
Kudo
et
al.
(
2002)
also
investigated
the
effect
of
sex
hormones
upon
PFOA
excretion
in
male
and
female
rats
(
strain
not
identified),
as
well
as
mRNA
levels
of
organic
anion
transporters
(
OAT).
Kudo
et
al.
treated
rats
with
probenecid
(
dose
not
identified),
and
found
that
renal
clearance
of
PFOA
was
"
markedly
reduced"
among
male
rats,
castrated
male
rats,
and
female
rats.
Kudo
et
al.
determined
that
the
level
of
OAT2
mRNA
in
male
rats
was
only
13%

that
in
female
rats,
and
concluded
that
OAT2
and
OAT3
are
responsible
for
urinary
elimination
of
PFOA
in
the
rat.
They
found
that
castration
or
estradiol
treatment
in
male
rats
increased
the
level
of
OAT2
mRNA
whereas
treatment
of
castrated
male
rats
with
testosterone
reduced
it.

Ovariectomy
of
female
rats
significantly
increased
the
level
of
OAT3
mRNA.
Collectively,
these
three
studies
demonstrate
that
female
rats
utilize
active
organic
anion
transporters
to
a
much
greater
degree
than
male
rats.
Therefore,
estimating
internal
PFOA
doses
based
on
OAT
mRNA
expression
is
not
likely
to
be
useful
for
male
rats.
Moreover,
an
accurate
estimation
of
internal
PFOA
doses
in
female
postweanling
rats
is
likely
to
be
precluded
by
the
fact
that
expression
of
the
primary
OAT
transporter
in
female
rats
(
OAT2)
is
not
mature
until
day
35
in
females.
It
would
be
difficult
to
estimate
the
internal
dose
in
postweanling
female
rats
(
i.
e.,
those
rats
older
8
than
22
days)
in
light
of
the
fact
that
the
primary
OAT
transporter
is
not
mature
until
at
least
day
35.

EPA
Response:
The
reviewer
agrees
that
maturation
of
the
OATs
will
influence
the
elimination
of
PFOA
in
the
prepubescent
rat.
However,
it
is
not
possible
to
quantify
the
impact
at
this
time.

EPA
agrees
and
has
addressed
this
in
section
5.5.

General
Comments
Relating
to
the
Preliminary
Risk
Assessment
Adequacy
of
metabolism
and
pharmacokinetic
study
descriptions:
Many
of
the
studies
described
in
Section
3.2
are
missing
important
details
that
would
assist
in
the
evaluation
of
each
study.
For
example,
the
specific
strain
of
animal
and
specific
numbers
of
animals
evaluated
are
left
out
of
the
study
summaries.

EPA
Response:
This
information
was
added
to
the
text.

REFERENCES
Hanhijarvi,
H;
Ophaug,
RH;
Singer,
L.
(
1982).
The
sex
related
difference
in
perfluorooctanoate
excretion
in
the
rat.
Proc
Soc
Exp
Biol
Med
171:
50­
55.

Kudo,
N;
Katakura,
M;
Sato,
Y;
Kawashima,
Y.
(
2002).
Sex
hormone­
regulated
renal
transport
of
perfluorooctanoic
acid.
Chem
Biol
Interact
139:
301­
316.

United
States
Environmental
Protection
Agency
(
U.
S.
EPA).
(
1991).
Guidelines
for
developmental
toxicity
risk
assessment.
EPA/
600/
FR­
91/
001.

United
States
Environmental
Protection
Agency
(
U.
S.
EPA).
(
2002).
Draft
hazard
assessment
of
perfluorooctanoic
acid
and
its
salts.
Office
of
Pollution
Prevention
and
Toxics.
Risk
Assessment
Division.
November
4,
2002.

United
States
Environmental
Protection
Agency
(
U.
S.
EPA).
(
2003).
Preliminary
risk
assessment
of
the
developmental
toxicity
associated
with
exposure
to
perfluorooctanoic
acid
and
its
salts.
Office
of
Pollution
Prevention
and
Toxics.
Risk
Assessment
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April
4,
2003.
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Vanden
Heuvel,
JP;
Davis,
JW;
Sommers,
R;
et
al.
(
1992).
Renal
excretion
of
perfluorooctanoic
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in
male
rats:
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of
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J
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7(
1):
31
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Ylinen,
M;
Kojo,
A;
Hanhijdrvi,
H;
et
al.
(
1990).
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perfluorooctanoic
acid
in
the
rat
after
single
and
subchronic
administration.
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Environ
Contam
Toxicol
44:
46­
53.

York,
R.
G.
(
2002).
Oral
(
gavage)
two­
generation
(
one
litter
per
generation)
reproduction
study
of
ammonium
perfluorooctanoic
(
APFO)
in
rats.
Argus
Research
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Protocol
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418­
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T­
6889.6,
March
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