Task
Force
June
14,
2002
P.
O.
Box
331,
Miflwood,
VA
22646
(
540)
837­
1602
Charles
M.
Auer
Environmental
Protection
Agency
1200
Pennsylvania
Avenue,
NW
Room
4146­
A
Washington,
DC
20004
Re:
~

Dear
Mr.
Auer:

The
HAP
Task
Force
has
addressed
the
technical
issues
raised
in
your
March
1,
2002
letter.
To
resolve
these
remaining
technical
issues,
a
meeting
was
held
at
the
recent
Society
of
Toxicology
(
SOT)
meeting
in
Nashville
between
EPA
scientists
and
our
contractor.
As
a
consequence,
appropriate
changes
and
additions
have
b~
ee~~~
made
to
Appendix
C
and
additional
references
regarding
the
PBPK
modeling
have
been
added.
These
are
enclosed.

With
these
changes,
I
look
forward
to
seeing
a
final
draft
ofthe
enforceable
consent
agreement
(
ECA)
for
ethylene
dichioride.

Enclosure
S
incer
ly
your~

nIA1
/
1~
~

Peter
E.
Voytek,
PhD
Manager
cc:
W.
C,
Norman,
Esq.
RECEIVED
OPPT
NCIC
2003
MAR11
5:
03PM
OPPT­
2003­
0010­
0052
APPENDIX
C
PK/
MECH
PROCEDURES
AND
ROUTE
TO
ROUTE
EXTRAPOLATION
REPORTING
FOR
ETHYLENE
DICHLORIDE
This
Appendix
contains
detailed
procedures
for
the
kinetic
studies
and
route­
to­
route
extrapolations
to
be
conducted
for
ethylene
dichioride,
and
is
organized
into
the
following
sections:

C.
1
Pharmacokinetic
Studies
for
F344
Rats
C.
2
Subchronic
Toxicity
C.
3
Subchronic
Neurotoxicity
C.
4
Reproductive
Toxicity
C,
5
PBPK
Model
Description
and
Coding
C,
6
General
Outline
for
Route­
to­
Route
Extrapolation
Reports
C:\
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2002
C.!.
Pharmacokinetic
Studies
for
F344
Rats
A
total
of4
pharmacokinetic
studies
(
designated
Study
1,
Study
2,
Study
3
and
Study
4)
will
be
conducted
to
support
PBPK
modeling
activities
for
ethylene
dichloride,
as
described
in
this
section
(
C.
1.).
It
is
anticipated
that
the
pharmacokinetic
studies
will
start
one
year
after
the
ECAis
signed,
with
an
interim
report
generated
six
months
later,
and
the
final
report
submitted
three
months
later
(
see
Appendix
A
for
complete
schedule).

Demonstration
ofPeriodicity
Following
Repeated
Inhalation
Exposures
Dose(
s)
Route
Exposure
Frequency
Observations
ProDosed
Rat
F344
Young
adult
132
animals
(
3
controls
at
beginning
(
t
=

0)
and
3
at
end
ofexposure
(
t
=
6
ix)
on
days
1,
3,
and
5;
3
exposed
animals
at
the
beginning
of
exposure
(
t
0)
on
days
3
and
5,
3
exposed
animals/
time
point
at
t
0.25,
0.5,
6.25
and
16
hr
during
and
after
exposure
on
days
1,
3,
and
5,
and
6
exposed
animals/
time
point
at
t
=
1,
3,
6,
and
8
hr
during
and
after
exposure
on
days
1,
3,
5)
To
be
determined
Inhalation
6
hrs/
day,
5
consecutive
days
Liver
and
lung
GSH
in
controls
(
n
3)
at
beginning
and
end
ofexposure
(
0
and
6
hrs)
on
days
1
,3
and
5.
Lung
and
liver
GSH
in
exposed
animals
(
n
3)
at
beginning
of
exposure
on
days
3
and
5.
Venous
blood
concentrations
ofEDC
(
n
3)
at
0.25,
0.5,
6.25,
and
16
hrs
after
exposure
starts
on
days
1,
3,
and
5.
Venous
blood
concentrations
ofEDC
(
n
=
6)
and
lung
and
liver
GSH
concentrations
(
n
3)
at
1,
3,
6,
and
8
hours
after
exposure
starts
on
days
1,
3,
and
5.
Parameter
Species
Strain
Age
Number
of
animals
C:\
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April
24,
2002
Kinetic
studies
wifi
be
conducted
for
demonstrating
periodicity
in
the
rat
following
inhalation
exposures
with
EDC.
Measurements
ofEDC
in
blood
and
tissue:
blood
partition
coefficients
are
anticipated
to
be
sufficient
to
describe
tissue
concentration
time
courses
ofEDC
based
onthe
results
ofSpreafico
eta!.
(
1980)
where
concentrations
in
brain,
kidney,
spleen,
liver,
lung,
andadiposetissue
were
found
to
parallel
the
concentrations
in
blood
ofrats
exposed
to
EDC
byiv,
oral,
and
inhalation
routes.
Measurements
oflung
and
liver
GSH
will
be
used
to
further
calibrate
and
validate
the
GSHdepletion
portion
ofthe
PBPK
model.
These
data
will
allow
the
model
to
compute
the
flux
through
both
metabolism
pathways
that
are
dependent
onGSH
conjugation
(
see
section
C.
5).
The
depletion
ofGSH
in
various
tissues
has
been
used
to
determine
and
validate
the
kinetic
constants
for
GSH
conjugation
reactions
for
a
number
ofchemicals
and
their
metabolites.
These
include
vinylidene
chloride
(
D'Souzaand
Andersen,
1988),
EDC
(
D'Souza
eta!.,
1988),
ethyl
acrylate
(
Frederick
eta!.,
1992),
ethylene
oxide
(
Krishnan
et
al.,
1992)
and
ally!
chloride
(
Clewell
and
Andersen,
1994).
In
contrast,
elimination
ofEDC
metabolites
via
the
GSH
pathway,
measured
as
urinary
thiodiglycolic
acid
(
TDGA)
is
not
anoptimal
biomarker
ofGSH
metabolism.
Departures
from
a
linear
relationship
between
dose
and
24­
hour
TDGAmay
be
observed
as
doseincreases
(
Payan
et
a!.,
1993)
in
therange
ofdoses
used
in
existing
toxicity
studies
(
e.
g.,
NCI,
1978).
A
possible
lag
in
urinary
elimination
as
TDGA,
extending
>
24
hours
after
dosing,
would
compromise
use
ofthis
biomarker
as
validation
for
GSH
metabolism
during
a
repeated
dosing
study.

