Document ID: EPA-HQ-OPPT-2003-0010-0045
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-03-11T05:00Z

273
Environmental
Protection
Agency
§
799.9135
(
2)
Subchronic
toxicity
 
(
i)
Required
testing.
(
A)
An
oral
90­
day
subchronic
toxicity
test
shall
be
conducted
with
1,3,5­
trimethylbenzene
in
accordance
with
§
798.2650
of
this
chapter
except
for
the
provisions
in
§
798.2650
(
e)(
3),
(
e)(
7)(
i),
and
(
e)(
11)(
v).
The
tests
shall
be
performed
using
drinking
water.
However,
if,
due
to
poor
stability
or
palatability,
a
drinking
water
test
is
not
feasible
for
a
given
substance,
that
substance
shall
be
administered
either
by
oral
gavage,
in
the
diet,
or
in
capsules
(
B)
For
the
purpose
of
this
section,
the
following
provisions
also
apply:
(
1)
Satellite
group
(
Rodent
only).
A
satellite
group
of
20
animals
(
10
animals
per
sex)
shall
be
treated
with
the
high
dose
level
for
90
days
and
observed
for
reversibility,
persistence,
and
delayed
occurrence
of
toxic
effects
for
a
posttreatment
period
of
appropriate
length,
normally
not
less
than
28
days.
(
2)
Histopathology.
Histopathology
of
the
lungs
of
all
animals
shall
be
performed
Special
attention
to
examination
of
the
lungs
of
rodents
shall
be
made
for
evidence
of
infection
since
this
provides
a
convenient
assessment
of
the
state
of
health
of
the
animals.
(
ii)
Reporting
requirements.
(
A)
The
subchronic
testing
for
chloroethane
shall
be
completed
and
the
final
report
submitted
to
EPA
by
June
27,
1995.
The
subchronic
testing
for
1,1­
dichloroethane
and
1,1,2,2­
tetrachlorethane
shall
be
completed
and
the
final
report
submitted
to
EPA
by
August
27,
1995.
The
subchronic
testing
for
1,3,5­
trimethylbenzene
shall
be
completed
and
the
final
report
submitted
to
EPA
by
April
10,
1995.
(
B)
For
each
test,
a
progress
report
shall
be
submitted
to
EPA
beginning
9
months
after
the
date
specified
in
paragraph
(
d)(
1)
of
this
section
and
at
6
 
month
intervals
thereafter
until
the
final
report
is
submitted
to
EPA.
(
d)
Effective
date.
(
1)
This
section
is
effective
on
December
27,
1993,
except
for
paragraphs
(
a)(
1),
(
a)(
2),
(
c)(
1)(
i)(
A),
(
c)(
1)(
ii)(
A),
(
c)(
1)(
ii)(
B),
(
c)(
2)(
i)(
A),
and
(
c)(
2)(
ii)(
A).
The
effective
date
for
paragraphs
(
a)(
2),
(
c)(
1)(
ii)(
B),
and
(
c)(
2)(
ii)(
A)
is
September
29,
1995.
The
effective
date
for
paragraphs
(
a)(
1),
(
c)(
1)(
i)(
A),
and
(
c)(
2)(
i)(
A)
is
February
27,
1996.
The
effective
date
for
paragraph
(
c)(
1)(
ii)(
A)
is
June
30,
1997.
(
2)
The
guidelines
and
other
test
methods
cited
in
this
section
are
referenced
as
they
exist
on
the
effective
date
of
the
final
rule.

[
58
FR
59681,
Nov.
10,
1993;
58
FR
1992,
Jan.
13,
1994,
as
amended
at
60
FR
56956,
Nov.
13,
1995;
61
FR
7223,
Feb.
27,
1996;
62
FR
35105,
June
30,
1997]

Subparts
E
 
G
[
Reserved]

Subpart
H
 
Health
Effects
Test
Guidelines
SOURCE:
62
FR
43824,
Aug.
15,
1997,
unless
otherwise
noted.

§
799.9135
TSCA
acute
inhalation
toxicity
with
histopathology.

(
a)
Scope.
This
section
is
intended
to
meet
the
testing
requirements
under
section
4
of
the
Toxic
Substances
Control
Act
(
TSCA).
In
the
assessment
and
evaluation
of
the
potential
human
health
effects
of
chemical
substances,
it
is
appropriate
to
test
for
acute
inhalation
toxic
effects.
The
goals
of
this
test
are
to
characterize
the
exposureresponse
relationship
for
sensitive
endpoints
following
acute
exposure
and
to
characterize
toxicologic
response
following
acute
high
exposures.
The
latter
is
of
particular
concern
in
relation
to
spills
and
other
accidental
releases
This
testing
is
designed
to
determine
the
gross
pathology
and
histopathology
resulting
from
acute
inhalation
exposure
to
a
substance.
Because
toxic
effects
on
the
respiratory
tract
are
of
particular
concern
following
inhalation
exposure,
several
indicators
of
respiratory
toxicity
consisting
of
histopathology
on
fixed
tissue
and
evaluation
of
cellular
and
biochemical
parameters
in
bronchoalveolar
lavage
fluid
should
be
employed.
The
respiratory
histopathology
consists
of
specialized
techniques
to
preserve
tissues
of
the
respiratory
tract
in
order
to
allow
detailed
microscopic
examination
to
identify
adverse
effects
of
chemical
substances
on
this
organ
system
The
bronchoalveolar
lavage
is
designed
to
be
a
rapid
screening
test
to
RECEIVED
OPPT
NCIC
2003
MAR11
5:
03PM
OPPT­
2003­
0010­
0045
274
40
CFR
Ch.
I
(
7
 
