Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
1
Final
Draft
Chapter
III.
Toxicokinetics
Limited
data
are
available
on
the
toxicokinetics
of
the
haloacetonitriles
(
HANs).
No
data
were
located
on
the
absorption
or
distribution
of
BCAN
following
oral
exposure.
A
comparative
toxicokinetics
and
metabolisms
study
in
mice
and
rats
has
been
conducted
for
DBAN
(
NTP,

2002).
However,
this
study
was
not
available
for
review
at
the
time
this
document
was
prepared.

No
studies
were
located
on
the
absorption,
distribution,
metabolism,
or
excretion
of
any
of
the
HANs
following
inhalation
or
dermal
exposure,
although
some
qualitative
information
can
be
inferred
from
toxicity
studies.

A.
Absorption
Roby
et
al.
(
1986)
administered
single
oral
gavage
doses
of
either
[
1­
14C]­
(
labeled
on
the
cyanide
group)
or
[
2­
14C]­
DCAN
(
labeled
on
the
dichloromethyl
group)
in
water
to
male
F344
rats
and
B6C3F1
mice.
The
administered
doses
were
0.2,
2,
or
15
mg/
kg
for
the
rats
and
2
or
15
mg/
kg
for
the
mice.
Appearance
of
label
in
feces,
urine
and
expired
air
was
monitored
until
at
least
70%
of
the
radioactivity
had
been
recovered.
The
amount
of
time
required
to
recover
70%

of
the
administered
radioactivity
differed
across
species.
In
rats,
the
collection
of
data
continued
for
6
days
for
[
1­
14C]­
DCAN
and
for
2
days
for
[
2­
14C]­
DCAN.
In
mice,
collection
was
terminated
at
24
hours
for
both
positions
of
radiolabel,
since
at
least
70%
of
the
dose
had
been
collected.
These
results
indicate
that
DCAN
in
water
is
well
absorbed
(
at
least
80%
to
90%)
from
the
gastrointestinal
tract,
since
only
8%
to
20%
of
the
total
dose
was
excreted
in
feces.
The
rate
of
absorption
was
not
determined,
and
data
on
the
blood
concentrations
of
radiolabel
over
time
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
2
Final
Draft
were
not
reported.
The
appearance
of
radiolabel
in
urine
and
exhaled
air
at
24
hours
suggests,

however,
that
DCAN
was
absorbed
rapidly.

Roth
et
al.
(
1990),
in
a
published
abstract,
reported
on
a
study
designed
to
test
whether
differential
absorption
and
distribution
kinetics
of
TCAN
in
tricaprylin
versus
corn
oil
was
responsible
for
observed
differences
in
developmental
toxicity
studies.
Pregnant
rats
(
strain
and
number
not
specified)
were
administered
a
single
oral
gavage
dose
of
55
mg
[
14C]­
TCAN/
kg
in
either
tricaprylin
or
corn
oil
on
gestation
day
10
or
11.
Levels
of
radiolabel
were
followed
in
the
maternal
stomach
and
intestinal
contents,
blood,
liver,
spleen,
heart,
adipose
tissue,
and
embryonic
tissue.
Maximal
blood
and
tissue
levels
were
observed
4
to
6
hours
following
exposure,

indicating
rapid
absorption
kinetics.
The
choice
of
solvent
vehicle
did
not
affect
the
absorption
kinetics.
No
data
on
the
degree
of
absorption
were
provided
in
this
published
abstract.
A
published
version
of
this
study
was
not
located,
and
the
data
presented
did
not
allow
an
independent
verification
of
the
results.

The
lethality
observed
in
acute
dermal
toxicity
tests
for
BCAN
(
Eastman
Kodak
Co.,

1992)
and
TCAN
(
Smyth
et
al.,
1962)
indicates
that
systemic
exposure
to
these
compounds
occurred,
demonstrating
that
HANs
can
be
absorbed
dermally.
These
studies
are
not
adequate
to
estimate
the
rate
and
degree
of
absorption
by
the
dermal
route.

In
summary,
the
existing
data
suggest
that
HANs
can
be
absorbed
following
either
oral
or
dermal
administration.
HANs
are
rapidly
absorbed
following
oral
administration,
based
on
the
observed
peak
blood
concentrations
4
to
6
hours
after
dosing
reported
by
Roth
et
al.
(
1990).
The
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
3
Final
Draft
strength
of
this
conclusion
is
limited,
however,
since
only
a
published
abstract
is
available.
The
degree
of
absorption
following
oral
dosing
is
nearly
complete,
based
on
the
small
fraction
of
the
administered
radioactivity
observed
in
feces
(
Roby
et
al.,
1986).
HANs
can
be
absorbed
through
the
skin,
but
the
existing
data
are
insufficient
to
estimate
the
rate
or
degree
of
absorption.
No
data
are
available
to
determine
whether
HANs
can
be
absorbed
following
inhalation
exposure.