Air
concentrations
should
be
measured
hourly.
Blood
samples
will
be
drawn
on
days
1,
3,
and
5.
Blood
samples
(
from
3
or
6
animals)
and
liver
and
lung
of3
animals
(
for
glutathione
determination)
will
be
collected
immediately
following
sacrifice.
Blood
samples
will
be
drawn
from
6
animals
only
at
sampling
times
where
lung
and
liver
GSH
are
also
being
measured
(
3
animals'
lungs
and
livers
for
GSH,
3
for
freezing).
Lung,
liver,
kidney,
brain,
adrenal
gland,
and
thyroid
will
be
frozen
for
possible
further
analysis.
A
positive
control
(
spiked
storage
sample)
wifi
be
required
to
demonstrate
the
recovery
ofEDC
in
tissues
following
freezing.
Tissues
wifi
be
stored
until
after
the
Tier
I
Program
Review.

C:\
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wpd
April
24,
2002
Demonstration
of
Periodicity
Following
Repeated
Oral
Exposures
to
Ethylene
Dichioride
by
Corn
Oil
Gavage
Parameter
Species
Strain
Age
Number
ofanimals
Dose(
s)
Route
Exposure
Frequency
Observations
Pronosed
Rat
F344
Young
adult
132
animals
(
3
controls
at
dosing
(
t
=
0)
and
3
after
dosing
(
t
=
2­
6
ix)
on
days
1,
3,
and
5;
3
exposed
animals
prior
to
dosing
(
t
=
0)
on
days
3
and
5;
3
exposed
animals
at
t
=
0.25,
4,
16,
and
24
hrs
after
dosing
on
days
1,
3,
and
5;
and
6
exposed
animals/
time
point
0.5,
1,
2,
and
8
hrs
after
dosing
on
days
1,
3,
5)
150
mg/
kg
Corn
oil
gavage
1
x/
day,
5
consecutive
days
Liver
and
lung
GSH
in
controls
(
n
=
3)
prior
to
dosing
(
at
0
hrs)
and
at
8
hrs
after
dosing.
Liver
and
lung
GSH
in
previously
dosed
animals
(
n
=
3)
prior
to
dosing
ofremaining
animals
(
t
=
0)
on
days
3
and
5.
Venous
blood
concentrations
ofEDC
(
n=
3)
at
0.25,
4,
16,
and
24
hrs
after
dosing
on
days
1,
3,
and
5.
Venous
blood
concentrations
of
EDC
(
n
6)
and
lung
and
liver
GSH
concentrations
(
n
=
3)
at
0.5,
1,
2,
and
8
hours
after
exposure
starts
on
days
1,
3,
and
5
Kinetic
studies
will
be
conducted
for
demonstrating
periodicity
in
the
rat
following
corn
oil
gavage
with
EDC.
Measurements
ofEDC
in
blood
and
tissue:
blood
partition
coefficients
are
anticipated
to
be
sufficient
to
describe
tissue
concentration
time
courses
ofEDC
based
on
the
results
ofSpreafico
et
al.
(
1980)
where
concentrations
in
brain,
kidney,
spleen,
liver,
lung,
and
adipose
tissue
were
found
to
parallel
the
concentrations
in
blood
of
rats
exposed
to
EDC
by
iv,
oral,
and
inhalation
routes.
Measurements
oflung
and
liver
GSH
wifi
be
usedto
further
calibrate
and
validate
theGSH­
depletion
portion
ofthe
PBPK
model.
These
data
wifi
allow
the
model
to
compute
the
flux
through
both
metabolism
pathways
that
are
dependent
on
GSH
conjugation
(
see
section
C.
5).
The
depletion
of
GSH
in
various
tissues
has
been
used
to
determine
and
validate
the
kinetic
constants
for
GSH
conjugation
reactions
for
a
number
of
chemicals
and
their
metabolites.
These
include
vinylidene
C:\
WINDOWS\
TemporaryInternet
Files\
Content.
1E5\
4EAADO4JA\
edc­
AppC_
rev.
wpd
April
24,
2002
chloride
(
D'Souza
and
Andersen,
1988),
EDC
(
D'Souza
et
al.,
1988),
ethyl
acrylate
(
Frederick
et
a!.,
1992),
ethylene
oxide
(
Krishnan
et
al.,
1992)
and
allyl
chloride
(
Clewell
and
Andersen,
1994).
In
contrast,
elimination
ofEDC
metabolites
via
the
GSH
pathway,
measured
as
urinary
thiodiglycolic
acid
(
TDGA)
is
not
an
optimal
biomarker
ofGSH
metabolism,
Departures
from
a
linear
relationship
between
dose
and
24­
hourTDGA
maybe
observed
as
dose
increases
(
Payan
et
aL,
1993)
in
the
range
ofdoses
used
in
existing
toxicity
studies
(
e.
g.,
NCI,
1978).
A
possible
lag
in
urinary
elimination
as
TDGA,
extending
>
24
hours
after
dosing,
would
compromise
use
ofthis
biomarker
as
validation
for
GSH
metabolism
during
a
repeated
dosing
study.