1
 
98
Edition)
§
799.9135
provide
an
early
indicator
of
pulmonary
toxicity
by
examining
biochemical
and
cytologic
endpoints
of
material
from
the
lungs
of
animals
exposed
to
potentially
toxic
chemical
substances.
These
acute
tests
are
designed
to
assess
the
relationship,
if
any,
between
the
animals'
exposure
to
the
test
substance
and
to
demonstrate
relationship
between
the
animals'
exposure
and
the
incidence
and
severity
of
observed
abnormalities,
including
gross
or
histopathologic
lesions,
body
weight
changes,
effects
on
mortality,
and
any
other
toxic
effects.
These
acute
tests
are
not
intended
to
provide
a
complete
evaluation
of
the
toxicologic
effects
of
a
substance,
and
additional
functional
and
morphological
evaluations
may
be
necessary
to
assess
completely
the
potential
effects
produced
by
a
chemical
substance.
Additional
tests
may
include
longer­
term
exposures,
or
more
in­
depth
evaluation
of
specific
organ
systems
as
indicated
by
signs
of
toxicity
following
acute
exposure
(
b)
Source.
This
a
new
section
developed
by
the
United
States
Environmental
Protection
Agency.
(
c)
Definitions.
The
following
definitions
apply
to
this
section.
Aerodynamic
diameter
(
dae)
refers
to
the
size
of
particles.
It
is
the
diameter
of
a
sphere
of
unit
density
that
behaves
aerodynamically
(
has
the
same
settling
velocity
in
air)
as
the
particle
of
the
test
substance.
It
is
used
to
compare
particles
of
different
size,
shape,
and
density,
and
to
predict
where
in
the
respiratory
tract
such
particles
may
be
primarily
deposited.
Exposure
response
is
the
relationship
between
the
exposure
concentration
and
the
measured
toxic
response,
whether
expressed
as
a
group
mean
±
standard
deviation)
in
the
case
of
a
continuous
variable
or
as
incidence
in
the
case
of
a
quantal
variable.
This
definiton
should
not
preclude
the
exploration
of
other
dose
metrics
in
establishing
this
relationship.
Geometric
standard
deviation
(
GSD)
is
a
dimensionless
number
equal
to
the
ratio
between
the
mass
median
aerodynamic
diameter
(
MMAD)
and
either
84%
or
16%
of
the
diameter
size
distribution
(
e.
g.,
MMAD
=
2
m
m;
84%
=
4
m
m;
GSD
=
4/
2
=
2.0.)
The
MMAD,
together
with
the
GSD,
describe
the
particle
size
distribution
of
an
aerosol.
Use
of
the
GSD
may
not
be
valid
for
non­
lognormally
distributed
aerosols.
(
If
the
size
distribution
deviates
from
the
lognormal,
it
shall
be
noted).
Inhalability
is
the
ratio
of
the
number
concentration
of
particles
of
a
certain
aerodynamic
diameter,
dae,
that
are
inspired
through
the
nose
or
mouth
to
the
number
concentration
of
the
same
dae
present
in
the
inspired
volume
of
ambient
air.
In
humans,
inhalability
can
exceed
15
m
m
dae,
whereas
inhalability
dramatically
decreases
for
particles
above
4
m
m
dae
in
small
laboratory
animals.
Lower
respiratory
tract
consists
of
those
structures
of
the
respiratory
tract
below
the
larynx.
Mass
geometric
mean
aerodynamic
diameter
or
the
mass
median
aerodynamic
diameter
(
MMAD)
is
the
calculated
aerodynamic
diameter
that
divides
the
particles
of
an
aerosol
(
a
gaseous
suspension
of
fine
liquid
or
solid
particles)
in
half,
based
on
the
weight
of
the
particles
By
weight,
50%
of
the
particles
will
be
larger
than
the
MMAD
and
50%
of
the
particles
will
be
smaller
than
the
MMAD.
Particle
regional
deposition
is
the
fraction
of
inhaled
particles
that
deposits
in
the
specific
region
of
the
respiratory
tract.
The
major
mechanisms
of
particle
deposition
in
the
respiratory
tract
include
impaction,
sedimentation,
diffusion
interception,
and
electrostatic
precipitation.
The
deposition
mechanism
that
is
dominant
for
a
given
region
depends
on
the
respiratory
tract
architecture
and
ventilation
rate
of
the
species
and
the
aerosol
particle
size
and
distribution.
The
respiratory
tract
in
both
humans
and
various
experimental
mammals
can
be
divided
into
three
regions
on
the
basis
of
structure,
size,
and
function:
(
1)
The
extrathoracic
region
or
upper
respiratory
tract
that
includes
the
nose,
mouth,
nasopharynx,
oropharynx,
laryngopharynx,
and
larynx.
(
2)
The
tracheobronchial
region
that
includes
the
trachea,
bronchi,
and
bronchioles
(
including
the
terminal
bronchioles).
(
3)
The
alveolar
region
that
includes
the
respiratory
bronchioles
(
if
present
275
Environmental
Protection
Agency
§
799.9135
in
the
species),
alveolar
ducts,
alveolar
sacs,
and
alveoli.
Respiratory
effects
are
any
adverse
effects
on
the
structure
or
functions
of
the
respiratory
system
related
to
exposure
to
a
chemical
substance.
Target
organ
is
any
organ
found
to
be
a
target
of
toxicity
in
the
4­
hour
(
hr)
high
concentration
group
as
a
result
of:
(
1)
The
initial
histopathologic
examination
(
respiratory
tract,
liver,
kidney
gross
lesions);
or
(
2)
The
retrospective
histopathologic
examination
of
archived
organs
triggered
by
their
identification
as
targets
of
toxicity
in
a
90­
day
study.
Toxic
effects
are
any
adverse
changes
(
a
change
that
is
statistically
and
biologically
significant)
in
the
structure
or
function
of
an
experimental
animal
as
a
result
of
exposure
to
a
chemical
substance.
Upper
respiratory
tract
consists
of
those
structures
of
the
respiratory
tract
above
and
including
the
larynx.
(
d)
Principle
of
the
test
method.
The
test
substance
shall
be
administered
to
several
groups
of
experimental
animals
one
concentration
level
and
duration
being
used
per
group.
Bronchoalveolar
lavage
shall
be
used
to
evaluate
early
effects
on
the
respiratory
system
by
examining
changes
in
the
content
of
the
lavage
fluid
of
the
lung.
At
24
hrs
following
exposure,
the
animals
shall
be
sacrificed
and
necropsied,
and
tissue
samples
from
the
respiratory
tract
and
other
major
organs
will
be
prepared
for
microscopic
examination.
The
exposure
levels
at
which
significant
toxic
effects
on
the
respiratory
organ
system
are
produced
are
compared
to
those
levels
that
produce
other
toxic
effects.
As
triggered
by
the
results
of
the
4­
hr
test,
additional
exposure
periods
of
1
hr
and
8
hrs
will
be
required
to
determine
the
effect
of
exposure
time
on
the
toxicity
observed.
A
1­
hr
exposure
study
can
be
elected
as
an
option
to
provide
data
suitable
for
risk
assessment
for
very
short
duration
exposures
as
may
occur
from
chemical
releases.
In
the
absence
of
adequate
toxicological
data
for
1­
hr
exposure,
the
Agency
will
extrapolate
to
shorter­
term
exposures
from
the
4­
hr
data
on
the
basis
of
concentration
alone.
This
is
a
conservative
method
of
extrapolation,
consistent
with
general
Agency
methods
for
deriving
criteria
for
short­
term
exposure
from
longerterm
studies
(
a
concentration
x
time
extrapolation
would
result
in
higher
concentration
for
a
shorter
duration).
(
e)
Test
procedures
 