B.
Distribution
Roby
et
al.
(
1986)
studied
the
tissue
distribution
of
radiolabel
following
administration
of
single
oral
gavage
doses
of
0.2
to
15
mg/
kg
of
[
1­
14C]­
or
[
2­
14C]­
DCAN
in
water
to
rats
and
mice.
Daily
excreta,
including
exhaled
volatile
organic
compounds
and
CO
2,
were
analyzed
until
at
least
70%
of
the
radioactivity
was
recovered.
The
amount
of
time
required
to
recover
70%
of
the
administered
radioactivity
differed
across
species:
6
days
following
oral
administration
of
[
1­
14C]­
DCAN
and
48
hours
for
[
2­
14C]­
DCAN
in
rats,
and
24
hours
regardless
of
the
position
of
the
radiolabel
in
mice.
After
at
least
70%
of
the
administered
dose
had
been
excreted,
the
animals
were
sacrificed
and
tissues
were
collected.
Label
was
detected
in
all
tissues
tested
(
see
Table
III­

1),
although
the
residual
tissue
levels
represented
a
small
portion
of
the
administered
dose.
Six
days
after
oral
administration
of
[
1­
14C]­
DCAN
to
rats,
the
tissue
distribution
of
the
label
as
percent
of
the
administered
dose
was:
blood
(
4.1
 
7.9%),
muscle
(
3.9
 
7.9%),
skin
(
3.3
 
6.3%)
and
liver
(
1.9
 
2.6%)
for
the
three
dose
groups.
For
[
2­
14C]­
DCAN,
the
liver
retained
the
largest
amount
of
radiolabel
(
approximately
5%
of
the
administered
dose
2
days
after
treatment),

followed
by
muscle
(
2.7
 
4.8%),
blood
(
2
 
4.6%)
and
skin
(
0.9
 
1.0%).
Most
other
tissues
contained
less
than
1%
of
the
dose.
These
tissue
distribution
data
for
rats
are
presented
in
Table
III­
1.
In
mice,
the
tissue
distribution
did
not
differ
greatly
between
the
alternately
labeled
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
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HECD
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4
Final
Draft
compounds.
The
largest
amount
of
radioactivity
was
present
in
the
liver,
3.5­
4.3%
of
the
administered
dose
for
[
1­
14C]­
DCAN
and
5.1­
5.4%
for
[
2­
14C]­
DCAN.
The
muscle
and
skin
also
contained
appreciable
amounts
of
radioactivity
as
shown
in
Table
III­
2.
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
5
Final
Draft
Dose
Table
III­
1.
Tissue
Levels
of
DCAN
After
Oral
Administration
to
Ratsa.
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
6
Final
Draft
Table
III­
2.
Tissue
Levels
of
DCAN
One
Day
After
Oral
Administration
to
Micea.
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
7
Final
Draft
These
data
do
not
provide
clear
evidence
for
significant
differences
in
distribution
for
the
dimethyl
and
cyanide
carbons,
since
only
small
differences
were
apparent
in
the
rat
study,
and
no
difference
was
observed
for
the
alternatively
labeled
compounds
in
mice.

A
study
to
assess
the
potential
of
TCAN
to
form
protein
and
DNA
adducts
provides
qualitative
evidence
for
wide
tissue
distribution
of
HANs.
Lin
et
al.
(
1992)
administered
single
oral
gavage
doses
ranging
from
7.2
to
69.3
mg/
kg
of
either
[
1­
14C]­
or
[
2­
14C]­
TCAN
in
tricaprylin
to
male
F344
rats.
DNA
was
isolated
from
liver,
stomach,
and
kidney,
and
several
proteins
were
isolated
from
blood.
The
tissues
were
analyzed
from
4
to
48
hours
following
dosing.
More
radiolabel
was
associated
with
DNA
when
the
trichloromethyl
carbon
[
2­
14C]
was
labeled
than
when
the
cyanide
group
carbon
[
1­
14C]
was
labeled.
The
study
authors
hypothesized
that
the
adducts
resulted
from
the
reaction
of
DNA
with
single
carbon
metabolites
formed
by
the
cleavage
of
TCAN.
DNA
binding
was
highest
in
the
stomach,
followed
by
liver
and
kidney.

Adducts
with
globin,
albumin,
and
globulins
were
also
identified,
and
similar
levels
were
observed
with
the
label
at
either
position.
In
addition,
the
HPLC
elution
profiles
for
the
radioactivity
was
the
same
regardless
of
the
position
of
the
label,
leading
the
authors
to
suggest
that
the
protein
adducts
are
formed
from
either
"
2­
carbon
metabolites
(
unsplit)
or
by
direct
reaction
with
TCAN."

Adduct
studies
provide
only
limited
information
on
tissue
distribution,
since
macromolecular
binding
may
also
be
dependent
on
metabolism
of
the
compound.
However,
the
observed
formation
of
serum
protein
adducts
and
the
appearance
of
DNA
adducts
in
all
three
tissues
measured
suggests
wide
distribution
of
TCAN.
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
8
Final
Draft
Additional
studies
have
been
conducted
to
address
the
potential
for
solvent
vehicle
to
alter
the
distribution
of
TCAN
and
have
been
reported
in
two
published
abstracts.
Roth
et
al.
(
1990)

administered
a
single
dose
of
55
mg
[
14C]­
TCAN/
kg
to
pregnant
rats
(
strain
and
number
not
specified)
in
either
tricaprylin
or
corn
oil
on
gestation
day
10
or
11.
The
radiolabel
(
the
identity
of
the
labeled
carbon
was
not
specified)
was
followed
for
48
hours
in
the
maternal
stomach
and
intestinal
contents,
blood,
liver,
spleen,
heart,
and
adipose
tissue,
and
in
embryos.
In
the
blood,