The
test
dose
was
selected
based
on
the
doses
administeredto
rats
in
the
high
dose
group
for
the
90­
day
study
conducted
by
Daniel
et
al.
(
1994).
Blood
samples
will
be
drawn
on
days
1,
3,
and
5.
Blood
samples
(
from
3
or
6
animals)
and
liver
and
lung
of3
animals
(
for
glutathione
determination)
will
be
collected
immediately
following
sacrifice.
Lung,
liver,
kidney,
brain,
adrenal
gland,
and
thyroid
will
be
frozen
for
possible
further
analysis.
A
positive
control
(
spiked
storage
sample)
will
be
required
to
demonstrate
the
recoveryofEDC
in
tissues
following
freezing.
Tissues
wifi
be
stored
until
after
the
Tier
I
Program
Review.

C:\
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Ten­
iporary
InternetFiles\
Content.
1E5\
4EAADO~
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edc­
AppCrev.
wpd
April
24,
2002
Demonstration
of
Periodicity
~
ridebAueousGavae
Parameter
Following
Repeated
Oral
Exposures
to
Ethylene
Proposed
Species
Rat
Strain
F344
Age
Young
adult
Number
of
animals
132
animals
(
3
controls
at
dosing
(
t
=
0)
and
3
after
dosing
(
t
2­
6
ix)
on
days
1,
3,
and
5;
3
exposed
animals
prior
to
dosing
(
t
=
0)
on
days
3
and
5;
3
exposed
animals
at
t
0.25,
4,
16,
and
24
hrs
after
dosing
on
days
1,
3,
and
5;
and
6
exposed
animals/
time
point
0.5,
1,
2,
and
8
hrs
after
dosing
on
days
1,
3,
5)
Doses
<
43
mg/
kg­
day
Route
Aqueous
gavage
Exposure
Frequency
ix/
day,
5
consecutive
days
Observations
Liver
and
lung
GSH
in
controls
(
n
=
3)
prior
to
dosing
(
at
0
hrs)
and
at
8
hrs
after
dosing.
Liver
and
lung
GSH
in
previously
dosed
animals
(
n
3)
prior
to
dosing
ofremaining
animals
(
t
0)
on
days
3
and
5.
Venous
blood
concentrations
ofEDC
(
n'=
3)
at
0.25,
4,
16,
and
24
hrs
after
dosing
on
days
1,
3,
and
5.
Venous
blood
concentrations
of
EDC
(
n
6)
and
lung
and
liver
GSH
concentrations
(
n
=
3)
at
0.5,
1,
2,
and
8
hours
after
exposure
starts
on
days
1,
3,
and
5
Kinetic
studies
will
be
conducted
for
demonstrating
periodicity
in
the
rat
following
aqueous
gavage
with
EDC.
Measurements
ofEDC
in
blood
and
tissue:
blood
partition
coefficients
are
anticipated
to
be
sufficient
to
describe
tissue
concentration
time
courses
ofEDC
based
on
the
results
ofSpreafico
et
al.
(
1980)
where
concentrationsin
brain,
kidney,
spleen,
liver,
lung,
and
adipose
tissue
were
found
to
parallel
the
concentrations
in
blood
ofrats
exposed
to
EDC
by
iv,
oral,
and
inhalation
routes.
Measurements
oflung
and
liver
GSH
will
be
used
to
further
calibrate
and
validate
the
GSH­
depletion
portion
of
the
PBPK
model.
These
data
will
allow
the
model
to
compute
the
flux
through
both
metabolism
pathways
that
are
dependent
on
GSH
conjugation
(
see
section
C.
5).
The
depletion
of
GSH
in
various
tissues
has
been
used
to
determine
and
validate
the
kinetic
constants
for
GSH
conjugation
reactions
for
a
number
of
chemicals
and
their
metabolites.
These
include
vinylidene
chloride
(
D'Souza
and
Andersen,
1988),
EDC
(
D'
Souza
et
aL,
1988),
ethyl
acrylate
(
Fredericket
al.,