(
1)
Animal
selection
 
(
i)
Species.
In
general,
the
laboratory
rat
and
mouse
should
be
used.
Under
some
circumstances,
other
species
such
as
the
hamster
or
guinea
pig,
may
be
more
appropriate,
and
if
these
or
other
species
are
used,
justification
should
be
provided.
(
ii)
Strain.
If
rats
and
mice
are
used,
the
use
of
the
F344
rat
and
the
B6C3F1
mouse
is
preferred
to
facilitate
comparison
with
existing
data.
(
iii)
Age.
Young
adults
shall
be
used.
The
weight
variation
of
animals
used
in
a
test
should
not
exceed
'
61'
20%
of
the
mean
weight
for
each
species.
(
iv)
Sex.
Equal
numbers
of
animals
of
each
sex
shall
be
used
for
each
concentration
level.
The
females
shall
be
nulliparous
and
nonpregnant.
(
v)
Health
status.
Body
weight
and
feed
consumption
are
not
sufficient
indicators
of
the
health
status
of
animals
prior
to
initiating
an
inhalation
toxicity
study.
Prior
to
initiating
the
study,
animals
shall
be
monitored
for
known
viral
and
bacterial
respiratory
pathogens
determined
by
conventional
microbiological
assays
(
e.
g.,
serology).
The
animals
shall
be
free
from
pathogens
at
the
start
of
exposure.
(
2)
Number
of
animals.
At
least
five
males
and
five
females
shall
be
used
in
each
concentration/
duration
and
control
group.
Animals
shall
be
randomly
assigned
to
treatment
and
control
groups.
(
3)
Control
groups.
The
control
group
shall
be
a
sham­
treated
group.
Except
for
treatment
with
the
test
substance,
animals
in
the
control
group
shall
be
handled
in
a
manner
identical
to
the
test­
group
animals.
Where
a
vehicle
is
used
to
help
generate
an
appropriate
concentration
of
the
substance
in
the
atmosphere,
a
vehicle
control
group
shall
be
used.
If
the
4­
and
8­
hr
exposure
studies
are
conducted
concurrently
a
concurrent
8­
hr
sham­
exposed
control
group
may
serve
as
the
control
group
for
both
the
4­
hr
and
the
8­
hr
exposure
studies,
provided
there
is
adequate
historical
control
data
showing
no
changes
in
histopathology
or
276
40
CFR
Ch.
I
(
7
 
