Peter
Voytek
To:
John
Schaoffer/
DC/
USEPA/
US
©
EPA
<
pvoytek@
crosshnk.
n
cc:
et>
Subject:
appendix
C
with
updated
changes
02/
12/
2003
02:
40
PM
John.
The
attached
file
contains
the
EDC
appendix
C
with
updated
changes
that
were
not
included
in
the
original
version
mailed
to
you
by
Caffey
Norman.
If
you
have
any
questions
let
me
know.

Peter
edc­
appCrevwpd
RECEIVED
OPPT
NCIC
2003
MAR11
5:
03PM
OPPT­
2003­
0010­
0019
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
dichloride,
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.
7
References
1
C.
1.
Pharmacokinetic
Studies
for
F344
Rats
A
total
of
4
pharmacokinetic
studies
(
designated
Study
1,
Study
2,
Study
3
and
Study
4)
will
be
conducted
to
support
PBPK
modeling
activities
for
ethylene
dichioride,
as
described
in
this
section
(
C.
1.).
It
is
anticipated
that
the
pharmacokinetic
studies
will
start
one
year
after
the
ECA
is
signed,
with
an
interim
report
generated
six
months
later,
and
the
final
report
submitted
three
months
later
(
see
Appendix
A
for
complete
schedule).

Study
1:
Demonstration
of
Periodicity
Following
Repeated
Inhalation
Exposures
__________________
Proposed
Rat
F344
Young
adult
132
animals
(
3
controls
at
beginning
(
t
=

0)
and
3
at
end
of
exposure
(
t
=
6
hr)
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
of
exposure
(
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
of
EDC
(
n
=
3)
at
0.25,
0.5,
6.25,
and
16
hrs
after
exposure
starts
on
days
1,
3,
and
5.
Venous
blood
concentrations
of
EDC
(
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
Dose(
s)
Route
Exposure
Frequency
Observations
Kinetic
studies
will
be
conducted
for
demonstrating
periodicity
in
the
rat
following
inhalation
exposures
with
EDC.
Measurements
of
EDC
in
blood
and
tissue:
blood
partition
coefficients
are
anticipated
to
be
sufficient
to
describe
tissue
concentration
time
courses
ofEDC
based
on
the
results
of
Spreafico
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
liverGSH
will
be
used
to
further
calibrate
and
validate
theGSH­
depletion
portion
ofthe
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
(
Frederick
et
al.,
1992),
ethylene
oxide
(
Krishnan
et
al.,
1992),
and
allyl
chloride
(
Clewell
and
Andersen,
1994).
In
contrast,
elimination
of
EDC
metabolites
via
the
GSH
pathway,
measured
as
urinary
thiodiglycolic
acid
(
TDGA)
is
not
an
optimal
biomarker
of
GSH
metabolism.
Departures
from
a
linear
relationship
between
dose
and
24­
hour
TDGA
may
be
observed
as
dose
increases
(
Payan
et
al.,
1993)
in
the
range
of
doses
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
(
forglutathione
determination)
will
be
collected
immediately
following
sacrifice.
Blood
samples
willbe
drawn
from
6
animals
only
at
sampling
times
where
lung
and
liverGSH
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.
Apositive
control
(
spiked
storage
sample)
will
be
required
to
demonstrate
the
recovery
of
EDC
in
tissues
following
freezing.
Tissues
will
be
stored
until
after
the
Tier
I
Program
Review.

3
Study
2:
Demonstration
of
Periodicity
Following
Repeated
Oral
Exposures
to
Ethylene
Dichloride
by
Corn
Oil
Gavage
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
hr)
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
ix/
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
of
EDC
(
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
of
EDC
in
blood
and
tissue:
blood
partition
coefficients
are
anticipated
to
be
sufficient
to
describe
tissue
concentration
time
courses
of
EDC
based
on
the
results
of
Spreafico
et
al.
(
1980)
where
concentrations
in
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
of
lung
and
liver
GSH
will
be
used
to
further
calibrate
and
validate
the
GSHdepletion
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
Parameter
Species
Strain
Age
Number
of
animals
4
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
(
Frederick
et
al.,
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
optimalbiomarker
ofGSH
metabolism.
Departuresfrom
a
linear
relationship
between
dose
and
24­
hourTDGAmaybe
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
afterdosing,
would
compromise
use
ofthis
biomarker
as
validation­
for
GSH
metabolism
during
a
repeated
dosing
study.