C:\
WJNDOWS\
Temporary
Internet
Files\
ContenLlE5\
4EAADO6JA\
ede"
AppC_
rev.
wpd
April
24,
2002
1992),
ethylene
oxide
(
Krishnan
et
al.,
1992)
and
allyl
chloride
(
Clewell
and
Andersen,
1994).
In
contrast,
elimination
ofEDC
metabolites
via
the
GSH
pathway,
measured
as
urinary
thiodiglycolic
acid
(
TDGA)
is
not
anoptimal
biomarker
ofGSHmetabolism.
Departures
from
a
linear
relationship
betweendose
and
24­
hourTDGA
may
be
observed
as
doseincreases
(
Payan
et
al.,
1993)
in
the
range
ofdoses
used
in
existing
toxicity
studies
(
e.
g.,
NCI,
1978).
A
possible
lag
in
urinary
elimination
as
TDGA,
extending
>
24
hours
after
dosing,
would
compromise
use
ofthis
biomarker
as
validation
for
GSH
metabolism
during
a
repeated
dosing
study.
The
rate
constant
for
oral
absorption
from
a
gavage
dose
of
EDC
in
water
(
to
be
determined
via
PBPK
modeling)
wifi
be
considered
appropriate
for
modeling
oftoxicity
studies
in
whichEDC
wasingested
via
adlibitum
access
to
drinkingwater
(
e.
g.,
O'Flaherty,
1996).

The
test
dose
will
be
selected
based
on
the
water
solubility
and
dosing
volume.
The
water
solubility
of
EDC
is
8.7
mg/
niL,
and
the
dosing
volume
for
a
rat
should
be
no
more
than
5
nil/
kg,
for
a
maximum
dose
of
43
mg/
kg/
d.
The
actual
dose
will
be
set
based
on
the
ability
of
the
lab
to
consistently,
homogeneously
solubilize
the
test
article.
Blood
samples
wifi
be
drawn
on
days
1,
3,
and
5.
Blood
samples
(
from
3
or
6
animals)
and
liver
and
lung
of
3
animals
(
for
glutathione
determination)
will
be
collected
immediately
following
sacrifice.
Lung,
liver,
kidney,
brain,
adrenal
gland,
and
thyroid
will
be
frozen
for
possible
further
analysis.
A
positive
control
(
spiked
storage
sample)
will
be
required
to
demonstrate
the
recovery
ofEDC
in
tissues
following
freezing.
Tissues
will
be
stored
until
after
the
Tier
I
Program
Review.

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2002
Study
4:
Determination
ofPartition
Coefficients
The
rat
brain:
air,
kidney:
air,
testes:
air,
and
ovary:
air
partition
coefficients
will
be
determined
using
the
vial
equilibration
technique
described
by
Gargas
et
al.
(
1989,
Toxicol.
Appl.
Pharmacol.
98,
87­
99).

A
total
of
5
rats
will
be
used,
ifpartition
coefficients
are
determined
individually.
A
higher
number
ofanimals
may
be
required
if
composite
samples
are
used.
It
is
preferred
that
partition
coefficients
be
determined
for
individual
animals,
however
if
tissue
volumes
are
insufficient
to
measure
individually
a
composite
sample
may
be
used.

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C.
2
Subchronic
Toxicity
Route­
To­
Route
Extrapolation
The
NOAEL/
LOAEL
values
obtained
from
the
existing
subchronic
study
ofDaniel
et
al.
(
1994)
will
be
extrapolated
from
the
oral
route
to
the
inhalation
route
using
a
PBPK
model
(
Appendix
C.
5).
The
internal
dose
metric
(
e.
g.,
peak
concentration
or
average
concentration
ofEDC,
or
amount
ofEDC
metabolized)
used
to
perform
this
extrapolation
will
be
determined
based
on
a
consideration
ofthe
effects
observed
and
a
plausible
mechanism
ofaction.

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C.
3
Subchronic
Neurotoxicity
~­
To­
RouteExtraolation
The
NOAEL/
LOAEL
values
obtained
from
the
subchronic
neurotoxicity
testing
will
be
extrapolated
from
the
oral
route
to
the
inhalation
route
using
a
PBPK
model
(
Appendix
C.
5).
The
internal
dose
metric
(
e.
g.,
peak
concentration
or
average
concentration
ofEDC,
or
amount
ofEDC
metabolized)
used
to
perform
this
extrapolation
will
be
determined
based
on
a
consideration
ofany
neurological
effects
observed
and
a
plausible
mechanism
ofaction.

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C.
4
Reproductive
Toxicity
~­
To­
RouteExtraolation
TheNOAEL/
LOAELvalues
obtained
from
the
reproductive
toxicity
testing
willbe
extrapolatedfrom
the
oral
route
to
the
inhalation
route
using
a
PBPK
model
(
Appendix
C.
5).
The
results
ofAlumot
et
al.
(
1976),
Rao
et
al.
(
1980)
and
Lane
et
al.
(
1982)
will
also
be
compared
to
the
current
reproductive
toxicity
testing.
The
internal
dose
metric
(
e.
g.,
peak
concentration
or
average
concentration
ofEDC,
or
amount
ofEDC
metabolized)
used
to
perform
this
extrapolation
will
be
determined
based
on
a
consideration
ofany
reproductive
effects
observed
and
a
plausible
mechanism
ofaction.