1
 
98
Edition)
§
799.9135
bronchoalveolar
lavage
of
controls
exposed
for
4
and
8
hrs.
Similarly,
if
the
optional
1­
hr
exposure
study
is
conducted
concurrently
with
the
4­
and/
or
8­
hr
study,
the
concurrent
control
group
for
those
studies
may
also
be
used
for
the
1­
hr
study,
provided
adequate
historical
control
data
show
no
changes
in
histopathology
or
bronchoalveolar
lavage
between
controls
exposed
for
these
time
periods.
(
4)
Concentration
level
and
concentration
selection.
For
the
4­
hr
study,
at
least
three
concentrations
shall
be
used
in
addition
to
the
control
group.
Ideally,
the
data
generated
from
the
test
should
be
sufficient
to
produce
an
exposure­
response
curve.
The
concentrations
can
either
be
linearly
or
logarithmically
spaced
depending
on
the
anticipated
steepness
of
the
concentration
response
curve.
A
rationale
for
concentration
selection
should
be
provided
to
indicate
that
the
selected
concentrations
will
maximally
support
detection
of
concentration­
response
relationship
The
high
concentration
should
be
clearly
toxic
or
a
limit
concentration
but
should
not
result
in
an
incidence
of
fatalities
that
would
preclude
a
meaningful
evaluation
of
the
data.
The
lowest
concentration
should
define
a
no­
observed­
adverse­
effects
level
(
NOAEL).
(
i)
Limit
concentration.
For
aerosols
and
particles,
the
high
concentrations
need
not
be
greater
than
2
mg/
L,
or
concentrations
that
cannot
maintain
a
particle
size
distribution
having
an
MMAD
between
1
and
4
m
m
(
i.
e.,
a
particle
size
that
permits
inhalability
and
deposition
throughout
the
respiratory
tract).
For
fibers,
the
bivariate
distribution
of
length
and
diameter
must
ensure
inhalability.
For
gases
and
vapors
the
concentrations
need
not
be
greater
than
50,000
ppm
or
50%
of
the
lower
explosive
limit,
whichever
is
lower.
If
a
test
at
an
aerosol
or
particulate
exposure
of
2
mg/
L
(
actual
concentration
of
respirable
substance)
for
4
hrs
or,
where
this
is
not
feasible,
the
maximum
attainable
concentration,
using
the
procedures
described
for
this
study,
produces
no
observable
toxic
effects
then
a
full
study
using
three
concentrations
will
not
be
necessary.
Similarly,
if
a
test
at
a
gas
or
vapor
exposure
of
50,000
ppm
or
50%
of
the
lower
explosive
limit,
whichever
is
lower,
produces
no
observable
toxic
effects,
then
a
full
study
using
three
concentrations
will
not
be
necessary.
(
ii)
8­
hr
study
and
optional
1­
hr
study.
If
the
8­
hr
study
is
triggered,
three
concentrations
shall
be
tested.
These
concentrations
should
allow
for
the
determination
of
an
effect
level
and
a
NOAEL.
If
the
option
to
perform
a
1­
hr
study
is
elected,
three
concentrations
shall
be
selected
and
tested
in
a
similar
manner.
(
5)
Inhalation
exposure.
Animals
can
be
exposed
to
the
substance
by
either
a
nose­
only
procedure
or
in
a
whole­
body
exposure
chamber.
(
i)
Inhalation
chambers.
The
animals
shall
be
tested
in
inhalation
equipment
designed
to
sustain
a
dynamic
airflow
for
nose­
only
exposures
of
at
least
300
ml/
minute/
animal
or
an
airflow
for
whole­
body
exposures
of
at
least
12
to
15
air
changes
per
hr
and
ensure
an
adequate
oxygen
content
of
at
least
19%
and
an
evenly
distributed
exposure
atmosphere.
Where
a
whole­
body
chamber
is
used,
its
design
shall
minimize
crowding
by
providing
individual
caging
As
a
general
rule,
to
ensure
stability
of
a
chamber
atmosphere,
the
total
``
volume''
of
the
test
animals
should
not
exceed
5%
of
the
volume
of
the
test
chamber.
(
ii)
Environmental
conditions.
The
temperature
at
which
the
test
is
performed
shall
be
maintained
at
22
°
C
(
±
2
°
C).
Ideally,
the
relative
humidity
should
be
maintained
between
40%
and
60%,
but
in
certain
instances
(
e.
g.,
tests
using
water
as
a
vehicle),
this
may
not
be
practical.
(
iii)
Exposure
periodicity.
For
acute
testing,
the
exposure
design
shall
enable
4
hrs
of
exposure
to
the
target
concentrations,
as
defined
by
an
average
of
±
 