The
testdose
was
selected
based
on
the
doses
administeredto
ratsin
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
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
recoveryofEDC
in
tissues
following
freezing.
Tissues
willbe
stored
until
after
the
Tier
I
Program
Review.

5
Study
3:
Demonstration
of
Periodicity
Following
Repeated
Oral
Exposures
to
Ethylene
Dichloride
by
Agueous
Gavage
Parameter
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
hr)
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
1
x/
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
of
EDC
(
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
of
EDC
in
blood
and
tissue:
blood
partition
coefficients
are
anticipated
to
be
sufficient
to
describe
tissue
concentration
time
courses
of
EDC
based
on
the
results
of
Spreafico
et
al.
(
1980)
where
concentrations
in
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
of
lung
and
liverGSH
willbe
used
to
further
calibrate
and
validate
the
GSHdepletion
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
6
chloride
(
D'Souza
and
Andersen,
1988),
EDC
(
D'Souza
et
al.,
1988),
ethyl
acrylate
(
Frederick
et
al.,
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
hourTDGAmaybe
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
afterdosing,
would
compromise
use
ofthis
biomarker
as
validation­
for
GSH
metabolism
during
a
repeated
dosing
study.
Therateconstant
for
oral
absorption
from
a
gavage
dose
of
EDC
in
water
(
to
be
determined
via
PBPK
modeling)
will
be
considered
appropriate
for
modeling
oftoxicity
studies
in
whichEDC
was
ingestedviaad
libitum
access
to
drinkingwater
(
e.
g.,
O'Flaherty,
1996).

The
test
dose
will
be
selected
based
on
the
water
solubility
and
dosing
volume.
Thewater
solubility
of
EDC
is
8.7
mg/
mL,
and
the
dosing
volume
for
a
rat
should
be
no
more
than
5
ml/
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
will
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
requiredto
demonstrate
the
recoveryof
EDC
in
tissues
following
freezing.
Tissues
will
be
stored
until
after
the
Tier
I
Program
Review.

7
Study
4:
Determination
of
Partition
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
etal.
(
1989,
Toxicol.
AppI.
Pharmacol.
98,
87­
99).

A
total
of5
rats
will
be
used,
if
partition
coefficients
are
determined
individually.
A
higher
number
of
animals
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.

8
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
routeusingaPBPK
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
of
action.

9
C.
3
Subchronic
Neurotoxicity
Route­
To­
Route
Extrapolation
The
NOAEL/
LOAELvalues
obtained
from
the
subchronic
neurotoxicity
testing
willbe
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
of
any
neurological
effects
observed
and
a
plausible
mechanism
ofaction.

10
C.
4
Reproductive
Toxicity
Route­
To­
Route
Extrapolation
The
NOAEL/
LOAEL
values
obtained
from
the
reproductive
toxicity
testing
will
be
extrapolated
from
the
oral
route
to
the
inhalation
route
using
a
PBPK
model
(
Appendix
C.
5).
The
results
Of
Alumot
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
of
EDC,
or
amount
ofEDC
metabolized)
used
to
perform
this
extrapolation
will
be
determined
based
on
a
consideration
ofany
reproductive
effectsobserved
and­
a
piausibiemechan~
sm
of
action.

11
C.
5
PBPK
Model
Description
and
Coding
The
preliminary
PBPK
model
(
to
be
refined
as
part
of
this
ECA)
is
a
modification
of
D'Souzaet
al.
(
1987,
1988)
to
include
periodic
consumption
of
drinking
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
of
glutathione
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
of
EDC
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
dichloride
Oxidative
metabolism
GSH
cl~~
Cl~
c1~~
°
(
VmaxL,
Km)
(
KOSM)

+
OSH
(
KGS)
OH
I'
(
KFEE)

Cl~
S­
G
Cl
Macromolecules,

­
other
reactions
The
PBPK
model
for
repeated
exposure
to
EDC,
to
be
refinedbased
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/
01'

INITIAL
12
`
SPECIAL
FLOW
RATES'
CONSTANT
QPC=
15
$`
alveolar
ventilation
rate
L/
hr/
kgAO.
74'
CONSTANT
QCC=
15
$`
Cardiac
output
L/
hr/
kg"
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
flow
to
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
=
0.3
16
$`
Fraction
rapid
tissue'
CONSTANT
VSC
=
0.48
$`
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
$
Fatlblood
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
$`
Blood/
air
partition
coefficient'