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C.
5
PBPK
Model
Description
and
Coding
The
preliminary
PBPK
model
(
to
be
refined
as
part
ofthis
ECA)
is
a
modification
ofD'Souza
et
al.
(
1987,
1988)
to
include
periodic
consumption
ofdrinking
water.
Rat
physiology
is
represented
by
five
tissue
groups
(
lung,
liver,
richly
perfused
tissues,
slowly
perfused
tissues,
and
adipose
tissues).
Because
conjugation
of
EDC
with
glutathione
in
the
lung
and
liver
is
an
important
pathway
of
elimination,
the
model
includes
normal
synthesis
and
breakdown
ofglutathione
in
the
lung
and
liver
and
a
time­
delayed
compensatory
increase
in
glutathione
synthesis
when
glutathione
concentrations
are
less
than
steady­
state
values.
The
oxidative
metabolite
ofEDC
is
assumed
to
stay
within
the
tissue
in
which
it
was
produced
(
lung
or
liver)
and
its
production
(
from
EDC)
and
further
reactions
(
GSH
conjugation
and
other
reactions)
are
described.
The
metabolic
scheme
is
depicted
below.

Ethylene
dichioride
Oxidative
metabolism
+
H
cl~
cl~
0
I
(
VmaxL,
Km)
(
KGSM)
I
+
GSH
I
(
KGS)
OH
(
KFEE)

CI~~

Macromolecules,
other
reactions
The
PBPK
model
for
repeated
exposure
to
EDC,
to
be
refined
based
on
the
studies
described
in
C.
1,
will
also
be
validated
against
other
relevant,
existing
pharmacokinetic
data
for
single
exposures
to
EDC
(
e.
g.,
Reitz
et
aL,
1982,
Spreafico
et
al.,
1980,
D'Souza
et
al.,
1987,
1988).

The
model
code
is
provided
below.

PROGRAM:
GLUTATHIONE
DEPLETION
MODEL
FOR
EDC
(
EDC­
GSH.
CSL)
`
Initial
parameter
values
for
the
rat
(
DSouza
et
al.
1987,
1988)'
`
Retyped,
documentation
modified
by
LMS
7/
19/
0
1'

INITIAL
`
SPECIAL
FLOW
RATES'

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ContenLlE5\
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edc­
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wpd
April
24,2002
CONSTANT
QPC'~=
l5
$`
alveolar
ventilationrate
L/
hr/
kg"
0.74'

CONSTANT
QCC=
15
$`
Cardiac
output
L/
hr/
kgt'
0.74'

`
FRACTIONAL
BLOOD
FLOW
TO
TISSUES'
CONSTANT
QLC
=
0.07
$`
Fractional
blood
flow
to
liver'
CONSTANT
QFC
=
0.05
$`
Fractional
blood
flow
to
fat'
CONSTANT
QRC
=
0.64
$`
Fractional
blood
flow
to
rapid'
CONSTANT
QSC
=
0.24
$`
Fractional
blood
flowto
slow'

`
BODY
WEIGHT'
CONSTANT
BW
=
0.22
$`
Body
weight
(
kg)'

`
FRACTIONAL
TISSUE
VOLUMES'
CONSTANT
VPC
=
0.004
$`
Fraction
lung
tissue'
CONSTANT
VLC
=
0.04
$`
Fraction
liver
tissue'
CONSTANT
VFC
=
0.07
$`
Fraction
fat
tissue'
CONSTANT
VRC
=
0.316
$`
Fraction
rapid
tissue'
CONSTANT
VSC
=
0.42
$`
Fraction
slow
tissue'

`
PARTITION
COEFFICIENTS'
CONSTANT
PP
=
1.1
$`
Lung/
blood
partition
coefficient'
CONSTANT
PL
=
1.1
$`
Liver/
blood
partition
coefficient'
CONSTANT
PF
=
12.2
$`
Fat/
blood
partition
coefficient'
CONSTANT
PS
=
0.8
$`
Slowly
perfused
tissue/
blood
partition
coefficient'
CONSTANT
PR
=
1.1
$`
Richly
perfused
tissue/
blood
partition
coefficient'
CONSTANT
PB
=
27.6
$`
Bloodlair
partition
coefficient'

CONSTANT
MW
=
98.96
$`
Molecular
weight
(
g/
mol)'

`
KINETIC
CONSTANTS'
CONSTANT
VMAX1C
=
3.15
$`
Maximum
velocity
ofmetabolism
(
mg/
hr­
kg"
0.7)'
CONSTANT
VMAXpC
=
3.15
$`
Maximum
velocity
ofmetabolism
(
mg/
hr­
kgt'
0.7)'
CONSTANT
KM
=
0.25
$`
Michaelis­
Menten
constant
(
mg/
L)'