5%
for
gases
and
vapors
and
±
15%
for
particles
and
aerosols.
If
triggered
by
the
results
of
the
4­
hr
exposure
additional
testing
shall
be
conducted
in
a
comparable
manner
using
an
8­
hr
exposure
period.
(
6)
Physical
measurements.
Measurements
or
monitoring
shall
be
made
of
the
following:
(
i)
Chemical
purity
of
the
test
material
shall
be
analyzed.
277
Environmental
Protection
Agency
§
799.9135
(
ii)
The
rate
of
airflow
shall
be
monitored
continuously,
but
shall
be
recorded
at
least
every
30
minutes.
(
iii)
The
actual
concentrations
of
the
test
substance
shall
be
measured
in
the
breathing
zone.
During
the
exposure
period,
the
actual
concentrations
of
the
test
substance
shall
be
held
as
constant
as
practical,
monitored
continuously
or
intermittently
depending
on
the
method
of
analysis,
and
recorded
at
least
at
the
beginning,
at
an
intermediate
time,
and
at
the
end
of
the
exposure
period.
Well­
established
and
published
monitoring
methods
should
be
used
where
available.
If
no
standard
methods
are
available,
then
accuracy
and
precision
information
must
be
supplied
(
iv)
During
the
development
of
the
generating
system,
appropriate
particle
size
analysis
shall
be
performed
to
establish
the
stability
of
the
aerosol.
During
exposure,
analysis
should
be
conducted
as
often
as
necessary
to
determine
the
consistency
of
particle
size
distribution.
The
particle
size
distribution
shall
have
an
MMAD
between
1
and
4
m
m.
The
particle
size
of
hygroscopic
materials
shall
be
small
enough
when
dry
to
assure
that
the
size
of
the
particle
at
saturation
will
still
have
an
MMAD
between
1
and
4
m
m.
Characterization
for
fibers
shall
include
the
bivariate
distribution
of
length
and
diameter
this
distribution
must
ensure
inhalability.
(
v)
If
the
test
substance
is
present
in
a
mixture,
the
mass
and
composition
of
the
entire
mixture,
as
well
as
the
principal
compound,
shall
be
measured.
(
vi)
Temperature
and
humidity
shall
be
monitored
continuously,
but
shall
be
recorded
at
least
every
30
minutes.
(
7)
Food
and
water
during
exposure
period
Food
shall
be
withheld
during
exposure
Water
may
also
be
withheld
in
certain
cases.
(
8)
Observation
period.
The
bronchoalveolar
lavage
and
respiratory
pathology
shall
be
conducted
24
hrs
following
exposure
to
allow
expression
of
signs
of
toxicity.
There
is
concern
that
some
latency
time
will
be
required
to
allow
migration
of
cells
and
macromolecules
into
the
lungs
following
exposure,
and
that
some
pathology
may
require
macromolecular
synthesis
or
degradation
before
cell
damage
develops
(
9)
Gross
pathology.
(
i)
All
animals
shall
be
subjected
to
a
full
gross
necropsy
which
includes
examination
of
orifices
and
the
cranial,
thoracic,
and
abdominal
cavities
and
their
contents.
(
ii)
At
least
the
lungs,
liver,
kidneys,
adrenals,
brain,
and
gonads
shall
be
weighed
wet,
as
soon
as
possible
after
dissection
to
avoid
drying.
(
iii)
The
following
organs
and
tissues,
or
representative
samples
thereof,
shall
be
preserved
in
a
suitable
medium
for
possible
future
histopathological
examination
All
gross
lesions;
brain­
including
sections
of
medulla/
pons;
cerebellar
cortex
and
cerebral
cortex;
pituitary;
thyroid/
parathyroid;
thymus;
heart;
sternum
with
bone
marrow;
salivary
glands;
liver;
spleen;
kidneys;
adrenals;
pancreas;
gonads;
accessory
genital
organs
(
epididymis,
prostrate,
and,
if
present,
seminal
vesicles);
aorta;
skin;
gall
bladder
(
if
present);
esophagus;
stomach;
duodenum;
jejunum;
ileum;
cecum;
colon;
rectum;
urinary
bladder;
representative
lymph
nodes;
thigh
musculature;
peripheral
nerve;
spinal
cord
at
three
levels
cervical,
midthoracic,
and
lumbar;
and
eyes.
Respiratory
tract
tissues
shall
also
be
preserved
in
a
suitable
medium.
(
10)
Histopathology.
The
following
histopathology
shall
be
performed:
(
i)
Full
histopathology
shall
be
performed
on
the
respiratory
tract,
liver
and
kidney
of
all
animals
in
the
control
and
high
concentration
groups.
The
histopathology
of
the
respiratory
tract
is
described
under
paragraph
(
e)(
11)
of
this
section.
(
ii)
All
gross
lesions
which
differ
from
controls
in
frequency,
distribution
type,
or
severity
in
all
concentration
groups.
(
iii)
Target
organs
in
all
animals,
as
indicated
by
the
observations
in
the
high
concentration
group
in
this
study.
Histopathologic
examination
of
target
organs
in
animals
at
all
concentration
levels
(
rather
than
only
to
the
extent
necessary
to
define
the
NOAEL)
can
support
the
application
of
exposure­
response
analyses
such
as
the
benchmark
concentration
approach.
(
iv)
Archived
organs
identified
as
targets
of
toxicity
from
results
of
the
90­
day
study
(
if
a
90­
day
study
is
required
278
40
CFR
Ch.
I
(
7
 