most
counts
were
bound
to
red
blood
cells,
and
in
the
plasma
up
to
50%
of
the
radiolabel
was
protein­
bound.
Tissue
levels
were
highest
in
the
liver
(
approximately
40
µ
g
TCAN
equivalents/
g
at
6
hours
post­
exposure).
Radiolabel
was
also
detected
in
embryos.
According
to
the
study
authors,
there
were
no
solvent­
related
differences
reported
for
any
of
the
toxicokinetic
parameters
evaluated
for
TCAN
when
administered
in
tricaprylin
versus
corn
oil.

In
a
follow
up
study
to
assess
the
impact
of
repeated
dosing
on
the
effect
of
solvent
vehicle
that
was
reported
in
a
published
abstract,
Gordon
et
al.
(
1991)
administered
one,
two,
or
three
successive
daily
doses
(
dose
not
specified)
of
[
1­
14C]
or
[
2­
14C]­
labeled
TCAN
in
tricaprylin
or
corn
oil
to
groups
of
pregnant
rats
(
strain
not
specified)
in
mid­
gestation.
The
animals
were
sacrificed
on
gestation
day
13
and
maternal
and
embryo
levels
of
radiolabel
were
evaluated.
The
14C
levels
in
the
embryos
from
the
tricaprylin
groups
were
described
as
much
greater
than
in
the
corn
oil
vehicle
group
following
3
daily
doses,
but
no
quantitative
estimate
was
reported.
After
three
doses,
maternal
blood
14C
levels
were
higher
for
the
tricaprylin
vehicle
group
compared
to
the
corn
oil
group.
The
abstract
did
not
report
the
effects
of
solvent
vehicle
on
the
embryo
or
maternal
blood
levels
of
TCAN­
associated
radioactivity
after
one
or
two
daily
doses,
precluding
an
analysis
of
trends
in
the
relationship
between
the
number
of
days
of
dosing
and
tissue
Drinking
Water
Criteria
Document
for
Haloacetonitriles
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III­
9
Final
Draft
accumulation.
In
the
maternal
liver,
14C
accumulation
of
both
[
1­
14C]
and
[
2­
14C]
was
greater
in
the
tricaprylin
group
than
in
the
corn
oil
group
after
two
daily
treatments,
but
after
three
doses
with
[
2­
14C]­
labeled
TCAN,
accumulation
was
higher
from
corn
oil.
While
solvent­
related
differences
in
the
accumulation
of
radiolabel
were
reported
in
this
study,
the
degree
of
difference
between
solvent
vehicle
groups
was
not
provided
for
any
of
the
findings
and
apparent
inconsistencies
observed
across
the
dosing
regimens
were
not
adequately
explained.
Based
on
inconsistent
results,
the
absence
of
quantitative
data,
and
the
lack
of
peer
review,
these
data
should
be
viewed
as
preliminary.
In
addition,
the
higher
embryonic
accumulation
of
TCAN
with
tricaprylin
in
this
study
appears
to
be
inconsistent
with
the
results
of
Roth
et
al.
(
1990)
with
TCAN
in
tricaprylin
and
corn
oil,
although
the
latter
study
used
a
shorter
dosing
regimen.

Therefore,
it
remains
unclear
if
solvent
vehicle
affects
tissue
distribution.
Nevertheless,
both
abstracts
provide
qualitative
evidence
for
wide
tissue
distribution
of
TCAN,
in
support
of
the
better­
documented
study
on
DCAN
by
Roby
et
al.
(
1986).

In
summary,
the
two
compounds
tested,
DCAN
and
TCAN,
are
widely
distributed
following
oral
dosing.
Radiolabeled
parent
compound
or
metabolites
have
been
identified
to
varying
degrees
in
blood
and
a
host
of
tissues,
including
in
embryos,
with
no
single
tissue
dominating
HAN
uptake.
The
role
of
solvent
vehicle
on
distribution
of
HANs
also
remains
unresolved,
with
contradictory
results
being
reported
within
a
single
published
abstract
(
Gordon
et
al.,
1991).
(
See
Chapter
VII
for
additional
analysis
of
potential
solvent
vehicle
effects.)
No
data
were
identified
to
evaluate
distribution
of
HANs
following
dermal
or
inhalation
exposure.
Drinking
Water
Criteria
Document
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10
Final
Draft
C.
Metabolism
Pereira
et
al.
(
1984)
administered
a
single
gavage
dose
of
0.75
mmol/
kg
BCAN
(
116
mg/
kg),
DBAN
(
149
mg/
kg),
DCAN
(
82
mg/
kg),
or
TCAN
(
108
mg/
kg)
in
tricaprylin
to
male
Sprague
Dawley
rats.
Urinary
thiocyanate,
the
only
metabolite
measured,
accounted
for
2.25%
to
12.8%
of
the
dose
by
24
hours.
The
excretion
of
thiocyanates
in
the
urine
was
in
the
order
of
BCAN>
DCAN>
DBAN>
TCAN.
The
authors
also
measured
the
effects
of
the
HANs
on
dimethylnitrosamine
demethylase
(
DMN)
activity
(
a
measure
of
CYP2E1
activity)
as
a
marker
of
protein
binding.
Based
on
dose­
responses
from
in
vitro
incubations
with
rat
liver
microsomes,