CONSTANT
KGSC='
0.0012
$`
conj
rate
const
w
parent(
l/(
uM­
ix­
kg'~'­
0.7))'
CONSTANT
KGSMC
=
0.15
$`
conj
rate
const
w
metab
(
l/(
uM­
hr­
kg'~­
0.7))'
CONSTANT
KFEEC=
4500
$`
conj
rate
const
w
non
gsh
(
l/(
hr­
kg'~'­
0.7))'
CONSTANT
KOalC
=
3.8
$`
GSH
synthase
synthesis
liv(
umol/
hr/
hr/
kg'~­
0.7)'
CONSTANT
KOapC
=
0.22
$`
GSH
synthase
synthesis
lu(
umol/
hr/
hr/
kgt'­
0.7)'
CONSTANT
K1CO.
1
16
$`
GSH
breakdown
(
1/
hr/
kg"­
0.7)'
CONSTANT
Ki
aC'
0.095
$`
GSH
synthase
breakdown(
1
/
hr/
kg"­
O.
7)'
CONSTANT
KS
=
1000
$`
Maximum
GSH
induction
(
uM)'
CONSTANT
TD
=
1.5
$`
Time
delay
(
ix)'

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rev.
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April
24,
2002
CONSTANT
GSO1
=
7000
$`
Initial
GSH
concentration
(
uM)'
CONSTANT
GSOp
=
1200
$`
Initial
GSH
concentration
(
uM)'
CONSTANT
PLRATIOO.
14
$`
MFO
ratio
lung/
liver'
CONSTANT
KOOl
=
11.254
$`
liver
(
umo]
Ihr)'
CONSTANT
KOOp
=
11.254
$`
liver
(
umol/
hr)'

DOSING
INFO'
CONSTANT
CONC
=
10
$
Inhaled
concentration
(
ppm)'

`
Periodic
drinking
water
exposure
section'
`
assume
t=
0
is
7
am
for
reference'
INTEGER
I
$
I'=
l
$`
Counter
for
drinking
arrays'
CONSTANT
DRCONC=
0.0
$`
Conc
ofEDC
in
water
(
mg/
L)'
CONSTANT
KA
=
S
$
rate
const
absorp
EDC
from
stomach'
ARRAY
DRTIME(
6)
$`
store
drinking
times
in
array'
ARRAY
DRPCT(
6)
$`
store
drinking
percentages'
CONSTANT
DRTIME=
1.0,5.0,9.0,13.0,17.0,21,0
CONSTANT
DRPCT=
0.233,0.
1,0.1,0.1,0.233,0.234
`
Assume
70
kg
man
drinks
2
liters/
day,
calc
rodent
allometrically'
DRVOL
=
0.
102*
BW**
0.7
$`
calc
vol
water
drunk
from
BW'
DRDOSE
=
DRVOL*
DRCONC
$`
total
dose
from
water
each
day
(
mg)'
ODOSE
=
0.0
$`
to
calculate
input
to
stom
(
mg)'
NEWDAY
=
0,0
$`
to
reset
arrays
each
24
hrs'

`
TIMTNG
PARAMETERS'
CONSTANT
TSTART
=
48.
$`
Start
ofexposure
(
hrs)'
CONSTANT
TPER=
24
CONSTANT
TSTOP=
l20
CONSTANT
POINTS=
1200
$`
Number
ofpoints
in
plot'
CINT
=
TSTOP/
POINTS
$`
Commun'
tion
interval'
TCHNG=
6
`
SCALED
PARAMETERS'
QC
=
QCC*
BW**
0.74
QP
=
QPC*
BW**
0.74
QL
=
QLC*
QC
QF
=
QFC*
QC
QS
=
QSC*
QC
QR=
QRC*
QC
VP
=
VPC*
BW
VL
=
VLC*
BW
VF
=
VFC*
BW
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April
24,
2002
VS
=
0.82*
BW~
VF
VR
=
0,09*
BW~
VL
`
Liver
metabolism'
VMAX1=
VMAXIC*
BW**
07
KOal
~
r~
K0alC*
BW**
0.7
Kgs
KgsC*
bw**(~
0.3)
Kgsm
KgsmC*
bw**(~
0.3)
Kfee
=
KfeeC*
BW**(~
0.3)
Ki
=
K1C*
BW**(~
0.3)
Kia
K1aC*
BW**(~
0,3)
AGSOI=
GSO1*
VL
`
Lung
metabolism
VMAXp=
PLRATIO*
VMAXpC*
BW**
0.7
KOap
=
KOapC*
BW**
0,7
AGSOpGSOp*
VP
P1=
0
P1R=
0
P2=
0
P2R=
0
P3=
100
P3R=
100
END
$`
End
ofinitial'

DYNAMIC
ALGORITHM
IALG'=
2
$~
Gearmethod
for
stiff
systems'

DERIVATIVE
`
CI=
Concentration
in
inhaled
air
(
mg/
l)'
CI=
MW/
24450*
CONC*
pulse(
tstart,
tper,
tchng)