1
 
98
Edition)
§
799.9135
for
this
substance)
should
be
elevated
in
high
concentration
animals
of
the
4­
hr
acute
study
to
determine
if
they
are
also
targets
of
acute
toxicity.
(
11)
Respiratory
tract
histopathology.
(
i)
Representative
sections
of
the
respiratory
tract
shall
be
examined
histologically
These
shall
include
the
trachea
major
conducting
airways,
alveolar
region,
terminal
and
respiratory
bronchioles
(
if
present),
alveolar
ducts
and
sacs,
and
interstitial
tissues.
(
ii)
Care
shall
be
taken
that
the
method
used
to
kill
the
animal
does
not
result
in
damage
to
the
tissues
of
the
upper
or
lower
respiratory
tract.
The
lungs
shall
be
infused
with
a
fixative
while
in
an
inflated
state
of
fixed
pressure.
(
iii)
The
upper
respiratory
tract
shall
be
examined
for
histopathologic
lesions
This
examination
shall
use
a
minimum
of
four
sections
located
as
specified
under
paragraphs
(
e)(
11)(
iii)(
A)
through
(
e)(
11)(
iii)(
D)
of
this
section.
An
evaluation
of
the
nasal
vestibule
shall
be
conducted.
The
method
described
by
the
reference
under
paragraph
(
h)(
11)
of
this
section
should
be
given
consideration.
The
use
of
additional
sections
shall
be
left
to
the
discretion
of
the
study
pathologist,
but
consideration
should
be
given
to
additional
sections
as
recommended
in
the
reference
under
paragraph
(
h)(
8)
of
this
section
to
ensure
adequate
evaluation
of
the
entire
upper
respiratory
tract,
particularly
the
nasopharyngeal
meatus.
The
following
transverse
sections
shall
be
examined:
(
A)
Immediately
posterior
to
the
upper
incisor
teeth.
(
B)
At
the
incisor
papilla.
(
C)
At
the
second
palatal
ridge.
(
D)
At
the
level
of
the
first
upper
molar
teeth.
(
iv)
The
laryngeal
mucosa
shall
be
examined
for
histopathologic
changes.
Sections
of
the
larynx
to
be
examined
include
the
epithelium
covering
the
base
of
the
epiglottis,
the
ventral
pouch,
and
the
medial
surfaces
of
the
vocal
processes
of
the
arytenoid
cartilages
(
12)
Bronchoalveolar
lavage.
(
i)
Animals
can
be
exposed
to
the
substance
by
either
a
nose­
only
procedure
or
in
a
whole­
body
exposure
chamber.
(
ii)
Care
should
be
taken
that
the
method
used
to
kill
the
animal
results
in
minimum
changes
in
the
fluid
of
the
lungs
of
the
test
animals.
(
iii)
At
the
appropriate
time,
the
test
animals
shall
be
killed
and
the
heartlung
including
trachea
removed
in
bloc.
Alternatively,
lungs
can
be
lavaged
in
situ.
If
the
study
will
not
be
compromised,
one
lobe
of
the
lungs
may
be
used
for
lung
lavage
while
the
other
is
fixed
for
histologic
evaluation.
The
lungs
should
be
lavaged
using
physiological
saline.
The
lavages
shall
consist
of
two
washes,
each
of
which
consists
of
approximately
80%
(
e.
g.,
5
ml
in
rats
and
1
ml
in
mice)
of
the
total
lung
volume.
Additional
washes
merely
tend
to
reduce
the
concentrations
of
the
material
collected.
The
lung
lavage
fluid
shall
be
stored
on
ice
at
5
°
C
until
assayed.
(
iv)
The
following
parameters
shall
be
determined
in
the
lavage
fluid
as
indicators
of
cellular
damage
in
the
lungs:
total
protein,
cell
count,
and
percent
leukocytes.
In
addition,
a
phagocytosis
assay
shall
be
performed
to
determine
macrophage
activity.
Assay
methods
described
in
the
references
under
paragraphs
(
h)(
1)
and
(
h)(
3)
of
this
section
may
be
used.
(
13)
Combined
protocol.
The
tests
described
may
be
combined
with
any
other
toxicity
study,
as
long
as
none
of
the
requirements
of
either
are
violated
by
the
combination.
(
f)
Triggered
testing.
If
no
adverse
effects
are
seen
in
the
4­
hr
study
as
compared
with
controls,
no
further
testing
is
necessary.
If
the
4­
hr
study
shows
positive
effects
in
histopathology
or
the
bronchoalveolar
lavage,
an
8­
hr
study
shall
be
conducted.
Only
those
tissues
showing
positive
results
in
the
4­
hr
study
must
be
pursued
in
the
follow
up
8­
hr
study.
Similarly,
if
the
option
to
perform
a
1­
hr
study
is
exercised
only
those
tissues
showing
positive
results
in
the
4­
hr
study
shall
be
pursued.
(
g)
Data
reporting
and
evaluation.
The
final
test
report
shall
include
the
following
information:
(
1)
Description
of
equipment
and
test
methods.
A
description
of
the
general
design
of
the
experiment
and
any
equipment
used
shall
be
provided.
279
Environmental
Protection
Agency
§
799.9135
(
i)
Description
of
exposure
apparatus,
including
design,
type,
dimensions,
source
of
air,
system
for
generating
particles,
aerosols,
gasses,
and
vapors,
method
of
conditioning
air,
treatment
of
exhaust
air,
and
the
method
of
housing
animals
in
a
test
chamber.
(
ii)
Description
of
the
equipment
for
measuring
temperature,
humidity,
and
particulate
aerosol
concentration
and
size.
(
iii)
Exposure
data
shall
be
tabulated
and
presented
with
mean
values
and
measure
of
variability
(
e.
g.,
standard
deviation)
and
should
include:
(
A)
Chemical
purity
of
the
test
material
(
B)
Airflow
rates
through
the
inhalation
equipment.
(
C)
Temperature
and
humidity
of
air.
(
D)
Nominal
concentration
(
total
amount
of
test
substance
fed
into
the
inhalation
equipment
divided
by
the
volume
of
air).
(
E)
Actual
concentration
in
test
breathing
zone.
(
F)
Particle
size
distribution
(
e.
g.,
MMAD
with
GSD)
and
the
bivariate
distribution
of
fiber
length
and
diameter
where
appropriate.
(
2)
Results
 