DBAN
and
BCAN
were
more
potent
inhibitors
of
this
enzyme
than
DCAN
or
TCAN.
In
contrast
to
the
in
vitro
results,
a
single
gavage
dose
of
0.75
mmol/
kg
TCAN,
but
not
0.75
mmol/
kg
DBAN,

significantly
inhibited
DMN
activity
in
liver
microsomes
of
rats
sacrificed
3
or
18
hours
after
dosing.
Results
for
other
HANs
were
not
presented.
The
mechanism
of
inhibition
was
considered
to
be
noncompetitive
or
uncompetitive
(
noncompetitive
inhibition
is
characterized
by
an
altered
ratio
of
K
m:
V
max
with
a
decrease
in
V
max,
while
uncompetitive
inhibition
is
characterized
by
a
constant
ratio
of
K
m:
V
max
with
a
decrease
in
the
V
max).
The
results
suggest
that
HANs
are
not
competing
for
the
active
site
of
CYP2E1.
Although
Pereira
et
al.
(
1984)
suggest
that
oxidative
dehalogenation
is
an
initial
step
in
the
metabolism
of
HANs,
the
data
are
inadequate
to
identify
the
specific
enzymes
involved.
Based
on
their
results
and
earlier
work
on
formation
of
cyanide
from
nitrile
compounds
presented
by
Silver
et
al.
(
1982),
Pereira
et
al.
(
1984)
proposed
a
metabolic
scheme
to
explain
the
formation
of
thiocyanates
from
HANs
(
Figure
III­
1).
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
11
Final
Draft
X
H
H
CN
C
OH
H
H
CN
C
H
CN­
H
C
O
X
X
X
CN
C
OH
X
X
CN
C
X
CN­
X
C
O
X
X
H
CN
C
OH
X
H
CN
C
X
CN­
H
C
O
C
H
O
CN
CO
CNC
O
CN
CO
CN­
X
C
O
CN
O
H
+

+
+

+

+
2
GST?

GST?

GST?
6
CYP?

CYP?
1
2
3
4
5
Glutathione
conjugate
Figure
III­
1
Proposed
Metabolism
of
Haloacetonitriles
Figure
Legend.
Proposed
metabolic
pathways
for
HANs.
Two
distinct
pathways
are
proposed:
conjugation
with
glutathione
and
oxidative
metabolism.
The
glutathione
pathway
is
shown
with
dashed
lines
to
indicate
that
direct
identification
of
these
conjugates
or
other
downstream
metabolites
has
not
been
demonstrated
in
vivo,
as
described
further
in
the
text.
It
is
not
clear
if
the
proposed
glutathione
conjugation
is
catalyzed
by
glutathione­
S­
transferases
(
GST)
or
is
nonenzymatic.
For
the
oxidative
pathway,
dehalogenation
is
thought
to
be
catalyzed
by
CYPs,
although
the
isoforms
that
mediate
this
reaction
have
not
been
identified.

Intermediate
metabolites
labeled
in
the
figure
with
numbers
1
through
6
are
as
follows:
1)
haloacetonitrile;
2)
halocyanomethanol;
3)
haloformaldehyde;
4)
cyanoformaldehyde;
5)
halocyanoformaldehyde;
6)
unidentified
glutathione
conjugates.
Presentation
of
the
oxidative
metabolism
pathway
adapted
from
Pereira
et
al.
(
1984).
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
12
Final
Draft
As
shown
in
this
figure,
metabolism
of
HANs
is
hypothesized
to
occur
by
oxidative
dehalogenation
to
yield
halocyanomethanols.
These
reactions
are
catalyzed
by
mixed­
function
oxidases
such
as
cytochrome
p450s
(
CYP),
although
the
identity
of
the
isozyme(
s)
that
carries
out
the
individual
reactions
has
not
been
determined.
The
halocyanomethanols
are
proposed
to
then
dehydrate
to
form
halocyanoformaldehydes
or
lose
cyanide
to
form
haloformaldehydes,
including
phosgene.
The
cyanoformaldehydes
undergo
further
oxidative
metabolism
to
form
CO
2
and
cyanide
(
which
can
then
be
further
metabolized
to
thiocyanate).

Pereira
et
al.
(
1984)
did
not
measure
the
intermediate
metabolites
proposed
in
their
metabolic
pathway.
However,
a
study
by
Roby
et
al.,
(
1986)
provides
additional
indirect
evidence
for
the
oxidative
metabolism
of
HANs.
Roby
et
al.
(
1986)
administered
single
oral
doses
of
0.2,
2,