`
Algebraic
solution
for
CAl
after
gas
exchange'
CA1=
(
QC*
CV
+
QP*
CI)/(
QC
+
QP/
PB)
CX
=
CAl/
PB
`
Mass
balance
for
the
lung
tissue
compartment'
RAP
=
QC*(
CA1~
CA)­
RAMp
AP
=
INTEG(
RAP,
0.0)
CP
=
AP/
VP
AUCP=
INTEG(
CP,
0.0)
CA=
CP/
PP
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April
24,
2002
AUCB=
INTEG(
CA,
0.0)

`
UPTAKE
BY
ORAL
ROUTE'
RSTOM
=
~
KA*
STOM
$`
dSTOM/
dT'
STOM
=
INTEG(
RSTOM,
0.0)
+
ODOSE
$`
amount
in
stomach
(
mg)'

`
AS
=
Amount
in
slowly
perfused
tissues
(
mg)'
RAS
=
QS*(
CA~
CVS)
AS
=
INTEG(
RAS,
0.0)
CVS
=
AS/(
VS*
PS)
CS
=
AS/
VS
`
AR=
Amount
in
richly
perfused
tissues
(
mg)'
RAR
=
QR*(
CA~
CVR)
AR
=
INTEG(
RAR,
0.0)
CVR
=
AR/(
VR*
PR)
CR
=
ARIVR
`
ÀY
=
Amount
in
fat
tissues
(
mg)'
RAF
=
QF*(
CA~
CVF)
AF
=
INTEG(
RAF,
0.0)
CVF
=
AF/(
VF*
PF)
CF
=
AF/
VF
CV
=
Mixed
venous
blood
concentration
(
mg/
l)'
CV
=
(
QF*
CVF
+
QL*
CVL
+
QS*
CVS
+
QR*
CVR)/
QC
`
LIVER
METABOLISM'
`
AL
=
Amount
in
liver
tissue
(
mg)'
RAL
=
QL*(
CA~
CVL)~
RAMl
+
KA*
STOM
AL
=
INTEG(
RAL,
0.0)
CVL
=
AL/(
VL*
PL)
CL
=
AL/
VL
AUCL
=
INTEG(
CL,
0.)

`
AM
=
Amount
metabolized
liver
(
mg)'
RAM1=(
VMAX1*
CVL)/(
KM+
CVL)+
RACPG1*
MW/
I000
AMI=
INTEG(
RAM1,0.)
AMPI=
AM1*
1000/
MW
RAMP1=
RAML*
1000/
MW
`
CMl=
OXIDATIVE
METABOLITE
LIVER
mg/
L)'
RAMMl=(
VMAX1*
CVL)/(
KM+
CVL)~
RACMGl*
MW/
l000~
RACMEEl*
MW/
1000
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April
24,
2002
AMM1=
INTEG(
RAMM1,0.)
CML=
AMMI/
VL
`
GSl
=
GLUTAHIONE
LIVER
(
uM)'
GSHtdl
=
DELAY(
GSI*
1
,
GS1,
TD,
10000)
$`
Time
delayed
GSH
levels'
RK0I~~
K0al*(
GSOl+
KS)/(
GSHtdl
+
KS)­
K1A*
K0l
KOl=
INTEG(
RKOL,
KOOl)
RAMGS1=
KOl­
K1
*
GS1*
VL.
RACMG1..
RACPGI
AMGS1=
INTEG(
RAMGS1,
AGSO1)
GS1=
AMGS1/
VL
`
ACMGlAMT
METABOLITE
CONJUGATED
WITH
GSH
LIVER
(
uMOLES)'
RACMGI=
KGSM*
GS1*
CMI*
1000/
MW
ACMG1=
INTEG(
RACMGI,
0.0)

`
ACMEEI=
AMT
METABOLITE
CONJUGATED
WITH
EVERYTHING
ELSE
LIVER'
`
uMOLES'
RACMEEI=
KFEE*
VL*
CM1*
1
000/
MW
ACMEEI=
INTEG(
RACMEE1,0.)

`
ACPGI
=
AMT
PARENT
CONJUGATED
WITH
GSH
LIVER
(
uMOLES)'
RACPG1=
KGS*
GS1*
CVL*
VL*
1000/
MW
ACPG1=
INTEG(
RACPG1,0.)

`
LUNG
METABOLISM'
`
AMp=
Amount
metabolized
lung
(
mg)'
RAMp=(
VMAXp*
CA)/(
KM+
CA)+
RACPGp*
MW/
i000
AMp"
INTEG(
RAMp,
O.)
AMPp=
AMp*
1000./
MW
pJ~=
p~
J~
4p*
i
000./
MW
`
CMpOXIDATIVE
METABOLITE
(
mg/
L)'
RAMMp=(
VMAXp*
CA)/(
KM+
CA)~
RACMGp*
MW/
1
000~
RACMEEp*
MW/
i000
AMMp=
INTEG(
RAMMp,
0.)
CMpAMMp/
VP
`
GSpGLUTAHIONE
LUNG
(
uM)'
GSHtdp=
DELAY(
GSp*
1
,
GSp,
TD,
1
0000)$'
Time
delayed
GSH
levels'
RK0p=
K0AP*(
GSOp+
KS)/(
GSHtdp+
KS)~
KlA*
KOp
KOpINTEG(
RKOp,
KOOp)
RAMGSp'KOp­
Kl*
GSp*
Vp..
RACMGpRACpGp
AMGSp='~
NTEG(
RAMGSp,
AGSOp)
GSp=
AMGSp/
VP
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2002
`
ACMGp=
AMT
METABOLITE
CONJUGATED
WITH
GSH
LUNG
(
uMOLES)'
RACMGp=
KGSM*
GSp*
CMp*
VP*
1000/
MW
ACMGp=
INTEG(
RACMGp,
0.)