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

`
KINETIC
CONSTANTS'
CONSTANT
VMAX1C
=
3.15
$`
Maximum
velocity
of
metabolism
(
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
S'conj
rate
const
w
parent(
l/(
uM­
hr­
kg'~­
0.7))'
CONSTANT
KGSMC
=
0.15
$`
conj
rate
const
w
metab
(
l/(
uM­
hr­
kgt'­
0.7))'
CONSTANT
KFEEC=
4500
$`
conj
rate
const
w
non
gsh
(
1/(
hr­
kg'~­
0.7))'
CONSTANT
KOa1C
=
3.8
$`
GSH
synthase
synthesis
liv(
umol/
hr/
hr/
kgt'­
0.7)'
CONSTANT
KOapC
=
0.22
$`
GSH
synthase
synthesis
lu(
umol/
hr/
hr/
kg"­
0.7)'
CONSTANT
KIC=
0.
l
16
$`
GSH
breakdown
(
l/
hr/
kg'~­
0.7)'
CONSTANT
K1
aC=
0.095
$`
GSH
synthase
breakdown(
1
/
hr/
kg'~'­
0.7)'

13
CONSTANT
KS
=
1000
$`
Maximum
GSH
induction
(
uM)'
CONSTANT
TD
=
1.5
$`
Time
delay
(
hr)'
CONSTANT
GSO1
=
7000
$`
Initial
GSH
concentration
(
uM)'
CONSTANT
GSOp
=
1200
$`
Initial
GSH
concentration
(
uM)'
CONSTANT
PLRATIO=
0.
14
$`
MFO
ratio
lung/
liver'
CONSTANT
KOOl
=
11.254
$
liver
(
umol/
hr)'
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=
1
$`
Counter
for
drinking
arrays'
CONSTANT
DRCONC=
0.0
$`
Conc
of
EDC
in
water
(
mg/
L)'
CONSTANTKA
=
5
$
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'

`
TIMING
PARAMETERS'
CONSTANT
TSTART
=
48.
$
Start
of
exposure
(
hrs)'
CONSTANT
TPER=
24
CONSTANT
TSTOP=
120
CONSTANT
POINTS=
1200
$`
Number
of
points
in
plot'
CINT
=
TSTOP/
POINTS
$`
Communication
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
14
VP
=
VPC*
BW
VL
=
VLC*
BW
VF
=
VFC*
BW
VS
=
0.82*
BWVF
VR
=
0.09*
BW~
VL
`
Liver
metabolism'
VMAX1=
VMAXIC*
BW**
0.7
KOal
=
KOaIC*
BW**
0.7
Kgs
=
KgsC*
bw**(~
0.3)
Kgsm
=
KgsmC*
bw**(~
0.3)
Kfee
=
KfeeC*
BW**(~
0.3)
Kl
=
K1C*
BW**(~
0.3)
Kia
=
KlaC*
BW**(~
0.3)
AGSOl=
GS01
*
VL
`
Lung
metabolism'
VMAXp=
PLRATIO*
VMAXpC*
BW*
*
0.7
KOap
=
KOapC*
BW**
0.7
AGSOp=
GSOp*
VP
P1=
0
P1R=
0
P2=
0
P2R=
0
P3=
100
P3R=
100
END
$`
End
of
initial'

DYNAMIC
ALGORITHM
IALG=
2
$`
Gear
method
for
stiff
systems
DERIVATIVE
`
CI=
Concentration
in
inhaled
air
(
mg/
I)'
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
15
AP
=
INTEG(
RAP,
0.0)
CP
=
AP/
VP
AUCP=
INTEG(
CP,
0.0)
CA=
CP/
PP
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*(
CACVS)
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
=
AR/
VR
`
AF
=
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!
1000
AM1=
INTEG(
RAMI,
0.)