or
15
mg/
kg
[
1­
14C]­
or
[
2­
14C]­
DCAN
to
rats,
and
2
or
15
mg/
kg
of
these
compounds
to
mice.
In
both
rats
and
mice,
labeling
of
the
cyanide
group
carbon
resulted
in
higher
amounts
of
radiolabel
in
urine
than
in
expired
air
(
i.
e.,
as
CO
2),
while
radioactivity
excreted
via
both
routes
was
nearly
equal
when
the
dichloromethyl
group
was
labeled.
Marginal
dose­
dependent
changes
in
the
amount
of
the
radiolabel
recovered
in
feces
and
expired
air
were
reported,
but
the
pattern
of
these
changes
were
not
consistent
across
species
or
with
position
of
the
radiolabel,
making
the
findings
difficult
to
interpret.
The
pattern
of
label
distribution
in
tissues
and
excreta
indicated
to
the
authors
that
DCAN
would
be
oxidized
to
dichlorocyanomethanol,
consistent
with
the
metabolic
scheme
proposed
by
Pereira
et
al.
(
1984).
Dichlorocyanomethanol
could
then
either
dehydrate
to
chlorocyanoformaldehyde
or
lose
cyanide
to
form
phosgene,
leading
to
terminal
degradation
products
including
chlorine,
formic
acid,
CO
2
and
cyanide.
The
authors
did
not,
however,
directly
measure
these
metabolites.
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
13
Final
Draft
Conjugation
with
glutathione
(
GSH)
also
appears
to
be
an
important
pathway
in
the
metabolism
of
HANs
as
shown
in
Figure
III­
1.
Lin
and
Guion
(
1989)
investigated
the
ability
of
BCAN,
DBAN,
DCAN,
and
TCAN
to
interact
with
GSH
and
glutathione­
s­
transferases
(
GST)
in
a
series
of
in
vitro
and
in
vivo
experiments.
The
in
vitro
experiments
tested
the
effect
of
various
incubation
conditions
on
the
direct
reactivity
of
HANs
with
GSH
as
measured
by
the
loss
of
GSH
in
the
incubation
mixture.
Direct
incubations
(
in
the
absence
of
GST)
revealed
that
HANs
have
the
potential
to
bind
to
GSH.
The
relative
reactivity
toward
GSH
was
DBAN>
BCAN>>
TCAN.
No
detectable
binding
with
DCAN
was
observed.
Addition
of
bovine
serum
albumin
to
the
incubations
reduced
the
degree
of
GSH
removal
by
TCAN,
but
had
no
modifying
effect
on
DBAN,

suggesting
that
at
least
for
DBAN,
binding
to
GSH
is
somewhat
specific
(
no
results
for
BCAN
were
reported).
The
presence
of
cytosol
(
a
cell
fraction
containing
GST
activity)
did
not
alter
GSH
loss
by
DBAN
or
TCAN
(
no
results
for
DCAN
or
BCAN
were
reported).
Therefore,
for
at
least
these
two
HANs,
GSH
conjugation
appears
to
be
nonenzymatic.
The
presence
of
microsomes
decreased
GSH
loss
with
DBAN
and
TCAN,
leading
the
authors
to
suggest
that
microsomal
metabolism
of
these
HANs
results
in
the
formation
of
metabolites
that
are
less
GSHreactive
than
the
parent
compounds.
No
results
were
presented
for
incubation
of
BCAN
with
microsomes.
Even
though
the
presence
of
cytosol
alone
did
not
alter
GSH
loss,
incubation
of
both
cytosol
and
microsome
with
DBAN
or
TCAN
decreased
GSH
loss
compared
to
that
observed
with
microsomes
alone.
Taken
together,
these
results
show
that
the
GSH
reactivity
of
HANs
varies
greatly
among
the
HANs
under
review
in
this
document.
These
in
vitro
findings
suggest
that
BCAN,
DBAN,
and
TCAN
appear
to
react
with
GSH
in
a
nonenzymatic
fashion
as
the
parent
compounds,
while
DCAN
shows
little
propensity
for
binding
to
GSH.
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
14
Final
Draft
In
a
second
part
of
this
study,
Lin
and
Guion
(
1989)
tested
the
ability
of
HANs
to
inhibit
GST
activity
(
as
measured
by
1­
chloro­
2,4­
dinitrobenzene
(
CDNB)
conjugation
with
GSH)
in
vitro
and
in
vivo.
All
the
HANs
decreased
GST
activity
in
vitro
in
the
order
of
TCAN>
BCAN=
DBAN>
DCAN,
with
a
4­
fold
difference
separating
the
level
of
GST
activity
in
the
presence
of
TCAN
and
DCAN.
For
the
in
vivo
studies,
male
Fischer
344
rats
were
administered
single
gavage
doses
of
0.75
mmol/
kg
BCAN
(
116
mg/
kg),
DBAN
(
149
mg/
kg),