`
ACMEEpAMT
METABOLITE
CONJUGATED
WITH
EVERYTHING
ELSE
LUNG
(
uMOLES)'
RACMEEp=
KFEE*
VP*
CMp*
1000/
MW
ACMEEp=
INTEG(
RACMEEp,
0.)

`
ACPGpAMT
PARENT
CONJUGATED
WITH
GSH
LUNG
(
uMOLES)'
RACPGp=
KGS*
GSp*
CP*
VP*
1000/
MW
ACPGPp=
INTEG(
RACPGp,
0.)

`
PCTGSH­
PERCENT
GSH
COMPARED
TO
CONTROL'
PCTGSHP=
GSp/
GSOp*
100
TERMT(
T.
GE.
TSTOP)

PROCEDURAL
(
P1
,
P
1R=
AMP,
RAMP)

IF(
T.
LT.(
DRTIME(
I)+
NEWDAY))
GO
TO
SKIP2
ODOSE=(
ODOSE+
DRPCT(
I)*
DRDOSE)*
PULSE(
TSTART,
TPER,
TPER)
1=
1+
1
IF(
I.
LT.
7)
GO
TO
SKIP2
1=
1­
6
NEWDAY=
NEWDAY+
24.0
SKIP2..
CONTINUE
IF
(
AMIN1
(
AMP1,
RAMP1,
ACMG1,
RACMG1,
ACMEE1,
RACMEE1).
LT.
1
E­
9)
GOTO
OUT
`
Pl=
PERCENT
PARENT
METABOLISM
THROUGH
GSH'
P1
=
ACPG1IAMP1*
100
P1
R=
RACPGJIRAMP1*
100
`
P2=
PERCENT
METABOLITE
CONJUGATED
WITH
GSH'
P2=
ACMGII(
ACMG1+
ACMEE1)*
100
P2R=
RACMGI1(
RACMGH~
RACMEEl)*
100
P3=
100­
P2
P3R=
100­
P2R
OUT.
.
CONTINUE
END
$`
End
ofprocedural'

C~\
WINDOWS\
TempararyInternet
Files\
ContentilE5\
4EAADd1&\
edc­
AppCrev.
wpd
April
24,
2002
END
$`
End
of
derivative'
END
$`
End
ofdynamic'
END
$`
End
of
program'

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Files\
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4EAADd19A\
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24,
2002
C.
6
General
Outline
for
Route­
to­
Route
Extrapolation
Reports
A
total
ofthree
route­
to­
route
extrapolation
reports
will
be
generated
for
EDC,
one
for
each
ofthe
following
endpoints:
subchronic
toxicity,
subchronic
neurotoxicity,
and
reproductive
toxicity.
At
a
minimum,
each
ofthese
reports
will
follow
the
general
outline
presented
below.

1.0
Introduction
Statement
of
objectives
for
a
specific
endpoint
route­
to­
route
extrapolation
relevant
to
the
HAPs
testing
for
EDC
Application
ofthe
PBPK
Model
for
EDC
relevant
to
specific
endpoint
2.0
Summary
ofKey
Study
(
ies)

For
subchronic
toxicity,
the
design
and
results
of(
Daniel
et
al.,
1994)
will
be
summarized
relevant
to
the
route­
to­
route
extrapolation
and
results
from
Tier
I
Program
Review
Testing.

For
subehronic
neurotoxicity,
the
design
and
results
ofthe
Tier
II
testing
will
be
summarized
relevant
to
the
route­
to­
route
extrapolation
and
results
from
Tier
I
Program
Review
Testing.

For
reproductive
toxicity,
the
design
and
results
ofthe
Tier
II
testing
wifi
be
summarized
and
the
results
of
Alumot
et
al.
(
1976),
Rao
et
al.
(
1980)
and
Lane
et
al.
(
1982)
will
be
summarized
relevant
to
the
route­
to­
route
extrapolation
and
results
from
Tier
I
Program
Review
Testing.

3.0
Selection
of
Critical
Endpoints
and
Dose
Measure(
s)

Forsubchronic
toxicity,
subchronic
neurotoxicityandreproductive­
toxicity,
theendpo
ints
and
dose
measures
will
be
determined
from
the
Tier
II
testing.

4~~
0
Route­
to­
Route
Extrapolation
Results
Quantitative
calculationofinhalation
NOAEL/
LOAEL
values
for
corresponding
oral
values.

5~
0
Sensitivity
Analysis
Assessment
ofthe
contribution
ofvariability/
uncertainty
in
each
parameter
to
PK
modeling
results.

6.0
Conclusions
7.0
References
C:\
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Files\
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4EAAD~
0A\
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AppC_
rev.
wpd
April
24,
2002