(
i)
General
group
animal
data.
The
following
information
shall
be
arranged
by
test
group
exposure
level.
(
A)
Number
of
animals
exposed.
(
B)
Number
of
animals
dying.
(
C)
Number
of
animals
showing
overt
signs
of
toxicity.
(
D)
Pre­
and
post­
exposure
body
weight
change
in
animals,
and
weight
change
during
the
observation
period.
(
ii)
Counts
and
incidence
of
gross
alterations
observed
at
necropsy
in
the
test
and
control
groups.
Data
shall
be
tabulated
to
show:
(
A)
The
number
of
animals
used
in
each
group
and
the
number
of
animals
in
which
any
gross
lesions
were
found.
(
B)
The
number
of
animals
affected
by
each
different
type
of
lesion,
and
the
locations
and
frequency
of
each
type
of
lesion.
(
iii)
Counts
and
incidence
of
general
histologic
alterations
in
the
test
group.
Data
shall
be
tabulated
to
show:
(
A)
The
number
of
animals
used
in
each
group
and
the
number
of
animals
in
which
any
histopathologic
lesions
were
found.
(
B)
The
number
of
animals
affected
by
each
different
type
of
lesion,
and
the
locations,
frequency,
and
average
grade
of
each
type
of
lesion.
(
iv)
Counts
and
incidence
of
respiratory
histopathologic
alterations
by
the
test
group.
Data
shall
be
tabulated
to
show:
(
A)
The
number
of
animals
used
in
each
group
and
the
number
of
animals
in
which
any
histopathologic
lesions
were
found.
(
B)
The
number
of
animals
affected
by
each
different
type
of
lesion,
and
the
locations,
frequency,
and
average
grade
of
each
type
of
lesion.
(
v)
Results
of
the
bronchoalveolar
lavage
study.
Data
shall
be
tabulated
to
show:
(
A)
The
amount
of
administered
lavage
fluid
and
recovered
lavage
fluid
for
each
test
animal.
(
B)
The
magnitude
of
change
of
biochemical
and
cytologic
indices
in
lavage
fluids
at
each
test
concentration
for
each
animal.
(
C)
Results
shall
be
quantified
as
amount
of
constituent/
mL
of
lavage
fluid.
This
assumes
that
the
amount
of
lavage
fluid
recovered
is
a
representative
sample
of
the
total
lavage
fluid.
(
3)
Evaluation
of
data.
The
findings
from
this
acute
study
should
be
evaluated
in
the
context
of
preceding
and/
or
concurrent
toxicity
studies
and
any
correlated
functional
findings.
The
evaluation
shall
include
the
relationship
between
the
concentrations
of
the
test
substance
and
the
presence
or
absence
incidence,
and
severity
of
any
effects.
The
evaluation
should
include
appropriate
statistical
analyses,
for
example
parametric
tests
for
continuous
data
and
non­
parametric
tests
for
the
remainder.
Choice
of
analyses
should
consider
tests
appropriate
to
the
experimental
design,
including
repeated
measures.
The
report
must
include
concentration
response
curves
for
the
bronchoalveolar
lavage
and
tables
reporting
observations
at
each
concentration
level
for
necropsy
findings
and
gross,
general,
and
respiratory
system
histopathology.
(
h)
Reference.
For
additional
background
information
on
this
test
guideline
the
following
references
should
be
consulted.
These
references
are
available
for
inspection
at
the
TSCA
Nonconfidential
Information
Center,
Rm.
280
40
CFR
Ch.
I
(
7
 
1
 
98
Edition)
§
799.9346
NE
 
B607,
Environmental
Protection
Agency,
401
M
St.,
SW.,
Washington,
DC,
12
noon
to
4
p.
m.,
Monday
through
Friday,
except
legal
holidays.
(
1)
Burleson,
G.
R.,
Fuller,
L.
B.,

´
e
nache,
M.
G.,
and
Graham,
J.
A.
Poly
(
I):
poly
(
C)­
enhanced
alveolar
peritoneal
macrophage
phagocytosis:
Quantification
by
a
new
method
utilizing
fluorescent
beads.
Proceedings
of
the
Society
of
Experimental
Biology
and
Medicine.
184:
468
 