16
AMPI=
AM1*
1000/
MW
RAMP1=
RAML*
1
000/
MW
`
CMI=
OXIDATIVE
METABOLITE
LIVER
mg/
L)'
RAMMI=(
VMAXI*
C
VL)/(
KM­
1­
CVL)­
RACMGI*
MW/
1
000­
RACMEE1*
MW/
1000
AMM1=
INTEG(
RAMM1,0.)
CML=
AMM1/
VL
`
GSI
=
GLUTAHIONE
LIVER
(
uM)'
GSHtd1
=
DELAY(
GS1*
1,
GS1,
TD,
10000)
$`
Time
delayed
GSH
levels'
RKO1=
KOal*(
GSO1+
KS)/(
GSHtd1
~
i~
KS)~
K1A*
K01
K0l=
INTEG(
RKOL,
K00l)
RAMGS1=
KOl­
K1
*
GS1*
VLRACMGI..
RACPG1
AMGS1=
INTEG(
RAMGS1,
AGSO1)
GS1=
AMGSI/
VL
`
ACMGl=
AMT
METABOLITE
CONJUGATED
WITH
GSH
LIVER
(
uMOLES)'
RACMG1=
KGSM*
GS1*
CM1*
1000/
MW
ACMGI=
INTEG(
RACMG1,0.0)

`
ACMEEl=
AMT
METABOLITE
CONJUGATED
WITH
EVERYTHING
ELSE
LIVER'
`
uMOLES'
RACMEE1=
KFEE*
VL*
CM1*
1000/
MW
ACMEE1=
INTEG(
RACMEE1,0.)

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

`
LUNG
METABOLISM'
`
AMp=
Amount
metabolized
lung
(
mg)
RAMp=(
VMAXp*
CA)/(
KM+
CA)+
RACPGp*
MW/
1000
AMp=
INTEG(
RAMp,
0.)
AMPp=
AMp*
1000./
MW
RAMPp=
RAMp*
1000./
MW
`
CMp=
OXIDATIVE
METABOLITE
(
mg/
L)'
RAMMp=(
VMAXp*
CA)/(
KM~
l~
CA)~
RACMGp*
MW/
1
000rnRACMEEp*
MW/
1000
AMMp=
INTEG(
RAMMp,
0.)
CMp=
AMMp/
VP
`
GSp=
GLUTAHIONE
LUNG
(
uM)'

17
GSHtdp=
DELAY(
GSp*
1
,
GSp,
TD,
1
0000)$'
Time
delayed
GSH
levels'
RKOp=
KOAP*(
GS
Op+
KS)/(
GSHtdp+
KS
)­
K
1
A*
KOp
KOp=
INTEG(
RKOp,
KOOp)
RAMGSp=
KOp­
K1
*
GSp*
VPRACMGpRACPGp
AMGSp=
INTEG(
RAMGSp,
AGSOp)
GSp=
AMGSp/
VP
`
ACMGp=
AMT
METABOLITE
CONJUGATED
WITH
GSH
LUNG
(
uMOLES)'
RACMGp=
KGSM*
GSp*
CMp*
VP*
1000/
MW
ACMGp=
INTEG(
RACMGp,
0.)

`
ACMEEp=
AMT
METABOLITE
CONJUGATED
WITHEVERYTHING
ELSE
LUNG(
uMOLES)'
RACMEEp=
KFEE*
VP*
CMp*
1000/
MW
ACMEEp=
1NTEG(
RACMEEp,
0.)

`
ACPGp=
AMT
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
1
R=
AMP,
RAMP)

IF(
T.
LT.(
DRTIME(
I)­
l­
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(
AMPI,
RAMP1,
ACMG1,
RACMGI,
ACMEE1,
RACMEEI).
LT.
lE­
9)
GOTO
OUT
`
Pl=
PERCENT
PARENT
METABOLISM
THROUGH
GSH'
P1=
ACPG1/
AMP1*
100
P1
R=
RACPGI/
RAMPI*
100
`
P2=
PERCENT
METABOLITE
CONJUGATED
WITH
GSH'
P2=
ACMGI/(
ACMG1+
ACMEEI)*
100
18
P2R=
RACMG1/(
RACMGI+
RACMEE1)*
100
P3=
100­
P2
P3R=
100­
P2R
OUT..
CONTINUE
END
$`
End
of
procedural'
END
$`
End
of
derivative'
END
$`
End
of
dynamic'
END
$`
End
of
program'

19
C.
6
General
Outline
for
Route­
to­
Route
Extrapolation
Reports
A
total
of
three
route­
to­
route
extrapolation
reports
will
be
generated
for
EDC,
one
for
each
of
the
following
endpoints:
subchronic
toxicity,
subchronic
neurotoxicity,
and
reproductive
toxicity.
At
a
minimum,
each
of
these
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
of
the
PBPK
Model
for
EDC
relevant
to
specific
endpoint
2.0
Summary
of
Key
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
subchronic
neurotoxicity,
the
design
and
results
of
the
TierII
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
TierII
testing
will
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)

For
subchronic
toxicity,
subchronic
neurotoxicity
and
reproductive
toxicity,
the
endpoints
and
dose
measures
will
be
determined
from
the
Tier
II
testing.