DCAN
(
82
mg/
kg),
or
TCAN
(
108
mg/
kg)
in
tricaprylin.
These
doses
represented
10
to
30%
of
reported
LD
50
for
the
individual
compounds.
Liver
GST
activities
and
GSH
concentrations
were
measured
1,
3,
and
18
hours
after
dosing.
GST
activity
was
slightly
decreased
at
3
hours
by
DBAN
and
TCAN,
and
was
significantly
decreased
by
DBAN
at
18
hours
(
data
not
shown).
The
authors
noted
that
there
are
several
potential
mechanisms
for
inhibition
of
GST
activity
by
HANs,

including
depletion
of
GSH
through
direct
conjugation,
by
competing
with
GSH
for
GST
catalytic
sites
(
competitive
inhibition),
or
through
noncompetitive
protein
binding
to
GST.
To
test
this
first
possibility,
the
authors
also
measured
liver
GSH
levels
following
in
vivo
administration
of
HANs
using
the
same
dosing
protocol
as
for
the
GST
activity
measurements.
For
BCAN,
DBAN,
and
DCAN,
GSH
levels
were
decreased
at
1
hour
post­
treatment,
recovered
to
control
levels
by
3
hours,
and
were
elevated
at
18
hours.
For
TCAN,
liver
GSH
levels
were
unchanged
at
1
hour,
and
were
elevated
at
3
and
18
hours
post­
treatment.
These
results
show
that
initial
decreases
in
GSH
levels
are
transient,
and
that
GSH
levels
return
to
control
levels
shortly
after
cessation
of
exposure,

with
a
rebound
to
higher
GSH
levels
within
a
day
post­
treatment.

The
effects
of
HANs
on
GST
and
GSH
levels
have
also
been
investigated
by
Ahmed
and
colleagues
(
Ahmed
et
al.,
1989;
Ahmed
et
al.,
1991).
In
an
in
vitro
study,
DBAN,
DCAN,
and
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
15
Final
Draft
TCAN
significantly
inhibited
GST
activity
(
CDNB
conjugation
with
GSH).
IC
50
(
the
concentration
of
inhibitor
resulting
in
50%
inhibition)
values
for
these
three
HANs
were
0.82,

2.49,
and
0.34
mM,
respectively.
This
result
suggests
that
TCAN
binds
more
readily
to
GST
than
DBAN
or
DCAN.
The
observed
inhibition
of
GST
was
reversible
upon
dialysis
of
the
enzyme,

suggesting
reversible
binding
of
HANs
to
GST
(
or
other
mechanisms
not
involving
direct
protein
reactivity).
Incubation
with
DBAN
or
DCAN
decreased
both
the
apparent
K
m
and
the
V
max
of
GST
activity
toward
GSH,
while
incubation
with
TCAN
increased
the
apparent
K
m
and
V
max.

Based
on
the
patterns
of
activity
of
GST
toward
GSH,
kinetic
interactions
were
described
as
intermediate
between
uncompetitive
and
noncompetitive
for
DBAN,
as
uncompetitive
for
DCAN,

and
as
competitive
for
TCAN.
The
effect
of
HANs
on
GST
activity
toward
its
substrate
CDNB
was
also
evaluated.
The
pattern
of
HAN
inhibition
of
GST­
dependent
conjugation
of
CDNB
was
described
as
mixed
by
the
study
authors
(
i.
e.,
showing
aspects
of
competitive,
uncompetitive,
and
noncompetitive
inhibition).
These
complex
patterns
of
inhibition
suggest
that
HANs
might
interact
with
GST
through
multiple
mechanisms.
For
example,
HANs
might
interact
at
the
catalytic
site
of
the
enzyme
(
consistent
with
competitive
inhibition)
as
well
as
other
protein
sites
(
consistent
with
uncompetitive,
and
noncompetitive
inhibition).

In
an
in
vivo
study
by
Ahmed
and
colleagues
(
1991),
GST
activity
and
GSH
levels
were
determined
in
time­
course
and
dose­
response
experiments
with
DBAN.
For
the
time­
course
experiment,
male
Sprague
Dawley
rats
were
administered
a
single
oral
dose
of
75
mg/
kg
DBAN
in
dimethyl
sulfoxide
(
75%
of
the
LD
50),
and
aortal
blood,
liver,
stomach,
and
kidney
were
harvested
at
0.5,
1,
2,
or
4
hours
after
treatment.
For
the
liver,
GSH
levels
were
significantly
(
p<
0.05)

decreased
at
0.5
hours
to
roughly
60%
of
controls
(
as
read
from
a
figure
in
the
paper),
recovered
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
16
Final
Draft
to
control
levels
by
2
hours,
and
were
increased
above
control
levels
at
4
hours.
The
mild
increase
at
4
hours
was
not
statistically
significant.
For
the
stomach,
GSH
levels
were
nearly
completely
depleted
by
0.5
hours
(
6%
of
control
levels),
and
remained
decreased
for
up
to
4
hours.
No
significant
change
in
blood
or
kidney
GSH
levels
was
detected.
The
effects
of
DBAN
on
GST
activity
closely
paralleled
the
effects
on
GSH
levels,
with
significant
decreases
beginning
at
0.5
hours
for
the
liver
and
stomach,
and
no
significant
effect
in
the
kidney.

For
the
dose­
response
experiment,
Ahmed
et
al.
(
1991)
administered
single
oral
gavage
doses
of
0,
25,
75,
or
100
mg/
kg
DBAN
in
dimethyl
sulfoxide
to
male
Sprague
Dawley
rats
and
harvested
blood
and
tissues
0.5
hours
after
dosing
as
described
for
the
time­
course
experiment.