476
(
1987).
(
2)
Gardner,
D.
E.,
Crapo,
J.
D.,
and
McClellan,
R.
O.
(
Eds.)
Toxicology
of
the
Lung.
(
Raven
Press,
New
York,
1993)
pp.
i
 
xii,
1
 
30.
(
3)
Gilmour,
G.
I.,
and
Selgrade,
M.
K.
A
comparison
of
the
pulmonary
defenses
against
streptococcal
infection
in
rats
and
mice
following
O3
exposure:
Differences
in
disease
susceptibility
and
neutrophil
recruitment.
Toxicology
and
Applied
Pharmacology.
123:
211
 
218
(
1993).
(
4)
Henderson,
R.
F.,
Benson,
J.
M.,
Hahn,
F.
F.,
Hobbs,
C.
H.,
Jones,
R.
K.,
Mauderly,
J.
L.,
McClellan,
R.
O.,
and
Pickrell,
J.
A.
New
approaches
for
the
evaluation
of
pulmonary
toxicity:
Bronchoalveolar
lavage
fluid
analysis.
Fundamental
and
Applied
Toxicology.
5:
451
 
458
(
1985).
(
5)
Henderson,
R.
F.
Use
of
bronchoalveolar
lavage
to
detect
lung
damage.
Environmental
Health
Perspectives
56:
115
 
129
(
1984).
(
6)
Henderson,
R.
F.,
Rebar,
A.
H.,
Pickrell,
J.
A.,
and
Newton,
G.
J.
Early
damage
indicators
in
the
lung.
III.
Biochemical
and
cytological
response
of
the
lung
to
inhaled
metal
salts.
Toxicology
and
Applied
Pharmacology.
50:
123
 
136
(
1979).
(
7)
McClellan,
R.
O.
and
Henderson,
R.
F.
(
Eds.)
Second
edition.
Concepts
in
Inhalation
Toxicology.
(
Taylor
and
Francis,
Washington,
DC,
1995)
pp.
i
 
xxiv,
1
 
24,
441
 
470.
(
8)
Mery,
S.,
Gross,
E.
A.,
Joyner,
D.
R.,
Godo,
M.,
and
Morgan,
K.
T.
Nasal
Diagrams:
A
Tool
for
Recording
the
Distribution
of
Nasal
Lesions
in
Rats
and
Mice.
Toxicologic
Pathology.
22:
353
 
372
(
1994).
(
9)
Phalen,
R.
F.
(
Ed)
Methods
in
Inhalation
Toxicology.
(
CRC
Press,
Boca
Raton,
FL,
1997)
pp.
i
 
xii,
1
 
12.
(
10)
Renne,
R.
A.,
Gideon,
K.
M.,
Miller
R.
A.,
Mellick,
P.
W.,
and
Grumbein,
S.
L.
Histologic
methods
and
interspecies
variations
in
the
laryngeal
histology
of
F344/
N
rats
and
B6C3F1
mice.
Toxicology
and
Pathology.
20:
44
 
51
(
1992).
(
11)
Young,
J.
T.
Histopathologic
examination
of
the
rat
nasal
cavity.
Fundamental
and
Applied
Toxicology.
1:
309
 
312
(
1981).

§
799.9346
TSCA
subchronic
inhalation
toxicity.

(
a)
Scope
This
section
is
intended
to
meet
the
testing
requirements
under
section
4
of
TSCA.
In
the
assessment
and
evaluation
of
the
toxic
characteristics
of
a
gas,
volatile
substance,
or
aerosol
particulate,
determination
of
subchronic
inhalation
toxicity
may
be
carried
out
after
initial
information
on
toxicity
has
been
obtained
by
acute
testing.
The
subchronic
inhalation
study
has
been
designed
to
permit
the
determination
of
the
no­
observed­
effect
level
(
NOEL)
and
toxic
effects
associated
with
continuous
or
repeated
exposure
to
a
test
substance
for
a
period
of
90
days.
This
study
is
not
capable
of
determining
those
effects
that
have
a
long
latency
period
for
development
(
e.
g.,
carcinogenicity
and
life
shortening).
Extrapolation
from
the
results
of
this
study
to
humans
is
valid
only
to
a
limited
degree.
It
can,
however
provide
useful
information
on
health
hazards
likely
to
arise
from
repeated
exposures
by
the
inhalation
route
over
a
limited
period
of
time.
It
will
provide
information
on
target
organs
and
the
possibilities
of
accumulation
and
can
be
of
use
in
selecting
concentration
levels
for
chronic
studies
and
establishing
safety
criteria
for
human
exposure.
Hazards
of
inhaled
substances
are
influenced
by
the
inherent
toxicity
and
by
physical
factors
such
as
volatility
and
particle
size.
(
b)
Source.
The
source
material
used
in
developing
this
TSCA
test
guideline
is
the
OPPTS
harmonized
test
guideline
870.3465
(
June
1996
Public
Draft).
This
source
is
available
at
the
address
in
paragraph
(
h)
of
this
section.
(
c)
Definitions.
The
following
definitions
apply
to
this
section.
Aerodynamic
equivalent
diameter
is
defined
as
the
diameter
of
a
unit
density