4.0
Route­
to­
Route
Extrapolation
Results
Quantitative
calculation
ofinhalation
NOAEL/
LOAELvalues
forcorresponding
oral
values.

5.0
Sensitivity
Analysis
Assessment
ofthe
contribution
ofvariability/
uncertainty
in
each
parameter
to
PK
modeling
results.

6.0
Conclusions
7.0
References
20
C.
7
References
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E,
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E,
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Clewell
HJ
3rd,
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1994;
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RW,
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1988;
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D'Souza
RW,
Francis
WR,
Bruce
RD,
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D'Souza
RW,
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WR,
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tissue
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and
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J
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Exp
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1988;
245(
2):
563­
568.

Daniel
FB,
Robinson
M,
Olson
GR,
York
RG,
Condie
LW,
Lane
RW,
Riddle
BL,
Borzelleca
JF.
Ten
and
ninety­
day
toxicity
studies
of
1
,2­
dichloroethane
in
Sprague­
Dawley
rats.
Drug
Chem
Toxicol
1994;
17(
4):
463­
477.

Frederick
CB,
Potter
DW,
Chang­
Mateu
MI,
Andersen
ME.
A
physiologically
based
pharmacokinetic
and
pharmacodynamic
model
to
describe
the
oral
dosing
of
rats
with
ethyl
acrylate
and
its
implications
for
risk
assessment.
Toxicol
AppI
Pharmacol
1992;
114:
246­
260.

Gargas
ML,
Burgess
RJ,
Voisard
DE,
Cason
GH,
Andersen
ME.
Partition
coefficients
of
low­
molecular
weight
volatile
chemicals
in
various
liquids
and
tissues.
Toxicol
Appl
Pharmacol
1989;
98:
87­
99.

Krishnan
K,
Gargas
ML,
Fennell
TR,
Andersen
ME.
A
physiologically
based
description
of
ethylene
oxide
dosimetry
in
the
rat.
Toxicol
Ind
Health.
1992;
8:
12
1­
40.

Lane
RW,
Riddle
BL,
Borzelleca
JF.
Effects
of
1,2­
dichioroethane
and
1,1,1­
thichioroethane
in
drinking
water
on
reproduction
and
development
in
mice.
Toxicol
Appl
Pharmacol
1982;
63(
3):
409­
42
1.

National
Cancer
Institute.
Bioassay
of
1
,2­
dichloroethane
for
21
possible
carcinogenicity,
NCI
Carcinogenesis
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No.
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DHEWPublication
No.
(
NIH)
1978;
78­
1361.
Washington,
D.
C.

O'Flaherty
EJ.
A
physiologically
based
model
of
chromium
kinetics
in
the
rat.
Toxicol
AppI
Pharmacol
1996;
138:
54­
64.

Payan
JP,
Beydon
D,
Fabry
J,
Brondeau
MT,
Ban
M,
de
Ceaurriz
J.
Urinary
thiodiglycolic
acid
and
thioether
excretion
in
male
rats
dosed
with
1
,2­
dichloroethane.
J
Appl
Toxicol
1993;
13(
6):
417­
422.

Rao
KS,
Murray
JS,
Deacon
MM,
John
JA,
Calhoun
LL,
Young
JT
Teratogenicity
and
reproduction
studies
in
animals
inhaling
ethylene
dichloride.
Banbury
Report
5:
149­
166
1980
Reitz
RH,
Fox
TR,
Ramsey
JC,
Quast
JF,
Langvardt
PW,
Watanabe
PG.
Pharmacokinetics
and
macromolecular
interactions
of
ethylene
dichloride
in
rats
after
inhalation
or
gavage.
Toxicol
AppI
Pharmacol
1982;
62:
190­
204.

Spreafico
F,
Zuccato
E,
Marcucci
F,
Sironi
M,
Paglialunga
S,
Madonna
M,
Mussini
E.
Pharmacokinetics
of
ethylene
dichloride
in
rats
treated
by
different
routes
and
its
longterm
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In:
Ames
B,
Infante
P,
Reitz
RH,
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1980:
107­
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22