Hepatic
and
gastric
GSH
levels
were
decreased
in
a
dose­
dependent
fashion,
and
were
significantly
(
p<
0.05)
decreased
beginning
at
25
mg/
kg.
For
the
liver,
GSH
levels
were
decreased
by
23%
at
25
mg/
kg,
by
38%
by
50
mg/
kg,
by
46%
at
75
mg/
kg,
and
by
57%
at
100
mg/
kg.
GSH
levels
in
the
stomach
tissue
were
decreased
by
43%
at
25
mg/
kg,
by
75%
at
50
mg/
kg,
by
84%
by
75
mg/
kg,
and
86%
by
100
mg/
kg.
No
significant
effect
on
blood
or
kidney
GSH
levels
was
observed.
Liver
and
stomach
GST
activities
were
also
decreased
in
a
dose­
dependent
fashion,
but
this
effect
was
less
severe
than
the
decreases
in
GSH
levels.
The
degree
of
inhibition
(
enzyme
activity
decreased
to
60%
of
control
levels
in
the
liver,
and
decreased
to
71%
of
control
levels
in
the
stomach)
was
statistically
significant
(
p<
0.05)
beginning
at
50
mg/
kg.
The
authors
suggested
that
the
likely
mechanism
for
GSH
depletion
was
direct
conjugation
with
HANs
due
to
their
electrophilic
nature.
They
further
noted
that
the
depletion
of
GSH,
coupled
with
protein
binding
to
the
GST
enzyme
itself,
could
lead
to
the
observed
inhibition
of
GST
activity.
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
17
Final
Draft
The
results
of
Lin
and
Guion
(
1989)
and
Ahmed
et
al.
(
1989;
1991)
suggested
that
HANs
inhibit
liver
GST
activity.
However,
NTP
(
2002)
reported
an
increase
in
liver
GST
activity
in
male
F344
rats
following
exposure
to
DBAN
in
their
drinking
water
for
14
days.
The
increase
of
126%

over
controls
was
only
significant
in
the
male
rats
exposed
to
drinking
water
containing
200
mg/
L
DBAN
(
18
mg/
kg/
day).
One
possible
explanation
for
the
opposite
effects
of
HANs
on
GST
activity
reported
among
these
studies
is
the
duration
of
dosing
that
was
employed
in
each
study.

The
studies
by
Lin
and
Guion
(
1989)
and
Ahmed
et
al.
(
1989;
1991)
were
single
dose
gavage
studies.
Even
in
these
studies
a
rebound
in
GSH
levels
or
GST
activity
was
noted
within
a
period
of
hours
that
often
exceeded
control
levels.
Therefore,
it
is
possible
that
longer­
term
exposure
(
such
as
in
the
14­
day
study)
enhances
GST
activity
due
to
a
rebound
effect
after
an
initial
decrease.
A
time
course
experiment
measuring
GSH
levels
and
GST
activities
over
acute
and
subchronic
periods
would
be
needed
to
determine
if
this
is
the
case.

The
results
of
Lin
and
Guion
(
1989),
Ahmed
et
al.
(
1989;
1991),
and
NTP
(
2002)
suggest
that
conjugation
with
GSH
may
be
an
important
source
of
HAN
detoxification
in
animal
toxicity
studies.
The
in
vitro
results
suggest
that
BCAN,
DBAN,
and
TCAN
are
conjugated
with
GSH
in
a
non­
enzymatic
fashion,
and
at
least
for
DBAN,
this
interaction
is
somewhat
selective.
The
decreases
in
GSH
levels
following
oral
dosing
of
rats
with
HANs
further
supports
the
in
vitro
findings.
In
in
vitro
experiments
HANs
also
appear
to
inhibit
GST
activity,
although
a
rebound
effect
after
longer
periods
of
exposure
could
be
possible.
Multiple
mechanisms
are
likely
involved
in
the
observed
inhibition
following
acute
dosing.
It
is
noteworthy
that
the
concentrations
of
HANs
used
in
these
studies
to
demonstrate
GSH
depletion
and
altered
GST
activity
are
orders
of
magnitude
greater
than
measured
human
exposures
to
these
compounds
in
drinking
water.
Since
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
18
Final
Draft
GSH
depletion
is
clearly
dose­
dependent
(
Ahmed
et
al.,
1991),
the
importance
of
this
pathway
in
human
exposure
situations
is
probably
minimal.

D.
Excretion
Pereira
et
al.
(
1984)
studied
urinary
excretion
of
thiocyanate
in
rats
following
single
oral
doses
of
0.75
mmol/
kg
of
several
HANs
in
tricaprylin.
The
percentages
of
the
administered
dose
excreted
as
thiocyanate
after
24
hours
were
12.8%,
7.67%,
9.28%,
and
2.25%
of
BCAN,
DBAN,

DCAN,
and
TCAN,
respectively.
No
data
were
presented
for
other
urinary
metabolites;
thus
the
total
contribution
of
urinary
excretion
cannot
be
estimated.
This
issue
was,
however,
more
fully
evaluated
in
the
kinetics
study
of
Roby
et
al.
(
1986),
in
which
single
oral
doses
of
[
1­
14C]­
or
[
2­
14C]­
DCAN
were
administered
in
water
to
male
rats
and
mice.
Urine,
feces,
and
expired
air
were
collected
and
the
radioactive
content
measured
until
at
least
70%
of
the
label
had
been
recovered.
In
rats,
this
required
6
days
for
[
1­
14C]­
DCAN
and
2
days
for
[
2­
14C]­
DCAN.
In
mice,

excretion
was
more
than
70%
complete
within
24
hours
for
both
locations
of
the
label.
In
both
animal
species,
the
label
of
[
1­
14C]­
DCAN
was
excreted
mostly
in
the
urine
(
42
 
70%),
with
lower
amounts
in
feces
(
9
 
20%,
some
of
which
may
have
been
unabsorbed)
and
expired
air
(
3
 
8%).
For
[
2­
14C]­
DCAN,
label
was
excreted
both
as
carbon
dioxide
in
air
(
33
 
37%)
and
in
urine
(
35
 
43%),

with
8
 
13%
in
feces
(
Shown
in
Table
III­
3
measured
as
radiolabel
excretion).
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
19
Final
Draft
Table
III­
3.
Excretion
of
DCAN
in
Rats
and
Mice.
.

Based
on
these
data,
excretion
of
HANs
is
nearly
complete
over
a
period
of
days.
The
rate
of
excretion
may
differ
across
species,
since
mice
excrete
DCAN
more
rapidly
than
rats.

Differences
in
excretion
of
thiocyanate
for
different
HANs
was
observed
by
Pereira
et
al.
(
1984),

with
TCAN
being
excreted
as
thiocyanate
to
a
lesser
degree
than
the
other
HANs.
It
is
not
clear
if
this
reflects
differences
in
metabolism
or
differences
in
excretion,
since
total
urinary
excretion
of
the
radiolabel
was
not
determined.
The
dispensation
of
the
two
carbons
also
differs
(
Roby
et
al.,

1986).
The
cyanide
group
tends
to
be
more
readily
excreted
in
the
urine
and
the
halomethyl
carbon
is
excreted
nearly
equally
in
expired
air
and
in
urine.
Excretion
in
the
feces
appears
to
be
limited.
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
20
Final
Draft
E.
Bioaccumulation
and
Retention
No
studies
were
located
that
provided
data
on
long­
term
accumulation
and
retention
of
BCAN,
DBAN,
DCAN,
or
TCAN
in
the
body.
The
existing
kinetic
studies
did
not
determine
halflives
and
were
not
conducted
for
sufficiently
long
periods
to
evaluate
long­
term
accumulation.

However,
the
results
of
Roby
et
al.
(
1986)
that
showed
relatively
rapid
excretion
of
DCANassociated
radioactivity
(
at
least
70%
of
the
administered
dose
excreted
within
6
days
in
rats
or
within
24
hours
in
mice)
suggests
limited
potential
for
the
bioaccumulation
of
the
HANs.

F.
Summary
Limited
data
are
available
on
the
toxicokinetics
of
the
HANs,
with
a
comprehensive
toxicokinetic
study
for
oral
dosing
available
only
for
DCAN.
However,
the
existing
toxicokinetic
data
suggest
that
HANs
can
be
rapidly
and
nearly
completely
absorbed
following
oral
dosing
(
Roby
et
al.,
1986;
Roth
et
al.,
1990).
Systemic
toxicity
data
suggest
that
HANs
are
absorbed
by
the
dermal
route.
Once
absorbed,
HANs
appear
to
be
widely
distributed.
The
two
compounds
tested,
DCAN
(
Roby
et
al.,
1986)
and
TCAN
(
Lin
et
al.,
1992),
were
widely
distributed
following
oral
dosing,
with
no
clear
preferences
in
tissue
distribution
apparent
based
on
the
limited
data.
No
data
were
available
on
tissue­
dependent
metabolism,
but
an
overall
metabolic
scheme
for
HANs
involving
an
initial
oxidative
dehalogenation
step
has
been
proposed
based
on
the
propensity
for
these
compounds
to
form
cyanide
and
metabolism
studies
for
other
nitriles
(
Pereira
et
al.,
1984).

Proposed
intermediate
metabolites
have
not
been
measured
directly,
and
the
identity
of
enzymes
responsible
for
steps
in
the
pathway
have
not
been
identified.
Conjugation
with
GSH,
at
least
at
high
doses,
might
be
a
second
important
route
of
metabolism
for
HANs
(
Ahmed
et
al.,
1989;
Lin
and
Guion,
1989;
Ahmed
et
al.,
1991;
NTP,
2002).
Excretion
of
HANs
is
nearly
complete
over
a
Drinking
Water
Criteria
Document
for
Haloacetonitriles
EPA/
OW/
OST/
HECD
III­
21
Final
Draft
period
of
days,
largely
in
urine
and
in
exhaled
air.
The
rate
of
excretion
may
differ
across
species,

since
mice
excrete
DCAN
more
rapidly
than
rats
(
Roby
et
al.,
1986).
Differences
in
urinary
excretion
of
thiocyanate
for
different
HANs
was
observed
by
Pereira
et
al.
(
1984),
with
TCAN
being
excreted
as
thiocyanate
to
a
lesser
degree
than
the
other
HANs.
The
results
of
Roby
et
al.

(
1986)
that
showed
relatively
rapid
excretion
of
DCAN­
associated
radioactivity
suggests
limited
potential
for
the
bioaccumulation
of
the
HANs.
However,
no
studies
were
located
that
provided
data
on
long­
term
accumulation
and
retention
of
any
of
the
HANs.
